Exploring Syndecan-4 and MLP and Their Interaction in Primary Cardiomyocytes and H9c2 Cells.

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.

Characterization of wooden breast myopathy: a focus on syndecans and ECM remodeling.

Introduction: 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. Methods: In this study, we have categorized four groups of WB myopathy in broiler chickens and performed a comprehensive characterization of the molecular and histological profiles of two of them, with a special focus on the role of the syndecans and remodeling of the extracellular matrix (ECM). Results and discussion: Our findings reveal differential expression and shedding of the four syndecan family members and increased matrix metalloproteinase activity. Additionally, we identified alterations in key signaling pathways such as MAPK, AKT, and Wnt. Our work provides novel insights into a deeper understanding of WB pathogenesis and suggests potential therapeutic targets for this condition.

Polyarginine Cell-Penetrating Peptides Bind and Inhibit SERCA2.

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.

Two Transient Receptor Potential Channels at Focal Adhesions.

Recently there have been reports that identify two transient receptor potential channels in cell-matrix junctions known as focal adhesions. These are the calcium channel TRP canonical 7 and the calcium-activated monovalent ion channel, TRP melastatin (TRPM) 4. Here, we report on the occurrence of TRPM4 in focal adhesions of fibroblasts. Of three commercial antibodies recognizing this channel, only one yielded focal adhesion staining, while the other two did not. The epitope recognized by the focal adhesion-localizing antibody was mapped to the extreme C-terminus of the TRPM4 protein. The other two antibodies bind to N-terminal regions of the TRPM4 proteins. Deletion of the TRPM4 gene by CRISPR/cas9 techniques confirmed that this channel is a bona fide focal adhesion component, while expression of full-length TRPM4 proteins suggested that processing may occur to yield a form that localizes to focal adhesions. Given the reports that this channel may influence migratory behavior of cells and is linked to cardiovascular disease, TRPM4 functions in adhesion should be explored in greater depth. (J Histochem Cytochem 71: 495-508, 2023).

BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.

BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

Inhibition of the extracellular enzyme A disintegrin and metalloprotease with thrombospondin motif 4 prevents cardiac fibrosis and dysfunction.

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)-ß 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-ß levels in the medium. Simultaneously, ADAMTS4 elicited a not previously known cleavage of TGF-ß-binding proteins, i.e. latent-binding protein of TGF-ß 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-ß availability. Targeting ADAMTS4 may serve as a novel strategy in heart failure treatment, in particular, in heart failure with fibrosis and diastolic dysfunction.

Disruption of Phosphodiesterase 3A Binding to SERCA2 Increases SERCA2 Activity and Reduces Mortality in Mice With Chronic Heart Failure.

BACKGROUND: Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca2+ ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators. METHODS: Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays. RESULTS: PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding. CONCLUSIONS: Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.

Cardiomyocyte-specific overexpression of syndecan-4 in mice results in activation of calcineurin-NFAT signalling and exacerbated cardiac hypertrophy.

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 Ca2+ fluxes, suggesting that syndecan-4 regulated Ca2+ 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 Ca2+-dependent calcineurin-NFAT signalling pathway, hypertrophic remodelling and dysfunction in cardiomyocytes in response to pressure overload.

The female syndecan-4-/- heart has smaller cardiomyocytes, augmented insulin/pSer473-Akt/pSer9-GSK-3ß signaling, and lowered SCOP, pThr308-Akt/Akt and GLUT4 levels.

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-3ß. 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-3ß 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.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

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.

Design of a Proteolytically Stable Sodium-Calcium Exchanger 1 Activator Peptide for In Vivo Studies.

The cardiac sodium-calcium exchanger (NCX1) is important for normal Na+- and Ca2+-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 (PLMcyt) and pSer68-PLM (pSer68-PLMcyt) were found to bind strongly to the intracellular loop of NCX1 (NCX1cyt) with similar K D values of 4.1 ± 1.0 nM and 4.3 ± 1.9 nM, but the PLMcyt-NCX1cyt 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-PLMcyt with a K D of 129 nM. Excess NOPT also reduced the PLMcyt-NCX1cyt 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.

