Expression Of Myofilament Proteins Research Articles

The role of TRPV2 on physiological and pathological remodeling of myocytes

Hemodynamic stress and extracellular matrix stiffness provide essential information for homeostatic regulation and functional adaptation on the physiological and pathological remodeling of myocytes. Previously, we have reported that transient receptor potential cation channel vanilloid-family type 2 (TRPV2) is a candidate of myocyte mechanosensor, and crucial for the maintenance of cardiac structure and function. Here, we show the role of TRPV2 in the physiological and pathological remodeling of myocytes. TRPV2-deficient neonatal cardiomyocytes exhibited no sarcomere formation, reduced Ca2+ contents of sarcoplasmic reticulum, or spontaneous beating, suggesting that TRPV2-dificiency affects myocyte maturation. The elimination of TRPV2 from juveniles showed the reduction of myofilaments and mildly chamber dilation in adult stage. Isolated myocytes from these mice showed the impaired Ca2+ handling with excitation-contraction coupling and contractile dysfunction. In hypertrophic response, increased actin-myosin cross-bridge formation enhance passive cardiomyocytes stiffness in the transverse direction. However, TRPV2-deficent hearts showed no change of myofilament protein expression and severe cardiac dysfunction to pressure-overload. Thus, TRPV2 is critical for compensate hypertrophic response to mechanical stress.

Suppression of myofilament cross-bridge kinetic in the heart of orchidectomized rats

The increased incidence of heart failure with reduced ejection fraction in men compared with women suggests that male sex hormones significantly impact myocardial contractile activation. This study aims to examine associations among molecular alterations, cellular modulations and in vivo cardiac contractile function upon deprivation of testicular hormones. Myocardial structure and functions were compared among sham-operated control and twelve-week orchidectomized (ORX) male rats with and without testosterone supplementation. Echocardiography and pressure-volume relationships demonstrated a decreased left ventricular ejection fraction compared with sham-operated controls. The percentage of contractility reduction was generally similar to the decrease in tension development detected in both right ventricular trabeculae and skinned isolated left ventricular cardiomyocytes of ORX rats. Reductions in tension cost and the rate constant of tension redevelopment (ktr) in ORX samples suggested a decrease in the rate of cross-bridge formation, reflecting a reduced number of cross-bridges. Slow cross-bridge detachment in ORX rat hearts could result from a shift of myosin heavy chain isoforms towards a slower ATPase activity β-isoform and reductions in the phosphorylation levels of cardiac troponin I and myosin binding protein-C. All the changes in the ORX rat heart, including ejection fractions and myofilament protein expression and phosphorylation, were completed attenuated by a physiological dose of testosterone. Testosterone plays a critical role in regulating the mechanical and contractile dynamics of the heart. Deprivation of male sex hormones cause the loss of normal preserved cardiac contractile function leading to a high risk of severe cardiomyopathy progression.

Very Low-Density Lipoproteins of Metabolic Syndrome Modulates STIM1, Suppresses Store-Operated Calcium Entry, and Deranges Myofilament Proteins in Atrial Myocytes.

Individuals with metabolic syndrome (MetS) are at high risk for atrial myopathy and atrial fibrillation. Very low-density lipoproteins (VLDLs) of MetS (MetS-VLDLs) are cytotoxic to atrial myocytes in vivo and in vitro. The calcineurin–nuclear factor of activated T-cells (NFAT) pathway, which is regulated by stromal interaction molecule 1 (STIM1)/ calcium release-activated calcium channel protein 1 (Orai1)–mediated store-operated Ca2+ entry (SOCE), is a pivotal mediator of adaptive cardiac hypertrophy. We hypothesized that MetS-VLDLs could affect SOCE and the calcineurin–NFAT pathway. Normal-VLDL and MetS-VLDL samples were isolated from the peripheral blood of healthy volunteers and individuals with MetS. VLDLs were applied to HL-1 atrial myocytes for 18 h and were also injected into wild-type C57BL/6 male mouse tails three times per week for six weeks. After the sarcoplasmic reticulum (SR) Ca2+ store was depleted, SOCE was triggered upon reperfusion with 1.8 mM of Ca2+. SOCE was attenuated by MetS-VLDLs, along with reduced transcriptional and membranous expression of STIM1 (P = 0.025), and enhanced modification of O-GlcNAcylation on STIM1 protein, while Orai1 was unaltered. The nuclear translocation and activity of calcineurin were both reduced (P < 0.05), along with the alteration of myofilament proteins in atrial tissues. These changes were absent in normal-VLDL-treated cells. Our results demonstrated that MetS-VLDLs suppressed SOCE by modulating STIM1 at the transcriptional, translational, and post-translational levels, resulting in the inhibition of the calcineurin–NFAT pathway, which resulted in the alteration of myofilament protein expression and sarcomere derangement in atrial tissues. These findings may help explain atrial myopathy in MetS. We suggest a therapeutic target on VLDLs to prevent atrial fibrillation, especially for individuals with MetS.

