Dystrophin-glycoprotein Complex Research Articles

Systemic delivery of full-length dystrophin in Duchenne muscular dystrophy mice

Current gene therapy for Duchenne muscular dystrophy (DMD) utilizes adeno-associated virus (AAV) to deliver micro-dystrophin (µDys), which does not provide full protection for striated muscles as it lacks many important functional domains of full-length (FL) dystrophin. Here we develop a triple vector system to deliver FL-dystrophin into skeletal and cardiac muscles. We split FL-dystrophin into three fragments linked to two orthogonal pairs of split intein, allowing efficient assembly of FL-dystrophin. The three fragments packaged in myotropic AAV (MyoAAV4A) restore FL-dystrophin expression in both skeletal and cardiac muscles in male mdx4cv mice. Dystrophin-glycoprotein complex components are also restored at the sarcolemma of dystrophic muscles. MyoAAV4A-delivered FL-dystrophin significantly improves muscle histopathology, contractility, and overall strength comparable to µDys, but unlike µDys, it also restores defective cavin 4 localization and associated signaling in mdx4cv heart. Therefore, our data support the feasibility of a mutation-independent FL-dystrophin gene therapy for DMD, warranting further clinical development.

The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research.

Sarcospan (SSPN) is a 25-kDa transmembrane protein that is broadly expressed at the cell surface of many tissues, including, but not limited to, the myofibers from skeletal and smooth muscles, cardiomyocytes, adipocytes, kidney epithelial cells, and neurons. SSPN is a core component of the dystrophin-glycoprotein complex (DGC) that links the intracellular actin cytoskeleton with the extracellular matrix. It is also associated with integrin α7β1, the predominant integrin expressed in skeletal muscle. As a tetraspanin-like protein with four transmembrane spanning domains, SSPN functions as a scaffold to facilitate protein-protein interactions at the cell membrane. Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy are caused by the loss of dystrophin at the muscle cell surface and a concomitant loss of the entire DGC, including SSPN. SSPN overexpression ameliorates Duchenne muscular dystrophy in the mdx murine model, which supports SSPN being a viable therapeutic target. Other rescue studies support SSPN as a biomarker for the proper assembly and membrane expression of the DGC. Highly specific and robust antibodies to SSPN are needed for basic research on the molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluations in human samples. The development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. To address the significant barrier presented by limited commercially available antibodies, we aimed to generate a panel of robust SSPN-specific antibodies that can serve as a resource for the research community. We created antibodies to three SSPN protein epitopes, including the intracellular N- and C-termini as well as the large extracellular loop (LEL) between transmembrane domains 3 and 4. We developed a panel of rabbit antibodies (poly- and monoclonal) against an N-terminal peptide fragment of SSPN. We used several assays to show that the rabbit antibodies recognize mouse SSPN with a high functional affinity and specificity. We developed mouse monoclonal antibodies against the C-terminal peptide and the large extracellular loop of human SSPN. These antibodies are superior to commercially available antibodies and outperform them in various applications, including immunoblotting, indirect immunofluorescence analysis, immunoprecipitation, and an ELISA. These newly developed antibodies will significantly improve the quality and ease of SSPN detection for basic and translational research.

Examining the Impact of Environmental and Genetic Factors that Influence Sarcospan Expression in the Heart

