Cathrine Rein Carlson

• Pejšková, Lucie; Solberg, Nina; Lunde, Marianne; Carlson, Cathrine Rein; Pedersen, Mona Elisabeth & Rønning, Sissel Beate (2026). Dual roles of syndecan-4 in regulating chicken fibrosis in vitro. Frontiers in Physiology. 17, p. 1–15. doi: 10.3389/fphys.2026.1782914. Full text in Research Archive Show summary

  Introduction: Wooden Breast (WB) is a myopathy affecting the skeletal breast muscle (Pectoralis major) in broiler chickens and is characterized by muscle fiber damage and varying degrees of fibrosis, ECM remodeling and inflammation. Several key factors such as pro-inflammatory cytokines like TGF-β1 and IL-1β, drive fibrosis in WB myopathy. We have previously shown that the expression of syndecan-4 (SDC4), a transmembrane proteoglycan, was increased in WB poultry skeletal muscle tissue. Furthermore, the ectodomain shedding of SDC4 by matrix metalloproteinases (MMPs) differed in the skeletal muscle satellite cells from isolated affected chickens compared with normal. While SDC4 has been previously implicated as a key driver for regulating myofibroblast activity in mechanically induced fibrosis in cardiac tissue, its specific role and shedding activity in chicken fibroblasts in relation to WB myopathy remain poorly understood.Methods: In vitro the overexpression system was used to mimic the previously detected increased SDC4 levels in WB and to further investigate fibrotic markers and syndecans at the gene and protein levels. Furthermore, we used blocking peptides derived from the SDC4 ectodomain and investigated their effect on SDC4 shedding and fibrotic markers. Additionally, TGF-β1 treatment, the main trigger of myofibroblasts, fibrosis, and cytokines, was used to investigate the connection between SDC4 shedding and fibrosis.Results and discussion: Overexpression of SDC4 in chicken fibroblasts reduced the gene and protein expression of fibrotic markers such as collagen I, collagen III and MMP-2. At the same time, we observed an increase in the gene expression of TGFB1 and IL1B. SDC4 overexpression also modulated intracellular proteins connected to fibrosis-relevant signaling pathways, with increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt. Moreover, we could observe a decreased production of ribosomal protein S6 and β-catenin. SDC4 overexpression induced shedding of a 15 kDa SDC4 fragment, while a 20 kDa fragment was produced at similar level regardless of overexpression. Modulation of ectodomain shedding using blocking peptides targeting various ectodomain regions (amino acids 76–106 and 100–130) significantly reduced the production of the 20 kDa endogenous fragment. However, it did not affect the expression level of the 15 kDa fragment. This contrasts with what we have seen with the same blocking peptides in chicken skeletal muscle satellite cells where the expression level of the 15 kDa fragment was reduced. When we stimulated the cells with TGF-β1, an increase in gene expression of fibrotic marker was observed as expected. No change in SDC4 gene expression was observed, while SDC4 fragment of 20 kDa was increased. Taken together, our results reveal a complex role of SDC4 and shedding in modulating fibrotic responses in chicken fibroblasts, suggesting a potential dual function where the full-length SDC4, produced by overexpression, may act as an anti-fibrotic regulator, while the TGF-β1 induced shed ectodomain fragments could promote pro-fibrotic and inflammatory processes.

• Mathiesen, Sabrina Bech; Støle, Thea Parsberg; Romaine, Andreas; Lunde, Marianne; Martinsen, Marita & Aronsen, Jan Magnus [Show all 10 contributors for this article] (2025). Syndecan-4 interacts directly with β-parvin and regulates the ILK-PINCH-β-parvin complex, the β-parvin-β-PIX-Rac1 axis, and cardiomyocyte geometry in a sex-dependent manner. Frontiers in Cell and Developmental Biology. 13. doi: 10.3389/fcell.2025.1569185. Full text in Research Archive Show summary

  Syndecan-4 is a ubiquitously expressed transmembrane proteoglycan that links the extracellular matrix to intracellular protein networks. It is located at stress-sensing structures in cardiomyocytes, including costameres and Z-discs, and in male mice, it is involved in the hypertrophic response to cardiac pressure overload. We have recently found female syndecan-4 KO cardiomyocytes, without challenge, to be smaller in area. Smaller cardiomyocytes with elongation defects have been observed in animal models with β-parvin deficiency, where the loss of this mechano-sensor disrupts the guanine nucleotide exchange factor (GEF) β-PIX-GTPase Rac1 axis, which is essential for proper cell elongation. β-parvin, together with integrin-linked kinase (ILK) and particularly interesting new cysteine-histidine-rich protein (PINCH), constitutes the IPP complex (ILK-PINCH-parvin), which is part of the integrin consensus adhesome. Interestingly, in a previous large cardiac interactome study, we have identified β-parvin, as well as ILK, β-PIX, and Rac1 as potential syndecan-4 partners. To better understand the syndecan-4-β-parvin association, we mapped their interaction and investigated the effect of syndecan-4 ablation on the IPP complex, the β-parvin-β-PIX-Rac1 axis, and cardiomyocyte geometry in both females and males. Interestingly, genetic ablation of syndecan-4 resulted in shorter cardiomyocytes in females only. The syndecan-4-β-parvin interaction was mapped to accessible sequences within the N-terminal, linker, and CH2 domains of β-parvin and the unique variable C2 cytoplasmic region of syndecan-4. Syndecan-4 ablation resulted in lower levels of membrane-localized β-parvin in both sexes and sex-specific differences in its associated partners ILK and PINCH, suggesting that syndecan-4 is linked to integrin signaling through the IPP complex. Finally, Rac1, known for its involvement in cell size regulation, and some of its regulators, β-PIX, RhoGDIα, and the serine/threonine kinase PAK, showed sex-specific alterations following syndecan-4 ablation. Altogether, our data suggest that syndecan-4 binds directly to β-parvin and regulates cardiomyocyte length, the IPP complex, and the β-parvin-β-PIX-Rac1 in a sex-dependent manner. These findings highlight a sex-specific role for syndecan-4 in cardiomyocyte structure, offering new insight into the molecular basis for sex differences in cardiac biology.

• Dahl, Anna Bergan; Carlson, Cathrine Rein; Laasmaa, Martin; Subramanian, Hariharan; Basson, Adelle & Li, Jia [Show all 16 contributors for this article] (2025). Activated CaMKIIδ translocates to the RyR nanodomain in cardiomyocytes. Cardiovascular Research (CVR). ISSN 0008-6363. 121(14), p. 2177–2188. doi: 10.1093/cvr/cvaf187. Full text in Research Archive Show summary

  Abstract Aims The heartbeat is triggered by the coordinated release of Ca2+ from the ryanodine receptor type-2 (RyR) in cardiomyocytes. Phosphorylation of RyR by Ca2+/calmodulin-dependent kinase IIδ (CaMKIIδ) fine-tunes this process in health, while hyperphosphorylation causes excessive, pathological Ca2+ release. We investigated how CaMKIIδ is spatially recruited and anchored to RyRs to achieve this functional regulation. Methods and results We employed confocal and dSTORM microscopy to investigate the macro- and nanoscale distribution of CaMKIIδ across cardiomyocytes, respectively. We linked positional rearrangement of the kinase during β-adrenergic stimulation (isoproterenol, Iso) to alterations in RyR phosphorylation and function (Ca2+ sparks), and the requirement of the CaMKIIδ anchoring protein AKAP18δ by knockdown/knockout. Confocal microscopy revealed that macroscale CaMKIIδ localization was not markedly altered during Iso-treatment, although a narrowing of its distribution around the Z-lines occurred, where the RyR reside. Higher resolution dSTORM imaging confirmed that local mobilization of CaMKIIδ by Iso decreased the distance from Z-lines and RyRs to the nearest CaMKIIδ by 28 and 12%, respectively. Functionally, kinase translocation into the RyR nanodomain was accompanied by increased channel phosphorylation and Ca2+ spark frequency. These actions were dependent on CaMKIIδ activity, since kinase translocation, RyR phosphorylation, and activation were all mimicked by the upstream activator of CaMKIIδ (8-CPT) and prevented by direct CaMKIIδ inhibitors (AIP, N1 peptide). A critical role of AKAP18δ in this mechanism was supported by immunoprecipitation experiments, which showed greater kinase binding to AKAP18δ during Iso-stimulation. Furthermore, loss of AKAP18δ by viral-mediated AKAP18δ knockdown or knockout prevented CaMKIIδ translocation to Z-lines. Microtubular disruption also blocked CaMKIIδ translocation. Conclusion Collectively, our results indicate that nanoscale movement of CaMKIIδ is closely associated with RyR activation following β-adrenergic stimulation. This translocation depends on an intact microtubular network and kinase binding to AKAP18δ.

• Rugolo, Ilde; Shen, Xin; Støle, Thea Parsberg; Dahl, Anna Bergan; Manfra, Ornella & Lunde, Marianne [Show all 12 contributors for this article] (2025). Identification of a Small Secretoneurin Derivative That Inhibits CaMKIIδ Activity. Journal of Cellular and Molecular Medicine. ISSN 1582-1838. 29(20). doi: 10.1111/jcmm.70900. Full text in Research Archive Show summary

  ABSTRACT Ventricular arrhythmias, a major cause of sudden cardiac death, are driven by Ca 2+ imbalance in cardiac myocytes, often linked to the overactivation of CaMKIIδ (Ca 2+ /calmodulin‐dependent protein kinase II delta). As such, inhibiting CaMKIIδ represents a promising therapeutic strategy. Based on our previous finding that native secretoneurin (SN) is a weak CaMKIIδ inhibitor, we aimed to develop a more potent derivative of SN to effectively counter aberrant Ca 2+ handling and arrhythmia risk. Various regions of SN were tested for CaMKII binding, identifying the core region as the sequence with the strongest binding capacity. This region was subsequently optimised with two phenylalanine substitutions, resulting in the SN derivative SN‐db‐short. Structural homology modeling and ELISA‐based assays revealed that SN‐db‐short bound both the substrate‐binding (S‐site) region of CaMKIIδ, in addition to the ATP‐binding region, with 8‐fold stronger binding compared to SN. Surface plasmon resonance experiments confirmed that SN‐db‐short exhibited a higher association rate and affinity for CaMKIIδ compared to SN. Consistent with only a partial calmodulin binding motif, SN‐db‐short showed no calmodulin binding, indicating selective CaMKIIδ inhibition. In functional studies, SN‐db‐short inhibited CaMKIIδ‐mediated phosphorylation of ryanodine receptor 2 and appeared more effective than SN in reducing the incidence of Ca 2+ sparks and Ca 2+ waves. SN‐db‐short also more markedly inhibited CaMKIIδ phosphorylation of phospholamban, slowed Ca 2+ reuptake, and reduced the magnitude of Ca 2+ transients during isoproterenol stimulation. SN‐db‐short effectively inhibits CaMKIIδ and significantly counters aberrant Ca 2+ handling in cardiomyocytes. Thus, this optimised peptide holds therapeutic potential for reducing the risk of ventricular arrhythmias.

