Turnover of myosin heavy and light chains in cultured embryonic chick cardiac and skeletal muscle

The relative rates of synthesis and breakdown of myosin heavy and light chains were studied in primary cell cultures of embryonic chick cardiac and skeletal muscle. Measurements were made after 4 days in culture, at which time both skeletal and cardiac cultures were differentiated and contracted spontaneously. Following a 4-hr pulse of radioactive leucine, myosin and its heavy and light chains were extracted to 90% or greater purity and the specific activities of the proteins were determined. In cardiac muscle, myosin heavy chains were synthesized approximately 1.6 times the rate of myosin light chains, and in skeletal muscle, heavy chains were synthesized at approximately 1.4 times the rate of light chains. Relative rates of degradation of muscle proteins were determined using a dual-isotope technique. In general, the soluble and myofibrillar proteins of both types of muscle had decay rates proportional to their molecular weights (larger proteins generally had higher decay rates) based on analyses utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A notable exception to this general rule was myosin heavy chains, which had decay rates only slightly higher than the myosin light chains. Direct measurements on purified proteins indicated that the heavy chains of myosin were turning over at a slightly greater rate (approximately 20%) than the myosin light chains in both cardiac and skeletal muscle. The reasons for the apparent discrepancy between these measurements of myosin heavy and light chain synthesis and degradation are discussed.     

Troponin subunits contribute to altered myosin ATPase activity in diabetic cardiomyopathy

Our group has documented that myocardial performance is impaired in the hearts of chronically diabetic rats and rabbits. Abnormalities in the contractile proteins and regulatory proteins may be responsible for the mechanical defects in the streptozotocin (STZ)-diabetic hearts. Previously, the major focus of our research on contractile proteins in abnormal states has concentrated on myosin ATPase and its isoenzymes. Our present study is based on the overall hypothesis that regulatory proteins, in addition to contractile protein, myosin contribute to altered cardiac contractile performance in the rat model of diabetic cardiomyopathy. The purpose of our research was to define the role of cardiac regulatory proteins (troponin-tropomyosin) in the regulation of actomyosin system in diabetic cardiomyopathy.For baseline data, myofibrillar ATPase studies were conducted in the myofibrils from control and diabetic rats. To focus on the regulatory proteins (troponin and tropomyosin), individual proteins of the cardiac system were reconstituted under controlled conditions. By this approach, myosin plus actin and troponin-tropomyosin from the normal and diabetic animals could be studied enzymatically. The proteins were isolated from the cardiac muscle of control and STZ-diabetic (4 weeks) rats. Sodium dodecyl sulfate gel electrophoretic patterns demonstrate differences in the cardiac TnT and TnI regions of diabetic animals suggesting the different amounts of TnT and/or TnI or possibly different cardiac isozymes in the regulatory protein complex. Myofibrils probed with a monoclonal antibody TnI-1 (specific for adult cardiac TnI) show a downregulation of cardiac TnI in diabetics when compared to its controls. Enzymatic data confirm a diminished calcium sensitivity in the regulation of the cardiac actomyosin system when regulatory protein(s) complex was recombined from diabetic hearts. Actomyosin ATPase activity in the hearts of diabetic animals was partially reversed when myosin from diabetic rats was regulated with the regulatory protein complex isolated from control hearts. To our knowledge, this is the first study which demonstrates that the regulatory proteins from normal hearts can upregulate cardiac myosin isolated from a pathologic rat model of diabetes. This diminished calcium sensitivity along with shifts in cardiac myosin heavy chain (V1V3) may be partially responsible for the impaired cardiac function in the hearts of chronic diabetic rats. (Mol Cell Biochem151: 165–172, 1995)     

Eyestalk ablation has little effect on actin and myosin heavy chain gene expression in adult lobster skeletal muscles

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.     

Myofibrillar protein turnover. Synthesis of protein-bound 3-methylhistidine, actin, myosin heavy chain and aldolase in rat skeletal muscle in the fed and starved states.

