Sarcomere Assembly Research Articles

Development, structure, and maintenance of C. elegans body wall muscle.

In C. elegans, mutants that are defective in muscle function and/or structure are easy to detect and analyze since: 1) body wall muscle is essential for locomotion, and 2) muscle structure can be assessed by multiple methods including polarized light, electron microscopy (EM), Green Fluorescent Protein (GFP) tagged proteins, and immunofluorescence microscopy. The overall structure of the sarcomere, the fundamental unit of contraction, is conserved from C. elegans to man, and the molecules involved in sarcomere assembly, maintenance, and regulation of muscle contraction are also largely conserved. This review reports the latest findings on the following topics: the transcriptional network that regulates muscle differentiation, identification/function/dynamics of muscle attachment site proteins, regulation of the assembly and maintenance of the sarcomere by chaperones and proteases, the role of muscle-specific giant protein kinases in sarcomere assembly, and the regulation of contractile activity, and new insights into the functions of the dystrophin glycoprotein complex.

Tropomyosin 1: Multiple roles in the developing heart and in the formation of congenital heart defects

Tropomyosin 1 (TPM1) is an essential sarcomeric component, stabilising the thin filament and facilitating actin's interaction with myosin. A number of sarcomeric proteins, such as alpha myosin heavy chain, play crucial roles in cardiac development. Mutations in these genes have been linked to congenital heart defects (CHDs), occurring in approximately 1 in 145 live births. To date, TPM1 has not been associated with isolated CHDs. Analysis of 380 CHD cases revealed three novel mutations in the TPM1 gene; IVS1+2T>C, I130V, S229F and a polyadenylation signal site variant GATAAA/AATAAA. Analysis of IVS1+2T>C revealed aberrant pre-mRNA splicing. In addition, abnormal structural properties were found in hearts transfected with TPM1 carrying I130V and S229F mutations. Phenotypic analysis of TPM1 morpholino-treated embryos revealed roles for TPM1 in cardiac looping, atrial septation and ventricular trabeculae formation and increased apoptosis was seen within the heart. In addition, sarcomere assembly was affected and altered action potentials were exhibited. This study demonstrated that sarcomeric TPM1 plays vital roles in cardiogenesis and is a suitable candidate gene for screening individuals with isolated CHDs.

Stk38 Modulates Rbm24 Protein Stability to Regulate Sarcomere Assembly in Cardiomyocytes

RNA-binding protein Rbm24 is a key regulator of heart development and required for sarcomere assembly and heart contractility. Yet, its underlying mechanism remains unclear. Here, we link serine/threonine kinase 38 (Stk38) signaling to the regulation of Rbm24 by showing that Rbm24 phosphorylation and its function could be modulated by Stk38. Using co-immunoprecipitation coupled with mass spectrometry technique, we identified Stk38 as an endogenous binding partner of Rbm24. Stk38 knockdown resulted in decreased Rbm24 protein level in cardiomyocytes. Further studies using Stk38 kinase inhibitor or activator showed that Rbm24 protein stability was regulated in a kinase activity-dependent manner. Deficiency of Stk38 caused reduction of sarcomere proteins and disarrangement of sarcomere, suggesting that Stk38 is essential for Rbm24 to regulate sarcomere assembly. Our results revealed that Stk38 kinase catalyzes the phosphorylation of Rbm24 during sarcomerogensis and this orchestrates accurate sarcomere alignment. This furthers our understanding of the regulatory mechanism of cardiac sarcomere assembly in both physiologic and pathologic contexts, and uncovers a potential novel pathway to cardiomyopathy through modulating the Stk38/Rbm24 protein activity.

From Ribosome to Sarcomere - Titin Dynamics in Striated Muscle Cells

The giant sarcomeric protein titin (up to 3.7 MDa) spans the half-sarcomere and has been regarded as blueprint for sarcomere assembly. Only little is known about the kinetics of titin's integration into the myofilament and its mechanistic role in sarcomere assembly. We study sarcomere dynamics by recombining fluorophores into the titin filament. In cardiomyocytes derived from knock-in mice expressing a titin fusion protein with eGFP attached to M-Band exon 6, FRAP experiments show an unexpected level of mobility of M-band titin that is calcium dependent. To investigate titin's role in sarcomere assembly and disassembly we inserted dsRed into titin's Z-disc region. The insertion of the fluorescent proteins at the N- and C-terminus of titin does not interfere with normal cellular function and double heterozygous knock-in animals have no obvious phenotype. The animals survive, are fertile, and have normal body and heart weights. Their myocytes allow us to simultaneously visualize titin's N- and the C-terminus using live cell imaging. Towards elucidating titin's role in myofilament dynamics we follow titin expression as well as sarcomere assembly and disassembly in differentiated mouse ES-cells, embryonic and neonatal myocytes, and satellite cells derived from titin-eGFP and -dsRed double heterozygous mice. During myofibrillogenesis in cardiomyocytes and skeletal muscle cells, titin integrates simultaneously into the Z-disc and M-band of the sarcomere. Finally, we use our animal models to follow titin synthesis, transport and degradation in developing and mature myocytes. The combination of fluorescent proteins, mouse genetics, live imaging, and super resolution microscopy provides a deeper understanding of sarcomere assembly and dynamics.

A Zebrafish Model for a Human Myopathy Associated with Mutation of the Unconventional Myosin MYO18B.

Myosin 18B is an unconventional myosin that has been implicated in tumor progression in humans. In addition, loss-of-function mutations of the MYO18B gene have recently been identified in several patients exhibiting symptoms of nemaline myopathy. In mouse, mutation of Myo18B results in early developmental arrest associated with cardiomyopathy, precluding analysis of its effects on skeletal muscle development. The zebrafish, frozen (fro) mutant was identified as one of a group of immotile mutants in the 1996 Tübingen genetic screen. Mutant embryos display a loss of birefringency in their skeletal muscle, indicative of disrupted sarcomeric organization. Using meiotic mapping, we localized the fro locus to the previously unannotated zebrafish myo18b gene, the product of which shares close to 50% identity with its human ortholog. Transcription of myo18b is restricted to fast-twitch myocytes in the zebrafish embryo; consistent with this, fro mutant embryos exhibit defects specifically in their fast-twitch skeletal muscles. We show that sarcomeric assembly is blocked at an early stage in fro mutants, leading to the disorganized accumulation of actin, myosin, and α-actinin and a complete loss of myofibrillar organization in fast-twitch muscles.

Titin-truncating mutations in dilated cardiomyopathy: the long and short of it.

Truncating variants in the TTN gene (TTNtv) are frequently identified in patients with dilated cardiomyopathy (DCM) but are also present in apparently healthy people in the general population. Consequently, there is considerable uncertainty about what it means for any single individual if a TTNtv is found. The aim of this review is to summarize current evidence implicating TTNtv in DCM pathogenesis and to provide some interpretative guidelines for clinical management. Next-generation sequencing studies have recently demonstrated that TTNtv are present in approximately one in five patients with DCM but also in up to 3% of individuals in the general population. These observations question whether TTNtv are sufficient alone to cause DCM and whether some TTNtv may be more deleterious than others. It has been suggested that functional effects of TTNtv can be predicted by their location in the titin protein, with DCM-associated variants typically occurring in the A-band region and/or in exons that are highly utilized across the range of titin isoforms. Recent data from animal and cell models suggest that developmental defects in the structural assembly of titin-deficient sarcomeres provide a template for mechanical stress-induced myocardial dysfunction during later life. Not all TTNtv are equal, and variants in constitutively expressed exons have the greatest likelihood of pathogenicity. The clinical significance of high-impact TTNtv is likely to differ according to patient context and each individual's unique suite of background genetic factors, comorbidities, and lifestyle factors. TTNtv identified in patients with DCM can be expected to have a major role in disease pathogenesis, but whether unaffected individuals with TTNtv will develop DCM is less certain.

Structural and Biophysical Analysis of Hypertrophic Cardiomyopathy-Linked Titin Missense Variants

Cardiomyopathies are an important cause of premature death from heart failure, ventricular arrhythmia and stroke. The most common subtype, hypertrophic cardiomyopathy (HCM), is an inherited disease that results in the thickening of the myocardium, whilst at the cellular level the sarcomere - the contractile unit of cardiac and skeletal muscle - is disrupted. The advent of high throughput sequencing has enabled the identification of variants in physiologically relevant genes, aiding the search for causative mutations of HCM. One gene that is now amenable to sequencing in HCM patients is TTN, which encodes Titin, a multidomain, multifunctional filament that spans half a sarcomere in striated muscle. Titin is essential for sarcomere assembly and plays a variety of roles in the heart, including providing passive muscle elasticity during the contraction-relaxation cycle and signaling via its kinase domain. The protein contains over 300 immunoglobulin and fibronectin III domains, the majority of which are located along the myosin-associated A-band region of the sarcomere. Whole exome sequencing of a cohort of HCM patients has identified a large number of missense variants in Titin, particularly in the A-band region of the protein. Whilst many of the variants are likely neutral, a number are enriched when compared to their frequency in control cohorts. We present here the structural and biophysical analysis of five Titin A-band fibronectin III domains and their associated variants enriched in either the HCM cohort or in the general population. The crystal structures of two wild type and HCM-linked variant domains have been determined, and thermal denaturation experiments demonstrate both variants have a reduced stability when compared to their wild type counterparts, suggesting a causal role in HCM and hinting at a method to distinguish pathogenic from non-pathogenic Titin missense variants.

Myo18b is essential for sarcomere assembly in fast skeletal muscle.

Congenital myopathies are muscle degenerative disorders with a broad clinical spectrum. A number of myopathies have been associated with molecular defects within sarcomeres, the force-generating component of the muscle cell. Whereas the highly regular organization of the myofibril has been studied in detail, in vivo assembly of sarcomeres remains a poorly understood process. Therefore, a more detailed knowledge of sarcomere assembly is crucial to better understand the pathogenic mechanisms within myopathies. Recently, mutations in myosin XVIIIB (MYO18B) have been associated with cases of myopathies, although the underlying mechanism for the resulting pathology remains to be defined. To analyze the role of myosin XVIIIB in skeletal muscle disease, zebrafish mutants for myo18b were generated. Full loss of myo18b function results in a complete lack of sarcomeric structure, revealing a highly surprising and essential role for myo18b in sarcomere assembly. Importantly, scattered thin and thick filaments assemble throughout the sarcoplasm; but fail to organize into recognizable sarcomeric structures in myo18b null mutants. In myo18b partial loss-of-function mutants sarcomeric structures are assembled, but thin and thick filaments remain misaligned within these structures. These observations suggest a novel model of sarcomere assembly where Myo18b coordinates the integration of preformed thick and thin filaments into the sarcomere. Disruption of this highly coordinated process results in a block in sarcomere biogenesis and the onset of myopathic pathology.

Titin and Nebulin in Thick and Thin Filament Length Regulation.

In this review we discuss the history and the current state of ideas related to the mechanism of size regulation of the thick (myosin) and thin (actin) filaments in vertebrate striated muscles. Various hypotheses have been considered during of more than half century of research, recently mostly involving titin and nebulin acting as templates or 'molecular rulers', terminating exact assembly. These two giant, single-polypeptide, filamentous proteins are bound in situ along the thick and thin filaments, respectively, with an almost perfect match in the respective lengths and structural periodicities. However, evidence still questions the possibility that the proteins function as templates, or scaffolds, on which the thin and thick filaments could be assembled. In addition, the progress in muscle research during the last decades highlighted a number of other factors that could potentially be involved in the mechanism of length regulation: molecular chaperones that may guide folding and assembly of actin and myosin; capping proteins that can influence the rates of assembly-disassembly of the myofilaments; Ca2+ transients that can activate or deactivate protein interactions, etc. The entire mechanism of sarcomere assembly appears complex and highly dynamic. This mechanism is also capable of producing filaments of about the correct size without titin and nebulin. What then is the role of these proteins? Evidence points to titin and nebulin stabilizing structures of the respective filaments. This stabilizing effect, based on linear proteins of a fixed size, implies that titin and nebulin are indeed molecular rulers of the filaments. Although the proteins may not function as templates in the assembly of the filaments, they measure and stabilize exactly the same size of the functionally important for the muscles segments in each of the respective filaments.

Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection

SummaryThe sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.

Aberrant developmental titin splicing and dysregulated sarcomere length in Thymosin β4 knockout mice

Sarcomere assembly is a highly orchestrated and dynamic process which adapts, during perinatal development, to accommodate growth of the heart. Sarcomeric components, including titin, undergo an isoform transition to adjust ventricular filling. Many sarcomeric genes have been implicated in congenital cardiomyopathies, such that understanding developmental sarcomere transitions will inform the aetiology and treatment. We sought to determine whether Thymosin β4 (Tβ4), a peptide that regulates the availability of actin monomers for polymerization in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 null mice, immunofluorescence-based sarcomere analyses revealed shortened thin filament, sarcomere and titin spring length in cardiomyocytes, associated with precocious up-regulation of the short titin isoforms during the postnatal splicing transition. By magnetic resonance imaging, this manifested as diminished stroke volume and limited contractile reserve in adult mice. Extrapolating to an in vitro cardiomyocyte model, the altered postnatal splicing was corrected with addition of synthetic Tβ4, whereby normal sarcomere length was restored. Our data suggest that Tβ4 is required for setting correct sarcomere length and for appropriate splicing of titin, not only in the heart but also in skeletal muscle. Distinguishing between thin filament extension and titin splicing as the primary defect is challenging, as these events are intimately linked. The regulation of titin splicing is a previously unrecognised role of Tβ4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction.

Calcium handling precedes cardiac differentiation to initiate the first heartbeat.

The mammalian heartbeat is thought to begin just prior to the linear heart tube stage of development. How the initial contractions are established and the downstream consequences of the earliest contractile function on cardiac differentiation and morphogenesis have not been described. Using high-resolution live imaging of mouse embryos, we observed randomly distributed spontaneous asynchronous Ca2+-oscillations (SACOs) in the forming cardiac crescent (stage E7.75) prior to overt beating. Nascent contraction initiated at around E8.0 and was associated with sarcomeric assembly and rapid Ca2+ transients, underpinned by sequential expression of the Na+-Ca2+ exchanger (NCX1) and L-type Ca2+ channel (LTCC). Pharmacological inhibition of NCX1 and LTCC revealed rapid development of Ca2+ handling in the early heart and an essential early role for NCX1 in establishing SACOs through to the initiation of beating. NCX1 blockade impacted on CaMKII signalling to down-regulate cardiac gene expression, leading to impaired differentiation and failed crescent maturation.

G9a inhibits MEF2C activity to control sarcomere assembly.