Objective: Susceptibility to cardiac injury can be hereditary and the most common causes include genetic variations in proteins regulating lipid metabolism, arterial hypertension, diabetes, body-mass index as well as environmental causes such as smoking. Sarcospan (SSPN) is an integral member of the dystrophin-glycoprotein complex (DGC) and has an important role in stabilizing the muscle membrane of striated muscle. Genetic variants in the SSPN gene have been linked with increased cardiovascular disease risk and studies are ongoing to identify underlying mechanisms. Our studies using perfused mouse hearts subjected to acute ischemia/reperfusion injury indicate that SSPN deletion increases infarct size and arrhythmia risk (Hwang et al, IJMS, 2023). In addition, adult SSPN−/− cardiomyocytes were found to have decreased antioxidative capacity and heightened susceptibility to oxidative stress. Therefore, we hypothesize that genetic factors that decrease cardiac SSPN expression will increase cardiac susceptibility of mice exposed to conditions of prolonged oxidative stress. In this study we examine the role of SSPN to heart injury caused by chronic oxidative stress mediated by cigarette smoke (CS). Methods: To assess the impact of chronic oxidative stress conditions on SSPN expression in the heart 2 month-old WT C57BL/6J mice were exposed to 4 months CS. We found that SSPN expression was significantly reduced the CS exposed hearts. Since CS increases oxidative damage and deteriorates antioxidant defenses of the heart and vascular system, we examined the combined impact of SSPN deficiency and chronic oxidative stress. To address this, 7-month-old WT and SSPN−/− male and female mice were exposed to CS for 4 months. The mice were assessed after CS for body composition (EchoMRI), glucose tolerance testing and echocardiography. Tissue histology, immunoblotting of oxidative and inflammatory stress markers, serum inflammatory markers were assessed. Lung fibroblasts were isolated from the smoked and non-smoked groups to evaluate inflammatory, fibrotic, and oxidative stress pathway activation to respective stimuli. Results: Preliminary findings indicate that after CS SSPN−/− hearts exhibit larger areas of myocardial damage than WT mice. Surprisingly, after CS the SSPN−/− mice showed better glucose tolerance than WT mice and no significant changes in body mass. Minor decrements were found in the cardiac function of the CSE mice. Exposure of lung fibroblasts obtained from non-smoked SSPN−/− mice to innate immune ligands revealed activation of specific pro-inflammatory pathways.Conclusions: SSPN-deficiency increases susceptibility to cardiac injury in mice subjected to CS exposure, likely through crosstalk mechanisms that exist between oxidative and inflammatory pathways that SSPN appears to influence. Funding Sources: Florida Department of Health, James and Esther King #21K12. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Computational analysis of missense mutations in dystrophin protein: Insights into domain-specific effects and functional implications

BackgroundDystrophin is a structural protein primarily found in skeletal muscles that connects trans-membrane component of dystrophin-glycoprotein complex to the intracellular cytoskeleton network. N-terminal domain of dystrophin binds to F-actin and its C-terminal domain attaches to dystrophin-associated complex (DAG) in the membrane. It serves as the bridge connecting the extracellular matrix to the actin based cytoskeleton of muscle cells across the plasma membrane. This complex works together to strengthen muscle fibers and shield them from damage as they contract and relax. ObjectiveOur Objective is to investigate the different pathogenic mutations in four distinct domains of dystrophin protein and to decipher how these mutations impact protein structure and function. In this study, we investigated the impact of disease causing missense mutations in isoforms of dystrophin – P.11532–1 (K18N.A165V, D3187G, F3228L); P11532–4 (Y223N, D3179G) and P-11532-11 (Y227N,D3183G).Occurrence of pathogenic mutations in dystrophin might compromise structural stability, interfere with protein-protein interactions or alter cellular signaling pathways collectively. All these can contribute to the progressive muscle degeneration observed in Duchene or Becker muscular dystrophy(DMD or BMD) and X-lined dilated cardiomyopathy(CMD3B) affected individuals. MethodsEvolutionary conservation analysis using multiple sequence alignment followed by pathogenicity prediction using web based server was performed. Homology modeling of mutants was performed bySWISS MODEL a fully automated homology-modeling server, accessible through the Expasy web server. These models were then analyzed using various servers such as Dynamut. Pymol was used for visualization and structural analysis. ResultsAnalyzed mutations cause structural instability by either gaining or losing specific interactions involved in the folding of protein. Loss of pi-pi stacking interactions has the potential to significant impact the overall stability of the protein. It is important to note that the majority of the mutations lead to stability defects rather than functional defects, ultimately contributing to different forms of muscular dystrophy. ConclusiónThe mutations in different domains of Dystrophin protein significantly alters the protein structure. This may in turn impact its ability to interact with other proteins. Understanding the specific impacts of these mutations can pave the way for development of targeted therapies that aims at restoring functionality of dystrophin and ameliorating the debilitating effects of muscular dystrophy.

ZAP70: A Key Gene Identified by Differential Expression Analysis for Early Diagnosis of Fetuses with Emanuel Syndrome.

Emanuel syndrome is a rare autosomal disorder characterized by microcephaly, heart defects, cleft palate and developmental delay. However, there is a lack of specific prenatal screening for Emanuel syndrome. To screen for early diagnostic marker genes in fetuses with karyotype+der[22]t(11;22)(q23;q11) of Emanuel syndrome. Transcriptome sequencing and clinical trait data of t(11;22)(q23;q11) translocation samples were screened from the GEO database. The differentially expressed genes (DEGs) were screened by principal component analysis of gene expression by R package, and intersections were taken with balanced and unbalanced DEGs. Then, the correlation with clinical traits was determined by WGCNA analysis, GO and KEGG enrichment analysis, and then univariate Cox analysis and Lasso analysis were performed to obtain the key genes. The core regulatory genes were obtained after protein-protein interaction (PPI) network analysis. A total of 50 DEGs were obtained after differential analysis. WGCNA analysis showed that DEG was associated with the chromosomal imbalance and age module. GO and KEGG enrichment analyses showed candidate genes were associated with exocytic vesicle membrane, synaptic vesicle membranes, glycoprotein complex, dystrophin-associated glycoprotein complex and malaria. COX and Lasso analyses yielded 5 hub genes, including ZBED9, RGS20, SGCB, ETV5, and ZAP70. The results of PPI identified the key regulatory gene associated with chromosomal imbalance as the ZAP70 gene. ZAP70 may be a key gene for early diagnosis of Emanuel syndrome in fetuses with+der[22]t(11;22)(q23;q11) karyotype.