The turnover of 3-methylhistidine (N tau-methylhistidine) and in some cases actin, myosin heavy chain and aldolase in skeletal muscle was measured in a number of experiments in growing and adult rats in the fed and overnight-starved states. In growing fed rats in three separate experiments, measurements of the methylation rate of protein-bound 3-methylhistidine by either [14C]- or [3H]-methyl-labelled S-adenosylmethionine show that 3-methylhistidine synthesis is slower than the overall rate of protein synthesis indicated by [14C]tyrosine incorporation. Values ranged from 36 to 51%. However, in one experiment with rapidly growing young fed rats, acute measurements over 1 h showed that 3-methylhistidine synthesis could be increased to the same rate as the overall rate. After overnight starvation in these rats, the steady-state synthesis rate of 3-methylhistidine was 38.8% of the overall rate. This was a similar value to that in adult non-growing rats, in which measurements of the relative labelling of 3-methylhistidine and histidine after a single injection of [14C]histidine indicated that 3-methylhistidine synthesis was 37% of the overall rate in the fed or overnight-starved state. According to measurements of actin, myosin heavy-chain and aldolase synthesis in the over-night-starved state with young rats, with a variety of precursors, slow turnover of 3-methylhistidine results from the specific slow turnover of actin, since turnover rates of myosin heavy chain, mixed protein and aldolase were 2.5, 3 and 3.4 times faster respectively. However, in the fed state synthesis rates of actin were increased disproportionately to give similar rates for all proteins. These results show that (a) 3-methylhistidine turnover in muscle is less than half the overall rate in both young and adult rats, (b) slow 3-methylhistidine turnover reflects the specifically slow turnover of actin compared with myosin heavy chain and other muscle proteins, and (c) during growth the synthesis rate of actin is particularly sensitive to the nutritional state and can be increased to a similar rate to that of other proteins.     

Relative synthesis of cardiac contractile proteins. Evidence for synthesis from the same precursor pool.

The relative molar synthesis of cardiac contractile proteins has been measured in the perfused heart under control haemodynamic conditions. This synthesis, of myosin heavy chains, individual light chains (1 and 2), actin and tropomyosin, was determined from isolated guinea-pig hearts perfused for 3h simultaneously with constant specific radioactivities and concentrations of [3H]lysine and [3H]phenylalanine.The data strongly suggest that all of the proteins studied were synthesized from the same precursor pools of lysine and phenylalanine, since the ratio of the specific activities of the two labels was the same in all of the proteins. Measurement of molar synthesis of each contractile protein was the same with either labelled amino acid. Under control haemodynamic-perfusion conditions, the relative molar synthesis of the contractile proteins was actin greater than heavy chains greater than light chain 2 greater than light chain 1 greater than tropomyosin.     

Contrasting response of protein degradation to starvation and insulin as measured by release of N tau-methylhistidine or phenylalanine from the perfused rat heart.

An isotope-dilution method is described for the measurement of N tau-methylhistidine release from the perfused rat heart. We argue that release of N tau-methylhistidine is indicative of cardiac actin degradation. N tau-Methylhistidine release is compared with phenylalanine release in the presence of cycloheximide (phenylalanine release being a measure of degradation of mixed proteins). In hearts perfused with glucose plus acetate, the rate of actin degradation was increased by starvation and was not inhibited by insulin. In contrast, the rate of mixed-protein degradation was decreased by starvation and was inhibited by insulin. The fractional rate of degradation of mixed proteins in hearts from fed or starved rats was greater than that for actin. It is suggested that there are at least two pools of intracellular protein, the degradation rates of which differ in terms of their response to insulin and starvation.     

