Regulating Striated Muscle Contraction: Through Thick and Thin.

Force generation in striated muscle is primarily controlled by structural changes in the actin-containing thin filaments triggered by an increase in intracellular calcium concentration. However, recent studies have elucidated a new class of regulatory mechanisms, based on the myosin-containing thick filament, that control the strength and speed of contraction by modulating the availability of myosin motors for the interaction with actin. This review summarizes the mechanisms of thin and thick filament activation that regulate the contractility of skeletal and cardiac muscle. A novel dual-filament paradigm of muscle regulation is emerging, in which the dynamics of force generation depends on the coordinated activation of thin and thick filaments. We highlight the interfilament signaling pathways based on titin and myosin-binding protein-C that couple thin and thick filament regulatory mechanisms. This dual-filament regulation mediates the length-dependent activation of cardiac muscle that underlies the control of the cardiac output in each heartbeat.

Matriglycan maintains t-tubule structural integrity in cardiac muscle.

Maintaining the structure of cardiac membranes and membrane organelles is essential for heart function. A critical cardiac membrane organelle is the transverse tubule system (called the t-tubule system) which is an invagination of the surface membrane. A unique structural characteristic of the cardiac muscle t-tubule system is the extension of the extracellular matrix (ECM) from the surface membrane into the t-tubule lumen. However, the importance of the ECM extending into the cardiac t-tubule lumen is not well understood. Dystroglycan (DG) is an ECM receptor in the surface membrane of many cells, and it is also expressed in t-tubules in cardiac muscle. Extensive posttranslational processing and O-glycosylation are required for DG to bind ECM proteins and the binding is mediated by a glycan structure known as matriglycan. Genetic disruption resulting in defective O-glycosylation of DG results in muscular dystrophy with cardiorespiratory pathophysiology. Here, we show that DG is essential for maintaining cardiac t-tubule structural integrity. Mice with defects in O-glycosylation of DG developed normal t-tubules but were susceptible to stress-induced t-tubule loss or severing that contributed to cardiac dysfunction and disease progression. Finally, we observed similar stress-induced cardiac t-tubule disruption in a cohort of mice that solely lacked matriglycan. Collectively, our data indicate that DG in t-tubules anchors the luminal ECM to the t-tubule membrane via the polysaccharide matriglycan, which is critical to transmitting structural strength of the ECM to the t-tubules and provides resistance to mechanical stress, ultimately preventing disruptions in cardiac t-tubule integrity.

Endoplasmic Reticulum-Plasma Membrane Junctions as Sites of Depolarization-Induced Ca2+ Signaling in Excitable Cells.

Membrane contact sites between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are found in all eukaryotic cells. In excitable cells they play unique roles in organizing diverse forms of Ca2+ signaling as triggered by membrane depolarization. ER-PM junctions underlie crucial physiological processes such as excitation-contraction coupling, smooth muscle contraction and relaxation, and various forms of activity-dependent signaling and plasticity in neurons. In many cases the structure and molecular composition of ER-PM junctions in excitable cells comprise important regulatory feedback loops linking depolarization-induced Ca2+ signaling at these sites to the regulation of membrane potential. Here, we describe recent findings on physiological roles and molecular composition of native ER-PM junctions in excitable cells. We focus on recent studies that provide new insights into canonical forms of depolarization-induced Ca2+ signaling occurring at junctional triads and dyads of striated muscle, as well as the diversity of ER-PM junctions in these cells and in smooth muscle and neurons.

The MYPT2-regulated striated muscle-specific myosin light chain phosphatase limits cardiac myosin phosphorylation in vivo.