Glycative stress inhibits hypertrophy and impairs cell membrane integrity in overloaded mouse skeletal muscle.

Glycative stress, characterized by the formation and accumulation of advanced glycation end products (AGEs) associated with protein glycation reactions, has been implicated in inducing a decline of muscle function. Although the inverse correlation between glycative stress and muscle mass and strength has been demonstrated, the underlying molecular mechanisms are not fully understood. This study aimed to elucidate how glycative stress affects the skeletal muscle, particularly the adaptive muscle response to hypertrophic stimuli and its molecular mechanism. Male C57BL/6NCr mice were randomly divided into the following two groups: the bovine serum albumin (BSA)-treated and AGE-treated groups. Mice in the AGE-treated group were intraperitoneally administered AGEs (0.5mg/g) once daily, whereas those in the BSA-treated group received an equal amount of BSA (0.5mg/g) as the vehicle control. After 7days of continuous administration, the right leg plantaris muscle of mice in each group underwent functional overload treatment by synergist ablation for 7days to induce muscle hypertrophy. In in vitro studies, cultured C2C12 myocytes were treated with AGEs (1mg/mL) to examine cell adhesion and cell membrane permeability. Continuous AGE administration increased the levels of fluorescent AGEs, Nε-(carboxymethyl) lysine, and methylglyoxal-derived hydroimidazolone-1 in both plasma and skeletal muscle. Plantaris muscle weight, muscle fibre cross-sectional area, protein synthesis rate, and the number of myonuclei increased with functional overload in both groups; however, the increase was significantly reduced by AGE treatment. Some muscles of AGE-treated mice were destroyed by functional overload. Proteomic analysis was performed to explore the mechanisms of muscle hypertrophy suppression and myofibre destruction by AGEs. When principal component analysis was performed on 4659 data obtained by proteomic analysis, AGE treatment was observed to affect protein expression only in functionally overloaded muscles. Enrichment analysis of the 436 proteins extracted using the K-means method further identified a group of proteins involved in cell adhesion. Consistent with this finding, dystrophin-glycoprotein complex proteins and cell adhesion-related proteins were confirmed to increase with functional overload; however, this was attenuated by AGE treatment. Additionally, the treatment of C2C12 muscle cells with AGEs inhibited their ability to adhere and increased cell membrane permeability. This study indicates that glycative stress may be a novel pathogenic factor in skeletal muscle dysfunctions by causing loss of membrane integrity and preventing muscle mass gain.

Cortactin interacts with αDystrobrevin-1 and regulates murine neuromuscular junction morphology

Neuromuscular junctions transmit signals from the nervous system to skeletal muscles, triggering their contraction, and their proper organization is essential for breathing and voluntary movements. αDystrobrevin-1 is a cytoplasmic component of the dystrophin-glycoprotein complex and has pivotal functions in regulating the integrity of muscle fibers and neuromuscular junctions. Previous studies identified that αDystrobrevin-1 functions in the organization of the neuromuscular junction and that its phosphorylation in the C-terminus is required in this process. Our proteomic screen identified several putative αDystrobrevin-1 interactors recruited to the Y730 site in phosphorylated and unphosphorylated states. Amongst various actin-modulating proteins, we identified the Arp2/3 complex regulator cortactin. We showed that similarly to αDystrobrevin-1, cortactin is strongly enriched at the neuromuscular postsynaptic machinery and obtained results suggesting that these two proteins interact in cell homogenates and at the neuromuscular junctions. Analysis of synaptic morphology in cortactin knockout mice showed abnormalities in the slow-twitching soleus muscle and not in the fast-twitching tibialis anterior. However, muscle strength examination did not reveal apparent deficits in knockout animals.