The Cardiac Syndecan-2 Interactome.

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.

Syndecan-4-/- Mice Have Smaller Muscle Fibers, Increased Akt/mTOR/S6K1 and Notch/HES-1 Pathways, and Alterations in Extracellular Matrix Components.

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.

Syndecan-4 Protects the Heart From the Profibrotic Effects of Thrombin-Cleaved Osteopontin.

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.

Coupling of the Na+/K+-ATPase to Ankyrin B controls Na+/Ca2+ exchanger activity in cardiomyocytes.

AIMS: Ankyrin B (AnkB) is an adaptor protein that assembles Na+/K+-ATPase (NKA) and Na+/Ca2+ exchanger (NCX) in the AnkB macromolecular complex. Loss-of-function mutations in AnkB cause the AnkB syndrome in humans, characterized by ventricular arrhythmias and sudden cardiac death. It is unclear to what extent NKA binding to AnkB allows regulation of local Na+ and Ca2+ domains and hence NCX activity. METHODS AND RESULTS: To investigate the role of NKA binding to AnkB in cardiomyocytes, we synthesized a disruptor peptide (MAB peptide) and its AnkB binding ability was verified by pulldown experiments. As opposed to control, the correlation between NKA and NCX currents was abolished in adult rat ventricular myocytes dialyzed with MAB peptide, as well as in cardiomyocytes from AnkB+/- mice. Disruption of NKA from AnkB (with MAB peptide) increased NCX-sensed cytosolic Na+ concentration, reduced Ca2+ extrusion through NCX, and increased frequency of Ca2+ sparks and Ca2+ waves without concomitant increase in Ca2+ transient amplitude or SR Ca2+ load, suggesting an effect in local Ca2+ domains. Selective inhibition of the NKAα2 isoform abolished both the correlation between NKA and NCX currents and the increased rate of Ca2+ sparks and waves following NKA/AnkB disruption, suggesting that an AnkB/NKAα2/NCX domain controls Ca2+ fluxes in cardiomyocytes. CONCLUSION: NKA binding to AnkB allows ion regulation in a local domain, and acute disruption of the NKA/AnkB interaction using disruptor peptides lead to increased rate of Ca2+ sparks and waves. The functional effects were mediated through the NKAα2 isoform. Disruption of the AnkB/NKA/NCX domain could be an important pathophysiological mechanism in the AnkB syndrome.

Secretoneurin Is an Endogenous Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor That Attenuates Ca2+-Dependent Arrhythmia.

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.

The cardiac syndecan-4 interactome reveals a role for syndecan-4 in nuclear translocation of muscle LIM protein (MLP).

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.

Caspase-1 induces smooth muscle cell growth in hypoxia-induced pulmonary hypertension.

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-1ß. 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-1ß, and IL-6, which were also reduced in the medium surrounding caspase-1 abrogated pulmonary arteries. By adding IL-18 or IL-1ß 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.

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes.

KEY POINTS: Using 3D direct stochastic optical reconstruction microscopy (dSTORM), we developed novel approaches to quantitatively describe the nanoscale, 3D organization of ryanodine receptors (RyRs) in cardiomyocytes. Complex arrangements of RyR clusters were observed in 3D space, both at the cell surface and within the cell interior, with allocation to dyadic and non-dyadic pools. 3D imaging importantly allowed discernment of clusters overlapping in the z-axis, for which detection was obscured by conventional 2D imaging techniques. Thus, RyR clusters were found to be significantly smaller than previous 2D estimates. Ca2+ release units (CRUs), i.e. functional groupings of neighbouring RyR clusters, were similarly observed to be smaller than earlier reports. Internal CRUs contained more RyRs in more clusters than CRUs on the cell surface, and yielded longer duration Ca2+ sparks. ABSTRACT: 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.