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Query: UNIPROT:P06889 (Mol)
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The sequence of events that leads to irreversible injury of the ischaemic myocardium is poorly understood but it is axiomatic that lack of oxygen will impair regeneration of ATP. In the globally-ischaemic heart a contracture develops which is independent of raised cytoplasmic free Ca2+ and which has been attributed to activation of actomyosin by nucleotide-free actomyosin cross-bridges ('rigor complexes') which form at low ATP concentrations. Single, metabolically-poisoned or anoxic cardiomyocytes show comparable behaviour, shortening before a significant rise in cytoplasmic free Ca2+ occurs. To explain the close temporal relationship that exists between cell shortening and the onset of the free Ca2+ rise we have predicted that, during myocyte shortening, a precipitous fall in cytosolic ATP concentration occurs, the result of rigor-complexes activating myosin ATPase, which then perturbs ionic homeostasis. Here we show, by means of continuous measurements of cytosolic ATP using firefly luciferase microinjected into single, isolated cardiomyocytes, that cell shortening coincides with an abrupt fall in cytosolic ATP.
J Mol Cell Cardiol 1992 Mar
PMID:Bioluminescent measurement in single cardiomyocytes of sudden cytosolic ATP depletion coincident with rigor. 162 46

The purpose of this study was to determine if selected biochemical parameters representing the contractile and calcium regulating systems of cardiac muscle scaled among mammals having inherently different resting heart rates (RHR). Eight mammalian species with RHR ranging from 51 to 475 beats per minute (bpm) were studied. The oxidative capacity of the myocardium is highly correlated with the RHR. The hypothesis of the present study was that the capacities of the energy utilizing processes of contraction and calcium regulation would also be correlated to the functional demand imposed on the muscle as represented by the RHR. Myosin (M) and myofibrillar (MF) ATPase activities, myosin isoenzyme distribution and sarcoplasmic reticulum (SR) ATPase activity were determined. Animals with RHR above 300 bpm express V1 myosin while animals with lower RHR express primarily V3. M and MF ATPase activities correlated with RHR, but the major difference in activities occurred at the 'threshold' RHR of about 300 bpm at which the switch from V3 to V1 appears to occur. SR ATPase activity per mg of microsomal protein was for the most part constant among different mammals, but the SR ATPase activity per g of heart tissue was significantly correlated with RHR as slower beating hearts tended to yield less SR protein per unit mass. We conclude that both the contractile and calcium regulating systems are scaled to the functional parameter of RHR among different mammals. The contractile system uses a slow myosin ATPase isoform at low resting heart rates whereas above the postulated threshold RHR of about 300 bpm a switch in gene expression to a fast myosin ATPase isoform occurs.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1991 Aug 14
PMID:Contractile and calcium regulating capacities of myocardia of different sized mammals scale with resting heart rate. 165 10

The regulatory light chains (RLCs) located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation signals. The RLCs belong to a family of calcium binding proteins and are composed of four "EF hand" ancestral calcium binding motifs (numbered I to IV). To determine the role of the first EF hand (EF hand I) in the regulatory process, chimaeric light chains were constructed by protein engineering, by switching this region between smooth muscle and skeletal muscle myosin RLCs. For example, chimaera G(I)S consisted of EF hand I of the smooth muscle (gizzard) RLC and EF hands II to IV of the skeletal muscle RLC, whereas chimaera S(I)G consisted of EF hand I of the skeletal muscle RLC and EF hands II to IV of the smooth muscle RLC. The chimaeric RLCs were expressed in Escherichia coli using the pLcII expression system, and after isolation and purification their regulatory properties were compared with those of wild-type smooth and skeletal muscle myosin RLCs. The chimaeric RLCs bound to the myosin heads in scallop striated muscle myofibrils from which the endogenous RLCs had been removed ("desensitized" myofibrils) with similar affinities to those of the wild-type smooth and skeletal muscle RLCs. Both chimaeric RLCs were able to regulate the actin-activated Mg(2+)-ATPase activity of scallop myosin: G(I)S inhibited the ATPase in the presence and absence of Ca2+, like the wild-type skeletal muscle RLC, while S(I)G inhibited the myosin ATPase in the absence of Ca2+, and this inhibition was relieved on Ca2+ addition, in the same way as the wild-type smooth muscle RLC. Thus the type of regulation that the RLCs confer on the myosin is determined by the source of EF hands II to IV rather than that of EF hand I.
J Mol Biol 1991 Apr 20
PMID:Chimaeric myosin regulatory light chains: sub-domain switching experiments to analyse the function of the N-terminal EF hand. 182 64