Reduced cardiac antioxidant defenses mediate increased susceptibility to workload-induced myocardial injury in males with genetic cardiomyopathy

Ongoing cardiomyocyte injury is a major mechanism in the progression of heart failure, particularly in dystrophic hearts. Due to the poor regenerative capacity of the adult heart, cardiomyocyte death results in the permanent loss of functional myocardium. Understanding the factors contributing to myocyte injury is essential for the development of effective heart failure therapies. As a model of persistent cardiac injury, we examined mice lacking β-sarcoglycan (β-SG), a key component of the dystrophin glycoprotein complex (DGC). The loss of the sarcoglycan complex markedly compromises sarcolemmal integrity in this β-SG−/− model. Our studies aim to characterize the mechanisms underlying dramatic sex differences in susceptibility to cardiac injury in β-SG−/− mice. Male β-SG−/− hearts display significantly greater myocardial injury and death following isoproterenol-induced cardiac stress than female β-SG−/− hearts. This protection of females was independent of ovarian hormones. Male β-SG−/− hearts displayed increased susceptibility to exogenous oxidative stress and were significantly protected by angiotensin II type 1 receptor (AT1R) antagonism. Increasing general antioxidative defenses or increasing the levels of S-nitrosylation both provided protection to the hearts of β-SG−/− male mice. Here we demonstrate that increased susceptibility to oxidative damage leads to an AT1R-mediated amplification of workload-induced myocardial injury in male β-SG−/− mice. Improving oxidative defenses, specifically by increasing S-nitrosylation, provided protection to the male β-SG−/− heart from workload-induced injury. These studies describe a unique susceptibility of the male heart to injury and may contribute to the sex differences in other forms of cardiac injury.

Plasticity and structural alterations of mitochondria and sarcoplasmic organelles in muscles of mice deficient in α-dystrobrevin, a component of the dystrophin-glycoprotein complex.

The dystrophin-glycoprotein complex (DGC) plays a crucial role in maintaining the structural integrity of the plasma membrane and the neuromuscular junction. In this study, we investigated the impact of the deficiency of α-dystrobrevin (αdbn), a component of the DGC, on the homeostasis of intracellular organelles, specifically mitochondria and the sarcoplasmic reticulum (SR). In αdbn deficient muscles, we observed a significant increase in the membrane-bound ATP synthase complex levels, a marker for mitochondria in oxidative muscle fiber types compared to wild-type. Furthermore, examination of muscle fibers deficient in αdbn using electron microscopy revealed profound alterations in the organization of mitochondria and the SR within certain myofibrils of muscle fibers. This included the formation of hyper-branched intermyofibrillar mitochondria with extended connections, an extensive network spanning several myofibrils, and a substantial increase in the number/density of subsarcolemmal mitochondria. Concurrently, in some cases, we observed significant structural alterations in mitochondria, such as cristae loss, fragmentation, swelling, and the formation of vacuoles and inclusions within the mitochondrial matrix cristae. Muscles deficient in αdbn also displayed notable alterations in the morphology of the SR, along with the formation of distinct anomalous concentric SR structures known as whorls. These whorls were prevalent in αdbn-deficient mice but were absent in wild-type muscles. These results suggest a crucial role of the DGC αdbn in regulating intracellular organelles, particularly mitochondria and the SR, within muscle cells. The remodeling of the SR and the formation of whorls may represent a novel mechanism of the unfolded protein response (UPR) in muscle cells.

Ptpn23 Controls Cardiac T-Tubule Patterning by Promoting the Assembly of Dystrophin-Glycoprotein Complex.

Cardiac transverse tubules (T-tubules) are anchored to sarcomeric Z-discs by costameres to establish a regular spaced pattern. One of the major components of costameres is the dystrophin-glycoprotein complex (DGC). Nevertheless, how the assembly of the DGC coordinates with the formation and maintenance of T-tubules under physiological and pathological conditions remains unclear. Given the known role of Ptpn23 (protein tyrosine phosphatase, nonreceptor type 23) in regulating membrane deformation, its expression in patients with dilated cardiomyopathy was determined. Taking advantage of Cre/Loxp, CRISPR/Cas9, and adeno-associated virus 9 (AAV9)-mediated in vivo gene editing, we generated cardiomyocyte-specific Ptpn23 and Actn2 (α-actinin-2, a major component of Z-discs) knockout mice. We also perturbed the DGC by using dystrophin global knockout mice (DmdE4*). MM 4-64 and Di-8-ANEPPS staining, Cav3 immunofluorescence, and transmission electron microscopy were performed to determine T-tubule structure in isolated cells and intact hearts. In addition, the assembly of the DGC with Ptpn23 and dystrophin loss of function was determined by glycerol-gradient fractionation and SDS-PAGE analysis. The expression level of Ptpn23 was reduced in failing hearts from dilated cardiomyopathy patients and mice. Genetic deletion of Ptpn23 resulted in disorganized T-tubules with enlarged diameters and progressive dilated cardiomyopathy without affecting sarcomere organization. AAV9-mediated mosaic somatic mutagenesis further indicated a cell-autonomous role of Ptpn23 in regulating T-tubule formation. Genetic and biochemical analyses showed that Ptpn23 was essential for the integrity of costameres, which anchor the T-tubule membrane to Z-discs, through interactions with α-actinin and dystrophin. Deletion of α-actinin altered the subcellular localization of Ptpn23 and DGCs. In addition, genetic inactivation of dystrophin caused similar T-tubule defects to Ptpn23 loss-of-function without affecting Ptpn23 localization at Z-discs. Last, inducible Ptpn23 knockout at 1 month of age showed Ptpn23 is also required for the maintenance of T-tubules in adult cardiomyocytes. Ptpn23 is essential for cardiac T-tubule formation and maintenance along Z-discs. During postnatal heart development, Ptpn23 interacts with sarcomeric α-actinin and coordinates the assembly of the DGC at costameres to sculpt T-tubule spatial patterning and morphology.

Gene therapy delivered micro-dystrophins co-localize with transgenic utrophin in dystrophic skeletal muscle fibers

Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by the absence of functional dystrophin. There are multiple ongoing clinical trials for DMD that are testing gene therapy treatments consisting of adeno-associated viral (AAV) vectors carrying miniaturized versions of dystrophin optimized for function, termed micro-dystrophins (μDys). Utrophin, the fetal homolog of dystrophin, has repeatedly been reported to be upregulated in human DMD muscle as a compensatory mechanism, but whether µDys displaces full-length utrophin is unknown. In this study, dystrophin/utrophin-deficient mice with transgenic overexpression of full-length utrophin in skeletal muscles were systemically administered low doses of either AAV6-CK8e-Hinge3-µDys (μDysH3) or AAV6-CK8e-μDys5 (μDys5). We used immunofluorescence to qualitatively assess the localization of μDys with transgenic utrophin and neuronal nitric oxide synthase (nNOS) in quadriceps muscles. μDys protein resulting from both gene therapies co-localized at myofiber membranes with transgenic utrophin. We also confirmed the sarcolemmal co-localization of nNOS with μDys5, but not with transgenic utrophin expression or μDysH3. Transgenic utrophin expression and μDys proteins produced from both therapies stabilize the dystrophin-glycoprotein complex as observed by sarcolemmal localization of β-dystroglycan. This study suggests that µDys gene therapy will likely not inhibit any endogenous compensation by utrophin in DMD muscle.

Comparison of differentially expressed genes in longissimus dorsi muscle of Diannan small ears, Wujin and landrace pigs using RNA-seq.

Pig growth is an important economic trait that involves the co-regulation of multiple genes and related signaling pathways. High-throughput sequencing has become a powerful technology for establishing the transcriptome profiles and can be used to screen genome-wide differentially expressed genes (DEGs). In order to elucidate the molecular mechanism underlying muscle growth, this study adopted RNA sequencing (RNA-seq) to identify and compare DEGs at the genetic level in the longissimus dorsi muscle (LDM) between two indigenous Chinese pig breeds (Diannan small ears [DSE] pig and Wujin pig [WJ]) and one introduced pig breed (Landrace pig [LP]). Animals under study were from two Chinese indigenous pig breeds (DSE pig, n = 3; WJ pig, n = 3) and one introduced pig breed (LP, n = 3) were used for RNA sequencing (RNA-seq) to identify and compare the expression levels of DEGs in the LDM. Then, functional annotation, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) network analysis were performed on these DEGs. Then, functional annotation, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) network analysis were performed on these DEGs. The results revealed that for the DSE, WJ, and LP libraries, more than 66, 65, and 71 million clean reads were generated by transcriptome sequencing, respectively. A total of 11,213 genes were identified in the LDM tissue of these pig breeds, of which 7,127 were co-expressed in the muscle tissue of the three samples. In total, 441 and 339 DEGs were identified between DSE vs. WJ and LP vs. DSE in the study, with 254, 193 up-regulated genes and 187, 193 down-regulated genes in DSE compared to WJ and LP. GO analysis and KEGG signaling pathway analysis showed that DEGs are significantly related to contractile fiber, sarcolemma, and dystrophin-associated glycoprotein complex, myofibril, sarcolemma, and myosin II complex, Glycolysis/Gluconeogenesis, Propanoate metabolism, and Pyruvate metabolism, etc. In combination with functional annotation of DEGs, key genes such as ENO3 and JUN were identified by PPI network analysis. In conclusion, the present study revealed key genes including DES, FLNC, PSMD1, PSMD6, PSME4, PSMB4, RPL11, RPL13A, ROS23, RPS29, MYH1, MYL9, MYL12B, TPM1, TPM4, ENO3, PGK1, PKM2, GPI, and the unannotated new gene ENSSSCG00000020769 and related signaling pathways that influence the difference in muscle growth and could provide a theoretical basis for improving pig muscle growth traits in the future.

DAG1 haploinsufficiency is associated with sporadic and familial isolated or pauci-symptomatic hyperCKemia.

DAG1 encodes for dystroglycan, a key component of the dystrophin-glycoprotein complex (DGC) with a pivotal role in skeletal muscle function and maintenance. Biallelic loss-of-function DAG1 variants cause severe muscular dystrophy and muscle-eye-brain disease. A possible contribution of DAG1 deficiency to milder muscular phenotypes has been suggested. We investigated the genetic background of twelve subjects with persistent mild-to-severe hyperCKemia to dissect the role of DAG1 in this condition. Genetic testing was performed through exome sequencing (ES) or custom NGS panels including various genes involved in a spectrum of muscular disorders. Histopathological and Western blot analyses were performed on muscle biopsy samples obtained from three patients. We identified seven novel heterozygous truncating variants in DAG1 segregating with isolated or pauci-symptomatic hyperCKemia in all families. The variants were rare and predicted to lead to nonsense-mediated mRNA decay or the formation of a truncated transcript. In four cases, DAG1 variants were inherited from similarly affected parents. Histopathological analysis revealed a decreased expression of dystroglycan subunits and Western blot confirmed a significantly reduced expression of beta-dystroglycan in muscle samples. This study supports the pathogenic role of DAG1 haploinsufficiency in isolated or pauci-symptomatic hyperCKemia, with implications for clinical management and genetic counseling.

Regulation of mechanical force on cardiomyocytes beating

The mechanical behavior of cardiomyocytes plays an essential role in the maintenance of life and health. It is traditionally believed that both electrical and chemical signals modulate the cardiomyocytes behaviors. Recent discoveries have elucidated that the physical cues of microenvironment can regulate cell activities such as proliferation, spreading, migration, and differentiation. However, researches concerning the regulation of cardiomyocytes beating through mechanical force remains sparse. Here, we prepared different polyacrylamide gels coating with different cell adhesion ligand proteins to simulate the physical microenvironment of cardiomyocyte. Then the mechanical loading forces were loaded by employing a tungsten probe to stretch elastic hydrogels which can emulate the mechanical oscillations induced by the beating of adjacent cardiomyocytes. We investigated the responsive behavior of cardiomyocytes to external mechanical oscillations within various physical microenvironments. Firstly， we loaded 1 Hz mechanical oscillation on the matrix (E=11 kPa) with different kinds and concentrations of ligands (0, 5, 20, 100 μg/ml) to stimulate cardiomyocytes and observed it's mechanical response behavior. Our findings indicate that all kinds of ligands including Laminin, Fibronectin and Collagen I , can mediate the cardiomyocytes response to extrinsic mechanical oscillatory stimuli, which might be due to distinct mechanisms of mechanical force coupling (Fig.1b). This suggests that mechanical force signals can regulate the beating of cardiomyocytes through matrix-ligand-cell signaling pathway, thereby inducing intercellular coupled oscillations for rhythmic control of cardiomyocytes. Cardiomyoctes cultured on 20μg/ml Laminin show the highest and most stable response fraction. We hypothesized that there exist dual force transduction pathways for Laminin binding to integrin and dystrophin glycoprotein complex (DGC) (Fig.1a). We further analyzed the cardiomyocytes behaviors under mechanical oscillation with different substrate stiffness (E=1.8, 11, 27 kPa) and concentrations of Laminin (0, 5, 20, 100 μg/ml). We found that cardiomyoctes cultured on 1.8 kPa coated with 20 μg/ml Laminin show the highest response fraction (Fig.1c). Our results demonstrate that the stiffness of substrate, the type and density of cell adhesion ligands, as well as the strength and rhythm of the mechanical signals can synergetic influence the cardiomyocytes responses to external mechanical stimulations, which provides the foundation for understanding the diseases such as cardiac arrhythmias and heart failure following myocardial infarction.

Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature invivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/-) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.

Identification of Genetic Variants Associated with Severe Myocardial Bridging through Whole-Exome Sequencing.

Myocardial bridging (MB) is a congenital coronary artery anomaly and an important cause of angina. The genetic basis of MB is currently unknown. This study used a whole-exome sequencing technique and analyzed genotypic differences. Eight coronary angiography-confirmed cases of severe MB and eight age- and sex-matched control patients were investigated. In total, 139 rare variants that are potentially pathogenic for severe MB were identified in 132 genes. Genes with multiple rare variants or co-predicted by ClinVar and CADD/REVEL for severe MB were collected, from which heart-specific genes were selected under the guidance of tissue expression levels. Functional annotation indicated significant genetic associations with abnormal skeletal muscle mass, cardiomyopathies, and transmembrane ion channels. Candidate genes were reviewed regarding the functions and locations of each individual gene product. Among the gene candidates for severe MB, rare variants in DMD, SGCA, and TTN were determined to be the most crucial. The results suggest that altered anchoring proteins on the cell membrane and intracellular sarcomere unit of cardiomyocytes play a role in the development of the missed trajectory of coronary vessels. Additional studies are required to support the diagnostic application of cardiac sarcoglycan and dystroglycan complexes in patients with severe MB.

Maturation of iPSC-derived cardiomyocytes in a heart-on-a-chip device enables modeling of dilated cardiomyopathy caused by R222Q-SCN5A mutation

To better understand sodium channel (SCN5A)-related cardiomyopathies, we generated ventricular cardiomyocytes from induced pluripotent stem cells obtained from a dilated cardiomyopathy patient harbouring the R222Q mutation, which is only expressed in adult SCN5A isoforms. Because the adult SCN5A isoform was poorly expressed, without functional differences between R222Q and control in both embryoid bodies and cell sheet preparations (cultured for 29–35 days), we created heart-on-a-chip biowires which promote myocardial maturation. Indeed, biowires expressed primarily adult SCN5A with R222Q preparations displaying (arrhythmogenic) short action potentials, altered Na+ channel biophysical properties and lower contractility compared to corrected controls. Comprehensive RNA sequencing revealed differential gene regulation between R222Q and control biowires in cellular pathways related to sarcoplasmic reticulum and dystroglycan complex as well as biological processes related to calcium ion regulation and action potential. Additionally, R222Q biowires had marked reductions in actin expression accompanied by profound sarcoplasmic disarray, without differences in cell composition (fibroblast, endothelial cells, and cardiomyocytes) compared to corrected biowires. In conclusion, we demonstrate that in addition to altering cardiac electrophysiology and Na+ current, the R222Q mutation also causes profound sarcomere disruptions and mechanical destabilization. Possible mechanisms for these observations are discussed.

Sarcospan Deficiency Increases Oxidative Stress and Arrhythmias in Hearts after Acute Ischemia-Reperfusion Injury.

The protein sarcospan (SSPN) is an integral member of the dystrophin-glycoprotein complex (DGC) and has been shown to be important in the heart during the development and the response to acute stress. In this study, we investigated the role of SSPN in the cardiac response to acute ischemia-reperfusion (IR) injury in SSPN-deficient (SSPN-/-) mice. First, the hemodynamic response of SSPN-/- mice was tested and was similar to SSPN+/+ (wild-type) mice after isoproterenol injection. Using the in situ Langendorff perfusion method, SSPN-/- hearts were subjected to IR injury and found to have increased infarct size and arrhythmia susceptibility compared to SSPN+/+. Ca2+ handling was assessed in single cardiomyocytes and diastolic Ca2+ levels were increased after acute β-AR stimulation in SSPN+/+ but not SSPN-/-. It was also found that SSPN-/- cardiomyocytes had reduced Ca2+ SR content compared to SSPN+/+ but similar SR Ca2+ release. Next, we used qRT-PCR to examine gene expression of Ca2+ handling proteins after acute IR injury. SSPN-/- hearts showed a significant decrease in L-type Ca2+ channels and a significant increase in Ca2+ release channel (RyR2) expression. Interestingly, under oxidizing conditions reminiscent of IR, SSPN-/- cardiomyocytes, had increased H2O2-induced reactive oxygen species production compared to SSPN+/+. Examination of oxidative stress proteins indicated that NADPH oxidase 4 and oxidized CAMKII were increased in SSPN-/- hearts after acute IR injury. These results suggest that increased arrhythmia susceptibility in SSPN-/- hearts post-IR injury may arise from alterations in Ca2+ handling and a reduced capacity to regulate oxidative stress pathways.

Dystrophic cardiomyopathy: role of the cardiac myofilaments.

