Sarcomere Function Research Articles

Novel Mutation Glu98Lys in Cardiac Tropomyosin Alters Its Structure and Impairs Myocardial Relaxation.

We characterized a novel genetic variant c.292G > A (p.E98K) in the TPM1 gene encoding cardiac tropomyosin 1.1 isoform (Tpm1.1), found in a proband with a phenotype of complex cardiomyopathy with conduction dysfunction and slow progressive neuromuscular involvement. To understand the molecular mechanism by which this mutation impairs cardiac function, we produced recombinant Tpm1.1 carrying an E98K substitution and studied how this substitution affects the structure of the Tpm1.1 molecule and its functional properties. The results showed that the E98K substitution in the N-terminal part of the Tpm molecule significantly destabilizes the C-terminal part of Tpm, thus indicating a long-distance destabilizing effect of the substitution on the Tpm coiled-coil structure. The E98K substitution did not noticeably affect Tpm's affinity for F-actin but significantly impaired Tpm's regulatory properties. It increased the Ca2+ sensitivity of the sliding velocity of regulated thin filaments over cardiac myosin in an in vitro motility assay and caused an incomplete block of the thin filament sliding at low Ca2+ concentrations. The incomplete motility block in the absence of Ca2+ can be explained by the loosening of the Tpm interaction with troponin I (TnI), thus increasing Tpm mobility on the surface of an actin filament that partially unlocks the myosin binding sites. This hypothesis is supported by the molecular dynamics (MD) simulation that showed that the E98 Tpm residue is involved in hydrogen bonding with the C-terminal part of TnI. Thus, the results allowed us to explain the mechanism by which the E98K Tpm mutation impairs sarcomeric function and myocardial relaxation.

Mavacamten, a precision medicine for hypertrophic cardiomyopathy: From a motor protein to patients.

Hypertrophic cardiomyopathy (HCM) is a primary myocardial disorder characterized by left ventricular hypertrophy, hyperdynamic contraction, and impaired relaxation of the heart. These functional derangements arise directly from altered sarcomeric function due to either mutations in genes encoding sarcomere proteins, or other defects such as abnormal energetics. Current treatment options do not directly address this causal biology but focus on surgical and extra-sarcomeric (sarcolemmal) pharmacological symptomatic relief. Mavacamten (formerly known as MYK-461), is a small molecule designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin, the fundamental motor of the sarcomere. This review summarizes the mechanism and translational progress of mavacamten from proteins to patients, describing how the mechanism of action and pharmacological characteristics, involving both systolic and diastolic effects, can directly target pathophysiological derangements within the cardiac sarcomere to improve cardiac structure and function in HCM. Mavacamten was approved by the Food and Drug Administration in April 2022 for the treatment of obstructive HCM and now goes by the commercial name of Camzyos. Full information about the risks, limitations, and side effects can be found at www.accessdata.fda.gov/drugsatfda_docs/label/2022/214998s000lbl.pdf.

Small molecule drugs to improve sarcomere function in those with acquired and inherited myopathies.

Muscle weakness is a hallmark of inherited or acquired myopathies. It is a major cause of functional impairment and can advance to life-threatening respiratory insufficiency. During the past decade, several small-molecule drugs that improve the contractility of skeletal muscle fibers have been developed. In this review, we provide an overview of the available literature and the mechanisms of action of small-molecule drugs that modulate the contractility of sarcomeres, the smallest contractile units in striated muscle, by acting on myosin and troponin. We also discuss their use in the treatment of skeletal myopathies. The first of three classes of drugs discussed here increase contractility by decreasing the dissociation rate of calcium from troponin and thereby sensitizing the muscle to calcium. The second two classes of drugs directly act on myosin and stimulate or inhibit the kinetics of myosin-actin interactions, which may be useful in patients with muscle weakness or stiffness.NEW & NOTEWORTHY During the past decade, several small molecule drugs that improve the contractility of skeletal muscle fibers have been developed. In this review, we provide an overview of the available literature and the mechanisms of action of small molecule drugs that modulate the contractility of sarcomeres, the smallest contractile units in striated muscle, by acting on myosin and troponin.