• Støle, Thea Parsberg; Romaine, Andreas; Kleiberg, Thea; Høst, Vibeke; Lunde, Marianne & Hasic, Almira [Show all 14 contributors for this article] (2025). Cardiac implications of chicken wooden breast myopathy. Frontiers in Physiology. 16, p. 1–14. doi: 10.3389/fphys.2025.1547661. Full text in Research Archive Show summary

  Introduction: Wooden breast disease is a myopathy of the skeletal muscle in chickens of commercial breeding. Although the underlying pathophysiology remains unknown, we and others have previously shown that affected broilers display varying degrees of fibrosis, extracellular matrix (ECM) remodeling, inflammation, and alterations in various molecular signaling pathways. Other myopathy conditions, such as Duchenne muscular dystrophy, also affect the cardiac muscle and are associated with fibrosis and reduced cardiac function. To determine potential cardiac implications of wooden breast disease and identify whether molecular and fibrotic changes were similar to what we have previously found in the breast, we have investigated the hearts of commercial Ross 308 broilers. Methods: Hearts from male Ross 308 broiler chickens from mildly and severely wooden breast-affected chickens categorized in previous studies were analyzed. Ventricles from the hearts were analyzed by immunoblotting, real-time qPCR, near-infrared spectroscopy, Raman spectroscopy, and Masson`s trichrome histology. RNA sequencing was also conducted to identify the molecular footprint of the mildly and severely wooden breast-affected chickens. Results: Compared to mildly affected chickens, the severely wooden breast-affected chickens did not show an increase in heart weight, water-binding capacity, or macronutrient composition. The hearts did also not display any differences in fibrosis development, extracellular matrix gene expression, or typical cardiac and inflammatory markers. The severely affected chickens did, however, show a reduction in protein levels of biglycan and fibromodulin, as well as alterations in matrix metalloproteinase 2, Wnt ligands, mTOR signaling, heat shock protein 70, and muscle LIM protein. Functional enrichment analysis of RNA sequencing also suggested a different molecular footprint of biological processes and pathways between the two groups. Conclusion: Hearts from wooden breast-affected chickens did not display the same fibrotic alterations as those previously found in the breast. Despite few alterations detected in the markers and signaling molecules tested, RNA sequencing indicated a different molecular footprint in the hearts of severely compared to mildly wooden breast-affected chickens.

• Pejšková, Lucie; Pisconti, Addolorata; Lunde, Marianne; Ho, Ka Yi; Solberg, Nina & Koga, Shiori [Show all 10 contributors for this article] (2024). Wooden breast myopathy is characterized by satellite cell dysfunction and syndecan-4 shedding. Frontiers in Physiology. 15, p. 1–22. doi: 10.3389/fphys.2024.1513311. Full text in Research Archive Show summary

  Introduction: Skeletal muscle satellite cells (MuSCs or stem cells) play a crucial role in muscle development, maintenance, and regeneration, supporting both hypertrophy and regenerative myogenesis. Syndecans (SDCs) act as communication bridges within the muscle microenvironment, regulating interactions with extracellular matrix components and contributing significantly to tissue repair and inflammation. Specifically, syndecan-4 (SDC4) is involved in muscle regeneration at multiple stages. Methods: This study delves into the emerging challenge of wooden breast (WB) myopathy and its connection with SDC4. Our hypothesis proposes that disruptions in MuSC dynamics through SDC4 contribute to the increased incidence of breast myopathies observed in growing broilers. To test our hypothesis, non-affected and affected broilers were systematically selected, and the characteristics of WB myopathy were studied both in vitro and in vivo. SDC4 overexpression in MuSCs and blocking peptides (BPs) corresponding to the SDC4 ectodomain were used for investigating the role of SDC4 in muscle development and its shedding levels. Results and discussion: In vivo examination of affected muscles revealed smaller fibers and changes in metabolic pathways. In vitro studies unveiled disrupted proliferation of MuSCs in WB myopathy, accompanied by the downregulation of several muscle markers. Investigation of the potential role of SDC4 in the pathogenesis of WB myopathy revealed a decreased tendency in SDC4 gene expression and increased shedding of its ectodomain. Moreover, we showed that SDC4 overexpression is linked to reduced proliferation in MuSCs and affected myogenesis. We detected an impaired proliferation of WB-affected MuSCs, revealing critical insights into the dysfunctional state of these cells in myopathy. Additionally, by treating MuSCs with blocking peptides derived from the SDC4 ectodomain, we identified altered proliferation. Taken together, this work contributes with valuable knowledge on the molecular mechanisms underlying WB myopathy and the role of SDC4 in this chicken myopathy.

• Støle, Thea Parsberg; Lunde, Marianne; Gehmlich, Katja; Christensen, Geir; Louch, William Edward & Carlson, Cathrine Rein (2024). Exploring Syndecan-4 and MLP and Their Interaction in Primary Cardiomyocytes and H9c2 Cells. Cells. 13(11). doi: 10.3390/cells13110947. Full text in Research Archive Show summary

  The transmembrane proteoglycan syndecan-4 is known to be involved in the hypertrophic response to pressure overload. Although multiple downstream signaling pathways have been found to be involved in this response in a syndecan-4-dependent manner, there are likely more signaling components involved. As part of a larger syndecan-4 interactome screening, we have previously identified MLP as a binding partner to the cytoplasmic tail of syndecan-4. Interestingly, many human MLP mutations have been found in patients with hypertrophic (HCM) and dilated cardiomyopathy (DCM). To gain deeper insight into the role of the syndecan-4–MLP interaction and its potential involvement in MLP-associated cardiomyopathy, we have here investigated the syndecan-4–MLP interaction in primary adult rat cardiomyocytes and the H9c2 cell line. The binding of syndecan-4 and MLP was analyzed in total lysates and subcellular fractions of primary adult rat cardiomyocytes, and baseline and differentiated H9c2 cells by immunoprecipitation. MLP and syndecan-4 localization were determined by confocal microscopy, and MLP oligomerization was determined by immunoblotting under native conditions. Syndecan-4–MLP binding, as well as MLP self-association, were also analyzed by ELISA and peptide arrays. Our results showed that MLP-WT and syndecan-4 co-localized in many subcellular compartments; however, their binding was only detected in nuclear-enriched fractions of isolated adult cardiomyocytes. In vitro, syndecan-4 bound to MLP at three sites, and this binding was reduced in some HCM-associated MLP mutations. While MLP and syndecan-4 also co-localized in many subcellular fractions of H9c2 cells, these proteins did not bind at baseline or after differentiation into cardiomyocyte-resembling cells. Independently of syndecan-4, mutated MLP proteins had an altered subcellular localization in H9c2 cells, compared to MLP-WT. The DCM- and HCM-associated MLP mutations, W4R, L44P, C58G, R64C, Y66C, K69R, G72R, and Q91L, affected the oligomerization of MLP with an increase in monomeric at the expense of trimeric and tetrameric recombinant MLP protein. Lastly, two crucial sites for MLP self-association were identified, which were reduced in most MLP mutations. Our data indicate that the syndecan-4–MLP interaction was present in nuclear-enriched fractions of isolated adult cardiomyocytes and that this interaction was disrupted by some HCM-associated MLP mutations. MLP mutations were also linked to changes in MLP oligomerization and self-association, which may be essential for its interaction with syndecan-4 and a critical molecular mechanism of MLP-associated cardiomyopathy.

• Perdreau-Dahl, Harmonie; Lipsett, David Bruce; Frisk, Michael; Kermani, Fatemeh; Carlson, Cathrine Rein & Brech, Andreas [Show all 19 contributors for this article] (2023). BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes. Circulation Research. ISSN 0009-7330. 132(11), p. E188–E205. doi: 10.1161/CIRCRESAHA.122.321732. Full text in Research Archive
• Vistnes, Maria; Erusappan, Pugazendhi Murugan; Sasi, Athiramol; Nordén, Einar Sjaastad; Bergo, Kaja Knudsen & Romaine, Andreas [Show all 22 contributors for this article] (2023). Inhibition of the extracellular enzyme A disintegrin and metalloprotease with thrombospondin motif 4 prevents cardiac fibrosis and dysfunction. Cardiovascular Research (CVR). ISSN 0008-6363. 119(10), p. 1915–1927. doi: 10.1093/cvr/cvad078. Full text in Research Archive Show summary

  Abstract Aims Heart failure is a condition with high mortality rates, and there is a lack of therapies that directly target maladaptive changes in the extracellular matrix (ECM), such as fibrosis. We investigated whether the ECM enzyme known as A disintegrin and metalloprotease with thrombospondin motif (ADAMTS) 4 might serve as a therapeutic target in treatment of heart failure and cardiac fibrosis. Methods and results The effects of pharmacological ADAMTS4 inhibition on cardiac function and fibrosis were examined in rats exposed to cardiac pressure overload. Disease mechanisms affected by the treatment were identified based on changes in the myocardial transcriptome. Following aortic banding, rats receiving an ADAMTS inhibitor, with high inhibitory capacity for ADAMTS4, showed substantially better cardiac function than vehicle-treated rats, including ∼30% reduction in E/e′ and left atrial diameter, indicating an improvement in diastolic function. ADAMTS inhibition also resulted in a marked reduction in myocardial collagen content and a down-regulation of transforming growth factor (TGF)-β target genes. The mechanism for the beneficial effects of ADAMTS inhibition was further studied in cultured human cardiac fibroblasts producing mature ECM. ADAMTS4 caused a 50% increase in the TGF-β levels in the medium. Simultaneously, ADAMTS4 elicited a not previously known cleavage of TGF-β-binding proteins, i.e. latent-binding protein of TGF-β and extra domain A-fibronectin. These effects were abolished by the ADAMTS inhibitor. In failing human hearts, we observed a marked increase in ADAMTS4 expression and cleavage activity. Conclusion Inhibition of ADAMTS4 improves cardiac function and reduces collagen accumulation in rats with cardiac pressure overload, possibly through a not previously known cleavage of molecules that control TGF-β availability. Targeting ADAMTS4 may serve as a novel strategy in heart failure treatment, in particular, in heart failure with fibrosis and diastolic dysfunction.

• Mitsou, Ioli; Carlson, Cathrine Rein; Multhaupt, Hinke A B; Brakebusch, Cord & Couchman, John R. (2023). Two Transient Receptor Potential Channels at Focal Adhesions. Journal of Histochemistry and Cytochemistry. ISSN 0022-1554. 71(9), p. 495–508. doi: 10.1369/00221554231194119. Full text in Research Archive
• Pejšková, Lucie; Rønning, Sissel Beate; Kent, Matthew Peter; Solberg, Nina; Høst, Vibeke & To, Thu-Hien [Show all 13 contributors for this article] (2023). Characterization of wooden breast myopathy: a focus on syndecans and ECM remodeling. Frontiers in Physiology. 14, p. 1–17. doi: 10.3389/fphys.2023.1301804. Full text in Research Archive Show summary

  The skeletal muscle deformity of commercial chickens (Gallus gallus), known as the wooden breast (WB), is associated with fibrotic myopathy of unknown etiology. For future breeding strategies and genetic improvements, it is essential to identify the molecular mechanisms underlying the phenotype. The pathophysiological hallmarks of WB include severe skeletal muscle fibrosis, inflammation, myofiber necrosis, and multifocal degeneration of muscle tissue. The transmembrane proteoglycans syndecans have a wide spectrum of biological functions and are master regulators of tissue homeostasis. They are upregulated and shed (cleaved) as a regulatory mechanism during tissue repair and regeneration. During the last decades, it has become clear that the syndecan family also has critical functions in skeletal muscle growth, however, their potential involvement in WB pathogenesis is unknown.

• Skogestad, Jonas; Albert, Ingrid; Hougen, Karina; Lothe, Gustav B; Lunde, Marianne & Eken, Olav Søvik [Show all 25 contributors for this article] (2023). Disruption of Phosphodiesterase 3A Binding to SERCA2 Increases SERCA2 Activity and Reduces Mortality in Mice with Chronic Heart Failure. Circulation. ISSN 0009-7322. 147(16), p. 1221–1236. doi: 10.1161/CIRCULATIONAHA.121.054168. Full text in Research Archive
• Lunde, Per Kristian; Manfra, Ornella; Støle, Thea Parsberg; Lunde, Marianne; Martinsen, Marita & Carlson, Cathrine Rein [Show all 7 contributors for this article] (2023). Polyarginine Cell-Penetrating Peptides Bind and Inhibit SERCA2. Cells. 12(19). doi: 10.3390/cells12192358. Full text in Research Archive Show summary

  Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg9 and Arg11 exert off-target effects on cellular Ca2+ homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca2+ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca2+ leak remained unchanged, indicating that protracted Ca2+ removal from the cytosol stemmed from inhibition of the SR Ca2+ ATPase 2 (SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as SERCA2 is expressed in diverse cell types, the wide-ranging consequences of SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings.