Abstract 423: Cardiomyocyte-Specific Epidermal Growth Factor Receptor Deletion Differentially Alters Cardiac Contractility and Remodeling During Physiologic versus Pathologic Development

Epidermal Growth Factor Receptor (EGFR), a member of the Erbb receptor tyrosine kinase family, is essential to the development of multiple tissue and organs. Due to the embryonic lethality of global EGFR deletion, the impact of EGFR signaling in the adult heart, normally or in response to pathological stimuli, has not been well-explored. Using recently attained mice with floxed EGFR alleles crossed with αMHC-Cre mice, we have generated a cardiomyocyte-specific constitutive EGFR knockout mouse model (CM-EGFR KO) to address its role in the heart. Compared to their wild-type (WT) littermate controls, CM-EGFR-KO mice displayed age-related development of cardiac dysfunction and remodeling, occurring between 7 and 9 weeks of age, as monitored via echocardiography and immunohistochemistry analyses. Although contractile responsiveness to β-adrenergic receptor stimulation was unaffected by EGFR deletion, RNASeq analysis of CM-EGFR-KO hearts within this timeframe revealed alterations in myofilament protein expression profiles. Additionally, there was enhanced cardiomyocyte death in the 7-9 week window of development, which persisted to 10 months of age, at which time the CM-EGFR KO mice had progressively worsened cardiac function, dilation and cardiomyocyte loss. To investigate the impact of cardiomyocyte-specific EGFR deletion on the response to pathologic stress, we challenged 12-14 week old WT and CM-EGFR KO mice with chronic isoproterenol infusion. Notably, while chronic isoproterenol treatment diminished cardiac function and induced hypertrophic remodeling in WT mouse hearts, CM-EGFR-KO mouse hearts displayed progressively improved contractility and ablated hypertrophy over time, suggesting that EGFR differentially regulates cardiac function and remodeling processes during physiological versus pathological development.

Abstract 188: Contractile Function and Myofilament Proteins of the Naked Mole-rat Heart Show Resistance to Oxidative Stress

The naked mole-rat (NMR) is the longest-lived rodent, with a maximum lifespan of &gt;31 years. Unlike every other mammal studied to date, this species withstands cardiovascular structural and functional changes for at least 75% of its lifespan. Due to the intersection of oxidative stress, aging, and cardiovascular disease, we questioned if NMRs were more resistant to oxidative stress-induced cardiac dysfunction compared to short-lived mice. Echocardiography showed that 7 days after a 20 mg/kg dose of doxorubicin (DOX), mice had a 25% decline in fractional shortening (36 ± 1% to 27 ± 2%). In contrast, the fractional shortening of NMRs was unchanged with DOX treatment (27 ± 1%). Previously we observed that while basal cardiac function is low, NMRs have a robust cardiac reserve, displaying a 1.7 fold increase in fractional shortening under exercise-like conditions. DOX-treated NMRs had a significant reduction in their dobutamine response, signifying a diminished cardiac reserve. Intriguingly, we found no changes in phosphorylation or expression of myofilament proteins in the NMR heart with DOX treatment. Mice on the other hand, increased the phosphorylation of cardiac myosin binding protein-C and switched expression from predominantly α-myosin heavy chain to the β-isoform. Electron microscopy showed that DOX caused marked mitochondrial swelling and loss of cristae as well as massive cardiac myofibrillar disarray in mice. Conversely, DOX-treated NMRs had only slight alterations to myofilament structure. NMRs additionally had twofold higher levels of glutathione in their hearts, indicating a high antioxidant capacity. These findings reveal that long-lived NMRs are less susceptible to oxidative stress-induced cardiac dysfunction than mice. The NMR’s low basal cardiac function, unique regulation of myofilament proteins, and high glutathione levels may all be integral in the species’ ability to withstand oxidative damage and preserve cardiac function well into its third decade of life.