MYH7 in cardiomyopathy and skeletal muscle myopathy.

Myosin heavy chain gene 7 (MYH7), a sarcomeric gene encoding the myosin heavy chain (myosin-7), has attracted considerable interest as a result of its fundamental functions in cardiac and skeletal muscle contraction and numerous nucleotide variations of MYH7 are closely related to cardiomyopathy and skeletal muscle myopathy. These disorders display significantly inter- and intra-familial variability, sometimes developing complex phenotypes, including both cardiomyopathy and skeletal myopathy. Here, we review the current understanding on MYH7 with the aim to better clarify how mutations in MYH7 affect the structure and physiologic function of sarcomere, thus resulting in cardiomyopathy and skeletal muscle myopathy. Importantly, the latest advances on diagnosis, research models in vivo and in vitro and therapy for precise clinical application have made great progress and have epoch-making significance. All the great advance is discussed here.

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.

Regulation of BAG3 localization and sarcomeric protein turnover in the cardiac myocyte.

Bcl2-associated athanogene-3 (BAG3) is a pro-autophagy co-chaperone that we have previously shown localizes to the cardiac sarcomere and is critical for proteostasis and maintenance of normal sarcomeric function. Indeed, BAG3 loss in heart failure (HF) results in accumulation of ubiquitinated sarcomeric proteins, and depressed maximum force generating capacity (Fmax). However, how BAG3 is regulated in the cell and how it coordinates sarcomeric protein turnover is unknown. During our analysis of human heart tissue, BAG3 appears as a “doublet”, with one band at 74 kDa (BAG3-L) and a second at a higher 84 kDa (BAG3-H). Previous studies hypothesized the BAG3-H band was due to phosphorylation. To test this, we treated human myocardial tissue with 10 units of alkaline phosphatase for 1 hour, which decreased troponin I phosphorylation by ∼60%, but had no impact on the intensity of BAG3-H or BAG3-L bands, suggesting these two bands are not due to phosphorylation. When we ran purified human BAG3 protein, it ran near the same molecular weight as the BAG3-H band, suggesting the lighter BAG3-L band is the modified version. These results suggest the BAG3-L band has been cleaved at either the N- or C-termini, which would be around 90 amino acids to cause the observed mass shift. Mass spectrometry analysis is being performed to determine the cleavage site and the domains being interrupted by this cleavage. Importantly, the BAG3-H/BAG3-L is higher in the myofilament compared to the cytoplasm of cardiomyocytes, suggesting the heavier form of BAG3 binds with sarcomere with increased affinity. We are also using high resolution live-cell imaging reveal the kinetics of the sarcomeric protein quality control system orchestrated by BAG3. These findings reveal novel regulation of the critical BAG3 protein and provides potential for BAG3-based HF therapies.

The polyamine spermidine enhances contractile function and sarcomere dynamics post-doxorubicin treatment.

Doxorubicin (DOX) is a chemotherapy used to treat childhood and adult malignancies. However, despite being a cornerstone for oncological care, the use of DOX is limited due to dose-dependent cardiotoxicity, which can lead to contractile dysfunction and heart failure. The exact mechanism of DOX-induced cardiotoxicity is not fully understood. However, proposed mechanisms include sarcomere degradation and impairment of autophagy. To determine whether enhancing autophagy can restore contractile function, we treated µ-patterned single-cell human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), displaying sarcomere disarray and reduced contractile force post-DOX treatment with spermidine, an autophagy activator. The contractile force was quantified using traction force microscopy. There were five conditions (untreated control, DOX, maintenance medium post-DOX, spermidine post-DOX, and spermidine-only). We found that hiPSC-CMs treated with spermidine post-DOX produce 67% of the contractile force generated by control cells (0.22 µN vs. 0.33 µN, not significant) and significantly (0.22 µN vs. 0.11 µN, p < 0.0001) higher contractile force compared to cells in maintenance medium post-DOX. In the spermidine-only group, our results demonstrate that spermidine significantly (0.57 µN vs. 0.33 µN, p < 0.0001) increases contractile force compared to control. Spermidine-only treated cells were also bigger (spread area = ∼1716 µm2 vs. ∼1353 µm2, p < 0.00001) than the control and had a more robust and organized sarcomere network. Preliminary qPCR results suggest that spermidine upregulates genes that control sarcomere function (e.g., MYH7) and organization (e.g., ANKD1). These data demonstrate that spermidine can enhance contractile force post-DOX treatment and increase hiPSC-CMs size and sarcomere network. The data further suggests that sarcomere homeostasis is one of the mechanisms spermidine regulates in hiPSC-CMs. This work opens the door for developing therapies that target autophagy and sarcomere proteostasis to treat cardiac dysfunction.