• Lunde, Ida Gjervold; Aronsen, Jan Magnus; Melleby, Arne Olav; Strand, Mari Elen; Skogestad, Jonas & Bendiksen, Bård Andre [Show all 11 contributors for this article] (2022). Cardiomyocyte-specific overexpression of syndecan-4 in mice results in activation of calcineurin-NFAT signalling and exacerbated cardiac hypertrophy. Molecular Biology Reports. ISSN 0301-4851. 49(12), p. 11795–11809. doi: 10.1007/s11033-022-07985-y. Full text in Research Archive Show summary

  Abstract Background Cardiomyocyte hypertrophy is a hallmark of cardiac dysfunction in patients with aortic stenosis (AS), and can be triggered by left ventricular (LV) pressure overload in mice by aortic banding (AB). Syndecan-4 is a transmembrane heparan sulphate proteoglycan which is found increased in the myocardium of AS patients and AB mice. The role of syndecan-4 in cardiomyocyte hypertrophy is not well understood. Purpose of the study We developed mice with cardiomyocyte-specific overexpression of syndecan-4 ( Sdc4-Tg ) and subjected these to AB to examine the role of syndecan-4 in hypertrophy and activation of the pro-hypertrophic calcineurin-NFAT signalling pathway. Methods and results Sdc4-Tg mice showed exacerbated cardiac remodelling upon AB compared to wild type (WT). At 2–6 weeks post-AB, Sdc4-Tg and WT mice showed similar hypertrophic growth, while at 20 weeks post-AB, exacerbated hypertrophy and dysfunction were evident in Sdc4-Tg mice. After cross-breeding of Sdc4-Tg mice with NFAT-luciferase reporter mice, we found increased NFAT activation in Sdc4-Tg hearts after AB. Immunoprecipitation showed that calcineurin bound to syndecan-4 in Sdc4-Tg hearts. Isolated cardiomyocytes from Sdc4-Tg mice showed alterations in Ca 2+ fluxes, suggesting that syndecan-4 regulated Ca 2+ levels, and thereby, activating the syndecan-4-calcineurin complex resulting in NFAT activation and hypertrophic growth. Similarly, primary cardiomyocyte cultures from neonatal rats showed increased calcineurin-NFAT-dependent hypertrophic growth upon viral Sdc4 overexpression. Conclusion Our study of mice with cardiomyocyte-specific overexpression of Sdc4 have revealed that syndecan-4 is important for activation of the Ca 2+ -dependent calcineurin-NFAT signalling pathway, hypertrophic remodelling and dysfunction in cardiomyocytes in response to pressure overload.

• Støle, Thea Parsberg; Lunde, Marianne; Shen, Xin; Martinsen, Marita; Lunde, Per Kristian & Li, Jia [Show all 12 contributors for this article] (2022). The female syndecan-4^−/− heart has smaller cardiomyocytes, augmented insulin/pSer473-Akt/pSer9-GSK-3β signaling, and lowered SCOP, pThr308-Akt/Akt and GLUT4 levels. Frontiers in Cell and Developmental Biology. 10. doi: 10.3389/fcell.2022.908126. Full text in Research Archive Show summary

  Background: In cardiac muscle, the ubiquitously expressed proteoglycan syndecan-4 is involved in the hypertrophic response to pressure overload. Protein kinase Akt signaling, which is known to regulate hypertrophy, has been found to be reduced in the cardiac muscle of exercised male syndecan-4 −/− mice. In contrast, we have recently found that pSer473-Akt signaling is elevated in the skeletal muscle ( tibialis anterior, TA) of female syndecan-4 −/− mice. To determine if the differences seen in Akt signaling are sex specific, we have presently investigated Akt signaling in the cardiac muscle of sedentary and exercised female syndecan-4 −/− mice. To get deeper insight into the female syndecan-4 −/− heart, alterations in cardiomyocyte size, a wide variety of different extracellular matrix components, well-known syndecan-4 binding partners and associated signaling pathways have also been investigated. Methods: Left ventricles (LVs) from sedentary and exercise trained female syndecan-4 −/− and WT mice were analyzed by immunoblotting and real-time PCR. Cardiomyocyte size and phosphorylated Ser473-Akt were analyzed in isolated adult cardiomyocytes from female syndecan-4 −/− and WT mice by confocal imaging. LV and skeletal muscle (TA) from sedentary male syndecan-4 −/− and WT mice were immunoblotted with Akt antibodies for comparison. Glucose levels were measured by a glucometer, and fasting blood serum insulin and C-peptide levels were measured by ELISA. Results: Compared to female WT hearts, sedentary female syndecan-4 −/− LV cardiomyocytes were smaller and hearts had higher levels of pSer473-Akt and its downstream target pSer9-GSK-3β. The pSer473-Akt inhibitory phosphatase PHLPP1/SCOP was lowered, which may be in response to the elevated serum insulin levels found in the female syndecan-4 −/− mice. We also observed lowered levels of pThr308-Akt/Akt and GLUT4 in the female syndecan-4 −/− heart and an increased LRP6 level after exercise. Otherwise, few alterations were found. The pThr308-Akt and pSer473-Akt levels were unaltered in the cardiac and skeletal muscles of sedentary male syndecan-4 −/− mice. Conclusion: Our data indicate smaller cardiomyocytes, an elevated insulin/pSer473-Akt/pSer9-GSK-3β signaling pathway, and lowered SCOP, pThr308-Akt/Akt and GLUT4 levels in the female syndecan-4 −/− heart. In contrast, cardiomyocyte size, and Akt signaling were unaltered in both cardiac and skeletal muscles from male syndecan-4 −/− mice, suggesting important sex differences.

• Wanichawan, Pimthanya; Skogestad, Jonas; Lunde, Marianne; Støle, Thea Parsberg; Stensland, Maria & Nyman, Tuula Anneli [Show all 10 contributors for this article] (2021). Design of a proteolytically stable sodium-calcium exchanger 1 activator peptide for in vivo studies. Frontiers in Pharmacology. 12(638646). doi: 10.3389/fphar.2021.638646. Full text in Research Archive Show summary

  The cardiac sodium–calcium exchanger (NCX1) is important for normal Na + - and Ca 2+ -homeostasis and cardiomyocyte relaxation and contraction. It has been suggested that NCX1 activity is reduced by phosphorylated phospholemman (pSer68-PLM); however its direct interaction with PLM is debated. Disruption of the potentially inhibitory pSer68-PLM-NCX1 interaction might be a therapeutic strategy to increase NCX1 activity in cardiac disease. In the present study, we aimed to analyze the binding affinities and kinetics of the PLM-NCX1 and pSer68-PLM-NCX1 interactions by surface plasmon resonance (SPR) and to develop a proteolytically stable NCX1 activator peptide for future in vivo studies. The cytoplasmic parts of PLM (PLM cyt ) and pSer68-PLM (pSer68-PLM cyt ) were found to bind strongly to the intracellular loop of NCX1 (NCX1 cyt ) with similar K D values of 4.1 ± 1.0 nM and 4.3 ± 1.9 nM, but the PLM cyt -NCX1 cyt interaction showed higher on/off rates. To develop a proteolytically stable NCX1 activator, we took advantage of a previously designed, high-affinity PLM binding peptide (OPT) that was derived from the PLM binding region in NCX1 and that reverses the inhibitory PLM (S68D)-NCX1 interaction in HEK293. We performed N- and C-terminal truncations of OPT and identified PYKEIEQLIELANYQV as the minimum sequence required for pSer68-PLM binding. To increase peptide stability in human serum, we replaced the proline with an N-methyl-proline (NOPT) after identification of N-terminus as substitution tolerant by two-dimensional peptide array analysis. Mass spectrometry analysis revealed that the half-life of NOPT was increased 17-fold from that of OPT. NOPT pulled down endogenous PLM from rat left ventricle lysate and exhibited direct pSer68-PLM binding in an ELISA-based assay and bound to pSer68-PLM cyt with a K D of 129 nM. Excess NOPT also reduced the PLM cyt -NCX1 cyt interaction in an ELISA-based competition assay, but in line with that NCX1 and PLM form oligomers, NOPT was not able to outcompete the physical interaction between endogenous full length proteins. Importantly, cell-permeable NOPT-TAT increased NCX1 activity in cardiomyocytes isolated from both SHAM-operated and aorta banded heart failure (HF) mice, indicating that NOPT disrupted the inhibitory pSer68-PLM-NCX1 interaction. In conclusion, we have developed a proteolytically stable NCX1-derived PLM binding peptide that upregulates NCX1 activity in SHAM and HF cardiomyocytes.

• Carlson, Cathrine Rein; Aronsen, Jan Magnus; Dahl, Anna Bergan; Moutty, Marie Christine; Lunde, Marianne & Lunde, Per Kristian [Show all 22 contributors for this article] (2021). AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR. Circulation Research. ISSN 0009-7330. 130(1), p. 27–44. doi: 10.1161/CIRCRESAHA.120.317976. Full text in Research Archive Show summary

  Background: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. Methods: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. Results: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. Conclusions: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

• Rønning, Sissel Beate; Carlson, Cathrine Rein; Aronsen, Jan Magnus; Pisconti, Addolorata; Høst, Vibeke & Lunde, Marianne [Show all 11 contributors for this article] (2020). Syndecan-4–/– Mice Have Smaller Muscle Fibers, Increased Akt/mTOR/S6K1 and Notch/HES-1 Pathways, and Alterations in Extracellular Matrix Components. Frontiers in Cell and Developmental Biology. doi: 10.3389/fcell.2020.00730. Full text in Research Archive Show summary

  Background: Extracellular matrix (ECM) remodeling is essential for skeletal muscle development and adaption in response to environmental cues such as exercise and injury. The cell surface proteoglycan syndecan-4 has been reported to be essential for muscle differentiation, but few molecular mechanisms are known. Syndecan-4–/– mice are unable to regenerate damaged muscle, and display deficient satellite cell activation, proliferation, and differentiation. A reduced myofiber basal lamina has also been reported in syndecan-4–/– muscle, indicating possible defects in ECM production. To get a better understanding of the underlying molecular mechanisms, we have here investigated the effects of syndecan-4 genetic ablation on molecules involved in ECM remodeling and muscle growth, both under steady state conditions and in response to exercise. Methods: Tibialis anterior (TA) muscles from sedentary and exercised syndecan-4–/– and WT mice were analyzed by immunohistochemistry, real-time PCR and western blotting. Results: Compared to WT, we found that syndecan-4–/– mice had reduced body weight, reduced muscle weight, muscle fibers with a smaller cross-sectional area, and reduced expression of myogenic regulatory transcription factors. Sedentary syndecan-4–/– had also increased mRNA levels of syndecan-2, decorin, collagens, fibromodulin, biglycan, and LOX. Some of these latter ECM components were reduced at protein level, suggesting them to be more susceptible to degradation or less efficiently translated when syndecan-4 is absent. At the protein level, TRPC7 was reduced, whereas activation of the Akt/mTOR/S6K1 and Notch/HES-1 pathways were increased. Finally, although exercise induced upregulation of several of these components in WT, a further upregulation of these molecules was not observed in exercised syndecan-4–/– mice. Conclusion: Altogether our data suggest an important role of syndecan-4 in muscle development.