Lysosomal enzyme activities in muscle following starvation and refeeding in the saithe Pollachius virens L

Saithe (Pollachius virens L.) were starved for 66 days at 10 degrees C and activities of aryl sulfatase, acid proteinase, beta-glucuronidase, RNAase and acid phosphatase measured in homogenates prepared from fast and slow myotomal muscles. In fed fish, hydrolase activities were generally higher in slow than fast muscles. With the exception of acid proteinase activity in slow muscle, the activities of all the lysosomal enzymes increased by 70 to 100% during starvation. In general, there was a proportionally larger increase in the hydrolase activities in fast than in slow muscle. In a second experiment, fish were starved for 74 days, and refed for up to 52 days. The increases in aryl sulfatase and acid proteinase activity produced in fast muscle with starvation were found to be rapidly reversed by refeeding. Lysosomal enzyme activities in fish sampled after 10 days refeeding were not significantly different from fed controls. Membrane fractions enriched in aryl sulfatase activity were prepared from the fast muscle of 66-day starved fish. These were capable of degrading both myosin heavy chains and actin to lower molecular weight peptides at acid (pH 5.0), but not at neutral pH. The results suggest a role for lysosomal enzymes in the breakdown of myofibrillar proteins during starvation.     

Protein synthesis, myosin ATPase activity and myofibrillar protein composition in hearts from tumour-bearing rats and mice.

Growing rats and adult weight-stable mice bearing a transplantable methylcholanthrene-induced sarcoma were compared with animals with various states of malnutrition. Heart protein synthesis was measured in vivo. Myocardial RNA, myofibrillar protein composition and the Ca2+-activated ATPase activity in heavy chains of native myosin were measured. 'Fingerprints' were made from myosin by trypsin treatment to evaluate possible structural changes in the protein. Cardiac protein-synthesis rate was decreased by 20% in growing tumour-bearing rats, by 35% in protein-malnourished (rats) and by 47% in starved rats, compared with freely fed controls (P less than 0.05). Adult tumour-bearing mice showed no significant decrease in myocardial protein synthesis. Pair-weighed control mice had significantly depressed heart protein synthesis. Protein translational efficiency was maintained in both tumour-bearing rats and mice, but was decreased in several groups of malnourished control animals. The Ca2+-activated myosin ATPase activity was decreased in all groups of malnourished animals, including tumour-bearing mice and rats, without any evidence of a change in cardiac isomyosin composition. We conclude that loss of cardiac muscle mass in tumour disease is communicated by both depressed synthesis and increased degradation largely owing to anorexia and host malnutrition. Increased adrenergic sensitivity in hearts from tumour-bearing and malnourished animals is not communicated by increased Ca2+-activated ATPase activity. This may be down-regulated in all groups with malnutrition, without any observable alterations in the isomyosin profile.     

Canine cardiac myosin with special referrence to pressure overload cardiac hypertrophy. I. Subunit composition.

In studies of myosin from left and right ventricles of normal hearts and hypertrophic hearts at 5 weeks and 13 weeks after aortic banding, polyacrylamide gel electrophoresis shows intermediate molecular weight components which derive from heavy chains fragmented in the presence of dodecyl sulfate. The proportion of degraded heavy chains is greater in myosin from hypertrophic hearts than normal hearts, with comparable degradation in left and right ventricle myosin. The observed fragmentation of myosin results from proteolysis due to contaminant proteases or a thermally activated, heat-stable nonenzymatic process, or both. The susceptibility of heavy chains to crude myofibrillar proteases differs in normal and hypertrophic cardiac myosin; however, the kinetics of tryptic digestion are identical for both myosins. With precautions to minimize proteolytic artifacts on dodecyl sulfate-polyacrylamide gel electrophoresis, preparations of myosin from left and right ventricles of normal and hypertrophic hearts exhibit comparable subunit composition, with approximately molar ratios of heavy chains, light chain L1, and light chain L2. Comparable stoichiometry for the light chain fraction is determined by high speed sedimentation equilibrium at pH 11 and direct fractionation of the different cardiac myosins. We do not confirm reports (e.g. Wikman-Coffelt, J., Fenner, C., Smith, A., and Mason, D. T. (1975) J. Biol. Chem. 250, 1257-1262) of different proportions of light chains in left and right ventricle myosin of normal and hypertrophic canine hearts. The light chains display microheterogeneity, with L1 generating two isoelectric variants and L2 generating two major and two minor variants, but identical mobilities and isoelectric values are obtained in the different myosin preparations.     

