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The myofibrils in an isolated muscle fiber remain straight during the early part of a lightly loaded contraction initiated by membrane depolarization, but, as shortening continues, myofibrils in the core of the fiber become wavy, which suggests that their activation has been interrupted by shortening of the fiber. This may be a factor determining the length-tension relation at short muscle lengths.
Incorporation of [(3)H]thymidine into nuclei of heart cells of 2-day-old rats indicates that neonatal cardiac cells containing well-aligned myofibrils synthesize DNA. In these highly differentiated cells, neither the presence of contractile proteins nor their organization into myofibrils inhibits either DNA synthesis or mitosis.
AIMS: To compare the ultrastructural aspects of human extraocular muscles in two types of mitochondrial disease: chronic progressive external ophthalmoplegia (CPEO) and Leber's hereditary optic neuropathy (LHON). METHODS: Muscle samples of the medial rectus obtained from surgery in a sporadic case of CPEO associated with deleted mitochondrial DNA, and post mortem in a case of 3460/ND1 LHON were processed for electron microscopy (EM). The medial rectus from an autoptic time to fixation matched control was used to exclude postmortem artefacts. RESULTS: The CPEO specimen revealed focal areas of disruption and abnormalities of mitochondria in some muscle fibres, creating a "mosaic-like" pattern. In the LHON specimen a diffuse increase in both number and size of mitochondria (mean diameter 0.85 mum v 0.65 mum of control, p<0.0001) with swollen appearance and disorganised cristae filled all spaces of sarcoplasmic reticulum. In some areas the excessive number of mitochondria slightly distorted myofibrils. CONCLUSION: EM investigation of extraocular muscles in CPEO and LHON reveals marked differences. A "mosaic-like" pattern caused by a selective damage of muscle fibres was evident in CPEO, whereas a diffuse increase in mitochondria with preservation of myofibrils characterised the LHON case. These ultrastructural changes may relate to the different expression of the two diseases, resulting in ophthalmoplegia in CPEO and normal eye movements in LHON.
Cardiac contractile activity is usually controlled by intracellular Ca2+, but it can also be modified by oxidizing agents. Incubation of guinea pig heart myofibrils with diamide (3 mM, 1 h) increased basal (no Ca2+) ATPase activity by 580% and abolished Ca2+ dependence. The effect was proportional to diamide concentration (0.01-1 mM) and duration of preincubation (up to 2 h). Dithiothreitol (5 mM, 1 h) reversed most of the basal ATPase activation and restored Ca2+ sensitivity. Other sulfhydryl reagents produced a similar effect but also produced inhibition of total ATPase. In intact cell preparations, diamide produced a slow tonic contraction, consistent with myofibril activation. In the perfused rat heart, 1 mM diamide slowly increased diastolic ventricular pressure; this increase was partially reversed by dithioerythritol. In isolated rat heart myocytes, 1 mM diamide produced a slow tonic contraction, increased contractility in response to stimulation. Cardiocytes superfused for 1 h with buffer containing EGTA to deplete Ca2+ did not contract in response to stimulation but showed a slow tonic contraction with diamide. This contraction could be slowly and only partially reversed by dithioerythritol. Response to stimulation was restored by addition of Ca2+. The results show that diamide can produce contraction in viable cells. This contraction does not require extracellular Ca2+ and is unlikely to involve intracellular Ca2+. The direct activation of myofibrillar ATPase may contribute to the increased myocardial stiffness seen in ischemia and to ischemic contracture.
The effects of thyroid deficiency (Td) and of chemical sympathectomy (Sx) were studied on marker enzymes of energy metabolism in cardiac muscle of neonatal and of adult rats. Td prevented the normal development of neonatal body weight, relative heart mass, and cardiac levels of cytochrome c (-22%), citrate synthase (-27%), phosphofructokinase (-20%) and Mg2+- and Ca2+-ATPase activity of purified myofibrils (-33%, -44%). Exogenous thyroxin replacement restored those parameters studied to normal with the exception that it persistently elevated citrate synthase activity significantly above normal control levels. Responses similar to those of Td neonates occurred when adult rats were similarly treated. Sx produced no consistent effects on respiratory and glycogenolytic marker enzymes, but caused a 20% reduction in Ca2+-ATPase activity of both neonatal and adult cardiac myofibrils. These findings suggest that cardiac muscle cells require thyroxin for normal growth and enzyme development. Also, Sx may impair cardiac functional capacity by altering Ca2+ activity of actomyosin ATPase.