The physiological importance of cardiac myosin regulatory light chain (RLC) phosphorylation by its dedicated cardiac myosin light chain kinase has been established in both humans and mice. Constitutive RLC-phosphorylation, regulated by the balanced activities of cardiac myosin light chain kinase and myosin light chain phosphatase (MLCP), is fundamental to the biochemical and physiological properties of myofilaments. However, limited information is available on cardiac MLCP. In this study, we hypothesized that the striated muscle-specific MLCP regulatory subunit, MYPT2, targets the phosphatase catalytic subunit to cardiac myosin, contributing to the maintenance of cardiac function in vivo through the regulation of RLC-phosphorylation. To test this hypothesis, we generated a floxed-PPP1R12B mouse model crossed with a cardiac-specific Mer-Cre-Mer to conditionally ablate MYPT2 in adult cardiomyocytes. Immunofluorescence microscopy using the gene-ablated tissue as a control confirmed the localization of MYPT2 to regions where it overlaps with a subset of RLC. Biochemical analysis revealed an increase in RLC-phosphorylation in vivo. The loss of MYPT2 demonstrated significant protection against pressure overload-induced hypertrophy, as evidenced by heart weight, qPCR of hypertrophy-associated genes, measurements of myocyte diameters, and expression of ß-MHC protein. Furthermore, mantATP chase assays revealed an increased ratio of myosin heads distributed to the interfilament space in MYPT2-ablated heart muscle fibers, confirming that RLC-phosphorylation regulated by MLCP, enhances cardiac performance in vivo. Our findings establish MYPT2 as the regulatory subunit of cardiac MLCP, distinct from the ubiquitously expressed canonical smooth muscle MLCP. Targeting MYPT2 to increase cardiac RLC-phosphorylation in vivo may improve baseline cardiac performance, thereby attenuating pathological hypertrophy.

Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca2+ binding, altering transport Ca2+ dependence without actually affecting the equilibrium binding affinity of the Ca2+-coordinating sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP.

Striated muscle: an inadequate soil for cancers.

Many organs of the body are susceptible to cancer development. However, striated muscles-which include skeletal and cardiac muscles-are rarely the sites of primary cancers. Most deaths from cancer arise due to complications associated with the development of secondary metastatic tumours, for which there are few effective therapies. However, as with primary cancers, the establishment of metastatic tumours in striated muscle accounts for a disproportionately small fraction of secondary tumours, relative to the proportion of body composition. Examining why primary and metastatic cancers are comparatively rare in striated muscle presents an opportunity to better understand mechanisms that can influence cancer cell biology. To gain insights into the incidence and distribution of muscle metastases, this review presents a definitive summary of the 210 case studies of metastasis in muscle published since 2010. To examine why metastases rarely form in muscles, this review considers the mechanisms currently proposed to render muscle an inhospitable environment for cancers. The "seed and soil" hypothesis proposes that tissues' differences in susceptibility to metastatic colonization are due to differing host microenvironments that promote or suppress metastatic growth to varying degrees. As such, the "soil" within muscle may not be conducive to cancer growth. Gaining a greater understanding of the mechanisms that underpin the resistance of muscles to cancer may provide new insights into mechanisms of tumour growth and progression, and offer opportunities to leverage insights into the development of interventions with the potential to inhibit metastasis in susceptible tissues.

Biofabrication of Living Actuators.

The impact of tissue engineering has extended beyond a traditional focus in medicine to the rapidly growing realm of biohybrid robotics. Leveraging living actuators as functional components in machines has been a central focus of this field, generating a range of compelling demonstrations of robots capable of muscle-powered swimming, walking, pumping, gripping, and even computation. In this review, we highlight key advances in fabricating tissue-scale cardiac and skeletal muscle actuators for a range of functional applications. We discuss areas for future growth including scalable manufacturing, integrated feedback control, and predictive modeling and also propose methods for ensuring inclusive and bioethics-focused pedagogy in this emerging discipline. We hope this review motivates the next generation of biomedical engineers to advance rational design and practical use of living machines for applications ranging from telesurgery to manufacturing to on- and off-world exploration.

Glutamate 139 of tropomyosin is critical for cardiac thin filament blocked-state stabilization.

The cardiac thin filament proteins troponin and tropomyosin control actomyosin formation and thus cardiac contractility. Calcium binding to troponin changes tropomyosin position along the thin filament, allowing myosin head binding to actin required for heart muscle contraction. The thin filament regulatory proteins are hot spots for genetic mutations causing heart muscle dysfunction. While much of the thin filament structure has been characterized, critical regions of troponin and tropomyosin involved in triggering conformational changes remain unresolved. A poorly resolved region, helix-4 (H4) of troponin I, is thought to stabilize tropomyosin in a position on actin that blocks actomyosin interactions at low calcium concentrations during muscle relaxation. We have proposed that contact between glutamate 139 on tropomyosin and positively charged residues on H4 leads to blocking-state stabilization. In this study, we attempted to disrupt these interactions by replacing E139 with lysine (E139K) to define the importance of this residue in thin filament regulation. Comparison of mutant and wild-type tropomyosin was carried out using in-vitro motility assays, actin co-sedimentation, and molecular dynamics simulations to determine perturbations in troponin-tropomyosin function caused by the tropomyosin mutation. Motility assays revealed that mutant thin filaments moved at higher velocity at low calcium with increased calcium sensitivity demonstrating that tropomyosin residue 139 is vital for proper tropomyosin-mediated inhibition during relaxation. Similarly, molecular dynamic simulations revealed a mutation-induced decrease in interaction energy between tropomyosin-E139K and troponin I (R170 and K174). These results suggest that salt-bridge stabilization of tropomyosin position by troponin IH4 is essential to prevent actomyosin interactions during cardiac muscle relaxation.

On the role of dysferlin in striated muscle: membrane repair, t-tubules and Ca2+ handling.

Dysferlin is a 237 kDa membrane-associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)-tubule network and the tight coordination of excitation-contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t-tubules, and it is suspected that dysferlin regulates t-tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction.

Functional divergence of the sarcomeric myosin, MYH7b, supports species-specific biological roles.

Myosin heavy chain 7b (MYH7b) is an evolutionarily ancient member of the sarcomeric myosin family, which typically supports striated muscle function. However, in mammals, alternative splicing prevents MYH7b protein production in cardiac and most skeletal muscles and limits expression to a subset of specialized muscles and certain nonmuscle environments. In contrast, MYH7b protein is abundant in python cardiac and skeletal muscles. Although the MYH7b expression pattern diverges in mammals versus reptiles, MYH7b shares high sequence identity across species. So, it remains unclear how mammalian MYH7b function may differ from that of other sarcomeric myosins and whether human and python MYH7b motor functions diverge as their expression patterns suggest. Thus, we generated recombinant human and python MYH7b protein and measured their motor properties to investigate any species-specific differences in activity. Our results reveal that despite having similar working strokes, the MYH7b isoforms have slower actin-activated ATPase cycles and actin sliding velocities than human cardiac ß-MyHC. Furthermore, python MYH7b is tuned to have slower motor activity than human MYH7b because of slower kinetics of the chemomechanical cycle. We found that the MYH7b isoforms adopt a higher proportion of myosin heads in the ultraslow, super-relaxed state compared with human cardiac ß-MyHC. These findings are supported by molecular dynamics simulations that predict MYH7b preferentially occupies myosin active site conformations similar to those observed in the structurally inactive state. Together, these results suggest that MYH7b is specialized for slow and energy-conserving motor activity and that differential tuning of MYH7b orthologs contributes to species-specific biological roles.

Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Myosin Heavy Chain as a Novel Key Modulator of Striated Muscle Resting State.

After years of intense research using structural, biological, and biochemical experimental procedures, it is clear that myosin molecules are essential for striated muscle contraction. However, this is just the tip of the iceberg of their function. Interestingly, it has been shown recently that these molecules (especially myosin heavy chains) are also crucial for cardiac and skeletal muscle resting state. In the present review, we first overview myosin heavy chain biochemical states and how they influence the consumption of ATP. We then detail how neighboring partner proteins including myosin light chains and myosin binding protein C intervene in such processes, modulating the ATP demand in health and disease. Finally, we present current experimental drugs targeting myosin ATP consumption and how they can treat muscle diseases.

Low-Salt Diet Regulates the Metabolic and Signal Transduction Genomic Fabrics, and Remodels the Cardiac Normal and Chronic Pathological Pathways.

Low-salt diet (LSD) is a constant recommendation to hypertensive patients, but the genomic mechanisms through which it improves cardiac pathophysiology are still not fully understood. Our publicly accessible transcriptomic dataset of the left ventricle myocardium of adult male mice subjected to prolonged LSD or normal diet was analyzed from the perspective of the Genomic Fabric Paradigm. We found that LSD shifted the metabolic priorities by increasing the transcription control for fatty acids biosynthesis while decreasing it for steroid hormone biosynthesis. Moreover, LSD remodeled pathways responsible for cardiac muscle contraction (CMC), chronic Chagas (CHA), diabetic (DIA), dilated (DIL), and hypertrophic (HCM) cardiomyopathies, and their interplays with the glycolysis/glucogenesis (GLY), oxidative phosphorylation (OXP), and adrenergic signaling in cardiomyocytes (ASC). For instance, the statistically (p < 0.05) significant coupling between GLY and ASC was reduced by LSD from 13.82% to 2.91% (i.e., -4.75×), and that of ASC with HCM from 10.50% to 2.83% (-3.71×). The substantial up-regulation of the CMC, ASC, and OXP genes, and the significant weakening of the synchronization of the expression of the HCM, CHA, DIA, and DIL genes within their respective fabrics justify the benefits of the LSD recommendation.

Isolated Cardiac Ryanodine Receptor Function Varies Between Mammals.

Concerted robust opening of cardiac ryanodine receptors' (RyR2) Ca2+ release 1oplasmic reticulum (SR) is fundamental for normal systolic cardiac function. During diastole, infrequent spontaneous RyR2 openings mediate the SR Ca2+ leak that normally constrains SR Ca2+ load. Abnormal large diastolic RyR2-mediated Ca2+ leak events can cause delayed after depolarizations (DADs) and arrhythmias. The RyR2-associated mechanisms underlying these processes are being extensively studied at multiple levels utilizing various model animals. Since there are well-described species-specific differences in cardiac intracellular Ca2+ handing in situ, we tested whether or not single RyR2 function in vitro retains this species specificity. We isolated RyR2-rich heavy SR microsomes from mouse, rat, rabbit, and human ventricular muscle and quantified RyR2 function using identical solutions and methods. The single RyR2 cytosolic Ca2+ sensitivity was similar across these species. However, there were significant species differences in single RyR2 mean open times in both systole and diastole-like solutions. In diastole-like solutions, single rat/mouse RyR2 open probability and frequency of long openings (> 6 ms) were similar, but these values were significantly greater than those of either single rabbit or human RyR2s. We propose these in vitro single RyR2 functional differences across species stem from the species-specific RyR2 regulatory environment present in the source tissue. Our results show the single rabbit RyR2 functional attributes, particularly in diastole-like conditions, replicate those of single human RyR2 best among the species tested.

Danicamtiv affected isometric force and cross-bridge kinetics similarly in skinned myocardial strips from male and female rats.

Myotropes are pharmaceuticals that have recently been developed or are under investigation for the treatment of heart diseases. Myotropes have had varied success in clinical trials. Initial research into myotropes have widely focused on animal models of cardiac dysfunction in comparison with normal animal cardiac physiology-primarily using males. In this study we examined the effect of danicamtiv, which is one type of myotrope within the class of myosin activators, on contractile function in permeabilized (skinned) myocardial strips from male and female Sprague-Dawley rats. We found that danicamtiv increased steady-state isometric force production at sub-maximal calcium levels, leading to greater Ca2+-sensitivity of contraction for both sexes. Danicamtiv did not affect maximal Ca2+-activated force for either sex. Sinusoidal length-perturbation analysis was used to assess viscoelastic myocardial stiffness and cross-bridge cycling kinetics. Data from these measurements did not vary with sex, and the data suggest that danicamtiv slows cross-bridge cycling kinetics. These findings imply that danicamtiv increases force production via increasing cross-bridge contributions to activation of contraction, especially at sub-maximal Ca2+-activation. The inclusion of both sexes in animal models during the formative stages of drug development could be helpful for understanding the efficacy or limitation of a drug's therapeutic impact on cardiac function.

Targeted genetic therapies for inherited disorders that affect both cardiac and skeletal muscle.

Skeletal myopathies and ataxias with secondary cardiac involvement are complex, progressive and debilitating conditions. As life expectancy increases across these conditions, cardiac involvement often becomes more prominent. This highlights the need for targeted therapies that address these evolving cardiac pathologies. Musculopathies by and large lack cures that directly target the genetic basis of the diseases; however, as our understanding of the genetic causes of these conditions has evolved, it has become tractable to develop targeted therapies using biologics, to design precision approaches to target the primary genetic causes of these varied diseases. Using the examples of Duchenne muscular dystrophy, Friedreich ataxia and Pompe disease, we discuss how the genetic causes of such diseases derail diverse homeostatic, energetic and signalling pathways, which span multiple cellular systems in varied tissues across the body. We outline existing therapeutics and treatments in the context of emerging novel genetic approaches. We discuss the hurdles that the field must overcome to deliver targeted therapies across the many tissue types affected in primary myopathies.

Pulsation waves along the Ciona heart tube reverse by bimodal rhythms expressed by a remote pair of pacemakers.

The heart of ascidians (marine invertebrate chordates) has a tubular structure, and heartbeats propagate from one end to the other. The direction of pulsation waves intermittently reverses in the heart of ascidians and their relatives; however, the underlying mechanisms remain unclear. We herein performed a series of experiments to characterize the pacemaker systems in isolated hearts and their fragments, and applied a mathematical model to examine the conditions leading to heart reversals. The isolated heart of Ciona robusta autonomously generated pulsation waves at ∼20 to 25 beats min-1 with reversals at ∼1 to 10 min intervals. Experimental bisections of isolated hearts revealed that independent pacemakers resided on each side and also that their beating frequencies periodically changed as they expressed bimodal rhythms, which comprised an ∼1.25 to 5.5 min acceleration/deceleration cycle of a beating rate of between 0 and 25 beats min-1. Only fragments including 5% or shorter terminal regions of the heart tube maintained autonomous pulsation rhythms, whereas other regions did not. Our mathematical model, based on FitzHugh-Nagumo equations applied to a one-dimensional alignment of cells, demonstrated that the difference between frequencies expressed by the two independent terminal pacemakers determined the direction of propagated waves. Changes in the statuses of terminal pacemakers between the excitatory and oscillatory modes as well as in their endogenous oscillation frequencies were sufficient to lead to heart reversals. These results suggest that the directions of pulsation waves in the Ciona heart reverse according to the changing rhythms independently expressed by remotely coupled terminal pacemakers.

N-Terminal Fragment of Cardiac Myosin Binding Protein C Modulates Cooperative Mechanisms of Thin Filament Activation in Atria and Ventricles.

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.

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Induced pluripotent stem cells (iPSCs) are obtained by introducing exogenous genes or adding chemicals to the culture medium to induce somatic cell differentiation. Similarly to embryonic stem cells, iPSCs have the ability to differentiate into all three embryonic cell lines. iPSCs can differentiate into cardiac muscle cells through two-dimensional differentiation methods such as monolayer cell culture and co-culture, or through embryoid body and scaffold-based three-dimensional differentiation methods. In addition, the process of iPSCs differentiation into cardiac muscle cells also requires activation or inhibition of specific signaling pathways,such as Wnt, BMP, Notch signaling pathways to mimic the development of the heart in vivo. In recent years, suspension culturing in bioreactors has been shown to produce large number of iPSCs derived cardiac muscle cells (iPSC-CMs). Before transplantation, it is necessary to purify iPSC-CMs through metabolic regulation or cell sorting to eliminate undifferentiated iPSCs, which may lead to teratoma formation. The transplantation methods for iPSC-CMs are mainly injection of cell suspension and transplantation of cell patches into the infarcted myocardium. Animal studies have shown that transplantation of iPSC-CMs into the infarcted myocardium can improve cardiac function. This article reviews the progress in preclinical studies on iPSC-CMs therapy for acute myocardial infarction and discusses the limitations and challenges of its clinical application to provide references for further clinical research and application.

A novel Cbx1, PurB, and Sp3 complex mediates long-term silencing of tissue- and lineage-specific genes.

miRNA-based cellular fate reprogramming offers an opportunity to investigate the mechanisms of long-term gene silencing. To further understand how genes are silenced in a tissue-specific manner, we leveraged our miRNA-based method of reprogramming fibroblasts into cardiomyocytes. Through screening approaches, we identified three proteins that were downregulated during reprogramming of fibroblasts into cardiomyocytes: heterochromatin protein Cbx1, transcriptional activator protein PurB, and transcription factor Sp3. We show that knockdown of Cbx1, PurB, and Sp3 was sufficient to induce cardiomyocyte gene expression in fibroblasts. Similarly, gene editing to ablate Cbx1, PurB, and Sp3 expression induced fibroblasts to convert into cardiomyocytes in vivo. Furthermore, high-throughput DNA sequencing and coimmunoprecipitation experiments indicated that Cbx1, PurB, and Sp3 also bound together as a complex and were necessary to localize nucleosomes to cardiomyocyte genes on the chromosome. Finally, we found that the expression of these genes led to nucleosome modification via H3K27me3 (trimethylated histone-H3 lysine-27) deposition through an interaction with the polycomb repressive PRC2 complex. In summary, we conclude that Cbx1, PurB, and Sp3 control cell fate by actively repressing lineage-specific genes.