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.     

Degradation of myocardial structural proteins in myocardial infarcted dogs is reduced by Ep459, a cysteine proteinase inhibitor

The purpose of this study is to clarify whether cysteine proteinases play an important role in the degradation of myocardial proteins in the infarcted tissue. We studied the effects of a cysteine proteinase inhibitor, Ep459, on degradation of cardiac structural proteins caused by ischemia due to coronary artery ligation for 24 h. Proteolytic effects of purified cysteine proteinases on isolated cardiac tissue were also examined. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, degradation of cardiac structural proteins, particularly of myosin heavy chain, alpha-actinin and troponin-I was observed in the infarcted tissue. Treatment with Ep459 significantly reduced protein degradation and total activity of cathepsins B and L in the infarcted tissue, compared with the findings in the untreated group. The electrophoretic pattern of the infarcted myocardium was similar to that of myofibrillar proteins degraded by cathepsins B and L. These results suggest that cysteine proteinases, particularly cathepsins B and L, are involved in degradation of myofibrillar proteins in myocardial infarction.     

Superprecipitation of hybrid actomyosin containing pathologic actin from failing hearts of adults and infants

Superprecipitation (SP) of artificial actomyosin, obtained by hybridization of Straub actin from the human myocardium with myosin of normal animal hearts was studied. Actin was prepared from the myocardium of persons who died of congestive heart failure and various non-cardiac diseases, as well as of infants whose death resulted from toxic pneumonia complicated or not with heart failure. It was shown that, in the control hybrid actomyosin, the substitution of normal Straub actin by that from the failing heart resulted in decrease of both the rate and extent of SP. The conclusion was made that both changes in myosin properties and Straub actin underlie the reduced contractility of the myofibrillar protein system in acute and congestive heart failure.     

Mode of action of rabbit skeletal muscle cathepsin B towards myofibrillar proteins and the myofibrillar structure.

1. The mode of degradation of myofibrillar proteins and the structural changes in myofibrils due to the action of cathepsin B highly purified from rabbit skeletal muscle were studied. 2. Cathepsin B degraded myosin heavy chain, actin and troponin T, but not alpha-actinin, tropomyosin, troponin I or troponin C among myofibrillar proteins. 3. Cathepsin B optimally degraded myosin heavy chain, actin and troponin T at around pH 5. Degradation of myosin heavy chain produced 6 fragments, 180,000, 150,000, 87,000, 81,000, 75,000 and 69,000 Da, respectively. Actin was hydrolyzed into fragments of 41,000, 38,000 and 30,000 Da. Troponin T was degraded into fragments of 21,000, 12,000 and 10,000 Da. 4. Cathepsin B caused the fragmentation of myofibrils and disturbance of the lateral arrangement of myofibrils. 5. Cathepsin B partly disintegrated the Z-line and the M-line, and induced disordering of the arrangement of filaments in the I-band.     

Peroxynitrite inhibits myofibrillar protein function in an in vitro assay of motility

We determined the effects of peroxynitrite (ONOO-) on cardiac myosin, actin, and thin filaments in order to more clearly understand the impact of this reactive compound in ischemia/reperfusion injury and heart failure. Actin filaments, native thin filaments, and alpha-cardiac myosin from rat hearts were exposed to ONOO- in the presence of 2 mM bicarbonate. Filament velocities over myosin, calcium sensitivity, and relative force generated by myosin were assessed in an in vitro motility assay in the absence of reducing agents. ONOO- concentrations > or =10 microM significantly reduced the velocities of thin filaments or bare actin filaments over alpha-cardiac myosin when any of these proteins were exposed individually. These functional deficits were linearly related to the degree of tyrosine nitration, with myosin being the most sensitive. However, at 10 microM ONOO- the calcium sensitivity of thin filaments remained unchanged. Cotreatment of myosin and thin filaments, analogous to the in vivo situation, resulted in a significantly greater functional deficit. The load supported by myosin after ONOO- exposure was estimated using mixtures experiments to be increased threefold. These data suggest that nitration of myofibrillar proteins can contribute to cardiac contractile dysfunction in pathologic states in which ONOO- is liberated.     