Dystrophic cardiomyopathy arises from mutations in the dystrophin gene. Dystrophin forms part of the dystrophin glycoprotein complex and is postulated to act as a membrane stabilizer, protecting the sarcolemma from contraction-induced damage. Duchenne muscular dystrophy (DMD) is the most severe dystrophinopathy, caused by a total absence of dystrophin. Patients with DMD present with progressive skeletal muscle weakness and, because of treatment advances, a cardiac component of the disease (i.e., dystrophic cardiomyopathy) has been unmasked later in disease progression. The role that myofilaments play in dystrophic cardiomyopathy is largely unknown and, as such, this study aimed to address cardiac myofilament function in a mouse model of muscular dystrophy. To assess the effects of DMD on myofilament function, isolated permeabilized cardiomyocytes of wild-type (WT) littermates and Dmdmdx-4cv mice were attached between a force transducer and motor and subjected to contractile assays. Maximal tension and rates of force development (indexed by the rate constant, k tr) were similar between WT and Dmdmdx-4cv cardiac myocyte preparations. Interestingly, Dmdmdx-4cv cardiac myocytes exhibited greater sarcomere length dependence of peak power output compared to WT myocyte preparations. These results suggest dystrophin mitigates length dependence of activation and, in the absence of dystrophin, augmented sarcomere length dependence of myocyte contractility may accelerate ventricular myocyte contraction-induced damage and contribute to dystrophic cardiomyopathy. Next, we assessed if mavacamten, a small molecule modulator of thick filament activation, would mitigate contractile properties observed in Dmdmdx-4cv permeabilized cardiac myocyte preparations. Mavacamten decreased maximal tension and k tr in both WT and Dmdmdx-4cv cardiac myocytes, while also normalizing the length dependence of peak power between WT and Dmdmdx-4cv cardiac myocyte preparations. These results highlight potential benefits of mavacamten (i.e., reduced contractility while maintaining exquisite sarcomere length dependence of power output) as a treatment for dystrophic cardiomyopathy associated with DMD.

Timed Release Ion Matrix Enhances Muscle Force in Dystrophic Mice

Background: Duchenne Muscular Dystrophy (DMD) is an x-linked, recessive chromosomal mutation of the gene dystrophin, affecting 1 in 3,500 males. DMD is associated with limb muscle dysfunction from repetitive damage to myofibers beginning at 2 years of age. In healthy muscle, dystrophin is responsible for maintaining the sarcolemma through the dystrophin-glycoprotein complex, while muscle with dystrophin deficiencies seen in DMD patients experience severe sarcolemmal disruption following muscle contraction. Despite major advances in genetic engineering, the functional outcomes of FDA-approved therapeutic strategies have failed to enhance the function of dystrophin-deficient myofibers. We have taken an alternative approach to apply a novel biomaterial, Time Release Ion Matrix (TRIM), that has the potential to release ions favorable to regeneration, restoring muscle structure and function in DMD mice. Hypothesis: We hypothesized that muscle force and myofiber cross-sectional area (CSA) of the tibialis anterior muscle (TA) in an established mouse model of muscle dystrophy [D2.B10 mdx (DBA. mdx)] would be enhanced 14 days post-injection of TRIM. Methods: The left TA of adult male DBA. mdx mice (n=3-9, age 6-7 months) was injected with 250 μg of TRIM suspended in saline or saline alone (vehicle control). At 14 days post injection, the TA muscle was isolated for in situ muscle force measurements using direct electrical stimulation. The TA was then removed and frozen in optimal cutting temperature compound (OCT) for histological analysis. Muscle cross-sections (thickness, 10 μm) were labeled with laminin to identify myofiber borders and myosin heavy chain as a marker of myofibers able to generate force. Images were analyzed using Nikon NIS-Elements software. To simulate body fluid, 300 mg of TRIM was suspended in physiological salt solution (PSS) for 120 hours. Results: The dissolution of TRIM in PSS over 120 hours was 40% (179 mg remaining). Peak isometric force of TAs injected with TRIM (60.0 ± 2.3 g) was 11% greater compared to saline alone (52.4 ± 2.4 g) (means ± SE, n=6-9 per group; P=0.048). There were fewer small diameter myofibers (< 400 μm2) in TAs injected with TRIM (10 ± 7%) compared to saline controls (24 ± 5 %) (n=3/group; P=0.03). Conclusion: 14 days following injection of TRIM into the TA of dystrophic mice, muscle force and myofiber CSA were enhanced by 11 and 14% respectively. We suggest that TRIM may be an effective treatment to restore muscle function in DMD and serve as a powerful tool for discovering therapeutic targets against DMD. University of Missouri Coulter Biomedical Accelerator (SSS, ABM), Texas A&M Department of Kinesiology and Sport Management (JAK, ABM) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.