In this study, we demonstrate that the lysine methyltransferase G9a inhibits sarcomere organization through regulation of the MEF2C-HDAC5 regulatory axis. Sarcomeres are essential for muscle contractile function. Presently, skeletal muscle disease and dysfunction at the sarcomere level has been associated with mutations of sarcomere proteins. This study provides evidence that G9a represses expression of several sarcomere genes and its over-expression disrupts sarcomere integrity of skeletal muscle cells. G9a inhibits MEF2C transcriptional activity that is essential for expression of sarcomere genes. Through protein interaction assays, we demonstrate that G9a interacts with MEF2C and its co-repressor HDAC5. In the presence of G9a, calcium signaling-dependent phosphorylation and export of HDAC5 to the cytoplasm is blocked which likely results in enhanced MEF2C-HDAC5 association. Activation of calcium signaling or expression of constitutively active CaMK rescues G9a-mediated repression of HDAC5 shuttling as well as sarcomere gene expression. Our results demonstrate a novel epigenetic control of sarcomere assembly and identifies new therapeutic avenues to treat skeletal and cardiac myopathies arising from compromised muscle function.

Deficient cMyBP-C protein expression during cardiomyocyte differentiation underlies human hypertrophic cardiomyopathy cellular phenotypes in disease specific human ES cell derived cardiomyocytes

AimsMutations of cardiac sarcomere genes have been identified to cause HCM, but the molecular mechanisms that lead to cardiomyocyte hypertrophy and risk for sudden death are uncertain. The aim of this study was to examine HCM disease mechanisms at play during cardiac differentiation of human HCM specific pluripotent stem cells. Methods and resultsWe generated a human embryonic stem cell (hESC) line carrying a naturally occurring mutation of MYPBC3 (c.2905 +1 G >A) to study HCM pathogenesis during cardiac differentiation. HCM-specific hESC-derived cardiomyocytes (hESC-CMs) displayed hallmark aspects of HCM including sarcomere disarray, hypertrophy and impaired calcium impulse propagation. HCM hESC-CMs presented a transient haploinsufficiency of cMyBP-C during cardiomyocyte differentiation, but by day 30 post-differentiation cMyBP-C levels were similar to control hESC-CMs. Gene transfer of full-length MYBPC3 during differentiation prevented hypertrophy, sarcomere disarray and improved calcium impulse propagation in HCM hESC-CMs. Conclusion(s)These findings point to the critical role of MYBPC3 during sarcomere assembly in cardiac myocyte differentiation and suggest developmental influences of MYBPC3 truncating mutations on the mature hypertrophic phenotype.

Molecular evolution of troponin I and a role of its N‐terminal extension in nematode locomotion

The troponin complex, composed of troponin T (TnT), troponin I (TnI), and troponin C (TnC), is the major calcium‐dependent regulator of muscle contraction, which is present widely in both vertebrates and invertebrates. Little is known about evolutionary aspects of troponin in the animal kingdom. Using a combination of data mining and functional analysis of TnI, we report evidence that an N‐terminal extension of TnI is present in most of bilaterian animals as a functionally important domain. Troponin components have been reported in species in most of representative bilaterian phyla. Comparison of TnI sequences shows that the core domains are conserved in all examined TnIs, and that N‐ and C‐terminal extensions are variable among isoforms and species. In particular, N‐terminal extensions are present in all protostome TnIs and chordate cardiac TnIs but lost in a subset of chordate TnIs including vertebrate skeletal‐muscle isoforms. Transgenic rescue experiments in Caenorhabditis elegans striated muscle show that the N‐terminal extension of TnI (UNC‐27) is required for coordinated worm locomotion but not in sarcomere assembly and single muscle‐contractility kinetics. These results suggest that N‐terminal extensions of TnIs are retained from a TnI ancestor as a functional domain. © 2016 Wiley Periodicals, Inc.