Contents of myofibrillar proteins in cardiac, skeletal, and smooth muscles

The in situ contents of myosin, actin, alpha-actinin, tropomyosin, troponin, desmin were estimated in dog cardiac, rabbit skeletal, and chicken smooth muscles. Whole muscle tissues were dissolved with 8 M guanidine hydrochloride and subjected to two-dimensional gel electrophoresis, which is a nonequilibrium pH gradient electrophoresis (Murakami, U. & Uchida, K. (1984) J. Biochem. 95, 1577-1584) with some modification. The amount of protein in a spot on a slab gel was determined by quantification of the extracted dye. Dye binding capacity of individual myofibrillar proteins was determined by using the purified protein. Myosin contents were 82 +/- 7 pmol/mg wet weight in cardiac muscle, 105 +/- 10 pmol/mg wet weight in skeletal muscle, and 45 +/- 4 pmol/mg wet weight in smooth muscle. Actin contents were 339 +/- 15 pmol/mg wet weight in cardiac muscle, 625 +/- 27 pmol/mg wet weight in skeletal muscle, and 742 +/- 13 pmol/mg wet weight in smooth muscle. The subunit stoichiometry of myosin in the three types of muscles was two heavy chains and four light chains, and there was one light chain 2 for every heavy chain. The molar ratio of actin to tropomyosin was 7/1 in the three types of muscles. Striking differences were seen in the molar ratio of myosin to actin: 1.0/4.1 in cardiac muscle, 1.0/6.0 in skeletal muscle, and 1.0/16.5 in smooth muscle.     

Inhibition of contraction of cultured muscle fibers results in increased turnover of myofibrillar proteins but not of intermediate- filament proteins

Muscle fibers are maintained in culture in a fully contractile state and are relaxed by the addition of 10(-7) M tetrodotoxin (TTX). This toxin binds to muscle membrane Na+- channels, abolishes spontaneous contractions and causes failure of the fiber to accumulate myosin heavy chains. These effects are reversible on removal of TTX. Synthesis and accumulation kinetics have been obtained for myofibrillar and for cytoplasmic filament proteins in normal, active muscle and in TTX- relaxed muscle fibers in culture. In relaxed fibers the synthesis of most proteins remained normal or slightly elevated. However, the accumulation of all myofibrillar proteins examined was markedly inhibited in TTX-treated cultures, whereas the accumulation of cytoplasmic filament proteins was normal or slightly elevated. Myofibrillar proteins examined were alpha-actin, troponin-C, myosin fast light chain 1, myosin fast light chain 2, alpha, beta-tropomyosins and the phosphorylated forms of tropomyosin and fast light chain 2. Cytoplasmic filament proteins studied were vimentin, alpha, beta-desmin and beta, alpha-actin. We also examined the synthesis and accumulation of six unidentified muscle-specific proteins and nine unidentified nonmuscle-specific proteins. Most of these proteins showed a normal accumulation pattern in TTX-relaxed fibers. We concluded that muscle fibers made inactive by TTX display an increased instability of all myofibrillar proteins while cytoplasmic filament proteins and cytoplasmic proteins in general are relatively unaffected. We suggest that TTX interferes, in a manner as yet unidentified, with assembly and normal stability of myofibrils. Decreased assembly and/or increased instability of myofibrils would lead to increased rates of myofibrillar protein degradation.     

Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart

The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy.