Components Of Sarcomere Research Articles

Modified mRNA-based gene editing reveals sarcomere-based regulation of gene expression in human induced-pluripotent stem cell-derived cardiomyocytes

Mutations in genes coding sarcomere components are the major causes of human inherited cardiomyopathy. Genome editing is widely applied to genetic modification of human pluripotent stem cells (hPSCs) before hPSCs were differentiated into cardiomyocytes to model cardiomyopathy. Whether genetic mutations influence the early hPSC differentiation process or solely the terminally differentiated cardiomyocytes during cardiac pathogenesis remains challenging to distinguish. To solve this problem, here we harnessed chemically modified mRNA (modRNA) and synthetic single-guide RNA to develop an efficient genome editing approach in hPSC-derived cardiomyocytes (hPSC-CMs). We showed that modRNA-based CRISPR/Cas9 mutagenesis of TNNT2, the coding gene for cardiac troponin T, results in sarcomere disassembly and contractile dysfunction in hPSC-CMs. These structural and functional phenotypes were associated with profound downregulation of oxidative phosphorylation genes and upregulation of cardiac stress markers NPPA and NPPB. These data confirmed that sarcomeres regulate gene expression in hPSC-CMs and highlighted the RNA technology as a powerful tool to achieve stage-specific genome editing during hPSC differentiation.

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal that MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development and prompting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility in the C-zone? Globular domains critical to MyBP-C's modulatory functions are absent from MyBP-H, suggesting that MyBP-H may be functionally silent. However, our results suggest an active role. In vitro motility experiments indicate MyBP-H shares MyBP-C's capacity as a molecular "brake." These results provide new insights and raise questions about the role of the C-zone during muscle development.

Abstract Or107: MyBP-HL nonsense mutations fail in sarcomere incorporation leads to modifications in myocyte contraction trough calcium modifications.

Introduction: Heart function depends on the cardiomyocyte contractile apparatus and proper sarcomere protein expression. Mutations in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation. AF is the most common cardiac arrhythmia and is associated with ischemic stroke, heart failure, and substantial morbidity and mortality. Recently, our group described a novel sarcomere component, myosin binding protein-H like (MyBP-HL), that is mainly expressed in the cardiac atria. MyBP-HL is composed of two immunoglobulin domains and one fibronectin domain, with homology to last three C-terminal domains of cardiac myosin binding protein-C (cMyBP-C). cMyBP-C nonsense mutations prevent his incorporation into the sarcomere. C-MyBP-C truncated peptides are identified by cells as poisoning peptides, and they are rapidly degraded. cMyBP-C nonsense mutations are causative of hypertrophic cardiomyopathy (HCM). The MyBP-HL Arg255stop variant has been described in humans and has been linked to ventricular conduction system abnormalities, atrial enlargement, dilated cardiomyopathy, and atrial and ventricular arrhythmias. The gnomAD database reports 9 nonsense mutations for MYBPHL in humans (Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255, and Tyr307). Hypothesis: MYBPHL nonsense mutations fail to incorporate into the sarcomere will lead to contractile disfunction. Results: We demonstrated that MyBP-HL overexpression showed to be able to incorporate in a similar pattern to cMyBP-C C-zone doublets. Nevertheless, nonsense mutations disrupt normal sarcomere localization. MyBP-HL nonsense mutations promote an increase in contraction amplitude and slower relaxation kinetics. Also, we observed an increase in sarcomere length. Similarly, MyBP-HL nonsense mutations lead to increase in amplitude peak of calcium transients, and reduction in velocity to maximal calcium peak. In accordance with calcium modifications, we observed through mass spectrometry an increase in calpain 6 abundance promoted by MyBP-HL nonsense mutations. Conclusion: Based on these data, we demonstrated that MYBPHL nonsense mutations prevent MyBP-HL incorporation into the thick filament. The failure of MyBP-HL truncating peptides to incorporate into the sarcomere leads to a deficiencies in myocytes contraction patterns, changes in calcium patterns and increase in degradation proteins abundance, which are related to AF prevalence.

Sarcomere. The structural unit of the myofibrillar complex of typical cardiomyocytes. Isometric aspects of sarcomere organization

Background. The complexity of understanding the structure and function of cardiomyocytes in the myocardium is a fundamental problem in the field of heart morphology. Sarcomeres are the main structural and functional units of myofibrils. Understanding the organization and dynamics of sarcomeres in cardiomyocytes is crucial for establishing the mechanisms responsible for the process of forming the contractile system of cardiomyocytes. Objective. The main purpose of this study is a review of literary sources that complement and expand the essential findings about the construction of myofibrils and their components when studied by effective methods of microscopic techniques. Materials. Literature review databases, systematic search of primary sources related to the research topic. Methods of visual examination of the components of sarcomeres and elements of their organization. Results. Transmission electron microscopy provides high-resolution imaging of sarcomeres, which is necessary for detailed analysis of their ultrastructure, including visualization of sarcomere components such as Z-discs, M-lines, thick and thin filaments. Morphometric methods are crucial for studying the compaction and orientation of myofibrils in cardiomyocytes. Conclusion. Morphometric methods provide valuable information about the structural remodeling of myofibrils in response to physiological stimuli or pathological conditions. These techniques are needed to improve our understanding of the mechanisms underlying muscle contraction and to develop methods to study cardiac remodeling processes in response to the adaptive response.

Titin: roles in cardiac function and diseases.

The giant protein titin is an essential component of muscle sarcomeres. A single titin molecule spans half a sarcomere and mediates diverse functions along its length by virtue of its unique domains. The A-band of titin functions as a molecular blueprint that defines the length of the thick filaments, the I-band constitutes a molecular spring that determines cell-based passive stiffness, and various domains, including the Z-disk, I-band, and M-line, serve as scaffolds for stretch-sensing signaling pathways that mediate mechanotransduction. This review aims to discuss recent insights into titin's functional roles and their relationship to cardiac function. The role of titin in heart diseases, such as dilated cardiomyopathy and heart failure with preserved ejection fraction, as well as its potential as a therapeutic target, is also discussed.

Sarcomeric tropomyosin expression during human iPSC differentiation into cardiomyocytes.

Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes. During the differentiation time course, we harvested cells on Days 0, 5, 10, 15, and 20 to analyze for various sarcomeric TPM transcripts by qRT-PCR and for sarcomeric TPM proteins using two-dimensional Western blot with sarcomeric TPM-specific CH1 monoclonal antibody followed by mass spectra analyses. Our results show increasing levels of total TPM transcripts and proteins during the period of differentiation, but varying levels of specific TPM isoforms during the same period. By Day 20, the rank order of TPM transcripts was TPM1α > TPM1κ > TPM2α > TPM1μ > TPM3α > TPM4α. TPM1α was the dominant protein produced with some TPM2 and much less TPM1κ and μ. Interestingly, small amounts of two lower molecular weight TPM3 isoforms were detected on Day 15. To the best of our knowledge this is the first demonstration of TPM1μ non-muscle isoform protein expression before and during cardiac differentiation.

Mechanisms of Sarcomere Assembly in Muscle Cells Inferred from Sequential Ordering of Myofibril Components

Voluntary movement in animals is driven by the contraction of myofibrils in striated muscle cells, which are linear chains of regular cytoskeletal units termed sarcomeres. While the ordered structure of mature sarcomeres, key to muscle contraction, is well established, the physical mechanism governing sarcomere assembly is still under debate. Here we put forward a theory of sarcomere self-assembly based on data. We measured the sequential ordering of sarcomere components in developing flight muscles, using a novel tracking-free algorithm. We find that myosin molecular motors, α-actinin cross-linkers, and the titin homologue Sallimus form periodic patterns before actin filaments become polarity sorted. Based on these, we propose that myosin, Sallimus, and sarcomere Z-disk proteins including α-actinin dynamically bind and unbind to an unordered bundle of actin to establish an initial periodic pattern. Periodicity of actin filaments is established only later. Our model proposes that nonlocal interactions between spatially extended myosin and titin/Sallimus containing complexes, and possibly tension-dependent feedback mediated by an α-actinin catch bond, drive this ordering process. We probe this hypothesis using mathematical models and derive predictive conditions for sarcomere pattern formation to guide future experiments. Published by the American Physical Society 2024

Cypher/ZASP drives cardiomyocyte maturation via actin-mediated MRTFA-SRF signalling.

Rationale: Cardiomyocytes (CMs) undergo dramatic structural and functional changes in postnatal maturation; however, the regulatory mechanisms remain greatly unclear. Cypher/Z-band alternatively spliced PDZ-motif protein (ZASP) is an essential sarcomere component maintaining Z-disc stability. Deletion of mouse Cypher and mutation in human ZASP result in dilated cardiomyopathy (DCM). Whether Cypher/ZASP participates in CM maturation and thereby affects cardiac function has not been answered. Methods: Immunofluorescence, transmission electron microscopy, real-time quantitative PCR, and Western blot were utilized to identify the role of Cypher in CM maturation. Subsequently, RNA sequencing and bioinformatics analysis predicted serum response factor (SRF) as the key regulator. Rescue experiments were conducted using adenovirus or adeno-associated viruses encoding SRF, both in vitro and in vivo. The molecular mechanisms were elucidated through G-actin/F-actin fractionation, nuclear-cytoplasmic extraction, actin disassembly assays, and co-sedimentation assays. Results: Cypher deletion led to impaired sarcomere isoform switch and morphological abnormalities in mitochondria, transverse-tubules, and intercalated discs. RNA-sequencing analysis revealed significant dysregulation of crucial genes related to sarcomere assembly, mitochondrial metabolism, and electrophysiology in the absence of Cypher. Furthermore, SRF was predicted as key transcription factor mediating the transcriptional differences. Subsequent rescue experiments showed that SRF re-expression during the critical postnatal period effectively rectified CM maturation defects and notably improved cardiac function in Cypher-depleted mice. Mechanistically, Cypher deficiency resulted in the destabilization of F-actin and a notable increase in G-actin levels, thereby impeding the nuclear localisation of myocardin-related transcription factor A (MRTFA) and subsequently initiating SRF transcription. Conclusion: Cypher/ZASP plays a crucial role in CM maturation through actin-mediated MRTFA-SRF signalling. The linkage between CM maturation abnormalities and the late-onset of DCM is suggested, providing further insights into the pathogenesis of DCM and potential treatment strategies.

Titin domains with reduced core hydrophobicity cause dilated cardiomyopathy

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the reduced hydrophobicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophobicity-reducing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.

Abstract 14515: Distinct Genetic Mechanisms for Bradyarrhythmias From Multi-Center Multi-Ancestry Meta-Analyses

Background: With an increasing prevalence in older adults, bradyarrhythmias pose a major public health problem. Hypothesis: Genetics can inform on disease pathophysiology and implicate targets for therapeutic development for bradyarrhythmias. Methods: We first performed ancestry-specific genome-wide association studies (GWAS) and combined the results in meta-analyses of up to 1.3 million individuals including 9,511 sinus node dysfunction (SND) cases, 34,086 distal conduction disease (DCD) cases, and 28,899 pacemaker implantation (PM) cases. We evaluated the biological relevance of bradyarrhythmia loci by analyses of transcriptomes, pleiotropy, and partitioned heritability based on cardiac single cell RNA sequencing data. Finally, we performed rare variant burden testing in 460,000 whole exome sequenced individuals (cases: 1,766 SND, 12,422 DCD & 5,646 PM) from UK Biobank and Mass General Brigham Biobank. Results: Common variant analyses identified 13, 28, and 21 loci for SND, DCD, and PM, respectively. SND and DCD were moderately genetically correlated (r g = 0.64). Common variant loci included genes underlying familial forms of bradyarrhythmias (HCN4 and SCN5A) and genes regulating population-level variation in electrocardiographic traits. Four well-known common variant arrhythmia loci ( SCN5A / SCN10A , CCDC141 , TBX20 , and CAMK2D ) were shared for SND and DCD, while other loci were more specific for SND ( PITX2 , HCN4 , ZFHX3 ) or DCD ( CAV1 and NKX2 - 5 ). We observed an inverse association between predicted cardiac expression of SCN10A and risk of SND and DCD, and positive associations of predicted cardiac expression of CEP68 and STRN with risk of SND and DCD, respectively. Among 9 cardiac cell types, cardiomyocyte-expressed genes were enriched for contributions to DCD heritability. Rare variant analyses implicated LMNA for all bradyarrhythmia subtypes; SMAD6 and SCN5A for DCD; and TTN , MYBPC3 , and SCN5A for PM. Conclusions: We identify novel genes and loci associated with bradyarrhythmias. The genetic architectures of SND and DCD are both overlapping and distinct. Multiple genetic mechanisms involving ion channels, sarcomeric components, cellular homeostasis, and cardiac development may influence the development of bradyarrhythmias.

Independent regulation of Z-lines and M-lines during sarcomere assembly in cardiac myocytes revealed by the automatic image analysis software sarcApp

Sarcomeres are the basic contractile units within cardiac myocytes, and the collective shortening of sarcomeres aligned along myofibrils generates the force driving the heartbeat. The alignment of the individual sarcomeres is important for proper force generation, and misaligned sarcomeres are associated with diseases, including cardiomyopathies and COVID-19. The actin bundling protein, α-actinin-2, localizes to the ‘Z-Bodies” of sarcomere precursors and the ‘Z-Lines’ of sarcomeres, and has been used previously to assess sarcomere assembly and maintenance. Previous measurements of α-actinin-2 organization have been largely accomplished manually, which is time-consuming and has hampered research progress. Here, we introduce sarcApp, an image analysis tool that quantifies several components of the cardiac sarcomere and their alignment in muscle cells and tissue. We first developed sarcApp to utilize deep learning-based segmentation and real space quantification to measure α-actinin-2 structures and determine the organization of both precursors and sarcomeres/myofibrils. We then expanded sarcApp to analyze ‘M-Lines’ using the localization of myomesin and a protein that connects the Z-Lines to the M-Line (titin). sarcApp produces 33 distinct measurements per cell and 24 per myofibril that allow for precise quantification of changes in sarcomeres, myofibrils, and their precursors. We validated this system with perturbations to sarcomere assembly. We found perturbations that affected Z-Lines and M-Lines differently, suggesting that they may be regulated independently during sarcomere assembly.

Independent regulation of Z-lines and M-lines during sarcomere assembly in cardiac myocytes revealed by the automatic image analysis software sarcApp.

Sarcomeres are the basic contractile units within cardiac myocytes, and the collective shortening of sarcomeres aligned along myofibrils generates the force driving the heartbeat. The alignment of the individual sarcomeres is important for proper force generation, and misaligned sarcomeres are associated with diseases, including cardiomyopathies and COVID-19. The actin bundling protein, α-actinin-2, localizes to the 'Z-Bodies" of sarcomere precursors and the 'Z-Lines' of sarcomeres, and has been used previously to assess sarcomere assembly and maintenance. Previous measurements of α-actinin-2 organization have been largely accomplished manually, which is time-consuming and has hampered research progress. Here, we introduce sarcApp, an image analysis tool that quantifies several components of the cardiac sarcomere and their alignment in muscle cells and tissue. We first developed sarcApp to utilize deep learning-based segmentation and real space quantification to measure α-actinin-2 structures and determine the organization of both precursors and sarcomeres/myofibrils. We then expanded sarcApp to analyze 'M-Lines' using the localization of myomesin and a protein that connects the Z-Lines to the M-Line (titin). sarcApp produces 33 distinct measurements per cell and 24 per myofibril that allow for precise quantification of changes in sarcomeres, myofibrils, and their precursors. We validated this system with perturbations to sarcomere assembly. We found perturbations that affected Z-Lines and M-Lines differently, suggesting that they may be regulated independently during sarcomere assembly.

Structure of the native myosin filament in the relaxed cardiac sarcomere

The thick filament is a key component of sarcomeres, the basic units of striated muscle1. Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2. Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-β chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-β chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components.

RBPMS regulates cardiomyocyte contraction and cardiac function through RNA alternative splicing.

RNA binding proteins play essential roles in mediating RNA splicing and are key post-transcriptional regulators in the heart. Our recent study demonstrated that RBPMS (RNA binding protein with multiple splicing) is crucial for cardiac development through modulating mRNA splicing, but little is known about its functions in the adult heart. In this study, we aim to characterize the post-natal cardiac function of Rbpms and its mechanism of action. We generated a cardiac-specific knockout mouse line and found that cardiac-specific loss of Rbpms caused severe cardiomyocyte contractile defects, leading to dilated cardiomyopathy and early lethality in adult mice. We showed by proximity-dependent biotin identification assay and mass spectrometry that RBPMS associates with spliceosome factors and other RNA binding proteins, such as RBM20, that are important in cardiac function. We performed paired-end RNA sequencing and RT-PCR and found that RBPMS regulates mRNA alternative splicing of genes associated with sarcomere structure and function, such as Ttn, Pdlim5, and Nexn, generating new protein isoforms. Using a minigene splicing reporter assay, we determined that RBPMS regulates target gene splicing through recognizing tandem intronic CAC motifs. We also showed that RBPMS knockdown in human induced pluripotent stem cell-derived cardiomyocytes impaired cardiomyocyte contraction. This study identifies RBPMS as an important regulator of cardiomyocyte contraction and cardiac function by modulating sarcomeric gene alternative splicing.

Abstract P2049: Myosin Heavy Chain-7 E848G Mutation-induced Sarcopenia And Hypocontractility In Induced Pluripotent Stem Cell Model Of Hypertrophic Cardiomyopathy Are Rescued By Muscle Ring Finger 1 Ablation

Hypertrophic cardiomyopathy (HCM) presents with idiopathic left ventricular hypertrophy and occasionally systolic dysfunction. In a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of HCM, we previously reported the pathogenic MYH7 E848G variant increased hiPSC-CM size and impaired contractility. To elucidate the mechanisms by which the E848G variant causes HCM, we now examined cardiac tissue from a MYH7 wt/E848G patient. We found reduced expression of the following sarcomeric proteins: β-myosin heavy chain (βMHC) (97.8 ± 1.1% reduction), α-sarcomeric actinin (αSA) (84.8 ± 7.3%), and ventricular myosin light chain (MLC-1v) (45.9 ± 13.9%) relative to healthy patient tissue. Isogenic MYH7 wt/E848G hiPSC-CMs also had reduced protein expression of βMHC (22.8%) and αSA (24.5%) relative to MYH7 wt/wt hiPSC-CMs and unchanged mRNA expression. These results suggest MYH7 E848G HCM induces broad sarcomere component deficiency. We tested hiPSC-CM contractility using single cell traction force microscopy (TFM) and engineered heart tissues (EHT). Single isogenic MYH7 wt/E848G hiPSC-CMs had lower maximum twitch force (80 ± 12 nN) than MYH7 wt/wt hiPSC-CMs (150 ± 20 nN) by TFM. Isogenic MYH7 wt/E848G EHTs had lower maximum twitch force (118.9 ± 23.7 μN) than MYH7 wt/wt EHTs (287.4 ± 27.7 μN). Because MuRF1, an E3 ubiquitin-protein ligase, is known to target βMHC for proteasomal degradation, we hypothesized MuRF1 mediates the sarcopenic and hypocontractile phenotype. Genetic ablation of TRIM63 (encoding MuRF1) significantly reduced MYH7 wt/E848G hiPSC-CM cell size by flow cytometry (6.8 ± 1.7%). MYH7 wt/E848G TRIM63 -/- hiPSC-CMs increased protein expression of βMHC (154.7 ± 51.9%), αSA (44.9 ± 25.6%), and MLC-1v (246.9 ± 57.3%) relative to MYH7 wt/E848G TRIM63 +/+ hiPSC-CMs. MYH7 wt/E848G TRIM63 -/- had significantly higher maximum twitch force by TFM (133 ± 14 nN) and EHTs (178.2 ± 9.8 μN) than MYH7 wt/E848G TRIM63 +/+ by TFM (67 ± 8.0 nN) and EHTs (118.9 ± 23.7 μN). Our results implicate sarcopenia in the pathogenesis of MYH7 E848G HCM and identify MuRF1 as a potential novel therapeutic target for HCM. Further study will determine if these findings are generalizable to other pathogenic HCM variants in MYH7 and other sarcomeric genes.

Abstract P3041: Stage-specific Alternative Rna Splicing Attunes Cardiomyocyte Proliferation And Contraction

During heart development, cardiomyocytes display reduced proliferation and enhanced contractility to support cardiac functional maturation, but the mechanistic basis is not fully understood. Alternative RNA splicing is an important mechanism of RNA posttranscriptional modification and gene regulation in the heart. In studying a cardiac RNA binding protein, RBPMS (RNA-binding protein with multiple splicing), we found that RBPMS mediates distinct RNA splicing profiles in adult hearts versus neonatal hearts, indicating its stage-specific modulation of alternative RNA splicing. We showed that RBPMS regulates the splicing of cytoskeletal genes to enable proliferation of neonatal cardiomyocytes. In adult hearts, RBPMS regulates the splicing of sarcomere genes to support cardiac contraction. Cardiac-specific loss of RBPMS causes severe dilated cardiomyopathy and early lethality in adult mice. Using both in vivo and in vitro approaches, we found that the absence of RBPMS caused aberrant splicing of some key sarcomeric components, including Titin and Nexilin, causing cardiomyocyte contractile defects. Intriguingly, we also showed that RBPMS interacts with other cardiac RNA binding proteins, such as RBM20, suggesting their synergistic effects on cardiomyocyte physiology. Our work provides novel insights into how RNA binding proteins regulate cardiomyocyte proliferation and contraction by mediating alternative RNA splicing.

Beneficial Effects of Tacrolimus on Brain-Death-Associated Right Ventricular Dysfunction in Pigs.

Right ventricular (RV) dysfunction remains a major problem after heart transplantation and may be associated with brain death (BD) in a donor. A calcineurin inhibitor tacrolimus was recently found to have beneficial effects on heart function. Here, we examined whether tacrolimus might prevent BD-induced RV dysfunction and the associated pathobiological changes. After randomized tacrolimus (n = 8; 0.05 mg·kg-1·day-1) or placebo (n = 9) pretreatment, pigs were assigned to a BD procedure and hemodynamically investigated 1, 3, 5, and 7 h after the Cushing reflex. After euthanasia, myocardial tissue was sampled for pathobiological evaluation. Seven pigs were used as controls. Calcineurin inhibition prevented increases in pulmonary vascular resistance and RV-arterial decoupling induced by BD. BD was associated with an increased RV pro-apoptotic Bax-to-Bcl2 ratio and RV and LV apoptotic rates, which were prevented by tacrolimus. BD induced increased expression of the pro-inflammatory IL-6-to-IL-10 ratio, their related receptors, and vascular cell adhesion molecule-1 in both the RV and LV. These changes were prevented by tacrolimus. RV and LV neutrophil infiltration induced by BD was partly prevented by tacrolimus. BD was associated with decreased RV expression of the β-1 adrenergic receptor and sarcomere (myosin heavy chain [MYH]7-to-MYH6 ratio) components, while β-3 adrenergic receptor, nitric oxide-synthase 3, and glucose transporter 1 expression increased. These changes were prevented by tacrolimus. Brain death was associated with isolated RV dysfunction. Tacrolimus prevented RV dysfunction induced by BD through the inhibition of apoptosis and inflammation activation.

Mechanisms of Sarcomere Protein Mutation-Induced Cardiomyopathies.

The pace of identifying cardiomyopathy-associated mutations and advances in our understanding of sarcomere function that underlies many cardiomyopathies has been remarkable. Here, we aim to synthesize how these advances have led to the promising new treatments that are being developed to treat cardiomyopathies. The genomics era has identified and validated many genetic causes of hypertrophic and dilated cardiomyopathies. Recent advances in our mechanistic understanding of sarcomere pathophysiology include high-resolution molecular models of sarcomere components and the identification of the myosin super-relaxed state. The advances in our understanding of sarcomere function have yielded several therapeutic agents that are now in development and clinical use to correct contractile dysfunction-mediated cardiomyopathy. New genes linked to cardiomyopathy include targets with limited clinical evidence and require additional investigation. Large portions of cardiomyopathy with family history remain genetically undiagnosed and may be due to polygenic disease.

Clinical and Genetic Screening for Hypertrophic Cardiomyopathy in Paediatric Relatives: Changing Paradigms in Clinical Practice.

Hypertrophic cardiomyopathy (HCM) is an important cause of morbidity and mortality in children. While the aetiology is heterogeneous, most cases are caused by variants in the genes encoding components of the cardiac sarcomere, which are inherited as an autosomal dominant trait. In recent years, there has been a paradigm shift in the role of clinical screening and predictive genetic testing in children with a first-degree relative with HCM, with the recognition that phenotypic expression can, and often does, manifest in young children and that familial disease in the paediatric age group may not be benign. The care of the child and family affected by HCM relies on a multidisciplinary team, with a key role for genomics. This review article summarises current evidence in clinical and genetic screening for hypertrophic cardiomyopathy in paediatric relatives and highlights aspects that remain to be resolved.

Interrogation of skeletal muscle activation in an intact system via a myofilament incorporated FRET biosensor.

It is important to understand the mechanisms involved in regulating skeletal muscle activation and contraction for advancing insights into acquired and inherited muscle disorders and for identifying potential targets for therapeutics. The field has elucidated many sarcomeric components needed for activation of contraction. We seek to expand this work using a fluorescent sarcomere biosensor that allows for monitoring of sarcomeric activation in live, intact skeletal muscles. This biosensor fuses fluorescent donor and acceptor proteins to the N- and C-terminals of fast skeletal troponin C protein, respectively. Using the biosensor, skeletal muscle activation is monitored in real time through Förster Resonance Energy Transfer (FRET) fluorescence. This system maintains all essential, physiological features of the muscle, including excitation-contraction coupling and load, which was not possible in reconstituted systems. Using a transgenic mouse line with the biosensor, we have demonstrated FRET fluorescence in intact muscles from transgenic animals, with the time to peak of the fluorescence preceding the force development and the biosensor kinetics uncoupled from intracellular calcium kinetics. Of note, the FRET signal has a prolonged return to baseline as compared to force in intact EDL muscles, something that is not observed in unloaded FDB myofibers, and which may be evidence of a “primed state” of myofilament activation. The “primed state” is uncoupled from Ca2+ and is modified using fast skeletal muscle specific small molecules that target either the thin filament or thick filament. As thick filament modulating small molecules could be detected by the TnC localized biosensor during single twitch contractions, this demonstrates intermyofilament signaling in live muscle. Additionally, the “primed state” duration can be impacted through varying stimulation protocols, including dual pulse stimulations. This unique phenomenon will be further investigated using mechanical and pharmacological manipulations.