Myosin Essential Light Chain Research Articles

A Positive Feedback Mechanism Drives Membrane Ingression During Cytokinesis in an Actomyosin-Ring Independent Manner

The molecular mechanism underlying actomyosin-ring (AMR) driven furrow ingression during cytokinesis is still unclear. We functionally separated sub-molecular domains of the AMR and associated proteins, which are important to maintain the integrity of the AMR, from domains that are important to drive plasma membrane ingression. Specific mutations that interrupt the interactions between Myosin II, the essential myosin light chain, IQGAP1 and the F-BAR protein Hof1 destabilize the cytokinetic AMR and prevent centripetal furrow ingression, but still allow asymmetric plasma membrane ingression with a velocity that is comparable to cells with a functional AMR. Our data indicate that the AMR directs ingression factors to the cytokinetic furrow to initiate a positive feedback mechanism that drives membrane ingression, which is independent of AMR contractility.

The Muscle Myosin Essential Light Chain is Not Essential for Muscle Function

The Muscle Myosin Essential Light Chain is Not Essential for Muscle Function

Long-term testosterone deficiency modifies myofilament and calcium-handling proteins and promotes diastolic dysfunction in the aging mouse heart.

The impact of long-term gonadectomy (GDX) on cardiac contractile function was explored in the setting of aging. Male mice were subjected to bilateral GDX or sham operation (4 wk) and investigated at 16-18 mo of age. Ventricular myocytes were field stimulated (2 Hz, 37°C). Peak Ca2+ transients (fura 2) and contractions were similar in GDX and sham-operated mice, although Ca2+ transients (50% decay time: 45.2 ± 2.3 vs. 55.6 ± 3.1 ms, P < 0.05) and contractions (time constant of relaxation: 39.1 ± 3.2 vs. 69.5 ± 9.3 ms, P < 0.05) were prolonged in GDX mice. Action potential duration was increased in myocytes from GDX mice, but this did not account for prolonged responses, as Ca2+ transient decay was slow even when cells from GDX mice were voltage clamped with simulated "sham" action potentials. Western blots of proteins involved in Ca2+ sequestration and efflux showed that Na+/Ca2+ exchanger and sarco(endo)plasmic reticulum Ca2+-ATPase type 2 protein levels were unaffected, whereas phospholamban was dramatically higher in ventricles from aging GDX mice (0.24 ± 0.02 vs. 0.86 ± 0.13, P < 0.05). Myofilament Ca2+ sensitivity at physiological Ca2+ was similar, but phosphorylation of essential myosin light chain 1 was reduced by ≈50% in ventricles from aging GDX mice. M-mode echocardiography showed no change in systolic function (e.g., ejection fraction). Critically, pulse-wave Doppler echocardiography showed that GDX slowed isovolumic relaxation time (12.9 ± 0.9 vs. 16.9 ± 1.0 ms, P < 0.05), indicative of diastolic dysfunction. Thus, dysregulation of intracellular Ca2+ and myofilament dysfunction contribute to deficits in contraction in hearts from testosterone-deficient aging mice. This suggests that low testosterone helps promote diastolic dysfunction in the aging heart. NEW & NOTEWORTHY The influence of long-term gonadectomy on contractile function was examined in aging male hearts. Gonadectomy slowed the decay of Ca2+ transients and contractions in ventricular myocytes and slowed isovolumic relaxation time, demonstrating diastolic dysfunction. Underlying mechanisms included Ca2+ dysregulation, elevated phospholamban protein levels, and hypophosphorylation of a myofilament protein, essential myosin light chain. Testosterone deficiency led to intracellular Ca2+ dysregulation and myofilament dysfunction, which may facilitate diastolic dysfunction in the setting of aging.

Bi-allelic mutations in MYL1 cause a severe congenital myopathy.