Abstract 127: Phosphorylation of Serum Responsive Factor by p90 Ribosomal S6 Kinase 3 Promotes Concentric Growth of Cardiac Myocytes

p90 Ribosomal S6 Kinase 3 (RSK3) is required for the induction of concentric hypertrophy in hearts subjected to pressure overload and catecholamine infusion, as well as in a model for Noonan Syndrome-associated hypertrophic cardiomyopathy. Serving important roles in both cardiac development and adult function, the transcription factor Serum Responsive Factor (SRF) regulates genes expression involved in myocyte growth and sarcomeric assembly. It has been reported that SRF can be phosphorylated by RSK protein kinases on residue Ser-103, but the function of that post-translational modification has remained uncertain. We now show that SRF is a substrate for RSK3 associated with muscle A-Kinase-Anchoring-Protein (mAKAP) scaffold in cardiac myocytes, contributing to the regulation of concentric myocyte growth. By co-immunoprecipitation assay, SRF and RSK3 were both associated with the mAKAP scaffold in heart extracts, such that a ternary complex could be detected when recombinant proteins were expressed in cells. Silencing RSK3 or mAKAP expression by siRNA transfection reduced phenylephrine-induced SRF(S103) phosphorylation in neonatal cardiac myocytes. Similar results were acquired following expression of a peptide comprising the mAKAP RSK3-binding domain (RBD) that competed the binding of the kinase to the scaffold. Overexpression of either a SRF S103A phosphoablative mutant or the RBD peptide diminished the increase in width of adult cardiac myocytes stimulated by phenylephrine. In contrast, overexpression of RSK3 or a SRF S103D phosphomimetic mutant promoted adult cardiac myocyte concentric hypertorphy in vitro. While baseline SRF S103 phosphorylation in the heart was inhibited by mAKAP or RSK3 gene knock-out in mice, acute transverse aortic constriction significantly increased SRF S103 phosphorylation in the heart. Based upon these results, we porpose that RSK3 phosphorylation of SRF at mAKAP signalosomes is an important regulator of concentric cardiac myocyte growth. These results are consistent with additional findings that an adeno-associated virus gene therapy vector that expresses RBD in the cardiac mycoyte attenuates pathological hypertrophy and prevents heart failure in response to pressure overload.

Nucleus-dependent sarcomere assembly is mediated by the LINC complex

Two defining characteristics of muscle cells are the many precisely positioned nuclei and the linearly arranged sarcomeres, yet the relationship between these two features is not known. We show that nuclear positioning precedes sarcomere formation. Furthermore, ZASP-GFP, a Z-line protein, colocalizes with F-actin in puncta at the cytoplasmic face of nuclei before sarcomere assembly. In embryos with mispositioned nuclei, ZASP-GFP is still recruited to the nuclei before its incorporation into sarcomeres. Furthermore, the first sarcomeres appear in positions close to the nuclei, regardless of nuclear position. These data suggest that the interaction between sarcomere proteins and nuclei is not dependent on properly positioned nuclei. Mechanistically, ZASP-GFP localization to the cytoplasmic face of the nucleus did require the linker of nucleoskeleton and cytoskeleton (LINC) complex. Muscle-specific depletion of klarsicht (nesprin) or klariod (SUN) blocked the recruitment of ZASP-GFP to the nucleus during the early stages of sarcomere assembly. As a result, sarcomeres were poorly formed and the general myofibril network was less stable, incomplete, and/or torn. These data suggest that the nucleus, through the LINC complex, is crucial for the proper assembly and stability of the sarcomere network.

Knockdown of DNA methyltransferase 3a alters gene expression and inhibits function of embryonic cardiomyocytes.

