Myosin Isozymes Research Articles

The positive inotropic effect of epinephrine on skeletal muscle: A brief review

Chronic and acute administration of epinephrine or related sympathomimetic agents are typically prescribed for the treatment of clinical disorders such as hypotension, anaphylactic and allergic reactions, and bronchial asthma. In addition to its effects on these infirmaties and on carbohydrate metabolism, epinephrine also exerts a positive inotropic effect on fast-contracting skeletal muscle in a variety of animal species. At present, the precise mechanisms responsible for the inotropic effect are not known. This communication reviews the positive inotropic effects of epinephrine on fast-contracting skeletal muscle and discusses possible mechanisms which might mediate this phenomenon. Epinephrine potentiates muscle twitches via the second messenger, cAMP, secondary to hormone binding to membrane-bound beta-receptors. Cyclic AMP then acts to increase carbohydrate metabolism, alter sodium/potassium exchange, phosphorylate myosin isozymes, and/or alter intracellular calcium exchange. Based on theoretical grounds, the first three mechanisms can be excluded. Therefore, it is tentatively hypothesized that the effect is due to cAMP-enhanced calcium exchange within the muscle fiber and/or to increased influx of extracellular calcium. This notion is consistent with the mechanism of the positive inotropic effects of epinephrine on cardiac tissue. If this hypothesis is correct, it would also suggest a role, at least under some conditions, for extracellular calcium in the process of skeletal muscle excitation-contraction coupling.

Skeletal Muscle Fiber Types and Myosin ATPase Activity Do Not Change With Age or Growth Hormone Administration

Fischer 344 rats at various ages throughout the life span have been treated with growth hormone, clonidine, and insulin-like growth factor-I to restore circulating somatomedin levels in old animals to levels found in younger rats. The injections were intended to reverse (2-week treatments) or prevent (6-month treatments) deleterious effects of aging on skeletal muscle--specifically the loss of fast fibers during the latter part of the life span. However, measurements of myosin ATPase (and subsequent histochemical fiber type determinations) revealed that the previously reported age-related decrease in ATPase activities and fast fiber content did not occur in barrier-protected specific pathogen free rats or mice. None of the treatments used had a significant effect on the ATPase activity or fiber composition of soleus, extensor digitorum longus, or diaphragm muscles, although parallel determinations in collaborating laboratories verified that the hormones had major effects in other systems. Previously reported decreases with age in ATPase activity of heart muscle were confirmed in our experiments, and these decreases were shown to reflect a change in myosin isozyme composition of the hearts, both at the protein and the mRNA levels of gene expression.

Minireview: Limitations Of Interpretation Of Age-Related Changes In Hormone Levels: Illustration By Effects Of Thyroid Hormones On Cardiac And Skeletal Muscle

Studies on age-related changes in hormone secretion or circulation are frequently interpreted to indicate changes in the functions of target tissues for that hormone. Such interpretations are not necessarily correct. For instance, the levels of thyroid hormone change modestly with age, and responses to this change differ dramatically in cardiac and skeletal muscles. There is a substantial change in myosin isozyme content of the heart, but little or no change in skeletal muscle fiber type distribution in specific pathogen free (SPF) barrier-protected rats. These changes (or lack of them) are reflected in functional differences (or lack of them) in the target tissues. The most obvious difference between the recent studies showing no differences with age in skeletal muscle fiber distribution and previous reports of general decreases with age in content of fast fibers is in animal housing conditions; all of the animals showing an age-related change had been housed in conventional colonies, while those exhibiting little or no change were in SPF barrier-protected facilities.

The Involvement of the Endocrine System in Regulating Cardiovascular Function: Emphasis on Vitamin D3*

Introduction DURING the last decade studies in a number of laboratories have provided evidence to suggest that the endocrine system plays an important role in regulating cardiovascular function. These studies have shown that the hormones insulin and thyroid hormone exert significant effects on key elements involved in regulating the activity of the cardiovascular system, including the distribution of myosin isozymes, the calcium pumping activity of the sarcoplasmic reticulum, and coronary flow. Recently, investigations have shown that the hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which is the active metabolite of vitamin D3, can also regulate cardiovascular function. The regulatory effects of 1,25(OH)2D3 include enhancement of cardiac and vascular muscle contractile function, modulation of blood pressure, and changes in myocardial collagen. The purpose of this review is to summarize current knowledge regarding the involvement of the endocrine system in regulating cardiovascular function, with empha...

Evidence for the presence of a distinct embryonic isoform of myosin heavy chain in chicken skeletat muscle

Abstract Immunochemical studies have identified a distinct myosin heavy chain (MHC) in the chicken embryonic skeletal muscle that was undetectable in this muscle in the posthatch period by both immunocytochemical and the immunoblotting procedures. This embryonic isoform, identified by antibody 96J, which also recognises the cardiac and SM1 myhosin heavy chains, differs from the embryonic myosin heavy chain belonging to the fast class described previously. Although the fast embryonic isoform is a major species present in the leg and pectoral embryonic muscles, slow embryonic isoform was present in significant amounts during early embryonic development. Immunocytochemical studies using another monoclonal antibody designated 9812, which is specific for SM1 MHC, showed this isoform to be restricted to only presumptive slow muscle cells. From these studies and those reported on the changes in SM2 MHC, it is proposed that as is the case for the fast class, there also exists a slow class of myosin heavy chains composed of slow embryonic, SM1 and SM2 isoforms. The differentiation of a muscle cell involoves transitions in series of myosin isozymes in both presumptive fast and slow skeletal muscle cells.

Intramuscular comparison of myosin isozymes and light chains in rat extensor digitorum longus muscle

Complete muscle cross sections were obtained from the proximal and distal third regions of ten rat extensor digitorum longus muscles. Electrophoretic methods were then used to quantify the various myosin isozymes and light chains in each muscle specimen. The results demonstrated that the relative distribution of the various myosin isozyme and light chain variables do not vary significantly between the two sampling regions.

Alteration of cardiac myosin isozymes after whole-body irradiation

Three myosin isozymes (Vl, V2, V3) have been shown to exist in the heart. V1 isozyme, containing two α heavy chains, has relatively higher actin- and calcium-stimulated myosin adenosine triphophatase (ATPase) activities than does V3, which is composed of two β heavy chains (Hoh et al, 1978; Martin et al, 1982; Litten et al, 1983). V2 isozyme appears to be a heterodimer composed of one α and one β heavy chain (Hoh et al, 1979; Everett et al, 1983a). The proportions of myosin isozymes are altered under various conditions of stress. During pressure-overload-induced hypertrophy, there is a shift toward the V3 isozyme (Mercadier et al, 1981; Litten et al, 1982). The amount of V3 isozyme is also increased with hypothyroidism and age and is decreased by hyperthyroidism and exercise (Litten et al, 1982; Everett et al, 1983b). Changes in the relative amounts of V1 and V3 isozymes are believed to be responsible, in part, for altered cardiac performance. A high proportion of V3 isozyme is associated with relatively slow, economic tension development within the heart, whereas a high proportion of V1 isozyme is associated with a faster, but less economical force development (Schwartz etal, 1981; Alpert et al, 1983). In this study, measurements of cardiac myosin isozymes were carried out in rats subjected to acute sublethal wholebody irradiation. The studies showed, for the first time, that there is a shift in the pattern of myosin isozymes 9 days after irradiation. This change may result in functional alterations of the heart following whole-body irradiation.

Fibre types inLimulus telson muscles: morphology and histochemistry

Using a variety of techniques, we have demonstrated the presence of at least two fibre types in Limulus median telson levator muscle. By light and electron microscopy, large (2,156 microns 2 mean cross-sectional area) fibres have A-bands of 4.1 microns, one-half I bands of 2.15 microns and Z lines less than or equal to 0.5 microns in width. Few mitochondria are found in these fibres, which comprise 54% of those present in a given microscope field and which occupy 82% of the total cross-sectional area. Small fibres (484 microns 2 mean cross-sectional area) have A bands of 6.3 microns, one-half I bands of 3.1 microns and Z lines between 0.5 and 1.0 microns in width and are rich in mitochondria. Although small fibres comprise nearly one-half (46%) of the fibres in a field, they occupy only 18% of the total cross-sectional area. Histochemical staining for alkaline-stable myofibrillar ATPase activity and mitochondrial reduced beta-nicotinamide adenine nucleotide (beta-NADH) tetrazolium reductase activity confirms the presence of two fibre types. The large fibres react positively for the myofibrillar ATPase activity and negatively for the mitochondrial enzyme activity. The reverse is seen with the small fibres. Some fibres of intermediate size, having intermediate staining characteristics, were also observed. Native gel electrophoresis of both myofibrillar and purified myosin preparations supports the observed differences in myofibrillar ATPase activity in that two myosin isozymes are resolved on pyrophosphate gels. Although the thick filaments isolated from unstimulated small fibres are longer (greater than 6.0 microns) than those isolated from unstimulated large fibres (4.26 microns), all have a similar appearance with respect to the arrangement of myosin heads on their surfaces, and similar diameters. The implications of the observed heterogeneity of fibre types is discussed with reference to previously reported phenomena in Limulus telson muscle, including changes in length of thick filaments on fibre stimulation and the shape of the length-tension curve obtained from fibre bundles.

Cardiac adaptations to endurance training in rats with a chronic myocardial infarction

The hemodynamic response to maximal exercise was determined in sedentary and trained rats with a chronic myocardial infarction (MI) produced by coronary artery ligation and in rats that underwent sham operations (SHAM). Infarct size in the MI groups of rats comprised 28-29% of the total left ventricle and resulted in both metabolic and hemodynamic changes that suggested that these animals had moderate compensated heart failure. The training regimen used in the present study produced significant increases in maximal O2 uptake (VO2max) when expressed in absolute terms (ml/min) or when normalized for body weight (ml.min-1.kg-1) and consisted of treadmill running at work loads that were equivalent to 70-80% of the animal's VO2max for a period of 60 min/day, 5 days/wk over an 8- to 10-wk interval. This training paradigm produced two major cardiocirculatory adaptations in the MI rat that had not been elicited previously when using a training paradigm of a lower intensity. First, the decrement in the maximal heart rate response to exercise (known as "chronotropic incompetence") found in the sedentary MI rat was completely reversed by endurance training. Second, the downregulation of cardiac myosin isozyme composition from the fast ATPase V1 isoform toward the slower ATPase (V2 and V3) isoforms in the MI rat was partially reversed by endurance training. These cardiac adaptations occurred without a significant increase in left ventricular pump function as an increase in maximal cardiac output (Qmax) and maximal stroke volume (SVmax) did not occur in the trained MI rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiological and pharmacological modulation of cardiac contractile proteins

AbstractThe contractile‐relaxation cycle of cardiac muscle is dependent upon the coordination of a variety of membrane and cytosolic events. The functional activities of the structures responsible for these events can be altered by endogenous modulators, on both a beat‐to‐beat or a long‐term basis, in order to optimize cardiac performance and meet the circulatory and nutritional needs of the body. This review focuses on the force‐generating structures, or myofibrils, of cardiac muscle, its activation by intracellular calcium, and the ways that activation of the myofibrillar proteins can be modulated by physiological and pharmacological mechanisms. Activation of the myofibrillar proteins is regulated by the troponin‐tropomyosin system of the actin (thin) filament; when troponin C binds calcium, sites along the thin filament are made available for myosin (thick) filament interaction. There are a number of physiological mechanisms that influence the affinity of troponin C for calcium, three of which include troponin I phosphorylation, intracellular pH, and muscle length. The relative affinity of troponin C for calcium decreases in parallel with a reduction in muscle length and intracellular pH and in parallel with an increase in troponin I phosphorylation. The type of myosin (isozyme) present in cardiac tissue also regulates myofibrillar activity. Ventricular muscle can contain up to three isozymes of myosin (V1, V2, and V3). Myofibrils containing only the V1 myosin isozyme can have an ATPase activity two to threefold hight than myofibrils containing only the V3 myosin isozyme. The type of myosin isozyme present in cardiac tissue is determined by a variety of factors; thyrotoxicosis and strenuous exercise tend to increase the relative amount of the V1 isozyme, whereas insulin and thyroxine insufficiency as well as aortic and pulmonary stenosis tend to decrease the relative amount of the V1 isozyme. Besides these physiologicall mechanisms, there are a variety of pharmacological agents that have been shown to alter the calcium activation (sensitize or desensitize) and/or maximal activity (potentiate or attenuate) of the cardiac contractile proteins. Some cardiotonic agents which increase the calcium sensitivity of the contractile proteins are AR‐L 115BS (Sulmazole), AR‐L 57, pimobendan, APP 201‐533, DPI 201‐106, and MCI‐154. However, some of these compounds are not selective and have ancillary activities, such as inhibiting cyclic nucleotide phosphodiesterase, so a single direct link to cardiotonic activity cannot be made. In addition to this class of agents, some calmodulin antagonists also can modulate cardiac contractile protein activity via modulation of calcium binding to troponin C. Some of the calcium‐binding protein modulators, such as calmidazolium, trifluoperazine, perhexiline, and bepridil are several times more potent in increasing myofibrillar ATPase activity than either AR‐L 115BS or APP 201‐533. The effect of these agents on altering calcium activation of the contractile proteins is also dependent upon the species and cardiovascular condition of the animal from which myofibrils are obtained. For example, calmidazolium increased basal and decreased maximal myofibrillar ATPase activity in both normal and myopathic hamster hearts but only increased the calcium sensitivity of myofibrils prepared from normal canine hearts. Studies are ongoing to evaluate the effect of these types of agents on ATPase activity of myofibrils from normal human hearts and hearts of patients with end‐stage failure. The relative advantages/disadvantages of using these types of agents to alter cardiac performance await future synthesis and identification of nanomolar potent compounds that selectively and specificaally alter the activity of cardiac contractile proteins.

Changes in Myosin Isozyme Expression during Cardiac Hypertrophy in Hyperthyroid Rabbits

The myosin isozyme distribution in the left ventricle and in the interventricular septum of rabbits was studied after 3, 7, 11, 14 and 21 days of L-thyroxine (500 micrograms/kg/day) administration. Histochemical procedures were employed to identify V1 and V3 by their Ca2+ ATPase activity and their proportions were quantified through polyacrylamide gel electrophoresis. In the left ventricle, the subepicardium was the first to show the shift from V3 to V1, followed by the subendocardium. The intermediate region became heterogeneous by 11 days and remained so until 21 days. The right subendocardial and the intermediate regions of the interventricular septum were heterogeneous in the normal rabbit and hyperthyroidism resulted in a shift from V3 to V1 in both the right and left subendocardial regions of the septum. Like the left ventricle, the intermediate region of the interventricular septum remained heterogeneous. Localized accumulations of collagen were seen in all regions of the left ventricle and interventricular septum. From these results we conclude that in thyrotoxic myocardial hypertrophy the isozymic shift from V3 to V1 is progressive, region-specific and is directly correlated with the period of hyperthyroidism in the first 2 weeks. Prolonged hyperthyroidism results in localized accumulation of collagen which does not exhibit any regional specificity.

Basis for myocardial mechanical defects associated with non-insulin-dependent diabetes

Hearts isolated from 12-mo non-insulin-dependent diabetic rats exhibited reduced rates of contractility and relaxation. Associated with the abnormality in contractility was a redistribution in myosin isozyme content to the least active V3 form. Defects in myocardial relaxation also occurred concomitantly with impaired handling of calcium. Total tissue calcium content rose 35% in the diabetic hearts. At the same time, the activity of the pump responsible for maintaining normal cytoplasmic calcium levels was reduced. At a free calcium concentration of 2.0 microM, the rates of sarcoplasmic reticular calcium uptake and adenosinetriphosphatase activity of the diabetic hearts were decreased approximately 30%. Diastolic ventricular stiffness increased dramatically. The net result of these abnormalities in calcium metabolism is a significant impairment in mechanical performance of the diabetic heart.

Effect of chronic sulfonylurea treatment on the myocardium of insulin-dependent diabetic rats

Adult rats treated with high doses of streptozocin became progressively more hyperglycemic during the first month of the diabetic condition. Treatment of these rats with the sulfonylurea glyburide halted, and in some cases, reversed this process in a high percentage of the diabetics. Associated with the glyburide-mediated improvement in fasting blood glucose levels was an increase in myocardial glucose utilization and lactate production. The stimulation of myocardial glucose utilization by insulin was greater in glyburide-treated hearts, indicating that the hyperglycemic agent increased insulin responsiveness. The sulfonylurea also partially restored insulin sensitivity to the normal range. In agreement with previous studies, myocardial mechanical function was significantly impaired in the diabetic heart. When treated with glyburide, the severity of the mechanical defect was significantly less. The sulfonylurea also promoted an increase in myosin ATPase activity and a shift in the myosin isozyme pattern in favour of the most active V1 form. These results imply that glyburide therapy can provide benefit to the diabetic heart by improving energy metabolism and promoting a shift in myosin towards the most active form.

Separation of cardiac myosin heavy chains by gradient SDS-PAGE.

Separation of alpha- and beta-myosin heavy chains (MHCs) in cardiac ventricles of rats by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was accomplished and compared with the separation of myosin isozymes obtained with pyrophosphate gels. Whole muscle homogenates were electrophoresed on a 4-9% linear gradient SDS polyacrylamide gel for 3-4 h. MHC bands were identified by the migration distance relative to a MHC standard and immunoblot results with a monoclonal antibody to MHC. The MHC bands were further identified as alpha and beta based on the electrophoretic mobility of ventricular homogenates from hypothyroid and hyperthyroid rats and ventricular and slow soleus skeletal muscle homogenates from control rats. The beta-MHC migrated faster than alpha-MHC, and laser densitometry revealed separate peaks when both MHCs were present. With homogenates containing MHC ranging from 0 to 100% alpha, the separation of MHCs with gradient SDS-PAGE correlated highly (r = 0.97) with separation of myosin isozymes by pyrophosphate gel electrophoresis. The SDS-PAGE technique reported herein is a quick, valid, and direct method for the identification and quantification of ventricular MHCs.

The effects of triiodothyronine on cultured neonatal rat cardiac myocytes

The purpose of this study was to determine whether thyroid hormone could directly affect the phenotypic expression of two isozymic systems [lactate dehydrogenase (LDH) and myosin] and the energy transducing potential of cultured neonatal heart cells. In addition we determined if these biochemical systems developed in culture as they normally do during in vivo post-natal development. Cells were maintained for 14 days in culture medium containing 10% horse serum and Earle's salts. Experimental cultures were supplemented with 10 nmol/l 3,3',5-triiodo-L-thyronine (T3). Hearts used to study in vivo development were excised from rats at the ages of 2 and 14 days post-natal to correspond with the time of isolating and harvesting the cultured heart cells, respectively. Adult hearts were used to represent the final developmental stage. Cultured cardiomyocytes without T3 administered to the culture medium showed no change in the isozymic profiles (myosin and LDH) or in metabolic potential during the 2 week culture period. The T3 treated cultures showed a complete shift to the V1 myosin isozyme. The glycolytic and aerobic metabolic potential [i.e., phosphofructokinase (PFK) and citrate synthase (CS) activities] and the LDH isozyme distribution were unaltered by T3 treatment. During in vivo development a shift toward the V1 myosin and H-LDH isozymes along with an increase in aerobic metabolism occurred in the rat heart. These findings indicate that the development of these selected biochemical systems in cultured cardiac myocytes does not result from an intrinsic myogenetic program and thus must be regulated in vivo by epigenetic factor(s). These results show that T3 has the potential to be the prime determinant of the phenotypic expression of the myosin isoforms, but does not have the potential to be the sole determinant for the expression of the LDH isozymes or the glycolytic (PFK) and aerobic (CS) capacities of cardiac muscle cells.

Myosin alkali light chain and heavy chain variations correlate with altered shortening velocity of isolated skeletal muscle fibers.

This study examines the myosin isozyme heterogeneity (in terms of both alkali light chains and myosin heavy chains) among skeletal muscle fibers of the rabbit and correlates these isozyme differences with the differences in a contractile property, the velocity of unloaded shortening, of the fibers. The mean velocities of unloaded shortening (pCa 4.3; 12 degrees C) were as follows: psoas IIb fibers, 2.07 fiber lengths/s (n = 25); tibialis anterior (IIb) fibers, 1.63 fiber lengths/s (n = 18); vastus intermedius IIa fibers, 0.98 fiber lengths/s (n = 15); fibers (IIa) from chronically stimulated tibialis anterior, 0.86 fiber lengths/s (n = 16). Peptide maps of the myosins showed that the myosin heavy chains of the two groups of IIb fibers were indistinguishable from each other, but different from the heavy chains of the IIa fibers. However, the difference in maximal shortening velocity of the two groups of IIb fibers was correlated with a difference in the alkali light chain ratio deduced from the intensity ratio of myosin isoforms separated by gel electrophoresis under nondenaturing conditions. The vastus intermedius (IIa) and chronically stimulated tibialis anterior (IIa) fibers were indistinguishable in terms of either velocities of unloaded shortening or myosin isozyme contents. Soleus fibers contained only slow-twitch myosin. Thus, among fibers that contained a variety of myosin isozymes, differences in shortening velocities were correlated with the alkali light chain ratio, myosin heavy chain type, or a combination of both.

Adult human masseter muscle fibers express myosin isozymes characteristic of development

Masseter muscle biopsies were obtained from nine patients undergoing orthognatic surgery or surgery for parotid tumors. A detailed enzyme-histochemical and immunocytochemical study of these muscles was performed using antibodies specific to the various isozymes of the myosin heavy chain (MHC) in order to identify the MHC isozymes that were present in the different fiber types. The contractile proteins in these same biopsies were analyzed by two-dimensional gel electrophoresis, native pyrophosphate gel electrophoresis, and by an immunopolypeptide mapping approach. These studies have shown that there is a very heterogeneous distribution of the myosin isozymes, with many fibers containing more than one myosin type. We also present evidence that in addition to adult fast and slow myosin, the human masseter muscle contains two proteins, neonatal MHC and embryonic myosin light chain, that are characteristic of developing muscle.

Differences in response of myosin isozyme transition of ordinary and specialized myocardium to overload.

To investigate the response of myosin isozyme transition in specialized myocardium to cardiac overload, we examined immunohistochemically the distribution of myosin isozymes in sinus node cells of overloaded canine atria, using the monoclonal antibodies CMA19 and HMC14, which are specific for atrial myosin heavy chain (alpha-HC) and ventricular myosin heavy chain (beta-HC), respectively. Overloading in canine right atria was induced by artificial tricuspid valve regurgitation and pulmonary stenosis. Right atrial mean pressure rose to 15-20 mm Hg (n = 4) 2 months after surgery. In the working myocardium, cardiac overload caused redistribution of myosin isozymes, alpha-HC to beta-HC. Compared with the normal right atria, fewer myocytes were labeled with CMA19, but more were labeled with HMC14. However, the reactivity of sinus node cells with CMA19 and HMC14 was not changed between normal and overloaded right atria, indicating no redistribution of myosin heavy chain isozymes, alpha-HC to beta-HC. These results suggest that isozymes in myosin heavy chains in the specialized myocardium are protected from overload effects by their firm cytoskeletal framework or other mechanisms.

Hemodynamic conditions regulate the performance of cardiac myosin isozymes

Hemodynamic conditions regulate the performance of cardiac myosin isozymes

Mechanical adaptation to chronic pressure overload.

According to Meerson, the adaptation to cardiac overload can be divided into three periods: the first stage, immediately after the initiation of the defect during which hypertrophy develops, followed by the stable hypertrophy phase (SHP), and a third phase of myocardial failure. Ventricular muscle contraction during SHP has been extensively studied both in vivo and in vitro with conflicting results. In isolated papillary muscles, most studies showed a normal or depressed contractility during chronic volume overload and a depressed inotropic state in pressure overload with a reduced maximal velocity of shortening which has been related to a myosin isozyme shift. In contrast, in conscious animals, haemodynamic status is usually described as preserved during SHP with a ventricular hyperfunction and a normal contractile function per unit of muscle. This was the basis of the concept of preload reserve and afterload mismatch described by Ross. However, mechanisms other than preload reserve may play a role during cardiac adaptation to pressure or volume overload. For instance, we recently showed in the early phase of pressure overload an increased inotropic state of the in situ heart with a change of the excitation contraction coupling evidenced by a modification of the force-frequency relations. Changes in the adrenergic receptors (density and/or affinity) may also contribute to the adaptation of the in situ heart to cardiac overload. They represent an important research area because they may explain, along with species and model differences, the discrepancies between in vivo and in vitro studies.