Congenital myopathies are typically characterised by early onset hypotonia, weakness and hallmark features on biopsy. Despite the rapid pace of gene discovery, ∼50% of patients with a congenital myopathy remain without a genetic diagnosis following screening of known disease genes. We performed exome sequencing on two consanguineous probands diagnosed with a congenital myopathy and muscle biopsy showing selective atrophy/hypotrophy or absence of type II myofibres. We identified variants in the gene (MYL1) encoding the skeletal muscle fast-twitch specific myosin essential light chain (ELC) in both probands. A homozygous essential splice acceptor variant (c.479-2A>G, predicted to result in skipping of exon 5 was identified in Proband 1, and a homozygous missense substitution (c.488T>G, p.(Met163Arg)) was identified in Proband 2. Protein modelling of the p.(Met163Arg) substitution predicted it might impede intermolecular interactions that facilitate binding to the IQ domain of myosin heavy chain, thus likely impacting on the structure and functioning of the myosin motor. MYL1 was markedly reduced in skeletal muscle from both probands, suggesting that the missense substitution likely results in an unstable protein. Knock down of myl1 in zebrafish resulted in abnormal morphology, disrupted muscle structure and impaired touch-evoked escape responses, thus confirming that skeletal muscle fast-twitch specific myosin ELC is critical for myofibre development and function. Our data implicate MYL1 as a crucial protein for adequate skeletal muscle function and that MYL1 deficiency is associated with severe congenital myopathy.

High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

We have used a novel time-resolved FRET (TR-FRET) assay to detect small-molecule modulators of actin-myosin structure and function. Actin-myosin interactions play crucial roles in the generation of cellular force and movement. Numerous mutations and post-translational modifications of actin or myosin disrupt muscle function and cause life-threatening syndromes. Here, we used a FRET biosensor to identify modulators that bind to the actin-myosin interface and alter the structural dynamics of this complex. We attached a fluorescent donor to actin at Cys-374 and a nonfluorescent acceptor to a peptide containing the 12 N-terminal amino acids of the long isoform of skeletal muscle myosin's essential light chain. The binding site on actin of this acceptor-labeled peptide (ANT) overlaps with that of myosin, as indicated by (a) a similar distance observed in the actin-ANT complex as in the actin-myosin complex and (b) a significant decrease in actin-ANT FRET upon binding myosin. A high-throughput FRET screen of a small-molecule library (NCC, 727 compounds), using a unique fluorescence lifetime readout with unprecedented speed and precision, showed that FRET is significantly affected by 10 compounds in the micromolar range. Most of these "hits" alter actin-activated myosin ATPase and affect the microsecond dynamics of actin detected by transient phosphorescence anisotropy. We conclude that the actin-ANT TR-FRET assay enables detection of pharmacologically active compounds that affect actin structural dynamics and actomyosin function. This assay establishes feasibility for the discovery of allosteric modulators of the actin-myosin interaction, with the ultimate goal of developing therapies for muscle disorders.

Essential Light Chains of Myosin and Their Role in Functioning of the Myosin Motor.

This review summarizes current data on the structure and functions of myosin essential light chains (ELCs) and on their role in functioning of the myosin head as a molecular motor. The data on structural and functional features of the N-terminal extension of myosin ELC from skeletal and cardiac muscles are analyzed; the role of this extension in the ATP-dependent interaction of myosin heads with actin in the molecular mechanism of muscle contraction is discussed. The data on possible interactions of the ELC N-terminal extension with the myosin head motor domain in the myosin ATPase cycle are presented, including the results of the authors' studies that are in favor of such interactions.

Differences between fast and slow muscles in scallops revealed through proteomics and transcriptomics