Insights from an electro-mechanical heart failure cell model: Role of SERCA enhancement on arrhythmogenesis and myocyte contraction

Background and objectiveStructural and electrical remodeling in heart failure predisposes the heart to ventricular arrhythmias. Computer modeling approaches, used to complement experimental results, can provide a more mechanistic knowledge of the biophysical phenomena underlying cardiac pathologies. Indeed, previous in-silico studies have improved the understanding of the electrical correlates of heart failure involved in arrhythmogenesis; however, information on the crosstalk between electrical activity, intracellular Ca2+ and contraction is still incomplete. This study aims to investigate the electro-mechanical behavior of virtual failing human ventricular myocytes to help in the development of therapies, which should ideally target pump failure and arrhythmias at the same time. MethodsWe implemented characteristic remodeling of heart failure with reduced ejection fraction by including reported changes in ionic conductances, sarcomere function and cell structure (e.g. T-tubules disarray). Model parametrization was based on published experimental data and the outcome of simulations was validated against experimentally observed patterns. We focused on two aspects of myocardial dysfunction central in heart failure: altered force-frequency relationship and susceptibility to arrhythmogenic early afterdepolarizations. Because biological variability is a major problem in the generalization of in-silico findings based on a unique set of model parameters, we generated and evaluated a population of models. ResultsThe population-based approach is crucial in robust identification of parameters at the core of abnormalities and in generalizing the outcome of their correction. As compared to non-failing ones, failing myocytes had prolonged repolarization, a higher incidence of early afterdepolarizations, reduced contraction and a shallower force-frequency relationship, all features peculiar of heart failure. Component analysis applied to the model population identified reduced SERCA function as a relevant contributor to most of these derangements, which were largely reverted or diminished by restoration of SERCA function alone. ConclusionsThese simulated results encourage the development of strategies comprising SERCA stimulation and highlight the need to evaluate both electrical and mechanical outcomes.

Cardiac Sarcomere Signaling in Health and Disease.

The cardiac sarcomere is a triumph of biological evolution wherein myriad contractile and regulatory proteins assemble into a quasi-crystalline lattice to serve as the central point upon which cardiac muscle contraction occurs. This review focuses on the many signaling components and mechanisms of regulation that impact cardiac sarcomere function. We highlight the roles of the thick and thin filament, both as necessary structural and regulatory building blocks of the sarcomere as well as targets of functionally impactful modifications. Currently, a new focus emerging in the field is inter-myofilament signaling, and we discuss here the important mediators of this mechanism, including myosin-binding protein C and titin. As the understanding of sarcomere signaling advances, so do the methods with which it is studied. This is reviewed here through discussion of recent live muscle systems in which the sarcomere can be studied under intact, physiologically relevant conditions.

Abstract 13073: Antisense Transcripts Regulate Titin Alternative Splicing and Sarcomere Function in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

The giant protein Titin is an essential structural component of the sarcomere and a key determinant of passive tension during diastole. Titin tension is regulated by alternative splicing within the spring-like domains in the I-band region of TTN pre-mRNA. The cardiac RNA-binding protein RBM20 has been shown to promote TTN exon skipping during splicing, but additional factors are likely involved. We hypothesized that a natural antisense transcript in the TTN locus ( TTN-AS1 ) could regulate TTN splicing in cis through recruitment of spliceosome components. Through single nuclei RNA-Seq and RNA fluorescence in situ hybridization (ISH) on human cardiac tissue and iPS-derived cardiomyocytes (iPS-CM), we showed that the expression of TTN-AS1 was restricted to cardiomyocyte nuclei. The percent spliced in (PSI) of each TTN exon was calculated based on RNA-Seq data from iPS-CM (n=3) and 26 exons (all in the I-band region) that are normally spliced out were included to a significantly higher extent (adjusted p&lt;0.05) upon siRNA-mediated TTN-AS1 knock down. Reduced exon skipping from TTN exon 50 was confirmed with exon-junction spanning qPCR probes (p&lt;0.05, n=3). Similar effects on splicing were seen in cells where RBM20 had been knocked down. The consequences of TTN-AS1 knock down on cardiomyocyte contractile function was assessed using sarcomere tracking. Transgenic expression of ACTN2-GFP was used to visualize z-disks in iPS-CM, sarcomeres (n=14,636) were recorded during contractions using wide-field epifluorescence microscopy and analyzed using Sarc-Graph software. The analysis revealed that in cells where TTN-AS1 had been knocked down, sarcomere length and fractional shortening, as well as contraction and relaxation time were increased (all p&lt;0.001). Again, similar effects were seen in iPS-CM where RBM20 had been knocked down. We observed co-localization of TTN-AS1 and RBM20 protein during active TTN splicing in iPS-CM nuclei using multiplex RNA ISH and immunofluorescence and confirmed interaction between TTN-AS1 and RBM20 using RNA Immunoprecipitation and qPCR. In conclusion, we show that TTN-AS1 can affect cardiomyocyte contractile properties and sarcomere dynamics through regulation of TTN alternative splicing.

Abstract 14213: The Polyamine Spermidine Enhances the Contractile Force of μ-Patterned Single-Cell Stem-Cell-Derived Cardiomyocytes Post Doxorubicin Treatment

Doxorubicin (DOX) is a chemotherapy used to treat childhood and adult malignancies, including pediatric leukemia and breast cancer. However, despite being a cornerstone for oncological care, the use of DOX is limited due to dose-dependent cardiotoxicity, which can lead to contractile dysfunction and heart failure. Yet, Dox is still widely used due to the lack of suitable replacements. The exact mechanism of Dox-induced cardiotoxicity is not fully understood. However, proposed mechanisms include sarcomere degradation and impairment of autophagy. To determine whether enhancing autophagy can restore contractile function, we treated μ-patterned single-cell human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), displaying sarcomere disarray and reduced contractile force post-Dox treatment with spermidine, an autophagy activator. The contractile force was quantified using traction force microscopy. There were five conditions (untreated control, Dox, maintenance medium post-Dox, spermidine post-Dox, and spermidine-only). We found that hiPSC-CMs treated with spermidine post-Dox produce 67% of the contractile force generated by control cells (.22 μN vs. .33 μN, not significant) and significantly (.22 μN vs. .11 μN, p &lt; .0001) higher contractile force compared to cells in maintenance medium post-Dox. In the spermidine-only group, our results demonstrate that spermidine significantly (.57 μN vs. .33 μN, p &lt; .0001) increases contractile force compared to control. Spermidine-only treated cells were also bigger (spread area = ~1716 μm 2 vs. ~1353 μm 2 , p &lt; .00001) than the control and had a more robust and organized sarcomere network. Preliminary qPCR results suggest that spermidine upregulates genes that control sarcomere function (e.g., MYH7) and organization (e.g., ANKD1). These data demonstrate that spermidine can enhance contractile force post-Dox treatment and increase cellular size and sarcomere network in hiPSC-CMs. The data further suggests that sarcomere homeostasis is one of the cellular mechanisms spermidine regulates in hiPSC-CMs. This work opens the door for developing therapies that target autophagy and sarcomere proteostasis to treat cardiac dysfunction.

Abstract 13724: Development of VS-041 as a Precision Medicine Targeting Cardiac-Relevant Matrix Metalloproteases (MMPs) in Heart Failure With Preserved Ejection Fraction (HFpEF)

Introduction. HFpEF is a heterogeneous syndrome, with several underlying etiologic and pathophysiologic factors. As “one size fits all” therapies are not equally efficacious across various HFpEF comorbidities, a precision medicine approach can offer a disease modifying treatment for HFpEF subpopulation stratified for specific molecular targets. Expression of MMP2, 9, and 13 in heart is elevated in HFpEF patients with hypertension, diabetes or coronary disease, and their levels in plasma correlate with survival, suggesting utility as predictive biomarkers. Genetic ablation of MMP2, 9, and 13 leads to a robust antifibrotic effect in the myocardium. Inhibition of MMP2, 9, and 13 directly regulates of Ca 2+ transients and protects sarcomere function. Hypothesis. We present preclinical profile of VS-041, a novel and narrow-spectrum MMP inhibitor, as a drug candidate for HFpEF patients with elevated levels of MMP2, 9, and 13. Methods and Results. VS-041, optimized with computer-aided drug design methods, shows single-digit IC 50 against MMP2 (1nM), MMP9 (2nM) and MMP13 (1nM), and maintains &gt;300-fold selectivity against MMP1 and TACE. In isolated cardiomyocytes VS-041 reduces contractility deficit due to fast pacing. With high predicted metabolic bioavailability (MF%=90), &gt;80% target occupancy by VS-041 is estimated after 50 mg/kg PO dose in rats. VS-041 demonstrated robust efficacy in Dahl Salt Sensitive rat model of hypertension and HFpEF after dosing at 25 or 50 mg/kg PO for 5 wks in a therapeutic mode (after 1-wk of high salt lead-in). VS-041 had no effect on blood pressure or ejection fraction confirming a direct effect on diastolic function. In Doppler mitral inflow, analysis of the isovolumic relaxation time and the E/A ratio of peak velocity of filling waves revealed 40% improvement in diastolic function. LV fibrosis was dose-proportionally decreased based on PSR morphometry. No adverse effects were observed after 5 weeks of dosing. Conclusions. Preclinical pharmacology and safety profile of VS-041 support its nomination as clinical candidate and initiation of IND-enabling studies. VS-041 has potential to be first-in-class precision medicine treatment for HFPEF patients with elevated levels of cardiac-relevant MMP2, 9 and 13.

Abstract 13258: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Introduction: Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 6.1 million . AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light chain phosphorylation in AF. Objective We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12CPP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

Sarcomere dynamics revealed by a myofilament integrated FRET-based biosensor in live skeletal muscle fibers

The sarcomere is the functional unit of skeletal muscle, essential for proper contraction. Numerous acquired and inherited myopathies impact sarcomere function causing clinically significant disease. Mechanistic investigations of sarcomere activation have been challenging to undertake in the context of intact, live skeletal muscle fibers during real time physiological twitch contractions. Here, a skeletal muscle specific, intramolecular FRET-based biosensor was designed and engineered into fast skeletal muscle troponin C (TnC) to investigate the dynamics of sarcomere activation. In transgenic animals, the TnC biosensor incorporated into the skeletal muscle fiber sarcomeres by stoichiometric replacement of endogenous TnC and did not alter normal skeletal muscle contractile form or function. In intact single adult skeletal muscle fibers, real time twitch contractile data showed the TnC biosensor transient preceding the peak amplitude of contraction. Importantly, under physiological temperatures, inactivation of the TnC biosensor transient decayed significantly more slowly than the Ca2+ transient and contraction. The uncoupling of the TnC biosensor transient from the Ca2+ transient indicates the biosensor is not functioning as a Ca2+ transient reporter, but rather reports dynamic sarcomere activation/ inactivation that, in turn, is due to the ensemble effects of multiple activating ligands within the myofilaments. Together, these findings provide the foundation for implementing this new biosensor in future physiological studies investigating the mechanism of activation of the skeletal muscle sarcomere in health and disease.