A previous study has shown that chronic chemical sympathectomy brought about by 6-hydroxydopamine injections results in a dpression in myocardial contractile function which is accompanied by reduced myofibrillar ATPase activity. To determine whether chronic beta-adrenergic receptor blockade elicits similar alterations in cardiac contractile-protein ATPase activity, adult rats were given twice-daily injections of propranolol 7 days/wk for 2 wk. Effective beta-adrenergic receptor blockade was verified by the lack of hemodynamic responsiveness to isoproterenol infusion. Myofibrils were prepared from left ventricular tissue and analyzed for ATPase activity. Myofibrillar ATPase activity was 295 +/- 8 nmol Pi.mg-1.min-1 in controls. Enzyme activity was not significantly different in propranolol-injected rats. The results demonstrate that chronic propranolol administration does not alter the ATPase activity of cardiac myofibrils. Therefore, it seems likely that the altered contractile-protein enzymatic properties resulting from chronic chemical sympathectomy do not occur as the result of a reducted level of cardiac beta-adrenergic receptor stimulation.
To change the levels of expression and isoenzyme distribution of creatine kinase (CK) in muscle, transgenic technology was used to express the B subunit of CK in mouse muscle. Normally, mammalian skeletal muscle contains the MM dimer of CK. The BB dimer and MB heterodimer of CK can be found in brain and heart, respectively. Heterologous genes consisting of skeletal and cardiac muscle-specific actin promoters fused to the genomic coding region of the B form of CK were used to create transgenic mice. Lines were established from the three highest expressing founders. Analysis of skeletal muscle extracts revealed that all three lines had an increase in total CK activity measured under maximal velocity conditions. The highest expressing line, 7001, had a CK activity 150% that of control muscle. Nuclear magnetic resonance saturation transfer was used to measure the in vivo rate of the CK reaction. In 7001 hindlimb muscles, the CK catalyzed reaction was 200% that of control muscle. The elevation in CK activity in transgenic muscle was accompanied by significant changes in the composition of the cytosolic isoenzyme ratio of CK. In control, 100% of CK was MM, whereas 7001 had 60 +/- 18% MM, 32 +/- 10% MB, and 8 +/- 2% BB. There were no changes in ATP, phosphocreatine, Pi, or creatine levels in transgenic muscle compared with control. Immunofluorescence of myofibrils isolated from control and transgenic muscle revealed specific association of CK to the M line. Small amounts of MB CK were detected on myofibrils from transgenic mice. Transgenic mice expressing the B subunit of CK in muscle represent a first step toward altering CK isoforms so as to elucidate the specific roles of these isoforms in energy metabolism.
Myofibrillar creatine kinase (CK) serves as one microcompartment of the phosphorylcreatine shuttle by providing ATP as substrate for adenosinetriphosphatase (ATPase). During perinatal heart development, augmentations of myofibrillar ATPase and CK occur in concert with increased contractile performance. The maximal reaction velocity (Vmax) for CK doubles during development in both intact native myofibril and enzyme extracted from myofibril. The absence of alterations in ADP and creatine phosphate substrate Michaelis constants (Km), isoenzyme composition, or total number of -SH groups suggests active site function (Vmax) is influenced indirectly via a subunit domain effect on enzyme conformation.
The kinetic properties of the myofibrillar system of atrial and ventricular myocardia of hyperthyroid rats were analyzed by determining ATPase activity and maximum shortening velocity. Hyperthyroidism was induced by daily subcutaneous injections of triiodothyronine (0.2 mg/kg body wt) for 2 wk. The treatment induced a marked atrial and ventricular hypertrophy and, in ventricular myocardium, an isomyosin shift toward a homogeneous V1 composition. Skinned trabeculae and purified myofibrils were prepared from atrial and ventricular myocardia. Enzymatic assays on the myofibrils showed that both Ca-stimulated ATPase activity and Ca-Mg-dependent ATPase activity had equal values in atrial and ventricular myocardia. In skinned trabeculae during maximal Ca activations, force-velocity curves were determined by load-clamp maneuvers, and unloaded shortening velocity (Vo) was obtained with the slack-test method. Both maximum shortening velocities extrapolated from the force-velocity curves (Vmax) and Vo were significantly higher (+68 and +52%, respectively) in atrial than in ventricular preparations. Developed tension was significantly greater in ventricular preparations. Maximum power output was not significantly different. Previous findings (V. Cappelli, R. Bottinelli, C. Poggesi, R. Moggio, and C. Reggiani. Circ. Res. 65: 446-457, 1989) had led to the conclusion that variations in ATPase activity and shortening velocity of ventricular myocardium can be accounted for by changes in isomyosin composition. In this light, the present results suggest that 1) ATPase activity is equal in atrial and ventricular myocardia as the two tissues contain the same myosin heavy chain isoform, 2) the difference in maximum speed of shortening between atrium and ventricle might be due to the presence of tissue-specific isoforms of myosin light chains.
The ATPase activity of myofibrils and myosin from hindlimb muscle was investigated in animals 4 wk after the induction of diabetes by an intravenous injection of streptozotocin (65 mg/kg). Ca2+-stimulated ATPase in myofibrils was increased in diabetic muscle at various times of incubation (1-7 min) as well as at different concentrations of free Ca2+ (10(-7)-10(-5) M Ca2+). Such an increase in Ca2+-stimulated ATPase was evident as early as 1 wk after streptozotocin injection, but Mg2+-ATPase activity remained unaltered. Treatment of diabetic animals with insulin Ca2+-ATPase and actin-activated ATPase activities of pure myosin were similarly increased in diabetic muscle. Myosin ATPase was also activated by K+- or NH4+-EDTA; these responses were more in diabetic muscle. However, sodium dodecyl sulfate gel electrophoresis failed to reveal differences in the patterns of contractile proteins, and pyrophosphate gels did not show significant changes in myosin isozyme patterns between diabetics and controls. The results of this study demonstrate an activation of contractile protein ATPase of skeletal muscle in diabetes and seem to indicate that such an alteration may be responsible for enhanced contractile function of skeletal muscle in this disease.
Myocardial stunning is a form of acute reversible cardiac dysfunction that occurs after brief periods of ischemia and reperfusion. In several animal models, stunning is associated with proteolytic truncation of troponin I (TnI). Mice expressing the same proteolytic TnI fragment [TnI-(1-193)] demonstrate cardiac depression with a decreased maximal calcium-activated tension. We therefore hypothesized preferential improvement in mice expressing TnI-(1-193) treated with the calcium-sensitizing drug EMD-57033. TnI-(1-193) and nontransgenic myofibrils exhibited significant sensitization to calcium in Mg-ATPase assays after EMD-57033 exposure. However, only transgenic myofibrils exhibited an increase in maximal activity (P = 0.023). EMD-57033 also increased maximal calcium-activated force in TnI-(1-193) muscle, such that it was comparable to nontransgenic cardiac muscle. EMD-57033 enhanced in vivo systolic function modestly in controls but had a marked effect in transgenic mice, with an almost threefold greater leftward shift of the end-systolic pressure-volume relation (P = 0.0005). These data indicate a targeted efficacy of EMD-57033 in offsetting the contractile defect in TnI-(1-193) mice, and this may have therapeutic implications in models displaying this myofilament defect.
We measured the functional activity of both myofibrils and fragmented sarcoplasmic reticulum (FSR) in microsomal fractions and homogenates of hearts of sedentary rats and rats exercised by swimming 75 min twice daily for 8 wk. Ca2+ transport was measured under conditions that restricted uptake of Ca2+ to the SR vesicles in the homogenates or microsomal fractions. There was a significant increase in myosin Ca2+-ATPase activity of myofibrils prepared from hearts of swimmers, indicating that a "training effect" had occurred. The mean rate of Ca2+ transport and mean storage capacity were the same for SR vesicles in the homogenates and microsomal fractions from hearts of controls and swimmers. At the same free Ca2+ concentration, the velocity of Ca2+ transport by FSR in homogenate preparations was inversely related to the myofibrillar ATPase activity in a series of preparations from hearts of swimmers, but there was little correlation between the same activities measured in preparations from a series of sedentary rats. Our results suggest that the increase in the rate of relaxation of hearts from exercised rats is not due to an increase in the rate of Ca2+ transport by the SR but may be due to other factors, which include an increase in the rate of cross-bridge cycling or an alteration in the relation and coordination between cross-bridge cycling and SR Ca2+ transport activity.
Electron probe microanalysis (EPMA) was carried out directly on myocardial cells and on the myofibrils and the mitochondria within them. A third subcellular compartment, which contains sarcoplasmic reticulum (SR), was measured indirectly. The percent of the total cell calcium content that resides within this "hidden" compartment was calculated from cell data minus weighted myofibril and mitochondria data. This approach was applied to control, ischemic, and reperfused myocardium, and other elements were also quantified. We found that the calcium content of this third compartment is little changed during global ischemia but is markedly depleted after 5 min reperfusion. We conclude that these changes are ascribable to changes in SR function.
Reperfusion of rabbit hearts after 15 min of global ischemia at 37 degrees C depressed developed pressure by 36% (myocardial stunning). Changes in myofilament function were investigated as causes of this depression. Kinetic analysis of the effects of stunning on myofibrillar catalyzed ATP hydrolysis showed that stunning lowered Michaelis constant (Km) slightly and left maximal enzyme reaction velocity unaltered in the stunned myofilaments. The myofilament end of the creatine kinase (CK) shuttle was also found to be unaffected in the stunned myofibrils. The Km ADP for myofibrillar CK from control and stunned hearts was 60.45 +/- 3.45 and 68.04 +/- 2.42 microM, respectively, and the CK activity at 100 microM ADP was 0.63 +/- 0.08 and 0.67 +/- 0.04 IU/mg myofibrillar protein from control and stunned hearts, a rate three times greater than the myofibrillar adenosinetriphosphatase (ATPase) rate and a rate sufficient to deliver ATP to the myofilaments. Myofilament Ca2+ sensitivity was assessed by measuring Ca2(+)-dependent myofibrillar Mg2(+)-ATPase activity at free [Ca2+] ranging from 10 nM to 32 microM and [Mg.ATP] of 0.8, 1.6, and 3.2 mM. The sensitivity of myofilaments to activation by Ca2+ was unaltered in the myofibrils isolated from stunned hearts. It is concluded from these analyses that the depression of pressure development observed in stunned hearts is not due to a defect in myofilament function.
We examined effects of temperature acclimation on ultrastructural characteristics of cardiac myocytes and maximal activities of metabolic enzymes in cardiac tissue of striped bass (Morone saxatilis). Ventricular mass and ventricular mass divided by body weight were significantly increased (29% and 40%, respectively) in animals acclimated to cold (5 degrees C) vs. warm temperatures (25 degrees C). Mean myocyte diameter was increased at cold temperature (3.47 +/- 0.14 vs. 2.98 +/- 0.08 microns), which is sufficient to explain the increase in ventricular mass. Ventricular enlargement did not alter volume densities of mitochondria, myofibrils, protein concentration, or citrate synthase activity. Thus total volume of mitochondria and myofibrils increased proportionately with cardiac mass in cold animals. Activities of hexokinase (34%) and carnitine palmitoyltransferase (42%) increased in cold animals, suggesting positive compensation and increased aerobic capacity for utilization of glucose and fatty acids for energy production. Enlargement of the ventricle and an increased capacity for ATP production in striped bass may help compensate for kinetic constraints at cold temperatures and maintain circulatory support to oxidative axial musculature for swimming activity.
Previous studies suggest that fast-twitch skeletal muscle overloaded by surgical removal of synergists contains a greater percent of slow-twitch fibers than normal muscle. Therefore we examined subcellular systems known to represent biochemical properties of slow-twitch skeletal muscle by measuring myosin ATPase, Ca2+ regulation of myofibril ATPase, Ca2+ uptake of sarcoplasmic reticulum (SR), and marker enzymes of glycogenolysis in normal soleus (NS) and in normal (NP) and surgically overloaded (OP) plantaris muscles of adult female rats. The OP muscles were 65% larger than NP muscles (P less than 0.001). Specific activity of myosin and myofibril ATPase was approximately 25% lower in OP compared with NP muscle (P less than 0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin revealed the presence of more slow and less fast myosin light-chain components in OP muscles. Although SR of NP muscle took up more Ca2+ than OP muscle during the initial for both groups. Marker regulatory enzymes of glycogenolysis collectively were reduced by 40% in OP compared with NP muscle (P less than 0.001). Collectively the data are consistent with the concept that some muscle fiber types were converted from "fast" to "slow" in the OP muscle.
The purpose of this study was to ascertain the time course of change during both compensatory growth (hypertrophy) and subsequent growth regression on myosin isoform expression in rodent fast-twitch plantaris muscle in response to functional overload (induced by removal of synergists). Peak hypertrophy of the plantaris muscle (92%) occurred after 9 wk of overload. After 7 wk of overload regression (induced by a model of hindlimb unweighting), muscle weight returned to within 30% of control values. Myofibril protein content (mg/g muscle) remained relatively constant throughout the overload period but became significantly depressed relative to control values after 7 wk of regression. However, when expressed on a per muscle basis (mg/muscle) no differences existed at this time point (t = 7 wk regression). The distribution of native myosin isoforms in the myofibril protein pool of the overloaded plantaris muscle reflected a progressive increase (23% at t = 9 wk; P less than 0.001) in the relative proportion of slow myosin (Sm). This change was also accompanied by increases in intermediate myosin (Im) as well as the repression of the fast myosin one (Fm1) isoform (P less than 0.001). These shifts in Sm and Fm1 isoform expression were gradually reversed during the regression period, whereas Im remained elevated relative to control values. These adaptive changes in myosin isoform expression during both hypertrophy and regression were further supported by concomitant shifts in both myosin adenosinetriphosphatase (ATPase) activity (decreased during overload) and slow myosin light chain (SLC) expression. However, during regression the changes in myosin isoform expression and myosin ATPase were not as synchronous as they were during overload. Estimation of the mixed myosin heavy chain (MHC) half-life (t 1/2), using a linear model that assumes zero-order synthesis and first-order degradation kinetics, revealed t 1/2 values of approximately 19 and 10 days for the overload and regression periods, respectively. Collectively these data suggest that 1) skeletal muscle myosin isoforms and corresponding ATPase activity are in a dynamic state of change, although not completely synchronous, in response to altered muscle stress, and 2) the kinetics of change in the mixed MHC protein pool are slower during compensatory growth compared with regression of growth.
The efficacy of anabolic steroid treatment [0.3 or 0.9 mg nandrolone decanoate (Deca-Durabolin) per day] was examined in the context of sparing rodent fast-twitch plantaris and slow-twitch soleus muscle weight, sparing subcellular protein, and altering isomyosin expression in response to hindlimb suspension. Female rats were assigned to four groups (7 rats/group for 6 wk): 1) normal control (NC), 2) normal steroid (NS), 3) normal suspension (N-SUS), and 4) suspension steroid (SUS-S). Compared with control values for the plantaris and soleus muscles, suspension induced 1) smaller body and muscle weight (P less than 0.05), 2) losses in myofibril content (mg/muscle, P less than 0.05), and 3) shifts in the relative expression (expressed as %of total isomyosin) of isomyosins which favored lesser slow myosin and greater fast myosin isotypes (P less than 0.05). Steroid treatment of suspended animals (SUS-S vs. N-SUS) partially spared body and muscle weight (P less than 0.05) and spared plantaris but not soleus myofibril content (mg/muscle, P less than 0.05). However, steroid treatment did not modify the isomyosin pattern induced by suspension. In normal rats (NS vs. NC), steroid treatment enhanced body and plantaris muscle weight but not soleus weight (P less than 0.05) and did not alter isomyosin expression in either muscle type. Collectively these data suggest that in young female rats anabolic steroids 1) enhance the body weight and the weight of a fast-twitch ankle extensor in normal rats, 2) ameliorate the loss in body weight, fast-twitch muscle weight and protein content and slow-twitch muscle weight associated with hindlimb suspension.(ABSTRACT TRUNCATED AT 250 WORDS)