Role of H2 Calponin in Myoblast Differentiation, Fusion and Myogenesis

Actin cytoskeleton plays a pivotal role in myoblast motility, adhesion, fusion and differentiation into myotubes. However, how the actin cytoskeleton is regulated during myogenesis is largely unknown. H2-calponin is a regulatory protein that stabilizes the actin cytoskeleton and regulates cell motility and adhesion. In the present study, we investigated the role of h2 calponin in the regulation of myoblast migration, differentiation and fusion. The results showed that h2-calponin is expressed at significant levels in undifferentiated mouse primary satellite cells and C2C12 myoblasts. In migrating satellite cells and C2C12 myoblasts, h2-calponin co-localizes with actin stress fibers at both the leading edge and the tail regions. In newly fused myotubes derived from primary satellite cells, h2-calponin is mainly present at the ends of the myotubes. The level of h2-calponin significantly decreases during satellite cell and myoblast differentiation and fusion, and becomes undetectable in mature myocytes. Primary satellite cells isolated from h2-calponin gene knockout mice had significantly decreased cell spreading area indicating reduced substrate adhesion and increased rate of cell proliferation as compared with wild type controls. Biochemical and histological studies revealed that skeletal muscles of h2-calponin knockout mice retain normal muscle weight and myofilament protein compositions. Myotubes derived from h2-calponin-null satellite cells showed apparently normal sarcomere striations and expression of myofilament proteins. Therefore, deletion of h2-calponin does not abolish the maximum capacity of myoblast function and myogenesis. However, in vitro differentiation studies revealed that h2-calponin-hull myoblasts had higher efficiency of fusion, as indicated by increased fusion index, than wild type controls. The data suggest that the function of h2-calponin provides a novel mechanism in regulating actin cytoskeleton activity during myoblast differentiation, fusion and myogenesis.

Skeletal muscle myofilament adaptations to aging, disease, and disuse and their effects on whole muscle performance in older adult humans.

Skeletal muscle contractile function declines with aging, disease, and disuse. In vivo muscle contractile function depends on a variety of factors, but force, contractile velocity and power generating capacity ultimately derive from the summed contribution of single muscle fibers. The contractile performance of these fibers are, in turn, dependent upon the isoform and function of myofilament proteins they express, with myosin protein expression and its mechanical and kinetic characteristics playing a predominant role. Alterations in myofilament protein biology, therefore, may contribute to the development of functional limitations and disability in these conditions. Recent studies suggest that these conditions are associated with altered single fiber performance due to decreased expression of myofilament proteins and/or changes in myosin-actin cross-bridge interactions. Furthermore, cellular and myofilament-level adaptations are related to diminished whole muscle and whole body performance. Notably, the effect of these various conditions on myofilament and single fiber function tends to be larger in older women compared to older men, which may partially contribute to their higher rates of disability. To maintain functionality and provide the most appropriate and effective countermeasures to aging, disease, and disuse in both sexes, a more thorough understanding is needed of the contribution of myofilament adaptations to functional disability in older men and women and their contribution to tissue level function and mobility impairment.