BackgroundScallops possess striated and catch adductor muscles, which have different structure and contractile properties. The striated muscle contracts very quickly for swimming, whereas the smooth catch muscle can keep the shells closed for long periods with little expenditure of energy. In this study, we performed proteomic and transcriptomic analyses of differences between the striated (fast) and catch (slow) adductor muscles in Yesso scallop Patinopecten yessoensis.ResultsTranscriptomic analysis reveals 1316 upregulated and 8239 downregulated genes in slow compared to fast adductor muscle. For the same comparison, iTRAQ-based proteomics reveals 474 differentially expressed proteins (DEPs), 198 up- and 276 downregulated. These DEPs mainly comprise muscle-specific proteins of the sarcoplasmic reticulum, extracellular matrix, and metabolic pathways. A group of conventional muscle proteins—myosin heavy chain, myosin regulatory light chain, myosin essential light chain, and troponin—are enriched in fast muscle. In contrast, paramyosin, twitchin, and catchin are preferentially expressed in slow muscle. The association analysis of proteomic and transcriptomic data provides the evidences of regulatory events at the transcriptional and posttranscriptional levels in fast and slow muscles. Among 1236 differentially expressed unigenes, 22.7% show a similar regulation of mRNA levels and protein abundances. In contrast, more unigenes (53.2%) exhibit striking differences between gene expression and protein abundances in the two muscles, which indicates the existence of fiber-type specific, posttranscriptional regulatory events in most of myofibrillar proteins, such as myosin heavy chain, titin, troponin, and twitchin.ConclusionsThis first, global view of protein and mRNA expression levels in scallop fast and slow muscles reveal that regulatory mechanisms at the transcriptional and posttranscriptional levels are essential in the maintenance of muscle structure and function. The existence of fiber-type specific, posttranscriptional regulatory mechanisms in myofibrillar proteins will greatly improve our understanding of the molecular basis of muscle contraction and its regulation in non-model invertebrates.

Dysfunction of Myosin Light‐Chain 4 (MYL4) Leads to Heritable Atrial Cardiomyopathy With Electrical, Contractile, and Structural Components: Evidence From Genetically‐Engineered Rats

BackgroundThere is increasing interest in the concept of atrial cardiomyopathy, but the underlying molecular and mechanistic determinants remain poorly defined. We identified a family with heritable atrial cardiomyopathy manifesting as progressive atrial‐selective electromechanical dysfunction, tachyarrhythmias, and bradyarrhythmias requiring pacemaker implantation. Myosin light‐chain 4 (MYL4), encoding the atrial‐selective essential myosin light chain, was identified as a candidate gene. We used genetically modified rat models to investigate the role of MYL4 in atrial cardiomyopathy.Methods and ResultsExome sequencing and systematic bioinformatic analyses identified a rare missense variant of MYL4 (c.31G>A [p.E11K]) in a large multiplex atrial cardiomyopathy family pedigree. The mutation cosegregated with atrial standstill (selected as the principal presenting trait) with a logarithm of the odds score of 5.3. The phenotype of rats with MYL4 mutation knock‐in confirmed the causative role of the mutation. MYL4 knockout rats showed a similar atrial cardiomyopathy phenotype, whereas rats with an adjacent 4‐amino‐acid deletion showed no phenotype. Both MYL4 p.E11K knock‐in rats and MYL4 knockout rats showed progressive atrial electrophysiological, contractile, and fibrotic abnormalities, similar to affected patients. Biochemical analyses of MYL4 p.E11K mutation rats showed activation of proapoptotic and profibrotic signaling, along with increased atrial‐cardiomyocyte terminal deoxynucleotidyl transferase dUTP nick end labeling staining, suggesting enhanced apoptotic cell death, findings that were mimicked by in vitro adenoviral transfer of the mutant gene to neonatal‐rat cardiomyocytes.ConclusionsLoss‐of‐function MYL4 gene variants cause progressive atrial cardiomyopathy in humans and rats. Our findings identify MYL4 as a key gene required for atrial contractile, electrical and structural integrity. These results improve our understanding of the molecular basis of atrial cardiomyopathy and introduce new models for further mechanistic analysis.

P1453Regulation of essential myosin light chain phosphorylation in dilated cardiomyopathy

P1453Regulation of essential myosin light chain phosphorylation in dilated cardiomyopathy

Molecular characterization and expression profiles of myosin essential light chain gene in the Pacific oyster Crassostrea gigas.

In molluscs, muscle contraction is triggered by a direct binding of Ca2+ to myosin. The myosin essential light chain (MELC) provides the Ca2+ binding site and is of importance for motor function and regulation. In this study, the complete cDNA sequence of MELC gene of the Pacific oyster, Crassostrea gigas, was obtained, and the expression profiles were performed in different tissues and different embryo-larval development stages. The results showed that the full length of C. gigas MELC (CgMELC) cDNA was 659bp, containing a 5'-untranslated region of 73 bp, a 3'-untranslated region of 112bp. The open reading frame encoded a 157 amino acid peptide. The protein sequence of CgMELC contained a conserved EF-hand calcium binding motif, and showed a high sequence identity (68.4-100%) with other bivalves. Quantitative analysis of CgMELC mRNA in tissues indicated that CgMELC was expressed at the highest level in the striated adductor muscle, followed by the smooth adductor muscle and mantle. During the development of embryos and larvae, quantitative analysis and whole-mount in situ hybridization revealed that CgMELC was expressed starting from the blastula stage and abundantly expressed in trochophore and D-shaped larvae, indicating that CgMELC might also be involved in regulation of larval muscle system development. Our data provided valuable information for further researches on the function of MELC and regulation of muscle contraction in oysters.

A Cardiomyopathy Mutation in the Myosin Essential Light Chain Alters Actomyosin Structure

We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepowerstroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin.

Auxotonic to isometric contraction transitioning in a beating heart causes myosin step-size to down shift.

Myosin motors in cardiac ventriculum convert ATP free energy to the work of moving blood volume under pressure. The actin bound motor cyclically rotates its lever-arm/light-chain complex linking motor generated torque to the myosin filament backbone and translating actin against resisting force. Previous research showed that the unloaded in vitro motor is described with high precision by single molecule mechanical characteristics including unitary step-sizes of approximately 3, 5, and 8 nm and their relative step-frequencies of approximately 13, 50, and 37%. The 3 and 8 nm unitary step-sizes are dependent on myosin essential light chain (ELC) N-terminus actin binding. Step-size and step-frequency quantitation specifies in vitro motor function including duty-ratio, power, and strain sensitivity metrics. In vivo, motors integrated into the muscle sarcomere form the more complex and hierarchically functioning muscle machine. The goal of the research reported here is to measure single myosin step-size and step-frequency in vivo to assess how tissue integration impacts motor function.A photoactivatable GFP tags the ventriculum myosin lever-arm/light-chain complex in the beating heart of a live zebrafish embryo. Detected single GFP emission reports time-resolved myosin lever-arm orientation interpreted as step-size and step-frequency providing single myosin mechanical characteristics over the active cycle. Following step-frequency of cardiac ventriculum myosin transitioning from low to high force in relaxed to auxotonic to isometric contraction phases indicates that the imposition of resisting force during contraction causes the motor to down-shift to the 3 nm step-size accounting for >80% of all the steps in the near-isometric phase. At peak force, the ATP initiated actomyosin dissociation is the predominant strain inhibited transition in the native myosin contraction cycle. The proposed model for motor down-shifting and strain sensing involves ELC N-terminus actin binding.Overall, the approach is a unique bottom-up single molecule mechanical characterization of a hierarchically functional native muscle myosin.

Hypercontractile mutant of ventricular myosin essential light chain leads to disruption of sarcomeric structure and function and results in restrictive cardiomyopathy in mice.

The E143K (Glu → Lys) mutation in the myosin essential light chain has been associated with restrictive cardiomyopathy (RCM) in humans, but the mechanisms that underlie the development of defective cardiac function are unknown. Using transgenic E143K-RCM mice, we sought to determine the molecular and cellular triggers of E143K-induced heart remodelling. The E143K-induced abnormalities in cardiac function and morphology observed by echocardiography and invasive haemodynamics were paralleled by augmented active and passive tension measured in skinned papillary muscle fibres compared with wild-type (WT)-generated force. In vitro, E143K-myosin had increased duty ratio and binding affinity to actin compared with WT-myosin, increased actin-activated ATPase activity and slower rates of ATP-dependent dissociation of the acto-myosin complex, indicating an E143K-induced myosin hypercontractility. E143K was also observed to reduce the level of myosin regulatory light chain phosphorylation while that of troponin-I remained unchanged. Small-angle X-ray diffraction data showed a decrease in the filament lattice spacing (d1,0) with no changes in the equatorial reflections intensity ratios (I1,1/I1,0) in E143K vs. WT skinned papillary muscles. The hearts of mutant-mice demonstrated ultrastructural defects and fibrosis that progressively worsened in senescent animals and these changes were hypothesized to contribute to diastolic disturbance and to mild systolic dysfunction. Gene expression profiles of E143K-hearts supported the histopathology results and showed an upregulation of stress-response and collagen genes. Finally, proteomic analysis evidenced RCM-dependent metabolic adaptations and higher energy demands in E143K vs. WT hearts. As a result of the E143K-induced myosin hypercontractility, the hearts of RCM mice model exhibited cardiac dysfunction, stiff ventricles and physiological, morphologic, and metabolic remodelling consistent with the development of RCM. Future efforts should be directed toward normalization of myosin motor function and the use of myosin-specific therapeutics to avert the hypercontractile state of E143K-myosin and prevent pathological cardiac remodelling.

In vitro actin motility velocity varies linearly with the number of myosin impellers

Cardiac myosin is the motor powering the heart. It moves actin with 3 step-size varieties generated by torque from the myosin heavy chain lever-arm rotation under the influence of myosin essential light chain whose N-terminal extension binds actin. Proposed mechanisms adapting myosin mechanochemical characteristics on the fly sometimes involve modulation of step-size selection probability via motor strain sensitivity. Strain following the power stroke, hypothetically imposed by the finite actin detachment rate 1/ton, is shown to have no effect on unloaded velocity when multiple myosins are simultaneously strongly actin bound in an in vitro motility assay. Actin filaments slide ∼2 native step-sizes while more than 1 myosin strongly binds actin probably ruling out an actin detachment limited model for imposing strain. It suggests that single myosin estimates for ton are too large, not applicable to the ensemble situation, or both. Parallel motility data quantitation involving instantaneous particle velocities (frame velocity) and actin filament track averaged velocities (track velocity) give an estimate of the random walk step-size, δ. Comparing δ for slow and fast motility components suggests the higher speed component has cardiac myosin upshifting to longer steps. Variable step-size characteristics imply cardiac myosin maintains a velocity dynamic range not involving strain.

17β-Estradiol-induced interaction of estrogen receptor α and human atrial essential myosin light chain modulates cardiac contractile function.

Chronicincreased workload of the human heart causes ventricular hypertrophy, re-expression of the atrial essential myosin light chain (hALC-1), and improved contractile function. Although hALC-1 is an important positive inotropic regulator of the human heart, little is known about its regulation. Therefore, we investigated the role of the sex hormone 17β-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function. We showed that E2 attenuated hALC-1 expression in human atrial tissues of both sexes and in human ventricular AC16 cells. E2 induced the nuclear translocation of estrogen receptor alpha (ERα) and hALC-1 in AC16 cells, where they cooperatively regulate the transcriptional activity of hALC-1 gene promoter. E2-activated ERα required the estrogen response element (ERE) motif within the hALC-1 gene promoter to reduce its transcriptional activity (vehicle: 15.55±4.80 vs. E2: 6.51±3.69; ~2 fold). This inhibitory effect was potentiated in the presence of hALC-1 (vehicle: 11.13±3.66 vs. E2: 2.18±1.10; ~5 fold), and thus, hALC-1 acts as a co-repressor of ERα-mediated transcription. Yeast two-hybrid screening of a human heart cDNA library revealed that ERα interacts physically with hALC-1 in the presence of E2. This interaction was confirmed by Co-Immunoprecipitation and immunofluorescence in human atrium. As a further novel effect, we showed that chronic E2-treatment of adult mouse cardiomyocytes overexpressing hALC-1 resulted in reduced cell-shortening amplitude and twitching kinetics of these cells independent of Ca2+ activation levels. Together, our data showed that the expression of hALC-1 gene is, at least partly, regulated by E2/ERα, while hALC-1 acts as a co-repressor. The inotropic effect of hALC-1 overexpression in cardiomyocytes can be significantly repressed by E2.

Clinical features, spectrum of causal genetic mutations and outcome of hypertrophic cardiomyopathy in South Africans.

SummaryBackgroundLittle is known about the clinical characteristics, spectrum of causal genetic mutations and outcome of hypertrophic cardiomyopathy (HCM) in Africans. The objective of this study was to delineate the clinical and genetic features and outcome of HCM in African patients.MethodsInformation on clinical presentation, electrocardiographic and echocardiographic findings, and outcome of cases with HCM was collected from the Cardiac Clinic at Groote Schuur Hospital over a mean duration of follow up of 9.1 ± 3.4 years. Genomic DNA was screened for mutations in 15 genes that cause HCM, i.e. cardiac myosinbinding protein C (MYBPC3), cardiac β-myosin heavy chain (MYH7), cardiac troponin T2 (TNNT2), cardiac troponin I (TNNI3), regulatory light chain of myosin (MYL2), essential light chain of myosin (MYL3), tropomyosin 1 (TPM1), phospholamban (PLN), α-actin (ACTC1), cysteine and glycine-rich protein 3 (CSRP3), AMP-activated protein kinase (PRKAG2), α-galactosidase (GLA), four-and-a-half LIM domains 1 (FHL1), lamin A/C (LMNA) and lysosomeassociated membrane protein 2 (LAMP2). Survival and its predictors were analysed using the Kaplan–Meier and Cox proportional hazards regression methods, respectively.ResultsForty-three consecutive patients [mean age 38.5 ± 14.3 years; 25 (58.1%) male; and 13 (30.2%) black African] were prospectively enrolled in the study from January 1996 to December 2012. Clinical presentation was similar to that reported in other studies. The South African founder mutations that cause HCM were not found in the 42 probands. Ten of 35 index cases (28.6%) tested for mutations in 15 genes had disease-causing mutations in MYH7 (six cases or 60%) and MYBPC3 (four cases or 40%). No disease-causing mutation was found in the other 13 genes screened. The annual mortality rate was 2.9% per annum and overall survival was 74% at 10 years, which was similar to the general South African population. Cox’s proportional hazards regression showed that survival was predicted by New York Heart Association (NYHA) functional class at last visit (p = 0.026), but not by the presence of a disease-causing mutation (p = 0.474).ConclusionsComprehensive genetic screening was associated with a 29% yield of causal genetic mutations in South African HCM cases, all in MYH7 and MBPC3 genes. A quarter of the patients had died after a decade of follow up, with NYHA functional class serving as a predictor of survival.

Age- and Activity-Related Differences in the Abundance of Myosin Essential and Regulatory Light Chains in Human Muscle.

Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM). We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping.

Essential light chain S195 phosphorylation is required for cardiac adaptation under physical stress.

Regulatory proteins of the sarcomere are pivotal for normal heart function and when affected by mutations are frequently causing cardiomyopathy. The exact function of these regulatory proteins and how mutations in these translate into distinct cardiomyopathy phenotypes remains poorly understood. Mutations in the essential myosin light chain (ELC) are linked to human cardiomyopathy characterized by a marked variability in disease phenotypes and high incidences of sudden death. Here we studied the role of the highly conserved S195 phosphorylation site of ELC using heterozygous adult zebrafish lazy susan (laz(m647)) in regulating contractile function in normal physiology and disease. Echocardiography revealed signs of systolic dysfunction in otherwise phenotypically unremarkable heterozygote mutants. However, after physical stress, heart function of laz heterozygous zebrafish severely deteriorated causing heart failure and sudden death. Mechanistically, we show that upon physical stress, ELCs become phosphorylated and lack of S195 dominant-negatively impairs ELC phosphorylation. In vitro motility analysis with native myosin from adult heterozygous hearts demonstrates that S195 loss, specifically following physical stress, results in altered acto-myosin sliding velocities and myosin binding cooperativity, causing reduced force generation and organ dysfunction. Using adult heterozygous zebrafish, we show that ELC S195 phosphorylation is pivotal for adaptation of cardiac function to augmented physical stress and we provide novel mechanistic insights into the pathogenesis of ELC-linked cardiomyopathy.

On the Rate of Synthesis of Individual Proteins within and between Different Striated Muscles of the Rat.

The turnover of muscle protein is responsive to different (patho)-physiological conditions but little is known about the rate of synthesis at the level of individual proteins or whether this varies between different muscles. We investigated the synthesis rate of eight proteins (actin, albumin, ATP synthase alpha, beta enolase, creatine kinase, myosin essential light chain, myosin regulatory light chain and tropomyosin) in the extensor digitorum longus, diaphragm, heart and soleus of male Wistar rats (352 ± 30 g body weight). Animals were assigned to four groups (n = 3, in each), including a control and groups that received deuterium oxide (2H2O) for 4 days, 7 days or 14 days. Deuterium labelling was initiated by an intraperitoneal injection of 10 μL/g body weight of 99.9% 2H2O-saline, and was maintained by administration of 5% (v/v) 2H2O in drinking water provided ad libitum. Homogenates of the isolated muscles were analysed by 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionisation time of flight mass spectrometry. Proteins were identified against the SwissProt database using peptide mass fingerprinting. For each of the eight proteins investigated, the molar percent enrichment (MPE) of 2H and rate constant (k) of protein synthesis was calculated from the mass isotopomer distribution of peptides based on the amino acid sequence and predicted number of exchangeable C–H bonds. The average MPE (2.14% ± 0.2%) was as expected and was consistent across muscles harvested at different times (i.e., steady state enrichment was achieved). The synthesis rate of individual proteins differed markedly within each muscle and the rank-order of synthesis rates differed among the muscles studied. After 14 days the fraction of albumin synthesised (23% ± 5%) was significantly (p < 0.05) greater than for other muscle proteins. These data represent the first attempt to study the synthesis rates of individual proteins across a number of different striated muscles.

Development of Physiologic versus Pathologic Hypertrophy in Mouse Models Expressing Mutations in Myosin Essential Light Chain

Mechanisms and signaling pathways underlying physiologic vs. pathological cardiac remodeling were investigated using transgenic Δ43, A57G and E143K mice expressing mutations in myosin essential light chain (ELC). In Δ43-hearts, the endogenous mouse ventricular-ELC is partially replaced with the N-terminal 43-amino-acid truncated human-ELC. The A57G (Alanine→Glycine) mutation was found to cause hypertrophic (HCM), while E143K (Glutamate→Lysine), restrictive cardiomyopathy (RCM). The hearts of Δ43-mice hypertrophy with age, but the ventricles do not show pathological remodeling or morphology vs. WT mice. In contrary, severe phenotypes were observed in Masson's trichrome-stained heart sections of HCM- and RCM-models. EM evaluation revealed abundant mitochondria in A57G-HCM and profound sarcomere defects (distorted Z- and M- lines) in E143K-RCM hearts. Normal in vivo function was recorded in Δ43-mice, but systolic and diastolic abnormalities were noted in HCM and RCM hearts. Functional studies showed elevated (E143K) or reduced (A57G) contractile force and increased Ca2+-sensitivity in RCM/HCM models vs. WT, but no change in Δ43-mice. Small-angle X-ray diffraction study showed decreased lattice spacing in pathological models of hypertrophy, but no changes in the physiological phenotype. Proteomic investigation using tandem-mass-tag-labeling showed changes in metabolic and cellular processes in all three phenotypes vs. WT and differential protein expression between physiologic and pathologic heart models. Compared with Δ43, E143K-RCM hearts showed upregulation of structural proteins, myomesin-1 and LIM-protein2, while Ca2+-binding proteins, sarcalumenin and mitochondrial protein Aralar1 were upregulated in A57G-HCM. Comparison of RCM and HCM hearts revealed upregulation of L-lactate dehydrogenase-B and isocitrate dehydrogenase-[NAD]-alpha, and downregulation of inorganic pyrophosphatase-2 in E143K vs. A57G hearts. In conclusion, in contrast to the physiologic model of hypertrophy, HCM and RCM mice show severe structural and metabolic abnormalities that trigger development of pathological hypertrophy and alter function. Supported by NIH-HL108343 (DSC).