• Herum, Kate Louise Møller; Romaine, Andreas; Wang, Ariel; Melleby, Arne Olav; Strand, Mari Elen & Pacheco, Julian [Show all 16 contributors for this article] (2020). Syndecan-4 Protects the Heart From the Profibrotic Effects of Thrombin-Cleaved Osteopontin. Journal of the American Heart Association (JAHA). 9(3). doi: 10.1161/JAHA.119.013518. Full text in Research Archive Show summary

  Background Pressure overload of the heart occurs in patients with hypertension or valvular stenosis and induces cardiac fibrosis because of excessive production of extracellular matrix by activated cardiac fibroblasts. This initially provides essential mechanical support to the heart, but eventually compromises function. Osteopontin is associated with fibrosis; however, the underlying signaling mechanisms are not well understood. Herein, we examine the effect of thrombin‐cleaved osteopontin on fibrosis in the heart and explore the role of syndecan‐4 in regulating cleavage of osteopontin. Methods and Results Osteopontin was upregulated and cleaved by thrombin in the pressure‐overloaded heart of mice subjected to aortic banding. Cleaved osteopontin was higher in plasma from patients with aortic stenosis receiving crystalloid compared with blood cardioplegia, likely because of less heparin‐induced inhibition of thrombin. Cleaved osteopontin and the specific osteopontin peptide sequence RGDSLAYGLR that is exposed after thrombin cleavage both induced collagen production in cardiac fibroblasts. Like osteopontin, the heparan sulfate proteoglycan syndecan‐4 was upregulated after aortic banding. Consistent with a heparan sulfate binding domain in the osteopontin cleavage site, syndecan‐4 was found to bind to osteopontin in left ventricles and cardiac fibroblasts and protected osteopontin from cleavage by thrombin. Shedding of the extracellular part of syndecan‐4 was more prominent at later remodeling phases, at which time levels of cleaved osteopontin were increased. Conclusions Thrombin‐cleaved osteopontin induces collagen production by cardiac fibroblasts. Syndecan‐4 protects osteopontin from cleavage by thrombin, but this protection is lost when syndecan‐4 is shed in later phases of remodeling, contributing to progression of cardiac fibrosis.

• Mathiesen, Sabrina Bech; Lunde, Marianne; Stensland, Maria; Martinsen, Marita; Nyman, Tuula Anneli & Christensen, Geir Arve [Show all 7 contributors for this article] (2020). The cardiac syndecan-2 interactome. Frontiers in Cell and Developmental Biology. 8:792, p. 1–18. doi: 10.3389/fcell.2020.00792. Full text in Research Archive Show summary

  The extracellular matrix (ECM) is important in cardiac remodeling and syndecans have gained increased interest in this process due to their ability to convert changes in the ECM to cell signaling. In particular, syndecan-4 has been shown to be important for cardiac remodeling, whereas the role of its close relative syndecan-2 is largely unknown in the heart. To get more insight into the role of syndecan-2, we here sought to identify interaction partners of syndecan-2 in rat left ventricle. By using three different affinity purification methods combined with mass spectrometry (MS) analysis, we identified 30 novel partners and 9 partners previously described in the literature, which together make up the first cardiac syndecan-2 interactome. Eleven of the novel partners were also verified in HEK293 cells (i.e., AP2A2, CAVIN2, DDX19A, EIF4E, JPH2, MYL12A, NSF, PFDN2, PSMC5, PSMD11, and RRAD). The cardiac syndecan-2 interactome partners formed connections to each other and grouped into clusters mainly involved in cytoskeletal remodeling and protein metabolism, but also into a cluster consisting of a family of novel syndecan-2 interaction partners, the CAVINs. MS analyses revealed that although syndecan-2 was significantly enriched in fibroblast fractions, most of its partners were present in both cardiomyocytes and fibroblasts. Finally, a comparison of the cardiac syndecan-2 and -4 interactomes revealed surprisingly few protein partners in common.

• Mathiesen, Sabrina Bech; Lunde, Marianne; Aronsen, Jan Magnus; Romaine, Andreas; Kaupang, Anita & Martinsen, Marita [Show all 11 contributors for this article] (2019). The cardiac syndecan-4 interactome reveals a role for syndecan-4 in nuclear translocation of muscle LIM protein (MLP). Journal of Biological Chemistry. ISSN 0021-9258. 294(22), p. 8717–8731. doi: 10.1074/jbc.RA118.006423. Full text in Research Archive Show summary

  Costameres are signaling hubs at the sarcolemma and important contact points between the extracellular matrix and cell interior, sensing and transducing biomechanical signals into a cellular response. The transmembrane proteoglycan syndecan-4 localizes to these attachment points and has been shown to be important in the initial stages of cardiac remodeling, but its mechanistic function in the heart remains insufficiently understood. Here, we sought to map the cardiac interactome of syndecan-4 to better understand its function and downstream signaling mechanisms. By combining two different affinity purification methods with MS analysis, we found that the cardiac syndecan-4 interactome consists of 21 novel and 29 previously described interaction partners. Nine of the novel partners were further validated to bind syndecan-4 in HEK293 cells (i.e. CAVIN1/PTRF, CCT5, CDK9, EIF2S1, EIF4B, MPP7, PARVB, PFKM, and RASIP). We also found that 19 of the 50 interactome partners bind differently to syndecan-4 in the left ventricle lysate from aortic-banded heart failure (ABHF) rats compared with SHAM-operated animals. One of these partners was the well-known mechanotransducer muscle LIM protein (MLP), which showed direct and increased binding to syndecan-4 in ABHF. Nuclear translocation is important in MLP-mediated signaling, and we found less MLP in the nuclear-enriched fractions from syndecan-4-/- mouse left ventricles but increased nuclear MLP when syndecan-4 was overexpressed in a cardiomyocyte cell line. In the presence of a cell-permeable syndecan-4-MLP disruptor peptide, the nuclear MLP level was reduced. These findings suggest that syndecan-4 mediates nuclear translocation of MLP in the heart

• Udjus, Camilla; Fadila, Cero; Halvorsen, Bente; Behmen, Dina; Carlson, Cathrine Rein & Bendiksen, Bård Andre [Show all 14 contributors for this article] (2019). Caspase-1 induces smooth muscle cell growth in hypoxia-induced pulmonary hypertension. American Journal of Physiology - Lung cellular and Molecular Physiology. 316(6), p. L999–L1012. doi: 10.1152/ajplung.00322.2018. Full text in Research Archive Show summary

  Lung diseases with hypoxia are complicated by pulmonary hypertension, leading to heart failure and death. No pharmacological treatment exists. Increased proinflammatory cytokines are found in hypoxic patients, suggesting an inflammatory pathogenesis. Caspase-1, the effector of the inflammasome, mediates inflammation through activation of the proinflammatory cytokines interleukin (IL)-18 and IL-1β. Here, we investigate inflammasome-related mechanisms that can trigger hypoxia-induced pulmonary hypertension. Our aim was to examine whether caspase-1 induces development of hypoxia-related pulmonary hypertension and is a suitable target for therapy. Wild-type (WT) and caspase-1−/− mice were exposed to 10% oxygen for 14 days. Hypoxic caspase-1−/− mice showed lower pressure and reduced muscularization in pulmonary arteries, as well as reduced right ventricular remodeling compared with WT. Smooth muscle cell (SMC) proliferation was reduced in caspase-1-deficient pulmonary arteries and in WT arteries treated with a caspase-1 inhibitor. Impaired inflammation was shown in hypoxic caspase-1−/− mice by abolished pulmonary influx of immune cells and lower levels of IL-18, IL-1β, and IL-6, which were also reduced in the medium surrounding caspase-1 abrogated pulmonary arteries. By adding IL-18 or IL-1β to caspase-1-deficient pulmonary arteries, SMC proliferation was retained. Furthermore, inhibition of both IL-6 and phosphorylated STAT3 reduced proliferation of SMC in vitro, indicating IL-18, IL-6, and STAT3 as downstream mediators of caspase-1-induced SMC proliferation in pulmonary arteries. Caspase-1 induces SMC proliferation in pulmonary arteries through the caspase-1/IL-18/IL-6/STAT3 pathway, leading to pulmonary hypertension in mice exposed to hypoxia. We propose that caspase-1 inhibition is a potential target for treatment of pulmonary hypertension.

• Skogestad, Jonas; Aronsen, Jan Magnus; Tovsrud, Nils; Wanichawan, Pimthanya; Hougen, Karina & Stokke, Mathis Korseberg [Show all 10 contributors for this article] (2019). Coupling of the Na+/K+-ATPase to Ankyrin B controls Na+/Ca2+ exchanger activity in cardiomyocytes. Cardiovascular Research (CVR). ISSN 0008-6363. 116(1), p. 78–90. doi: 10.1093/cvr/cvz087. Full text in Research Archive
• Ottesen, Anett Hellebø; Carlson, Cathrine Rein; Eken, Olav Søvik; Sadredini, Mani; Myhre, Peder Langeland & Shen, Xin [Show all 24 contributors for this article] (2019). Secretoneurin Is an Endogenous Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor That Attenuates Ca2+-Dependent Arrhythmia. Circulation: Arrhythmia and Electrophysiology. ISSN 1941-3149. 12(4). doi: 10.1161/CIRCEP.118.007045. Full text in Research Archive Show summary

  Background: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. Methods: Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia–induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9–induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes. Results: SN levels were elevated in patients with CPVT and following ventricular arrhythmia–induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9–induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. Conclusions: SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.

• Shen, Xin; Brink, Jonas van den; Hou, Yufeng; Colli, Dylan; Le, Christopher & Kolstad, Terje R Selnes [Show all 12 contributors for this article] (2018). 3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes. Journal of Physiology. ISSN 0022-3751. 597(2), p. 399–418. doi: 10.1113/JP277360. Full text in Research Archive Show summary

  Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z‐axis and obscured detection by 2D techniques. Non‐overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single ‘rogue’ RyR. Internal CRUs were more tightly packed along z‐lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t‐tubules, which enabled quantification of dyadic and non‐dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.

• Simon, Dan N.; Wriston, Amanda; Fan, Qiong; Shabanowitz, Jeffrey; Florwick, Alyssa & Dharmaraj, Tejas [Show all 12 contributors for this article] (2018). OGT (O-GlcNAC transferase) selectively modifies multiple residues unique to Lamin A. Cells. 7(44). doi: 10.3390/cells7050044. Full text in Research Archive Show summary

  The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in LMNA cause over 12 diseases (‘laminopathies’). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is β-O-linked N-acetylglucosamine-(O-GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified O-GlcNAc transferase (OGT) enzyme showed robust O-GlcNAcylation of recombinant mature lamin A tails (residues 385–646), with no detectable modification of lamin B1, lamin C, or ‘progerin’ (Δ50) tails. Using mass spectrometry, we identified 11 O-GlcNAc sites in a ‘sweet spot’ unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly O-GlcNAc-modified at seven sites. By contrast, O-GlcNAcylation was undetectable on tails bearing deletion Δ50, which causes Hutchinson–Gilford progeria syndrome, and greatly reduced by deletion Δ35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Δ35, which does not remove the majority of identified O-GlcNAc sites, does remove potential OGT-association motifs (lamin A residues 622–625 and 639–645) homologous to that in mouse Tet1. These biochemical results are significant because they identify a novel molecular pathway that may profoundly influence lamin A function. The hypothesis that lamin A is selectively regulated by OGT warrants future testing in vivo, along with two predictions: genetic variants may contribute to disease by perturbing OGT-dependent regulation, and nutrient or other stresses might cause OGT to misregulate wildtype lamin A.

• Fan, Qiong; Moen, Anders; Anonsen, Jan Haug; Bindesbøll, Christian; Sæther, Thomas & Carlson, Cathrine Rein [Show all 7 contributors for this article] (2018). O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase. Biochemical and Biophysical Research Communications - BBRC. ISSN 0006-291X. 499(2), p. 354–360. doi: 10.1016/j.bbrc.2018.03.164. Full text in Research Archive
• Ottesen, Anett Hellebø; Carlson, Cathrine Rein; Louch, William Edward; Dahl, Mai Britt; Sandbu, Ragnhild Askeland & Johansen, Rune Forstrøm [Show all 15 contributors for this article] (2017). Glycosylated Chromogranin A in Heart Failure: Implications for Processing and Cardiomyocyte Calcium Homeostasis. Circulation: Heart Failure. ISSN 1941-3289. 10(2). doi: 10.1161/CIRCHEARTFAILURE.116.003675. Full text in Research Archive Show summary

  Background—Chromogranin A (CgA) levels have previously been found to predict mortality in heart failure (HF), but currently no information is available regarding CgA processing in HF and whether the CgA fragment catestatin (CST) may directly influence cardiomyocyte function. Methods and Results—CgA processing was characterized in postinfarction HF mice and in patients with acute HF, and the functional role of CST was explored in experimental models. Myocardial biopsies from HF, but not sham-operated mice, demonstrated high molecular weight CgA bands. Deglycosylation treatment attenuated high molecular weight bands, induced a mobility shift, and increased shorter CgA fragments. Adjusting for established risk indices and biomarkers, circulating CgA levels were found to be associated with mortality in patients with acute HF, but not in patients with acute exacerbation of chronic obstructive pulmonary disease. Low CgA-to-CST conversion was also associated with increased mortality in acute HF, thus, supporting functional relevance of impaired CgA processing in cardiovascular disease. CST was identified as a direct inhibitor of CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, and CST reduced CaMKIIδ-dependent phosphorylation of phospholamban and the ryanodine receptor 2. In line with CaMKIIδ inhibition, CST reduced Ca2+ spark and wave frequency, reduced Ca2+ spark dimensions, increased sarcoplasmic reticulum Ca2+ content, and augmented the magnitude and kinetics of cardiomyocyte Ca2+ transients and contractions. Conclusions—CgA-to-CST conversion in HF is impaired because of hyperglycosylation, which is associated with clinical outcomes in acute HF. The mechanism for increased mortality may be dysregulated cardiomyocyte Ca2+ handling because of reduced CaMKIIδ inhibition.

• Lubner, Joshua M.; Dodge-Kafka, Kimberly L.; Carlson, Cathrine Rein; Church, George M.; Chou, Michael F. & Schwartz, Daniel (2017). Cushing's syndrome mutant PKAL205R exhibits altered substrate specificity. FEBS Letters. ISSN 0014-5793. 591(3), p. 459–467. doi: 10.1002/1873-3468.12562. Full text in Research Archive
• Weber, Jonasz J; Golla, Matthias; Guaitoli, Giambattista; Wanichawan, Pimthanya; Hayer, Stefanie N & Krahl, Ann-Christin [Show all 15 contributors for this article] (2017). A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3. Brain. ISSN 0006-8950. 140(5), p. 1280–1299. doi: 10.1093/brain/awx039. Full text in Research Archive
• Hafver, Tandekile Lubelwana; Wanichawan, Pimthanya; Manfra, Ornella; Souza, Gustavo Antonio De; Lunde, Marianne & Martinsen, Marita [Show all 9 contributors for this article] (2017). Mapping the in vitro interactome of cardiac sodium (Na+ )-calcium (Ca2+ ) exchanger 1 (NCX1). Proteomics. ISSN 1615-9853. 17(17-18). doi: 10.1002/pmic.201600417. Full text in Research Archive
• Wanichawan, Pimthanya; Hodne, Kjetil; Hafver, Tandekile Lubelwana; Lunde, Marianne; Martinsen, Marita & Louch, William Edward [Show all 8 contributors for this article] (2016). Development of a high affinity peptide that prevents phospholemman (PLM) inhibition of the sodium-calcium exchanger 1 (NCX1). Biochemical Journal. ISSN 0264-6021. 473(15), p. 2413–2423. doi: 10.1042/BCJ20160465. Full text in Research Archive
• Lunde, Ida Gjervold; Herum, Kate Louise Møller; Carlson, Cathrine Rein & Christensen, Geir Arve (2016). Syndecans in heart fibrosis. Cell and Tissue Research. ISSN 0302-766X. 365(3), p. 539–552. doi: 10.1007/s00441-016-2454-2. Full text in Research Archive
• Manotheepan, Ravinea; Danielsen, Tore Kristian; Sadredini, Mani; Anderson, Mark E.; Carlson, Cathrine Rein & Lehnart, Stefan [Show all 8 contributors for this article] (2016). Exercise training prevents ventricular tachycardia in CPVT1 due to reduced CaMKII-dependent arrhythmogenic Ca2+ release. Cardiovascular Research (CVR). ISSN 0008-6363. 111(3), p. 295–306. doi: 10.1093/cvr/cvw095. Full text in Research Archive
• Hafver, Tandekile Lubelwana; Hodne, Kjetil; Wanichawan, Pimthanya; Aronsen, Jan Magnus; Dalhus, Bjørn & Lunde, Per Kristian [Show all 14 contributors for this article] (2016). Protein phosphatase 1c associated with the cardiac sodium calcium exchanger 1 regulates its activity by dephosphorylating Serine 68-phosphorylated phospholemman. Journal of Biological Chemistry. ISSN 0021-9258. 291(9), p. 4561–4579. doi: 10.1074/jbc.M115.677898. Full text in Research Archive Show summary

  The sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis. Serine 68 phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na+/K+ ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1, however, the molecular basis of this association is unknown. In the present study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pull-down experiments and peptide arrays constrained PP1c anchoring to the K-I/V-F-F motif in the first Ca2+ binding domain (CBD1) in NCX1. This binding site is also partially in agreement with the extended PP1 binding motif; K-I/V-F-F-X5-8-Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K-I/V-F-F motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K-I/V-F-F motif was mutated or when the PLM and PP1c binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1-(F407P) (mutated K-I/V-F-F motif) resulted in NCX1 current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.

• Tinholt, Mari; Stavik, Benedicte; Louch, William Edward; Carlson, Cathrine Rein; Sletten, Marit & Ruf, Wolfram [Show all 9 contributors for this article] (2015). Syndecan-3 and TFPI colocalize on the surface of endothelial-, smooth muscle-, and cancer cells. PLOS ONE. 10(1). doi: 10.1371/journal.pone.0117404. Full text in Research Archive
• Ottesen, Anett Hellebø; Louch, William Edward; Carlson, Cathrine Rein; Landsverk, Ole J. B.; Kurola, Jouni & Johansen, Rune Forstrøm [Show all 18 contributors for this article] (2015). Secretoneurin is a novel prognostic cardiovascular biomarker associated with cardiomyocyte calcium handling. Journal of the American College of Cardiology. ISSN 0735-1097. 65(4), p. 339–351. doi: 10.1016/j.jacc.2014.10.065. Full text in Research Archive
• Singh, Arpita; Rigatti, Marc; Carlson, Cathrine Rein; Moraru, Ion I. & Dodge-Kafka, Kimberly L. (2015). Analysis of AKAP7y Dimerization. Journal of Signal Transduction. ISSN 2090-1739. doi: 10.1155/2015/371626. Full text in Research Archive
• Rønning, Sissel Beate; Carlson, Cathrine Rein; Stang, Espen; Kolset, Svein Olav; Hollung, Kristin & Pedersen, Mona Elisabeth (2015). Syndecan-4 regulates muscle differentiation and is internalized from the plasma membrane during myogenesis. PLOS ONE. 10(6). doi: 10.1371/journal.pone.0129288. Full text in Research Archive Show summary

  The cell surface proteoglycan syndecan-4 has been reported to be crucial for muscle differentiation, but the molecular mechanisms still remain to be fully understood. During in vitro differentiation of bovine muscle cells immunocytochemical analyses showed strong labelling of syndecan-4 intracellularly, in close proximity with Golgi structures, in membranes of intracellular vesicles and finally, in the nuclear area including the nuclear envelope. Chase experiments showed that syndecan-4 was internalized from the plasma membrane during this process. Furthermore, when syndecan-4 was knocked down by siRNA more myotubes were formed, and the expression of myogenic transcription factors, β1-integrin and actin was influenced. However, when bovine muscle cells were treated with a cell-penetrating peptide containing the cytoplasmic region of syndecan-4, myoblast fusion and thus myotube formation was blocked, both in normal cells and in syndecan-4 knock down cells. Altogether this suggests that the cytoplasmic domain of syndecan-4 is important in regulation of myogenesis. The internalization of syndecan-4 from the plasma membrane during muscle differentiation and the nuclear localization of syndecan-4 in differentiated muscle cells may be part of this regulation, and is a novel aspect of syndecan biology which merits further studies.

• Skrbic, Biljana; Engebretsen, Kristin V Thunheim; Strand, Mari Elen; Lunde, Ida Gjervold; Herum, Kate Louise Møller & Marstein, Henriette [Show all 12 contributors for this article] (2015). Lack of collagen VIII reduces fibrosis and promotes early mortality and cardiac dilatation in pressure overload in mice. Cardiovascular Research (CVR). ISSN 0008-6363. 106(1), p. 32–42. doi: 10.1093/cvr/cvv041. Full text in Research Archive
• Berk, Jason M.; Simon, Dan N.; Jenkins-Houk, Clifton R.; Westerbeck, Jason W.; Grønning-Wang, Line Mariann & Carlson, Cathrine Rein [Show all 7 contributors for this article] (2014). The molecular basis of emerin-emerin and emerin-BAF interactions. Journal of Cell Science. ISSN 0021-9533. 127(18), p. 3956–3969. doi: 10.1242/jcs.148247. Full text in Research Archive
• Vistnes, Maria; Aronsen, Jan Magnus; Lunde, Ida Gjervold; Sjaastad, Ivar; Carlson, Cathrine Rein & Christensen, Geir Arve (2014). Pentosan polysulfate decreases myocardial expression of the extracellular matrix enzyme ADAMTS4 and improves cardiac function in vivo in rats subjected to pressure overload by aortic banding. PLOS ONE. 9(3). doi: 10.1371/journal.pone.0089621. Full text in Research Archive
• Aspelin, Trude; Eriksen, Morten; Ilebekk, Arnfinn; Cataliotti, Alessandro; Carlson, Cathrine Rein & Lyberg, Torstein (2014). ß-blockade abolishes the augmented cardiac tPA release induced by transactivation of heterodimerised bradykinin receptor-2 and b2-adrenergic receptor in vivo. Thrombosis and Haemostasis. ISSN 0340-6245. 112(5), p. 951–959. doi: 10.1160/TH14-01-0059. Full text in Research Archive
• Wanichawan, Pimthanya; Lubelwana, Tandekile; Hodne, Kjetil; Aronsen, Jan Magnus; Lunde, Ida Gjervold & Dalhus, Bjørn [Show all 13 contributors for this article] (2014). Molecular basis of calpain cleavage and inactivation of the sodium-calcium exchanger 1 in heart failure. Journal of Biological Chemistry. ISSN 0021-9258. 289(49), p. 33984–33998. doi: 10.1074/jbc.M114.602581. Full text in Research Archive Show summary

  Cardiac sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is central to the maintenance of normal Ca2+ homeostasis and contraction. Studies indicate that the Ca2+-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca2+ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met369 antibody identified a novel calpain cleavage site at Met369. Engineering NCX1-Met369 into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met369 inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met369 cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.

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• Pejšková, Lucie; Solberg, Nina; Lunde, Marianne; Carlson, Cathrine Rein; Pedersen, Mona Elisabeth & Rønning, Sissel Beate (2025). Dual roles of syndecan-4 in regulating fibrosis. Full text in Research Archive
• Pejšková, Lucie; Pisconti, Addolorata; Lunde, Marianne; Solberg, Nina; Høst, Vibeke & Kent, Matthew Peter [Show all 13 contributors for this article] (2024). Understanding Syndecan Functions in the Development of "Wooden Breast" Myopathy. Full text in Research Archive
• Pejšková, Lucie; Pisconti, Addolorata; Lunde, Marianne; Ho, Ka Yi Kelly; Solberg, Nina & Koga, Shiori [Show all 12 contributors for this article] (2024). Deciphering Syndecan Dynamics in the Skeletal Muscle Myopathy “Wooden Breast”. Full text in Research Archive
• Aronsen, Jan Magnus; Skogestad, Jonas; Albert, Ingrid; Melleby, Arne Olav & Carlson, Cathrine Rein (2023). Response by Aronsen et al to Letter Regarding Article, "Disruption of Phosphodiesterase 3A Binding to SERCA2 Increases SERCA2 Activity and Reduces Mortality in Mice With Chronic Heart Failure". Circulation. ISSN 0009-7322. 148(10), p. 857–858. doi: 10.1161/CIRCULATIONAHA.123.065884. Full text in Research Archive
• Pejšková, Lucie; Rønning, Sissel Beate; Kent, Matthew Peter; Wold, Jens Petter; Mosleth, Ellen Færgestad & Høst, Vibeke [Show all 11 contributors for this article] (2023). Shedding of syndecans play a crucial role in the extensive remodelling of the extracellular matrix in skeletal muscle myopathy in chicken. Full text in Research Archive
• Pejšková, Lucie; Rønning, Sissel Beate; Kent, Matthew Peter; Høst, Vibeke; Solberg, Nina & Kolset, Svein Olav [Show all 9 contributors for this article] (2023). Syndecans as potential regulators of the severe skeletal muscle myopathy. Full text in Research Archive
• Tønnessen, Theis; Christensen, Geir; Carlson, Cathrine Rein; Skrbic, Biljana & Sjaastad, Ivar (2022). Calcineurin-NFAT dynamics corresponds to cardiac remodeling during aortic banding and debanding, mimicking aortic valve replacement. Frontiers in Molecular Medicine for Cardiology. doi: 10.3389/fmmed.2022.980717. Full text in Research Archive
• Rønning, Sissel Beate; Carlson, Cathrine Rein; Aronsen, Jan Magnus; Høst, Vibeke; Pisconti, Addolorate & Lunde, Marianne [Show all 11 contributors for this article] (2019). Extracellular Matrix remodeling and multiple signaling pathways are regulated by syndecan-4 during myogenesis. Full text in Research Archive
• Ottesen, Anett Hellebø; Carlson, Cathrine Rein; Laver, Derek R; Myhre, Peder Langeland; Dalhus, Bjørn & Lunde, Per Kristian [Show all 14 contributors for this article] (2017). Secretoneurin is an Endogenous CaMKII Inhibitor That Attenuates Ca2+-Dependent Arrhythmogenesis. Circulation. ISSN 0009-7322. 136, p. A13900–A13900. Full text in Research Archive
• Rønning, Sissel Beate; Carlson, Cathrine Rein; Aronsen, Jan Magnus; Høst, Vibeke; Liland, Kristian Hovde & Stang, Espen [Show all 10 contributors for this article] (2017). Syndecan-4 is a skeletal muscle regenerator and is important for normal skeletal muscle fibre development. Full text in Research Archive
• Mathiesen, Sabrina Bech; Lunde, Marianne; Christensen, Geir Arve & Carlson, Cathrine Rein (2016). Mass spectrometry analysis of cytoplasmic binding partners of cardiac syndecan-2. Full text in Research Archive
• Wanichawan, Pimthanya; Hafver, Tandekile Lubelwana; Lunde, Marianne; Martinsen, Marita; Louch, William Edward & Sejersted, Ole M [Show all 7 contributors for this article] (2016). The N-terminal membrane occupation and recognition nexus (MORN) domains of junctophilin-2 bind to Na+/Ca2+ exchanger. Full text in Research Archive
• Aronsen, Jan Magnus; Skogestad, Jonas; Hougen, Karina; Lunde, Marianne; Lothe, Gustav B & Albert, Ingrid [Show all 13 contributors for this article] (2016). Physical coupling of PDE3A regulates SERCA2 activity in cardiomyocytes. Full text in Research Archive
• Albert, Ingrid; Skogestad, Jonas; Carlson, Cathrine Rein; Lunde, Marianne; Marshall, Serena & Lunde, Per Kristian [Show all 8 contributors for this article] (2016). Development of PDE3A-SERCA2 disruptor agents for SERCA2 activation. Full text in Research Archive
• Rønning, Sissel Beate; Carlson, Cathrine Rein; Høst, Vibeke; Aronsen, Jan Magnus; Liland, Kristian Hovde & Stang, Espen [Show all 9 contributors for this article] (2016). Syndecan-4 is important for normal skeletal muscle fibre development. Full text in Research Archive
• Rønning, Sissel Beate; Pedersen, Mona Elisabeth & Carlson, Cathrine Rein (2016). Syndecan-4 in skeletal muscle development and regeneration. Full text in Research Archive
• Hafver, Tandekile Lubelwana; Souza, Gustavo Antonio De; Wanichawan, Pimthanya; Lunde, Marianne; Martinsen, Marita & Sejersted, Ole M [Show all 7 contributors for this article] (2016). In vivo protein-protein interaction map of cardiac NCX1. Full text in Research Archive
• Mathiesen, Sabrina Bech; Lunde, Marianne; Christensen, Geir Arve & Carlson, Cathrine Rein (2016). Mass spectrometry analysis of cytoplasmic binding partners of cardiac syndecan - 2. Full text in Research Archive
• Manotheepan, Ravinea; Danielsen, Tore Kristian; Saberniak, Jorg; Sadredini, Mani; Anderson, Mark e. & Carlson, Cathrine Rein [Show all 11 contributors for this article] (2016). Exercise training as anti-arrhythmic therapy in cathecholaminergic polymorphic ventricular tachycardia type 1. Full text in Research Archive
• Ottesen, Anett; Carlson, Cathrine; Edwards, Andrew; Landsverk, Ole; Johansen, Rune & Moe, Morten [Show all 10 contributors for this article] (2015). Secretoneurin, a Novel Endogenous CaMKII Inhibitor, Augments Cardiomyocyte Calcium Handling and Inhibits Arrhythmogenic Calcium Release. Full text in Research Archive
• Hafver, Tandekile Lubelwana; Souza, Gustavo Antonio De; Lunde, Marianne; Martinsen, Marita; Sejersted, Ole M & Carlson, Cathrine Rein (2015). The in vitro NCX1 interactome. Full text in Research Archive
• Carlson, Cathrine Rein; Nygård, Ståle; Sirnes, Solveig; Jarstadmarken, Hilde; Martinsen, Marita & Lunde, Marianne [Show all 9 contributors for this article] (2015). A protein-protein interaction (PPI) map of cardiac syndecan-4. Full text in Research Archive
• Marshall, Serena; Aronsen, Jan Magnus; Skogestad, Jonas; Albert, Ingrid; Lothe, Gustav B & Lunde, Per Kristian [Show all 13 contributors for this article] (2015). Dual PDE3A coupling to AKAP18 and SERCA2 Controls SERCA2 Activity in adult cardiomyocytes. Full text in Research Archive
• Hafver, Tandekile Lubelwana; Souza, Gustavo Antonio De; Lunde, Marianne; Martinsen, Marita; Sejersted, Ole M & Carlson, Cathrine Rein (2015). Identification of the in vitro NCX1 interactome. Full text in Research Archive
• Aronsen, Jan Magnus; Skogestad, Jonas; Hougen, Karina; Lunde, Marianne; Lothe, Gustav B & Albert, Ingrid [Show all 12 contributors for this article] (2015). Physical coupling between SERCA2 and PDE3A regulates SERCA2 activity in cardiomyocytes. Full text in Research Archive
• Ottesen, Anett Hellebø; Carlson, Cathrine Rein; Louch, William Edward; Johansen, Rune F.; Stridsberg, Mats & Jarstadmarken, Hilde [Show all 12 contributors for this article] (2015). Chromogranin A is a potent prognostic biomarker in acute heart failure, and its fragment catestatin regulates cardiomyocyte calcium homeostasis by CaMKII inhibition. Full text in Research Archive
• Ottesen, Anett Hellebø; Carlson, Cathrine Rein; Louch, William Edward; Dahl, Mai Britt; Sandbu, Ragnhild Askeland & Johansen, Rune F. [Show all 15 contributors for this article] (2015). Enhanced myocardial chromogranin A glycosylation in acute heart failure: reduced processing and potential impact on cardiomyocyte calcium homeostasis. Full text in Research Archive
• Albert, Ingrid; Børstad, Mira Halvorsen; Carlson, Cathrine Rein; Calejo, Anna; Skogestad, Jonas & Lunde, Marianne [Show all 10 contributors for this article] (2015). Development of SERCA2 activating agents. Full text in Research Archive
• Manotheepan, Ravinea; Danielsen, Tore Kristian; Sadredini, Mani Nikoo; Anderson, Mark e.; Carlson, Cathrine Rein & Lehnart, Stefan [Show all 8 contributors for this article] (2015). Exercise training reduced CaMKII-dependent arrhythmogenic Ca2+ release and prevents ventricular tachycardia in CPVT 1. Full text in Research Archive
• Rønning, Sissel Beate; Carlson, Cathrine Rein; Hollung, Kristin; Pedersen, Mona Elisabeth; Stang, Espen & Kolset, Svein Olav [Show all 7 contributors for this article] (2015). Syndecan-4 in skeletal muscle development and regeneration. Full text in Research Archive
• Rønning, Sissel Beate; Carlson, Cathrine Rein; Pedersen, Mona Elisabeth; Hollung, Kristin; Kolset, Svein Olav & Stang, Espen (2015). Syndecan-4 in skeletal muscle development and regeneration. Full text in Research Archive
• Wanichawan, Pimthanya; Hodne, Kjetil; Hafver, Tandekile Lubelwana; Lunde, Marianne; Martinsen, Marita & Louch, William Edward [Show all 8 contributors for this article] (2015). Development of a high affinity phospholemman binding peptide that reverses phosphorylated serine 68 phospholemman inhibition of the cardiac sodium-calcium exchanger 1. Full text in Research Archive
• Ottesen, Anett Hellebø; Carlson, Cathrine Rein; Edwards, Andrew; Landsverk, Ole J. B.; Johansen, Rune Forstrøm & Moe, Morten Kaare [Show all 12 contributors for this article] (2015). Secretoneurin,a novel endogenous CaMKII inhibitor, augments cardiomyocyte Calcium handling and inhibits arrhythmogenic Calcium release. Biophysical Journal. ISSN 0006-3495. 108, p. 340a–340a. Full text in Research Archive
• Hodne, Kjetil; Wanichawan, Pimthanya; Lubelwana, Tandekile; Louch, William Edward; Lunde, Marianne & Mathisen, Marita [Show all 8 contributors for this article] (2015). Liberation of Pser68-Plm inhibition of NCX1 by an optimized anchoring disruptor peptide. Biophysical Journal. ISSN 0006-3495. 108, p. 434a–434a. Full text in Research Archive
• Hafver, Tandekile Lubelwana; Souza, Gustavo Antonio De; Lunde, Marianne; Martinsen, Marita; Sejersted, Ole M & Carlson, Cathrine Rein (2015). Mapping the in vitro interactome of NCX1. Full text in Research Archive
• Rønning, Sissel Beate; Carlson, Cathrine Rein; Stang, Espen; Kolset, Svein Olav; Christensen, Geir Arve & Hollung, Kristin [Show all 7 contributors for this article] (2015). Syndecan-4 is important for skeletal muscle development. Full text in Research Archive
• Pedersen, Mona Elisabeth; Rønning, Sissel Beate; Carlson, Cathrine Rein; Stang, Espen; Kolset, Svein Olav & Hollung, Kristin (2015). Syndecan-4 is important for skeletal muscle development. Full text in Research Archive
• Børstad, Mira Halvorsen; Albert, Ingrid; Lunde, Marianne; Carlson, Cathrine Rein; Sjaastad, Ivar & Aronsen, Jan Magnus (2015). Molecular mechanisms for PDE3A mediated inhibition of SERCA2 activity. Full text in Research Archive
• Hafver, Tandekile Lubelwana; Wanichawan, Pimthanya; Hodne, Kjetil; Aronsen, Jan Magnus; Dalhus, Bjørn & Lunde, Marianne [Show all 12 contributors for this article] (2015). Anchoring onto NCX1 facilitates dephosphorylation of P-SER68-PLM. Biophysical Journal. ISSN 0006-3495. 108, p. 584a–584a. Full text in Research Archive
• Hafver, Tandekile Lubelwana; Hodne, Kjetil; Wanichawan, Pimthanya; Aronsen, Jan Magnus; Dalhus, Bjørn & Lunde, Per Kristian [Show all 12 contributors for this article] (2015). PP1 associated with cardiac NCX1 regulates exchange activity by dephosphorylating pSer-68-PLM. Full text in Research Archive
• Aronsen, Jan Magnus; Skogestad, Jonas; Hougen, Karina; Lunde, Marianne; Lothe, Gustav B & Lunde, Per Kristian [Show all 10 contributors for this article] (2014). Hypokalemia induce CA2+ waves by lowering NKA alpha 2 activity in rat ventricular cardiomyocytes. Full text in Research Archive
• Aronsen, Jan Magnus; Skogestad, Jonas; Hougen, Karina; Lunde, Marianne; Lothe, Gustav B & Lunde, Per Kristian [Show all 10 contributors for this article] (2014). Physical coupling of PDE3A to SERCA2 regulates SERCA2 activity in cardiomyoyctes. Full text in Research Archive
• Ottesen, Anett Hellebø; Louch, William Edward; Carlson, Cathrine Rein; Landsverk, Ole J. B.; Kurola, Jouni & Johansen, Rune Forstrøm [Show all 18 contributors for this article] (2014). Secretoneurin provides independent prognostic information in patients with heart failure and cardiac arrest, and has a direct inhibitory effect on diastolic Ca2+ leak and arrhythmogenic Ca2+ waves via CaMKII inhibition. Circulation Research. ISSN 0009-7330. 115. Full text in Research Archive
• Hafver, Tandekile Lubelwana; Wanichawan, Pimthanya; Hodne, Kjetil; Aronsen, Jan Magnus; Dalhus, Bjørn & Lunde, Marianne [Show all 10 contributors for this article] (2014). Molecular analysis of PP1 anchoring to NCX1 macromolecular complex. Full text in Research Archive Show summary

  Introduction: The cardiac Na+/Ca2+ exchanger 1 (NCX1) modulates excitation-contraction coupling and contributes to Ca2+ removal in cardiomyocytes. Altered expression and activity of NCX1 is linked to dysfunctional Ca2+ handling in chronic heart disease. Consequently, modulation of NCX1 activity has been proposed as a therapeutic target, but detailed understanding of NCX1 regulation is warranted. It has been shown that NCX1 exists in a macromolecular complex which would allow for regulation of its activity. NCX1 is also regulated by its accessory protein phospholemman (PLM) which inhibits NCX1 activity when phosphorylated at serine 68 (pSer68-PLM). Protein phosphatase 1 (PP1) dephosphorylates pSer68-PLM, and thus indirectly regulates NCX1 activity. Although no anchoring site is known, PP1 co-precipitates NCX1. We hypothesised that PP1 regulates NCX1 activity and that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer68-PLM dephosphorylation. We set out to identify PP1 binding sites to the pPLM-NCX1 complex and assess the biological function of the interaction. Methods and results: NCX1 co-precipitated PP1 and PLM in wild-type rat left ventricular lysates and PLM-NCX1 co transfected HEK 293 cells. NCX1 protein levels were upregulated, PP1/NCX1 protein levels were downregulated while pSer68-PLM/total PLM protein levels were upregulated in our pressure overload model. The proximity ligation assay showed that cardiac NCX1 and PP1 colocalize within <40nm in isolated cardiomyocytes. Bioinformatic analysis revealed three putative PP1 binding sites on NCX1. Overlay assays of NCX1 and PP1α showed that PP1α bound directly to the consensus sequence R/KVxF in CBD1 (calcium binding domain 1) in NCX1, which is preserved across isoforms. The reciprocal NCX1 binding site was identified within residues 235-260 in PP1α, a region which harbours the aspartic acid, leucine and arginine docking motif. Binding data were confirmed by immunoprecipitations, mutation analysis of NCX1-GFP and FLAG-HIS-PP1α deletion mutants expressed in HEK 293 cells and in pulldown assays using NCX1 biotinylated peptides. We have generated a peptide docking model of the R/KVxF motif bound close to the PP1α active site. Single and double mutations of residues in the docking motif confirmed the interaction. Biacore analysis indicated that the NCX1-PPIα binding is strong. In competition assay using NCX1, PP1 and PLM co transfected HEK cells a NCX1 peptide disruptor was able to displace the interaction, p<0.05, indicating that PP1 dephosphorylates pSer68-PLM when anchored to NCX1. Conclusion: The R/KVxF motif in CBD1 of NCX1 facilitates PP1 anchoring to NCX1 and is directly and functionally required for pSer68-PLM dephosphorylation.

• Hafver, Tandekile Lubelwana; Wanichawan, Pimthanya; Hodne, Kjetil; Aronsen, Jan Magnus; Dalhus, Bjørn & Lunde, Marianne [Show all 10 contributors for this article] (2014). Molecular analysis of the pPLM-NCX1-PP1 complex. Full text in Research Archive Show summary

  Introduction: The cardiac Na+/Ca2+ exchanger (NCX1) is an ion transporting membrane protein which modulates excitation-contraction coupling and contributes to Ca2+ removal in cardiomyocytes. Altered expression and activity of NCX1 has been linked to dysfunctional Ca2+ handling in chronic heart disease. Consequently, modulation of NCX1 activity has been proposed as a therapeutic target, but detailed understanding of NCX1 regulation is warranted. In the rat heart, NCX1 exists in a macromolecular complex comprising of kinases, phosphatases, anchoring and adaptor proteins, which enables fine tuning of signals which converge on NCX1. NCX1 is also regulated by its accessory protein phospholemman (PLM). When phosphorylated at Serine 68 by PKA and PKC, PLM binds to and inhibits NCX1 activity. It has been shown that phospho Serine 68-PLM (pSer68-PLM) is dephosphorylated by protein phosphatase 1 (PP1) and furthermore PP1 co-precipitate with NCX1, however, no anchoring site is known. We hypothesised that PP1 regulates NCX1 activity and that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer68-PLM dephosphorylation. We set out to identify PP1 binding sites to the pPLM-NCX1 complex and assess the biological function of the interaction. Methods and results: We have shown that NCX1 exists in a macromolecular complex with PP1 and PLM in wild-type rat left ventricular lysates and in transfected HEK cells. The proximity ligation assay was used to show that cardiac NCX1 and PP1 colocalize within <40nm in isolated cardiomyocytes. Bioinformatic analysis revealed three putative PP1 binding sites on NCX1. Overlay assays of NCX1 and PP1α showed that PP1α bound directly to the consensus sequence R/KVxF in CBD1 (calcium binding region 1) in NCX1, which is preserved across isoforms. The reciprocal NCX1 binding site was identified within residues 235-260 in PP1, a region which harbours the aspartic acid (Asp), leucine (Leu) and arginine (Arg) docking motif. Immunoprecipitations and mutation analysis of NCX1-GFP and FLAG-HIS-PP1α deletion mutants expressed in HEK 293 cells confirmed this interaction. A peptide docking model of the R/KVxF motif bound to the PP1α active site showed that the Asp 242 and Leu 243, identified from the PP1α overlay, make contact with the Valine (Val) and Phenylalanine (Phe) in the R/KVxF motif. Single and double mutations of Asp 242, Leu 243 and Arg 257 confirmed the interaction. Biacore analysis also showed that recombinant NCX1 cytoplasmic part bound to GST-PP1α recombinant protein. Conclusion: PP1 anchored directly to NCX1 through an R/KVxF motif in CBD1, whereas NCX1 bound to a DL motif within residues 235-260 in PP1. The biological role of the R/KVxF motif in PLM and NCX1 regulation is currently being assessed.

• Rønning, Sissel Beate; Carlson, Cathrine Rein; Kolset, Svein Olav; Stang, Espen; Hollung, Kristin & Pedersen, Mona Elisabeth (2014). The cell surface proteoglycan syndecan 4 is important for in vitro muscle cell differentiation. Full text in Research Archive
• Ottesen, Anett Hellebø; Carlson, Cathrine Rein; Louch, William Edward; Stridsberg, Mats; Høiseth, Arne Didrik & Brynildsen, Jon [Show all 10 contributors for this article] (2014). Chromogranin A is a potent risk marker in acute heart failure and directly associated with cardiomyocyTe Ca2+ handling. European Heart Journal. ISSN 0195-668X. 35, p. 328–328. Full text in Research Archive
• Hodne, Kjetil; Wanichawan, Pimthanya; Hafver, Tandekile Lubelwana; Aronsen, Jan Magnus; Lunde, Ida Gjervold & Lunde, Marianne [Show all 12 contributors for this article] (2014). Identification and functional role of calpain cleavage site in Na+-Ca2+ exchanger 1 (NCX1). Biophysical Journal. ISSN 0006-3495. 106, p. 762a–762a. Full text in Research Archive
• Rønning, Sissel Beate; Carlson, Cathrine Rein; Kolset, Svein Olav; Stang, Espen; Hollung, Kristin & Pedersen, Mona Elisabeth (2014). Syndecan-4 in muscel development. Full text in Research Archive
• Rønning, Sissel Beate; Carlson, Cathrine Rein; Kolset, Svein Olav; Stang, Espen; Hollung, Kristin & Pedersen, Mona Elisabeth (2014). Syndecan-4 is involved in regulation of muscle differentiation at multiple stages and by different mechanisms. Full text in Research Archive
• Ottesen, Anett Hellebø; Carlson, Cathrine Rein; Louch, William Edward; Høiseth, Arne Didrick; Brynildsen, Jon & Stridsberg, Mats [Show all 9 contributors for this article] (2014). Chromogranin A directly modifies cardiomyocyte Ca2+ homeostasis via CaMKII inhibition and is a strong prognostic biomarker in acute heart failure. Full text in Research Archive
• Hafver, Lubelwana; Wanichawan, Pimthanya; Hodne, Kjetil; Aronsen, Jan Magnus; Dalhus, Bjørn & Lunde, Marianne [Show all 10 contributors for this article] (2014). Molecular analysis of the phospho-PLM-NCX1-PP1 complex. Full text in Research Archive
• Wanichawan, Pimthanya; Hafver, Tandekile Lubelwana; Lunde, Ida Gjervold; Aronsen, Jan Magnus; Lunde, Marianne & Kvaløy, Heidi [Show all 10 contributors for this article] (2013). Calpain induces cleavage of the cardiac Na+ - Ca2+ exchanger 1 (NCX1) in pressure-overloaded hearts: Molecular interactions and identification of a calpain cleavage site. Full text in Research Archive
• Ølnes, Åsa Schawlann; Bjune, Katrine; Strøm, Thea Bismo & Carlson, Cathrine Rein (2026). Genetic variants in HDL cholesterol extremes: Functional characterization, population-based screening, and clinical interpretation. Universitetet i Oslo. ISSN 9788234808418. Full text in Research Archive Show summary

  Lipids are essential molecules in all living organisms, contributing to cell structure, energy storage, and signaling. Because they are insoluble in water, they must be transported in the bloodstream as lipoproteins, in a tightly regulated process. Disruption of this process can lead to abnormal lipid levels and increased risk of cardiovascular disease. High-density lipoprotein (HDL), one type of lipoprotein, has traditionally been considered protective against cardiovascular disease due to its role in reverse cholesterol transport. However, this view has been challenged, as HDL-cholesterol levels alone may not reflect the functional properties of HDL. This thesis is situated within the field of cardiovascular genetics and lipid metabolism and aims to investigate genetic causes of extreme HDL-cholesterol levels and their functional consequences. Specifically, we examined the prevalence and impact of rare variants in key HDL-related genes and assessed how these variants affect protein function. The study combines population-based data from the Tromsø Study and UK Biobank with functional analyses of genetic variants in CETP, ABCA1, APOA1, LCAT, PLTP and SCARB1. Participants with extremely low or high HDL-cholesterol levels were genetically characterized, and selected variants were investigated using in vitro assays to assess their effects on protein expression and activity. We found that monogenic causes of extreme HDL-cholesterol levels are rare. Functional analyses revealed that different variants disrupt HDL metabolism through distinct molecular mechanisms, including impaired protein trafficking and reduced cholesterol transport. These findings enabled reclassification of several genetic variants and improved interpretation of their clinical relevance. Overall, this work demonstrates that integrating functional data with population-based genetic information is essential for understanding HDL biology. The results contribute to improved diagnostics and interpretation of genetic variants in HDL-related disorders.

• Pejšková, Lucie; Carlson, Cathrine Rein; Rønning, Sissel Beate & Pedersen, Mona Elisabeth (2025). Chicken Health: Preventing wooden breast by identifying and editing regulators of severe chicken myopathy. Universitetet i Oslo. ISSN 9788234806667. Full text in Research Archive Show summary

  With the rapid growth and intensive breeding of chickens, a severe myopathy known as wooden breast (WB) has emerged during the last decade. This myopathy is a myodegenerative disease in chickens, characterized by hardened muscle fibers, extensive extracellular matrix (ECM) remodeling, skeletal muscle fibrosis, tissue degeneration, and disrupted metabolic processes. This PhD thesis aims to investigate the molecular mechanisms driving the progression of WB, with a special focus on ECM remodeling and the roles of syndecans, a family of transmembrane proteoglycans. Syndecan shedding, which involves the proteolytic release of their extracellular domains, is considered fundamental in regulating fibrotic processes, influencing cellular signaling pathways and ECM deposition. Syndecan shedding is also involved in muscle development, regeneration, and homeostasis. Using a multi-omics approach with immunostaining, histology, gene and protein expression analyses, transcriptomics, and proteomics, the research provides a comprehensive exploration of ECM proteins, metabolic processes, myogenesis, syndecan expression and their shedding in the pathophysiology of WB, both in vivo and in vitro. The involvement of syndecans in WB myopathy is a novel area of study. This thesis highlights their various roles in cell-specific activities. In Paper I, we investigated the histological and molecular characteristics of WB myopathy in chickens, revealing extensive ECM remodeling in muscle tissue. This included increased expression of collagens, small leucine-rich proteins, glycosaminoglycan biosynthesis, ECM-receptor interactions, and cell adhesion, along with a disrupted collagen structure. Additionally, the study characterized syndecan expression and their shedding in WB myopathy. The investigation revealed elevated gene expression of syndecan-1 (SDC1) and syndecan-4 (SDC4) in WB myopathy, with all syndecan family members exhibiting increased shedding in WB-affected chickens. This was accompanied by enhanced matrix metalloproteinase (MMP) activity and alterations in key signaling pathways, including MAPK, Akt, and Wnt. These findings suggest that syndecans contribute significantly to ECM remodeling and fibrosis in WB myopathy, highlighting them as a potential part of further research interest. Paper II in this thesis focused on the role of muscle dysfunction and the potential role of syndecan-4 in the development of WB myopathy, using both an in vivo and in vitro approach. In this study, significant metabolic alterations linked to glycolysis, oxidative phosphorylation and metabolic pathways were identified in WB. Affected chickens showed varied muscle fiber sizes, more PAX7-expressing muscle stem cells, but no change in the proliferation marker PCNA, suggesting ongoing regeneration, as well as a defect in proliferation. Muscle satellite cells (MuSCs) derived from WB-affected chickens showed impaired proliferation, retained differentiation potential, and had altered gene expression of myogenic markers. Furthermore, we observed increased shedding of syndecan-4 in WB-affected MuSCs. The overexpression of SDC4 in non-affected MuSCs resulted in decreased proliferation and lower levels of myogenesis markers. In this paper, we found that the use of blocking peptides derived from syndecan-4 ectodomain partially enhanced the proliferation of MuSCs and reduced syndecan-4 shedding, indicating the pivotal role of syndecan-4 in MuSCs with an impact on WB. Paper III focused on the hallmark of WB – fibrosis and the role of syndecan-4. An in vitro approach using embryonic chicken fibroblasts revealed that overexpression of SDC4 reduced the expression of fibrotic markers like collagen I and III, while inducing shedding of syndecan-4 and increasing gene expression of pro-inflammatory cytokines such as transforming growth factor-beta 1 (TGF-β1, TGFB1 gene) and interleukin-1 beta (IL-1β, IL1B gene). Additionally, the study explored the use of blocking peptides derived from the syndecan-4 ectodomain and found that they significantly decreased syndecan-4 shedding, as evidenced by the 20 kDa cytoplasmic remaining fragment on the Western blot, without affecting fibrotic markers. TGF-β1 stimulation on the other hand increased this syndecan-4 shedding and triggered production of fibrotic markers, identifying interactions between syndecan-4 and TGF-β1. Through these findings, this thesis extends the understanding of WB myopathy in broiler chickens and contributes to the development of strategies to improve poultry health while addressing broader challenges in research on skeletal myopathies and fibrotic diseases. Although various aspects of the investigations require further exploration, we suggest that this work will lay the foundation for new hypotheses and research directions regarding WB myopathy and the role of syndecans.

• Grønland, Mari Therese; Eijsink, Vincent; Dalhus, Bjørn; Bjørås, Magnar & Carlson, Cathrine Rein (2015). Human CaMKIIδ mutanter – proteinekspresjon, rensing, krystallisering og analyse av SN-peptid binding. Norges miljø- og biovitenskapelige universitet (NMBU). Full text in Research Archive Show summary

  I denne oppgaven var målet å løse den 3-dimensjonale strukturen av komplekset mellom et peptid fra Sekretoneurin (SN) og Calmodulin (CaM) avhengig kinase IIδ (CaMKIIδ). Det ble derfor laget flere ulike mutanter av CaMKIIδ, til uttrykking, rensing, bindingsanalyse mellom CaMKIIδ og SN-peptidet. Under normale forhold vil CaMKIIδ binde seg til Calmodulin og bli aktivert ved fosforylering av treonin 287 i det autoregulatoriske domenet, som er lokalisert fra aminosyre 281-316. Det er vist at hjertesvikt gir en økning av SN-nivået, SN binder til CaMKIIδ og hemmer aktiviteten til kinasen som er assosiert med hjertesvikt. Det er derfor av stor interesse å studere i detalj strukturen av komplekset mellom SN-peptidet og kinasen CaMKIIδ, også med henblikk på design av nye forbindelser som kan binde til og regulere aktiviteten til kinasen.For å se på proteinstrukturen til komplekset mellom SN-peptidet og CaMKIIδ valgte vi å følge 3 ulike strategier; design av trunkerte former av CaMKIIδ, design av aktivert CaMKIIδ ved hjelp av fosforyleringsmimikk, og design av mutanter av CaMKIIδ med SN-peptid motivet inkludert i det autoregulatoriske domenet. Det ble laget flere trunkerte mutanter av CaMKIIδ uten det autoregulatoriske domenet som kan forhindre binding av SN-peptidet; CaMKIIδ264 og CaMKIIδ275, hvor sekvensen stopper ved henholdsvis aminosyre 264 og 275. Forsøkene viste av disse mutantene ikke ble uttrykt fra plasmidet pNIC28-Bsa4, i motsetning til den noe lengre formen som stopper ved aminosyre 335, CaMKIIδ335. Det ble videre laget to konstrukter for mutanter av en annen trunkert form av CaMKIIδ, denne gang med stopp ved residue 280. Kinasen ble fusjonert med NusA, et protein som kan gi økt løselighet. Av de to konstruktene, NusA-CaMKIIδ280 og NusA-extended-CaMKIIδ280, var det ene ikke uttrykt mens det andre ga god ekspresjon, men ikke noe løselig protein. Det ble laget en siste trunkert mutant, CaMKIIδ291, hvor sekvensen stoppet midt i det autoregulatoriske domenet. Denne formen var uttrykt og løselig, men det var ikke tid til å utvikle en renseprotokoll. Det ble videre laget en mutant som skulle mimikere den aktive CaMKIIδ, hvor treonin er fosforylert, ved å erstatte treonin 287 med aspartat. Denne mutanten av CaMKIIδ, også i plasmidet pNIC28-Bsa4, hadde god ekspresjon, men var ikke løselig. Strukturen av CaMKIIδ335 bundet til CaM er tidligere publisert, og basert på en analyse av dette komplekset ble det besluttet å sette inn SN-peptidsekvensen i det autoregulatoriske domenet til proteinet. Dette er basert på en sekvenssammenstilling av SN-peptidet og nevnte domene i CaMKIIδ. Blant de konserverte residuene finner vi blant annet fosforyleringssetet treonin 287. Innsettingen ble gjort i to steg ved setedirigert mutagenese. Det ble dermed laget to mutanter med henholdsvis halve og hele SN-peptidet satt inn; CaMKIIδ1/2SN og CaMKIIδSN. Disse mutantene var godt uttrykt, men ved MS analyse viste det seg at CaMKIIδSN fragmenterte og aggregerte under rensing. Da ingen av de 3 strategiene ga rent løselig protein med høyt utbytte for krystallisering, gikk vi tilbake til den lengre formen CaMKIIδ335. Det ble etablert en rensemetode for CaMKIIδ335 som ga rent protein. Renset CaMKIIδ335 ble benyttet til bindingsanalyser ved hjelp av mikroskalatermoforese, men ingen binding mellom CaMKIIδ335 og SN-peptidet ble påvist under de gitte betingelser. Det ble i tillegg utført screening med over 800 krystalliseringsbetingelser av proteinkomplekset mellom CaMKIIδ og SN-peptid og proteinkomplekset CaMKIIδ, SN-peptidet og CaM. Det ble funnet 5 krystalliseringsbetingelser som må studeres nærmere og evt optimaliseres. Ettersom de ulike CaMKIIδ mutantene var vanskelig å fremstille og rense, og i mangel på en eksperimentell struktur av komplekset mellom CaMKIIδ og SN-peptidet ble det laget flere peptid-dockingmodeller av komplekset mellom SN-peptidet og CaMKIIδ som viser mulige strukturer av komplekset.

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