Physiological Contractility of Cardiomyocytes in the Wall of Mouse and Rat Azygos Vein

We recently demonstrated the abundant presence of cardiomyocytes in the wall of thoracic veins of adult mouse and rat (Kracklauer, Feng, Jiang, Lin, Lin, Jin, FEBS J. 280:880-91, 2013). The highly differentiated morphology and myofilament protein expression of the venous cardiomyocytes suggested contractile functions. Here we further investigated the contractility of mouse and rat azygos venous rings in comparison with that of atrial strips and ventricular papillary muscle. X-gal staining of transgenic mouse vessels expressing lacZ under cloned cardiac troponin T promoter demonstrated that the venous cardiomyocytes are discontinuous from atrial myocardium and longitudinally aligned in the wall of thoracic veins perpendicular to the vessel axis. Histology study showed striation patterns in the venous cardiomyocytes, which indicate encirclement orientation of myofibrils in the vessel wall. Mechanical studies found that mouse and rat azygos veins produce strong cardiac type twitch contractions when stimulated by electrical pacing in contrast to the weak and slow smooth muscle contractions induced using 90 mM KCl. The twitch contraction and relaxation of rodent azygos veins further exhibited cardiac type beta-adrenergic responses. Quantitative characterization showed that the contractions of venous cardiomyocytes are slightly slower than that of atrium muscle but significantly faster than that of ventricular papillary muscle. These novel findings indicate that the cardiomyocytes in rodent thoracic veins possess fully differentiated cardiac muscle phenotype despite their anatomical and functional separation from the heart.

Changes in myofilament proteins, but not Ca2+ regulation, are associated with a high-fat diet-induced improvement in contractile function in heart failure

Pathological conditions such as diabetes, insulin resistance, and obesity are characterized by elevated plasma and myocardial lipid levels and have been reported to exacerbate the progression of heart failure (HF). Alterations in cardiomyocyte Ca(2+) regulatory properties and myofilament proteins have also been implicated in contractile dysfunction in HF. However, our prior studies reported that high saturated fat (SAT) feeding improves in vivo myocardial contractile function, thereby exerting a cardioprotective effect in HF. Therefore, we hypothesized that SAT feeding improves contractile function by altering Ca(2+) regulatory properties and myofilament protein expression in HF. Male Wistar rats underwent coronary artery ligation (HF) or sham surgery (SH) and were fed normal chow (SHNC and HFNC groups) or a SAT diet (SHSAT and HFSAT groups) for 8 wk. Contractile properties were measured in vivo [echocardiography and left ventricular (LV) cannulation] and in isolated LV cardiomyocytes. In vivo measures of contractility (peak LV +dP/dt and -dP/dt) were depressed in the HFNC versus SHNC group but improved in the HFSAT group. Isolated cardiomyocytes from both HF groups were hypertrophied and had decreased percent cell shortening and a prolonged time to half-decay of the Ca(2+) transient versus the SH group; however, SAT feeding reduced in vivo myocyte hypertrophy in the HFSAT group only. The peak velocity of cell shortening was reduced in the HFNC group but not the HFSAT group and was positively correlated with in vivo contractile function (peak LV +dP/dt). The HFNC group demonstrated a myosin heavy chain (MHC) isoform switch from fast MHC-α to slow MHC-β, which was prevented in the HFSAT group. Alterations in Ca(2+) transients, L-type Ca(2+) currents, and protein expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase and phosphorylated phospholamban could not account for the changes in the in vivo contractile properties. In conclusion, the cardioprotective effects associated with SAT feeding in HF may occur at the level of the isolated cardiomyocyte, specifically involving changes in myofilament function but not sarcoplasmic reticulum Ca(2+) regulatory properties.

Transmural variation in myosin heavy chain isoform expression modulates the timing of myocardial force generation in porcine left ventricle