We previously found that in utero caffeine exposure causes down-regulation of DNA methyltransferases (DNMTs) in embryonic heart and results in impaired cardiac function in adulthood. To assess the role of DNMTs in these events, we investigated the effects of reduced DNMT expression on embryonic cardiomyocytes. siRNAs were used to knock down individual DNMT expression in primary cultures of mouse embryonic cardiomyocytes. Immunofluorescence staining was conducted to evaluate cell morphology. A video-based imaging assay and multielectrode array were used to assess cardiomyocyte contractility and electrophysiology, respectively. RNA-Seq and multiplex bisulfite sequencing were performed to examine gene expression and promoter methylation, respectively. At 72 h after transfection, reduced DNMT3a expression, but not DNMT1 or -3b, disrupted sarcomere assembly and decreased beating frequency, contractile movement, amplitude of field action potential, and cytosolic calcium signaling of cardiomyocytes. RNA-Seq analysis revealed that the DNMT3a-deficient cells had deactivated gene networks involved in calcium, endothelin-1, renin-angiotensin, and cardiac β-adrenergic receptor signaling, which were not inhibited by DNMT3b siRNA. Moreover, decreased methylation levels were found in the promoters of Myh7, Myh7b, Tnni3, and Tnnt2, consistent with the up-regulation of these genes by DNMT3a siRNA. These data show that DNMT3a plays an important role in regulating embryonic cardiomyocyte gene expression, morphology and function.-Fang, X., Poulsen, R. R., Wang-Hu, J., Shi, O., Calvo, N. S., Simmons, C. S., Rivkees, S. A., Wendler, C. C. Knockdown of DNA methyltransferase 3a alters gene expression and inhibits function of embryonic cardiomyocytes.

Variation in stiffness regulates cardiac myocyte hypertrophy via signaling pathways.

Much diseased human myocardial tissue is fibrotic and stiff, which increases the work that the ventricular myocytes must perform to maintain cardiac output. The hypothesis tested is that the increased load due to greater stiffness of the substrata drives sarcomere assembly of cells, thus strengthening them. Neonatal rat ventricular myocytes (NRVM) were cultured on polyacrylamide or polydimethylsiloxane substrates with stiffness of 10 kPa, 100 kPa, or 400 kPa, or glass with stiffness of 61.9 GPa. Cell size increased with stiffness. Two signaling pathways were explored, phosphorylation of focal adhesion kinase (p-FAK) and lipids by phosphatidylinositol 4,5-bisphosphate (PIP2). Subcellular distributions of both were determined in the sarcomeric fraction by antibody localization, and total amounts were measured by Western or dot blotting, respectively. More p-FAK and PIP2 distributed to the sarcomeres of NRVM grown on stiffer substrates. Actin assembly involves the actin capping protein Z (CapZ). Both actin and CapZ dynamic exchange were significantly increased on stiffer substrates when assessed by fluorescence recovery after photobleaching (FRAP) of green fluorescent protein tags. Blunting of actin FRAP by FAK inhibition implicates linkage from mechano-signalling pathways to cell growth. Thus, increased stiffness of cardiac disease can be modeled with polymeric materials to understand how the microenvironment regulates cardiac hypertrophy.

Influence of V54M mutation in giant muscle protein titin: a computational screening and molecular dynamics approach

Recent genetic studies have revealed the impact of mutations in associated genes for cardiac sarcomere components leading to dilated cardiomyopathy (DCM). The cardiac sarcomere is composed of thick and thin filaments and a giant muscle protein known as titin or connectin. Titin interacts with T-cap/telethonin in the Z-line region and plays a vital role in regulating sarcomere assembly. Initially, we screened all the variants associated with giant protein titin and analyzed their impact with the aid of pathogenicity and stability prediction methods. V54M mutation found in the hydrophobic core region of the protein associated with abnormal clinical phenotype leads to DCM was selected for further analysis. To address this issue, we mapped the deleterious mutant V54M, modeled the mutant protein complex, and deciphered the impact of mutation on binding with its partner telethonin in the titin crystal structure of PDB ID: 1YA5 with the aid of docking analysis. Furthermore, two run molecular dynamics simulation was initiated to understand the mechanistic action of V54M mutation in altering the protein structure, dynamics, and stability. According to the results obtained from the repeated 50 ns trajectory files, the overall effect of V54M mutation was destabilizing and transition of bend to coil in the secondary structure was observed. Furthermore, MMPBSA elucidated that V54M found in the Z-line region of titin decreases the binding affinity of titin to Z-line proteins T-cap/telethonin thereby hindering the protein–protein interaction.