Thea Parsberg Støle

• 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.

• 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.

• 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.

• 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.

• 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.

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