Imaging of Existing and Newly Translated Proteins Elucidates Mechanisms of Sarcomere Turnover.

BACKGROUND: How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres. METHODS: We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands. RESULTS: We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells. CONCLUSIONS: Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles.

PatternJ: an ImageJ toolset for the automated and quantitative analysis of regular spatial patterns found in sarcomeres, axons, somites, and more.

Regular spatial patterns are ubiquitous forms of organization in nature. In animals, regular patterns can be found from the cellular scale to the tissue scale, and from early stages of development to adulthood. To understand the formation of these patterns, how they assemble and mature, and how they are affected by perturbations, a precise quantitative description of the patterns is essential. However, accessible tools that offer in-depth analysis without the need for computational skills are lacking for biologists. Here, we present PatternJ, a novel toolset to analyze regular one-dimensional patterns precisely and automatically. This toolset, to be used with the popular imaging processing program ImageJ/Fiji, facilitates the extraction of key geometric features within and between pattern repeats in static images and time-lapse series. We validate PatternJ with simulated data and test it on images of sarcomeres from insect muscles and contracting cardiomyocytes, actin rings in neurons, and somites from zebrafish embryos obtained using confocal fluorescence microscopy, STORM, electron microscopy, and brightfield imaging. We show that the toolset delivers subpixel feature extraction reliably even with images of low signal-to-noise ratio. PatternJ's straightforward use and functionalities make it valuable for various scientific fields requiring quantitative one-dimensional pattern analysis, including the sarcomere biology of muscles or the patterning of mammalian axons, speeding up discoveries with the bonus of high reproducibility.

Exploring the Connection Between Relaxed Myosin States and the Anrep Effect.

The Anrep effect is an adaptive response that increases left ventricular contractility following an acute rise in afterload. Although the mechanistic origin remains undefined, recent findings suggest a two-phase activation of resting myosin for contraction, involving strain-sensitive and posttranslational phases. We propose that this mobilization represents a transition among the relaxed states of myosin-specifically, from the super-relaxed (SRX) to the disordered-relaxed (DRX)-with DRX myosin ready to participate in force generation. This hypothesis offers a unified explanation that connects myosin's SRX-DRX equilibrium and the Anrep effect as parts of a singular phenomenon. We underscore the significance of this equilibrium in modulating contractility, primarily studied in the context of hypertrophic cardiomyopathy, the most common inherited cardiomyopathy associated with diastolic dysfunction, hypercontractility, and left ventricular hypertrophy. As we posit that the cellular basis of the Anrep effect relies on a two-phased transition of myosin from the SRX to the contraction-ready DRX configuration, any dysregulation in this equilibrium may result in the pathological manifestation of the Anrep phenomenon. For instance, in hypertrophic cardiomyopathy, hypercontractility is linked to a considerable shift of myosin to the DRX state, implying a persistent activation of the Anrep effect. These valuable insights call for additional research to uncover a clinical Anrep fingerprint in pathological states. Here, we demonstrate through noninvasive echocardiographic pressure-volume measurements that this fingerprint is evident in 12 patients with hypertrophic obstructive cardiomyopathy before septal myocardial ablation. This unique signature is characterized by enhanced contractility, indicated by a leftward shift and steepening of the end-systolic pressure-volume relationship, and a prolonged systolic ejection time adjusted for heart rate, which reverses post-procedure. The clinical application of this concept has potential implications beyond hypertrophic cardiomyopathy, extending to other genetic cardiomyopathies and even noncongenital heart diseases with complex etiologies across a broad spectrum of left ventricular ejection fractions.

Considering impact of age and sex on cardiac cytoskeletal components.

The cardiac cytoskeletal components are integral to cardiomyocyte function and are responsible for contraction, sustaining cell structure, and providing scaffolding to direct signaling. Cytoskeletal components have been implicated in cardiac pathology; however, less attention has been paid to age-related modifications of cardiac cytoskeletal components and how these contribute to dysfunction with increased age. Moreover, significant sex differences in cardiac aging have been identified, but we still lack a complete understanding to the mechanisms behind these differences. This review summarizes what is known about how key cardiomyocyte cytoskeletal components are modified because of age, as well as reported sex-specific differences. Thorough consideration of both age and sex as integral players in cytoskeletal function may reveal potential avenues for more personalized therapeutics.

Nrg1 Regulates Cardiomyocyte Migration and Cell Cycle in Ventricular Development.