Recent studies have shown that the sequence and timing of mechanical activation of myocardium vary across the ventricular wall. However, the contributions of variable expression of myofilament protein isoforms in mediating the timing of myocardial activation in ventricular systole are not well understood. To assess the functional consequences of transmural differences in myofilament protein expression, we studied the dynamic mechanical properties of multicellular skinned preparations isolated from the sub-endocardial and sub-epicardial regions of the porcine ventricular midwall. Compared to endocardial fibres, epicardial fibres exhibited significantly faster rates of stretch activation and force redevelopment (k(tr)), although the amount of force produced at a given [Ca2+] was not significantly different. Consistent with these results, SDS-PAGE analysis revealed significantly elevated expression of alpha myosin heavy chain (MHC) isoform in epicardial fibres (13 +/- 1%) versus endocardial fibres (3 +/- 1%). Linear regression analysis revealed that the apparent rates of delayed force development and force decay following stretch correlated with MHC isoform expression (r2 = 0.80 and r2 = 0.73, respectively, P < 0.05). No differences in the relative abundance or phosphorylation status of other myofilament proteins were detected. These data show that transmural differences in MHC isoform expression contribute to regional differences in dynamic mechanical function of porcine left ventricles, which in turn modulate the timing of force generation across the ventricular wall and work production during systole.

Late-stage alterations in myofibrillar contractile function in a transgenic mouse model of dilated cardiomyopathy (Tgαq*44)

Mechanical and biochemical alterations were investigated in permeabilized cardiomyocytes along with the progression of dilated cardiomyopathy (DCM) in a transgenic mouse line overexpressing the activated Galphaq protein (Tgalphaq*44). The isometric force, its Ca(2+) sensitivity (pCa(50)) and the turnover rate of the actin-myosin cycle (k(tr)) were determined at sarcomere lengths (SLs) of 1.9 mum and 2.3 mum before (at 4 and 10 months of age) and after hemodynamic decompensation (at 14 and 18 months of age) in Tgalphaq*44 cardiomyocytes and in age-matched control cardiomyocytes. The SL-dependence of pCa(50) was not different in Tgalphaq*44 and control hearts. In contrast, a significant increase in pCa(50) was observed in the Tgalphaq*44 cardiomyocytes (DeltapCa(50): 0.10-0.15 vs. the controls) after 10 months of age that could be diminished by exposures to the catalytic subunit of protein kinase A (PKA). Accordingly, a decline in endogenous PKA activity and decreased troponin I phosphorylation were detected after 10 months in the Tgalphaq*44 hearts. Finally, the maximal Ca(2+)-activated force (F(o)) and k(tr) were lower and the passive force (F(passive)) was higher at 18 months in the Tgalphaq*44 cardiomyocytes compared to the control. These mechanical alterations were paralleled by a robust increase in beta-myosin heavy chain expression in the Tgalphaq*44 hearts. In conclusion, our data suggested that an initial decrease of PKA signaling and subsequent changes in myofilament protein expression may contribute to the development of dilated cardiomyopathy in Tgalphaq*44 hearts.

Differences in Ito and myofilament protein expression may underlie transmurally‐varying electromechanics in the canine left ventricle

Myocytes isolated from the endocardial, midmyocardial, and epicardial regions of the canine left ventricle (LV) possess distinct action potential morphologies, Ca2+ transients (CaTs), and timecourses of unloaded shortening. Some of these differences have been linked to variable expression of Ca2+‐handling and ion channel proteins. We used a computational model of myocyte electromechanics to test the hypothesis that identified variations in protein expression account for observed differences in mechanical function between epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) cells. The ENDO cell model displayed a greater delay to onset of shortening compared with EPI and MID cells, in agreement with experiments. Time to onset of cell shortening was strongly affected by the magnitude of the transient outward K+ current (Ito). Simulated shortening in response to experimentally measured ENDO and MID CaTs agreed well with experiments, while responses to the EPI CaT did not. Agreement between measured and simulated EPI shortening was improved by increasing the rate of crossbridge cycling. These results suggest important roles for Ito and altered crossbridge kinetics in modulating electromechanical function within the canine LV. Supported by NSF grant BES‐0506252 and NIH grant P41 RR‐08605.

Simulated microgravity effects on the rat carotid and femoral arteries: role of contractile protein expression and mechanical properties of the vessel wall

The goal of this study was to determine the effects of microgravity on myofilament protein expression and both passive and active length-force relationships in carotid and femoral arteries. Microgravity was simulated by 20-day hindlimb unweighting (HU) in Wistar male rats, and carotid and femoral artery segments were isolated from both HU and control (CTL) rats for Western blot and length-force analysis. Western blots revealed that HU significantly decreased myosin light chain-20 (MLC-20) protein levels in both carotid and femoral arteries and decreased myosin heavy chain (MHC) in femoral artery. alpha-Actin levels were not altered by HU treatment in either artery. Length-force analysis demonstrated that HU did not change either passive or active length-force relationships in the femoral artery. HU-treated arterial rings developed significantly less force to 100 mM K(+) than CTL, but optimal lengths were identical. In the carotid artery, length-active force curves were identical for both CTL and HU; however the length-passive force curve for HU-treated rings exhibited a steeper slope than CTL, suggesting decreased compliance of the artery wall. In conclusion, our data suggest that the HU-induced decreases in both MLC-20 and MHC in femoral artery are responsible for the decreased contraction to 100 mM K(+) in HU-treated femoral artery rings. In the carotid artery, the HU-induced decrease in vessel wall compliance may counter any decrease in contractility caused by the decreased MLC-20 levels.

Decreased Ca2+ sensitivity of isometric tension in skinned cardiac myocytes from tail-suspended rats.

Tail suspension in rats causes a cephalic shift in blood, resulting in a volume load on the heart similar to that observed during microgravity spaceflight or mild heart failure. The present study determined the influence of increased cardiac hemodynamic load on myofilament isometric tension as a function of Ca2+ concentration in skinned cardiac myocytes of control and 7-day head-down tilt Sprague-Dawley rats. Isometric force of single skinned myocytes was measured by attaching cells with adhesive to a force transducer and piezoelectric translator. A significant decrease in the Ca2+ sensitivity of tension was observed in cardiac myocytes from suspended rats [pCa of half-maximal tension (pCa50) of 5.83 +/- 0.03] compared with control rats (pCa50 of 5.94 +/- 0.03). Maximum tension generation and slope of the tension-pCa relationship were unaffected by head-down tilt. Electrophoretic analysis of myofilament proteins indicates differences in expression of proteins in the 50-60 and 100-120 kDa ranges; immunoblot analysis of tubulin (50 kDa) expression indicates no change in the ratio of beta-tubulin to light chain 1 or tropomyosin. Decreased force-producing ability at a given submaximum Ca2+ concentration in cardiac myocytes from suspended rats suggests a decrease in contractility possibly due to changes in cardiac myofilament protein expression following chronic elevated volume load on the heart.

C2C12 cells: biophysical, biochemical, and immunocytochemical properties.

We examined the myofibril biochemical, structural, and biophysical properties of C2C12, a mouse skeletal muscle cell line (American Type Culture Collection), to assess whether force development and the sensitivity of the myofilaments to calcium could be measured in C2C12 myotubes and whether a cardiac contractile protein, troponin T, is expressed and incorporated into C2C12 myofibrils. When myoblasts fused and differentiated into myotubes, expression of myofilament proteins was initiated. Multiple cardiac and skeletal muscle troponin T isoforms were coexpressed. Cardiac troponin T expression increased and then decreased with time. Fluorescence immunocytochemistry demonstrated incorporation of cardiac troponin T isoforms into the myofibrils. At the time of the biophysical studies, mean myotube diameter was 12 microns (range 5-25 microns), and mean length was 290 microns (range 130-520 microns). The estimated maximum force developed by chemically skinned myotubes at 6-7 days poststarvation, 0.88 +/- 0.12 microN (mean +/- 95% confidence interval, n = 5), was significantly less (P < 0.05) than that at 10-13 days poststarvation, 1.12 +/- 0.12 microN (n = 7). The force-pCa relation yielded a Hill coefficient of 2.9 +/- 0.6 (n = 7) and half-maximal activation at pCa of 5.77 +/- 0.20. The demonstration that the biophysical properties of C2C12 cells can be measured and that cardiac and skeletal muscle troponin T isoforms are incorporated and colocalized into myofibrils suggest that these cells could be a useful model to assess the effects of exogenous native and mutated cardiac and skeletal contractile protein isoforms on myofilament function.