Prospects for remodeling the hypertrophic heart with myosin modulators

Hypertrophic cardiomyopathy (HCM) is a complex but relatively common genetic disease that usually arises from pathogenic variants that disrupt sarcomere function and lead to variable structural, hypertrophic, and fibrotic remodeling of the heart which result in substantial adverse clinical outcomes including arrhythmias, heart failure, and sudden cardiac death. HCM has had few effective treatments with the potential to ameliorate disease progression until the recent advent of inhibitory myosin modulators like mavacamten. Preclinical investigations and clinical trials utilizing this treatment targeted to this specific pathophysiological mechanism of sarcomere hypercontractility in HCM have confirmed that myosin modulators can alter disease expression and attenuate hypertrophic remodeling. Here, we summarize the state of hypertrophic remodeling and consider the arguments for and against salutary HCM disease modification using targeted myosin modulators. Further, we consider critical unanswered questions for future investigative and therapeutic avenues in HCM disease modification. We are at the precipice of a new era in understanding and treating HCM, with the potential to target agents toward modifying disease expression and natural history of this most common inherited disease of the heart.

Sarcomere maturation: function acquisition, molecular mechanism, and interplay with other organelles.

During postnatal cardiac development, cardiomyocytes mature and turn into adult ones. Hence, all cellular properties, including morphology, structure, physiology and metabolism, are changed. One of the most important aspects is the contractile apparatus, of which the minimum unit is known as a sarcomere. Sarcomere maturation is evident by enhanced sarcomere alignment, ultrastructural organization and myofibrillar isoform switching. Any maturation process failure may result in cardiomyopathy. Sarcomere function is intricately related to other organelles, and the growing evidence suggests reciprocal regulation of sarcomere and mitochondria on their maturation. Herein, we summarize the molecular mechanism that regulates sarcomere maturation and the interplay between sarcomere and other organelles in cardiomyocyte maturation.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

Diabetes disturbs functional adaptation of the remote myocardium after ischemia/reperfusion

Diabetes mellitus type 2 is associated with adverse clinical outcome after myocardial infarction. To better understand the underlying causes we here investigated sarcomere protein function and its calcium-dependent regulation in the non-ischemic remote myocardium (RM) of diabetic mice (db/db) after transient occlusion of the left anterior descending coronary artery. Before and 24 h after surgery db/db and non-diabetic db/+ underwent magnetic resonance imaging followed by histological and biochemical analyses of heart tissue. Intracellular calcium transients and sarcomere function were measured in isolated cardiomyocytes. Active and passive force generation was assessed in skinned fibers and papillary muscle preparations. Before ischemia and reperfusion (I/R), beat-to-beat calcium cycling was depressed in diabetic cardiomyocytes. Nevertheless, contractile function was preserved owing to increased myofilament calcium sensitivity and higher responsiveness of myocardial force production to β-adrenergic stimulation in db/db compared to db/+. In addition, protein kinase C activity was elevated in db/db hearts leading to strong phosphorylation of the titin PEVK region and increased titin-based tension of myofilaments. I/R impaired the function of whole hearts and RM sarcomeres in db/db to a larger extent than in non-diabetic db/+, and we identified several reasons. First, the amplitude and the kinetics of cardiomyocyte calcium transients were further reduced in the RM of db/db. Underlying causes involved altered expression of calcium regulatory proteins. Diabetes and I/R additively reduced phospholamban S16-phosphorylation by 80% (P < 000.1) leading to strong inhibition of the calcium ATPase SERCA2a. Second, titin stiffening was only observed in the RM of db/+, but not in the RM of db/db. Finally, db/db myofilament calcium sensitivity and force generation upon β-adrenergic stimulation were no longer enhanced over db/+ in the RM. The findings demonstrate that impaired cardiomyocyte calcium cycling of db/db hearts is compensated by increased myofilament calcium sensitivity and increased titin-based stiffness prior to I/R. In contrast, sarcomere function of the RM 24 h after I/R is poor because both these compensatory mechanisms fail and myocyte calcium handling is further depressed.

LEFT VENTRICULAR REMODELING IN HEART FAILURE (PART I): CURRENT UNDERSTANDING OF PATHOMECHANISMS AND RELATED MYOCARDIAL DYSFUNCTION

Aim: to provide a literature review of the current data on various pathomechanisms of left ventricular (LV) remodeling in heart failure (HF) patients and their role in the development and progression of myocardial dysfunction. This paper is a first part of the review, devoted to the current state of pathophysiology of LV remodeling in HF.&#x0D; Material and methods. The thematic scientific papers, published during the last decade, constituted the study material. The research methodology involved bibliosemantic method and structural and logical analysis.&#x0D; Results and discussion. LV remodeling is the result of complex changes at the molecular, cellular and tissue levels, affecting the myocardial mass, geometry and performance, and ultimately leading to HF development and progression. LV systolic dysfunction occurs through the numerous mechanisms, including the defects in sarcomere function, abnormal excitation-contraction coupling and calcium homeostasis, ion channel dysfunction, mitochondrial and metabolic abnormalities, depressed cardiomyocytes survival signaling, redox pathobiology, inflammation and inadequate vasculogenesis. The term «LV diastolic dysfunction» covers the alterations in diastolic distensibility, filling or relaxation of the LV, regardless of whether LV (global) systolic function is normal or abnormal, and regardless of whether the patient has clinical manifestations of HF. The up-to-date pathophysiological paradigm of the development and progression of HF with LV diastolic dysfunction and preserved LV (global) systolic function considers systemic inflammation as a key pathomechanism of structural and functional changes of the myocardium, promoted by various cardiovascular and extracardiac conditions. In its turn, the systemic inflammation promotes endothelial dysfunction, contributing to multiple end-organ damage.&#x0D; Conclusion. The deepening one`s knowledge of various pathomechanisms of LV remodeling and related myocardial dysfunction in HF patients is an important prerequisite for identifying new perspectives on further fundamental research аnd more rational designing of future clinical trials.

A pathogenic mechanism associated with myopathies and structural birth defects involves TPM2-directed myogenesis.

Nemaline myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle–specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as cap myopathy, congenital fiber type disproportion, and distal arthrogryposis (DA). The in vivo pathomechanisms underlying TPM2-related disorders are unknown, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found 4 variants significantly affected muscle development and muscle function. Transient overexpression of the 4 variants also disrupted the morphogenesis of mouse myotubes in vitro and negatively affected zebrafish muscle development in vivo. We used transient overexpression assays in zebrafish to characterize 2 potentially novel TPM2 variants and 1 recurring variant that we identified in patients with DA (V129A, E139K, A155T, respectively) and found these variants caused musculoskeletal defects similar to those of known pathogenic variants. The consistency of musculoskeletal phenotypes in our assays correlated with the severity of clinical phenotypes observed in our patients with DA, suggesting disrupted myogenesis is a potentially novel pathomechanism of TPM2 disorders and that our myogenic assays can predict the clinical severity of TPM2 variants.

Genetic Basis of Left Ventricular Noncompaction.

Left ventricular noncompaction (LVNC) is the third most common pediatric cardiomyopathy characterized by a thinned myocardium and prominent trabeculations. Next-generation genetic testing has led to a rapid increase in the number of genes reported to be associated with LVNC, but we still have little understanding of its pathogenesis. We sought to grade the strength of the gene-disease relationship for all genes reported to be associated with LVNC and identify molecular pathways that could be implicated. Following a systematic PubMed review, all genes identified with LVNC were graded using a validated, semi-quantitative system based on all published genetic and experimental evidence created by the Clinical Genome Resource (ClinGen). Genetic pathway analysis identified molecular processes and pathways associated with LVNC. We identified 189 genes associated with LVNC: 11 (6%) were classified as definitive, 21 (11%) were classified as moderate, and 140 (74%) were classified as limited, but 17 (9%) were classified as no evidence. Of the 32 genes classified as definitive or moderate, the most common gene functions were sarcomere function (n=11; 34%), transcriptional/translational regulator (n=6; 19%), mitochondrial function (n=3; 9%), and cytoskeletal protein (n=3; 9%). Furthermore, 18 (56%) genes were implicated in noncardiac syndromic presentations. Lastly, 3 genetic pathways (cardiomyocyte differentiation via BMP receptors, factors promoting cardiogenesis in vertebrates, and Notch signaling) were found to be unique to LVNC and not overlap with pathways identified in dilated cardiomyopathy and hypertrophic cardiomyopathy. LVNC is a genetically heterogeneous cardiomyopathy. Distinct from dilated or hypertrophic cardiomyopathies, LVNC appears to arise from abnormal developmental processes.