BACKGROUND: Cardiac ventricles provide the contractile force of the beating heart throughout life. How the primitive endocardium-layered myocardial projections called trabeculae form and mature into the adult ventricles is of great interest for biology and regenerative medicine. Trabeculation is dependent on the signaling protein Nrg1 (neuregulin-1). However, the mechanism of action of Nrg1 and its role in ventricular wall maturation are poorly understood. METHODS: We investigated the functions and downstream mechanisms of Nrg1 signaling during ventricular chamber development using confocal imaging, transcriptomics, and biochemical approaches in mice with cardiac-specific inactivation or overexpression of Nrg1. RESULTS: Analysis of cardiac-specific Nrg1 mutant mice showed that the transcriptional program underlying cardiomyocyte-oriented cell division and trabeculae formation depends on endocardial Nrg1 to myocardial ErbB2 (erb-b2 receptor tyrosine kinase 2) signaling and phospho-Erk (phosphorylated extracellular signal-regulated kinase; pErk) activation. Early endothelial loss of Nrg1 and reduced pErk activation diminished cardiomyocyte Pard3 and Crumbs2 (Crumbs Cell Polarity Complex Component 2) protein and altered cytoskeletal gene expression and organization. These alterations are associated with abnormal gene expression related to mitotic spindle organization and a shift in cardiomyocyte division orientation. Nrg1 is crucial for trabecular growth and ventricular wall thickening by regulating an epithelial-to-mesenchymal transition-like process in cardiomyocytes involving migration, adhesion, cytoskeletal actin turnover, and timely progression through the cell cycle G2/M phase. Ectopic cardiac Nrg1 overexpression and high pErk signaling caused S-phase arrest, sustained high epithelial-to-mesenchymal transition-like gene expression, and prolonged trabeculation, blocking compact myocardium maturation. Myocardial trabecular patterning alterations resulting from above- or below-normal Nrg1-dependent pErk activation were concomitant with sarcomere actin cytoskeleton disorganization. The Nrg1 loss- and gain-of-function transcriptomes were enriched for Yap1 (yes-associated protein-1) gene signatures, identifying Yap1 as a potential downstream effector. Furthermore, biochemical and imaging data reveal that Nrg1 influences pErk activation and Yap1 nuclear-cytoplasmic distribution during trabeculation. CONCLUSIONS: These data establish the Nrg1-ErbB2/ErbB4-Erk axis as a crucial regulator of cardiomyocyte cell cycle progression and migration during ventricular development.

Danicamtiv Increases Myosin Recruitment and Alters Cross-Bridge Cycling in Cardiac Muscle.

BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. METHODS: Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficit. RESULTS: Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the ON state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ. CONCLUSIONS: As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy.

Microstructural and Microvascular Phenotype of Sarcomere Mutation Carriers and Overt Hypertrophic Cardiomyopathy.

BACKGROUND: In hypertrophic cardiomyopathy (HCM), myocyte disarray and microvascular disease (MVD) have been implicated in adverse events, and recent evidence suggests that these may occur early. As novel therapy provides promise for disease modification, detection of phenotype development is an emerging priority. To evaluate their utility as early and disease-specific biomarkers, we measured myocardial microstructure and MVD in 3 HCM groups-overt, either genotype-positive (G+LVH+) or genotype-negative (G-LVH+), and subclinical (G+LVH-) HCM-exploring relationships with electrical changes and genetic substrate. METHODS: This was a multicenter collaboration to study 206 subjects: 101 patients with overt HCM (51 G+LVH+ and 50 G-LVH+), 77 patients with G+LVH-, and 28 matched healthy volunteers. All underwent 12-lead ECG, quantitative perfusion cardiac magnetic resonance imaging (measuring myocardial blood flow, myocardial perfusion reserve, and perfusion defects), and cardiac diffusion tensor imaging measuring fractional anisotropy (lower values expected with more disarray), mean diffusivity (reflecting myocyte packing/interstitial expansion), and second eigenvector angle (measuring sheetlet orientation). RESULTS: Compared with healthy volunteers, patients with overt HCM had evidence of altered microstructure (lower fractional anisotropy, higher mean diffusivity, and higher second eigenvector angle; all P<0.001) and MVD (lower stress myocardial blood flow and myocardial perfusion reserve; both P<0.001). Patients with G-LVH+ were similar to those with G+LVH+ but had elevated second eigenvector angle (P<0.001 after adjustment for left ventricular hypertrophy and fibrosis). In overt disease, perfusion defects were found in all G+ but not all G- patients (100% [51/51] versus 82% [41/50]; P=0.001). Patients with G+LVH- compared with healthy volunteers similarly had altered microstructure, although to a lesser extent (all diffusion tensor imaging parameters; P<0.001), and MVD (reduced stress myocardial blood flow [P=0.015] with perfusion defects in 28% versus 0 healthy volunteers [P=0.002]). Disarray and MVD were independently associated with pathological electrocardiographic abnormalities in both overt and subclinical disease after adjustment for fibrosis and left ventricular hypertrophy (overt: fractional anisotropy: odds ratio for an abnormal ECG, 3.3, P=0.01; stress myocardial blood flow: odds ratio, 2.8, P=0.015; subclinical: fractional anisotropy odds ratio, 4.0, P=0.001; myocardial perfusion reserve odds ratio, 2.2, P=0.049). CONCLUSIONS: Microstructural alteration and MVD occur in overt HCM and are different in G+ and G- patients. Both also occur in the absence of hypertrophy in sarcomeric mutation carriers, in whom changes are associated with electrocardiographic abnormalities. Measurable changes in myocardial microstructure and microvascular function are early-phenotype biomarkers in the emerging era of disease-modifying therapy.

Stretch Harmonizes Sarcomere Strain Across the Cardiomyocyte.

BACKGROUND: Increasing cardiomyocyte contraction during myocardial stretch serves as the basis for the Frank-Starling mechanism in the heart. However, it remains unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres. We investigated sarcomere contractile synchrony and how intersarcomere dynamics contribute to increasing contractility during cell lengthening. METHODS: Sarcomere strain and Ca2+ were simultaneously recorded in isolated left ventricular cardiomyocytes during 1 Hz field stimulation at 37 °C, at resting length and following stepwise stretch. RESULTS: We observed that in unstretched rat cardiomyocytes, differential sarcomere deformation occurred during each beat. Specifically, while most sarcomeres shortened during the stimulus, ≈10% to 20% of sarcomeres were stretched or remained stationary. This nonuniform strain was not traced to regional Ca2+ disparities but rather shorter resting lengths and lower force production in systolically stretched sarcomeres. Lengthening of the cell recruited additional shortening sarcomeres, which increased contractile efficiency as less negative, wasted work was performed by stretched sarcomeres. Given the known role of titin in setting sarcomere dimensions, we next hypothesized that modulating titin expression would alter intersarcomere dynamics. Indeed, in cardiomyocytes from mice with titin haploinsufficiency, we observed greater variability in resting sarcomere length, lower recruitment of shortening sarcomeres, and impaired work performance during cell lengthening. CONCLUSIONS: Graded sarcomere recruitment directs cardiomyocyte work performance, and harmonization of sarcomere strain increases contractility during cell stretch. By setting sarcomere dimensions, titin controls sarcomere recruitment, and its lowered expression in haploinsufficiency mutations impairs cardiomyocyte contractility.

Estudio preliminar de correlación fenotipo-genotipo en miocardiopatías de pacientes derivados a un centro de alta complejidad del conurbano bonaerense / A Preliminary Study of the Phenotype-Genotype Correlation in Cardiomyopathies in Patients Referred to a Tertiary Healthcare Center in the Suburbs of Buenos Aires

RESUMEN Introducción : Las miocardiopatías se definen como un trastorno del miocardio en el que el músculo cardíaco es estructural y funcionalmente anormal, en ausencia de enfermedad arterial coronaria, hipertensión arterial (HTA), enfermedad valvular y enfermedad cardíaca congénita. Estas enfermedades son relativamente frecuentes, y suponen una importante causa de morbimortalidad a nivel global. Aunque el estudio genético se recomienda para el cribado familiar, la falta de datos robustos sobre asociaciones genotipo-fenotipo específicas ha reducido su impacto en el manejo clínico. Objetivos : El objetivo de este estudio es analizar la frecuencia de mutaciones en una población de pacientes con miocardiopatía derivados a un centro de alta complejidad y el análisis de la correlación genotipo-fenotipo en las mutaciones identificadas. Material y métodos: Se estudiaron en forma prospectiva 102 pacientes con sospecha de miocardiopatía hipertrófica (MCH) familiar, de los cuales 70 constituían casos índices, de una cohorte ambispectiva de pacientes con miocardiopatías controladas en un hos pital público de alta complejidad de tercer nivel de atención de la provincia de Buenos Aires, desde enero 2012 al 30 agosto 2022. Resultados : De 102 pacientes 83 fueron considerados afectados. De eelos, 31 eran MCH y 52 fenocopias, sin diferencia en el pronóstico. Se realizó estudio genético en 77 pacientes, de los cuales 57 presentaron mutaciones reconocibles, en el 80% de los casos coincidentes con un Score de Mayo ≥3. Se detectaron 28 variantes de significado incierto. Conclusiones : Se comprobó que realizar estudio molecular guiado por el Score de Mayo permitió obtener un alto grado de probabilidad de detectar mutaciones. Se evidenció la importancia del estudio molecular debido a la existencia de solapamiento fenotípico y genotípico de las miocardiopatías. El conocimiento de la variante genética causal actualmente no afecta el manejo clínico de la mayoría de los pacientes con MCH, pero es de ayuda ante un pequeño grupo de genes que tienen opciones de tratamiento.

ABSTRACT Background : Cardiomyopathies are defined as a disorder of the myocardium in which the heart muscle is structurally and functionally abnormal, in the absence of coronary artery disease, hypertension (HT), valvular heart disease and congenital heart disease. These diseases are relatively common and a major cause of morbidity and mortality worldwide. Although genetic testing is recommended for family screening, lack of solid data on specific genotype-phenotype associations has reduced its impact on clinical management. Objectives : This study aims to analyze the frequency of mutations in a population of patients with cardiomyopathy referred to a tertiary healthcare center and to analyze the genotype-phenotype correlation of the identified mutations. Methods : We prospectively included 102 patients with suspected familial hypertrophic cardiomyopathy (HCM), 70 of which were index cases, from an ambispective cohort of patients with cardiomyopathies treated in a tertiary healthcare public hos pital in the province of Buenos Aires, from January 2012 to August 30, 2022. Results : Of 102 patients, 83 were considered affected. Of these, 31 were HCM and 52 were phenocopies, with no difference in prognosis. A genetic study was carried out in 77 patients, of whom 57 presented recognizable mutations, in 80% of the cases coinciding with a Mayo Score ≥3. Twenty-eight variants of uncertain significance were detected. Conclusions : It was confirmed that molecular testing guided by the Mayo Score provided high probability of detecting mutations. Molecular testing proved to be important due to the phenotypic and genotypic overlap in cardiomyopathies. Understanding the causative genetic variant, nowadays, does not affect the clinical management of most HCM patients, but is helpful in a small group of genes with treatment options.

Circulating MicroRNAs Identify Early Phenotypic Changes in Sarcomeric Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy. Pathogenic germline variation in genes encoding the sarcomere is the predominant cause of disease. However diagnostic features, including unexplained left ventricular hypertrophy, typically do not develop until late adolescence or after. The early stages of disease pathogenesis and the mechanisms underlying the transition to a clinically overt phenotype are not well understood. In this study, we investigated if circulating microRNAs (miRNAs) could stratify disease stage in sarcomeric HCM. METHODS: We performed arrays for 381 miRNAs using serum from HCM sarcomere variant carriers with and without a diagnosis of HCM and healthy controls. To identify differentially expressed circulating miRNAs between groups, multiple approaches were used including random forest, Wilcoxon rank sum test, and logistic regression. The abundance of all miRNAs was normalized to miRNA-320. RESULTS: Of 57 sarcomere variant carriers, 25 had clinical HCM and 32 had subclinical HCM with normal left ventricular wall thickness (21 with early phenotypic manifestations and 11 with no discernible phenotypic manifestations). Circulating miRNA profile differentiated healthy controls from sarcomere variant carriers with subclinical and clinical disease. Additionally, circulating miRNAs differentiated clinical HCM from subclinical HCM without early phenotypic changes; and subclinical HCM with and without early phenotypic changes. Circulating miRNA profiles did not differentiate clinical HCM from subclinical HCM with early phenotypic changes, suggesting biologic similarity between these groups. CONCLUSIONS: Circulating miRNAs may augment the clinical stratification of HCM and improve understanding of the transition from health to disease in sarcomere gene variant carriers.

Cooperation between myofibril growth and costamere maturation in human cardiomyocytes.

Costameres, as striated muscle-specific cell adhesions, anchor both M-lines and Z-lines of the sarcomeres to the extracellular matrix. Previous studies have demonstrated that costameres intimately participate in the initial assembly of myofibrils. However, how costamere maturation cooperates with myofibril growth is still underexplored. In this work, we analyzed zyxin (costameres), α-actinin (Z-lines) and myomesin (M-lines) to track the behaviors of costameres and myofibrils within the cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). We quantified the assembly and maturation of costameres associated with the process of myofibril growth within the hiPSC-CMs in a time-dependent manner. We found that asynchrony existed not only between the maturation of myofibrils and costameres, but also between the formation of Z-costameres and M-costameres that associated with different structural components of the sarcomeres. This study helps us gain more understanding of how costameres assemble and incorporate into the cardiomyocyte sarcomeres, which sheds a light on cardiomyocyte mechanobiology.

Contractility and Calcium Transient Maturation in the Human iPSC-Derived Cardiac Microfibers.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are considered immature in the sarcomere organization, contractile machinery, calcium transient, and transcriptome profile, which prevent them from further applications in modeling and studying cardiac development and disease. To improve the maturity of hiPSC-CMs, here, we engineered the hiPSC-CMs into cardiac microfibers (iCMFs) by a stencil-based micropatterning method, which enables the hiPSC-CMs to be aligned in an end-to-end connection for prolonged culture on the hydrogel of physiological stiffness. A series of characterization approaches were performed to evaluate the maturation in iCMFs on both structural and functional levels, including immunohistochemistry, calcium transient, reverse-transcription quantitative PCR, cardiac contractility, and electrical pacing analysis. Our results demonstrate an improved cardiac maturation of hiPSC-CMs in iCMFs compared to micropatterned or random single hiPSC-CMs and hiPSC-CMs in a random cluster at the same cell number of iCMFs. We found an increased sarcomere length, better regularity and alignment of sarcomeres, enhanced contractility, matured calcium transient, and T-tubule formation and improved adherens junction and gap junction formation. The hiPSC-CMs in iCMFs showed a robust calcium cycling in response to the programmed and continuous electrical pacing from 0.5 to 7 Hz. Moreover, we generated the iCMFs with hiPSC-CMs with mutations in myosin-binding protein C (MYBPC3) to have a proof-of-concept of iCMFs in modeling cardiac hypertrophic phenotype. These findings suggest that the multipatterned iCMF connection of hiPSC-CMs boosts the cardiac maturation structurally and functionally, which will reveal the full potential of the application of hiPSC-CM models in disease modeling of cardiomyopathy and cardiac regenerative medicine.

What Can We Learn from Single Sarcomere and Myofibril Preparations?

Sarcomeres are the smallest functional contractile unit of muscle, and myofibrils are striated muscle organelles that are comprised of sarcomeres that are strictly aligned in series. Furthermore, passive forces in sarcomeres and myofibrils are almost exclusively produced by the structural protein titin, and all contractile, regulatory, and structural proteins are in their natural configuration. For these mechanical and structural reasons single sarcomere and myofibril preparations are arguably the most powerful to answer questions on the mechanisms of striated muscle contraction. We developed and optimized single myofibril research over the past 20 years and were the first to mechanically isolate and test single sarcomeres. The results from this research led to the uncovering of the crucial role of titin in muscle contraction, first molecular explanations for the origins of the passive and the residual force enhancement properties of skeletal and cardiac muscles, the discovery of sarcomere length stability on the descending limb of the force-length relationship, and culminating in the formulation of the three-filament theory of muscle contraction that, aside from actin and myosin, proposes a crucial role of titin in active force production. Aside from all the advantages and possibilities that single sarcomere and myofibril preparations offer, there are also disadvantages. These include the fragility of the preparation, the time-consuming training to master these preparations, the limited spatial resolution for length and force measurements, and the unavailability of commercial systems for single sarcomere/myofibril research. Ignoring the mechanics that govern serially linked systems, not considering the spatial resolution and associated accuracies of myofibril systems, and neglecting the fragility of myofibril preparations, has led to erroneous interpretations of results and misleading conclusions. Here, we will attempt to describe the methods and possible applications of single sarcomere/myofibril research and discuss the advantages and disadvantages by focusing on specific applications. It is hoped that this discussion may contribute to identifying the enormous potential of single sarcomere/myofibril research in discovering the secrets of muscle contraction.

An increase in force after stretch of diaphragm fibers and myofibrils is accompanied by an increase in sarcomere length nonuniformities and Ca2+ sensitivity.

When muscle fibers from limb muscles are stretched while activated, the force increases to a steady-state level that is higher than that produced during isometric contractions at a corresponding sarcomere length, a phenomenon known as residual force enhancement (RFE). The mechanisms responsible for the RFE are an increased stiffness of titin molecules that may lead to an increased Ca2+ sensitivity of the contractile apparatus, and the development of sarcomere length nonuniformities. RFE is not observed in cardiac myofibrils, which makes this phenomenon specific to certain preparations. The aim of this study was to investigate whether the RFE is present in the diaphragm, and its potential association with an increased Ca2+ sensitivity and the development of sarcomere length nonuniformities. We used two preparations: single intact fibers and myofibrils isolated from the diaphragm of mice. We investigated RFE in a variety of lengths across the force-length relationship. RFE was observed in both preparations at all lengths investigated and was larger with increasing magnitudes of stretch. RFE was accompanied by an increased Ca2+ sensitivity as shown by a change in the force-pCa2+ curve, and increased sarcomere length nonuniformities. Therefore, RFE is a phenomenon commonly observed in skeletal muscles, with mechanisms that are similar across preparations.

Sarcomere protein modulation: The new frontier in cardiovascular medicine and beyond.

Over the past decade, the constant progress in science and technologies has provided innovative drug molecules that address specific disease mechanisms thus opening the era of drugs targeting the underlying pathophysiology of the disease. In this scenario, a new paradigm of modulation has emerged, following the development of small molecules capable of interfering with sarcomere contractile proteins. Potential applications include heart muscle disease and various forms of heart failure, although promising targets also include conditions affecting the skeletal muscle, such as degenerative neuromuscular diseases. In cardiac patients, a cardiac myosin stimulator, omecamtiv mecarbil, has shown efficacy in heart failure with reduced systolic function, lowering heart failure related events or cardiovascular death, while two inhibitors, mavacamten and aficamten, in randomized trials targeting hypertrophic cardiomyopathy, have been shown to reduce hypercontractility and left ventricular outflow obstruction improving functional capacity. Based on years of intensive basic and translational research, these agents are the prototypes of active pipelines promising to deliver an array of molecules in the near future. We here review the available evidence and future perspectives of myosin modulation in cardiovascular medicine.

Sbk2, a Newly Discovered Atrium-Enriched Regulator of Sarcomere Integrity.

BACKGROUND: Heart development relies on tight spatiotemporal control of cardiac gene expression. Genes involved in this intricate process have been identified using animals and pluripotent stem cell-based models of cardio(myo)genesis. Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal line of conditionally immortalized neonatal rat atrial myocytes (NRAMs), which allows toggling between proliferative and differentiated (ie, excitable and contractile) phenotypes in a synchronized and homogenous manner. METHODS: In this study, the unique properties of conditionally immortalized NRAMs (iAMs) were exploited to identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation. RESULTS: Transcriptome analysis of iAM-1 cells at different stages during one cycle of differentiation and subsequent dedifferentiation identified ≈13 000 transcripts, of which the dynamic changes in expression upon cardiomyogenic differentiation mostly opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many with known (lineage-specific) functions in cardiac muscle formation. Filtering for cardiac-enriched low-abundance transcripts, identified multiple genes with an uncharacterized role during cardio(myo)genesis including Sbk2 (SH3 domain binding kinase family member 2). Sbk2 encodes an evolutionarily conserved putative serine/threonine protein kinase, whose expression is strongly up- and downregulated during iAM-1 cell differentiation and dedifferentiation, respectively. In neonatal and adult rats, the protein is muscle-specific, highly atrium-enriched, and localized around the A-band of cardiac sarcomeres. Knockdown of Sbk2 expression caused loss of sarcomeric organization in NRAMs, iAMs and their human counterparts, consistent with a decrease in sarcomeric gene expression as evinced by transcriptome and proteome analyses. Interestingly, co-immunoprecipitation using Sbk2 as bait identified possible interaction partners with diverse cellular functions (translation, intracellular trafficking, cytoskeletal organization, chromatin modification, sarcomere formation). CONCLUSIONS: iAM-1 cells are a relevant and suitable model to identify (lowly expressed) genes with a hitherto unidentified role in cardiomyocyte differentiation as exemplified by Sbk2: a regulator of atrial sarcomerogenesis.