Previous studies have shown that dietary provision of carbohydrate can alter cardiac isomyosin distribution in hormonally deficient rats. The main objective of this study was to determine if varying the heart's potential to utilize carbohydrate for energy provision can influence the cardiac isomyosin expression in normal weanling rats. Animals were assigned to one of five groups according to dietary and/or metabolic treatment: (1) mixed-control--(M); (2) high carbohydrate--(H); (3) low carbohydrate--(L); (4) mixed-diet supplemented with oxfenicine, a cardiospecific fatty acid oxidation inhibitor--(MO); and (5) high carbohydrate diet supplemented with oxfenicine--(HO). The results show that 4 weeks of dietary manipulations aimed to either increase or decrease carbohydrate supply to the heart, failed to induce any alterations in either cardiac myosin ATPase activity or isoenzyme pattern. However, extremes in carbohydrate provision altered the metabolic properties of both heart and skeletal muscle. A low carbohydrate diet increased 3-hydroxyacyl CoA dehydrogenase (P less than 0.05) and citrate synthase activities (P less than 0.05) and decreased glycogen content in both heart and soleus muscle; whereas, a high carbohydrate diet, in conjunction with oxfenicine, tended to increase hexokinase activity in these same tissues. These alterations provide indirect evidence that the contributions of both fat and carbohydrate to the energy balance of the heart and skeletal muscle were altered by the imposed dietary interventions. Collectively, these results suggest that although the substrate utilization patterns of the normal weanling heart can be modified via dietary manipulation, such shifts do not exert any regulatory influence on cardiac isomyosin expression.
J Mol Cell Cardiol 1990 Mar
PMID:Dietary effects on cardiac metabolic properties in rodents. 214 63

The cardiac effects of excess growth hormone (GH) were studied in the intact adult rat and in tissues prepared from the rat. Female Wistar-Furth rats were inoculated with a clonal cell line of pituitary cells which secrete GH. Five weeks later, heart weight had increased 37% compared to control (P less than 0.01) due to concomitant increases in left and right ventricular weight. Hemodynamic measurements in the anesthetized rat showed that GH stimulated rats had a decrease in blood pressure and heart rate and a small increase of left ventricular end-diastolic pressure (P less than 0.05). Measurement of left ventricular contractility and relaxation, and response to beta-adrenergic stimulation were decreased in GH compared to control (P less than 0.05). Contractile protein biochemistry showed an 18% reduction in Ca2(+)-myosin ATPase activity of the left ventricle (P less than 0.05) and non-denaturing pyrophosphate gels of purified myosin demonstrated a significant shift of isoforms from the exclusive V1 pattern to both V1 and V3 isomyosins in both ventricles (P less than 0.05). In contrast to the physiological and protein biochemistry adaptations, left ventricular morphology by light microscopy and ultrastructure by electron microscopy were normal in the GH stimulated heart. There were no significant changes in myofibril fraction, in the myofibril to mitochondria ratio or in the capillary numerical density of the hypertrophied left ventricle (P = N.S.). This study demonstrates that under prolonged and extreme stimulation by GH, the heart undergoes considerable growth/hypertrophy. Although cardiac morphology remains normal during this growth, there are alterations of the isomyosins such that ATPase activity is diminished and ventricular function is decreased.
J Mol Cell Cardiol 1990 Apr
PMID:Cardiac physiology, biochemistry and morphology in response to excess growth hormone in the rat. 214 88

Scallop adductor myosin is regulated by its subunits; the regulatory light chain (R-LC) and essential light chain (E-LC). Myosin light chains suppress muscle activity in the absence of calcium and are responsible for relaxation. The binding of Ca2+ to the myosin triggers contraction by releasing the inhibition imposed on myosin by the light chains. To map the functional domains of the R-LC, we have carried out mutagenesis followed by bacterial expression. Both wild-type and mutant proteins were hybridized to scallop myosin heavy chain/E-LC to map the regions of the light chain that are responsible for the binding to the myosin heavy chain/E-LC, for restoring the specific calcium-binding site, and controlling the myosin ATPase activity. The R-LC is expressed in Escherichia coli using the pKK223-3 (Pharmacia) expression vector and has been purified to greater than 90% purity. E. coli-expressed wild-type R-LC differs from the native R-LC by having the initiating methionine residue and an unblocked NH2 terminus. The wild-type R-LC restores Ca2+ binding and Ca2+ sensitivity when hybridized to scallop myosin. A point mutation of the sixth Ca2(+)-liganding position of domain I (Asp39----Ala39) results in a R-LC that binds more weakly to the heavy chain/E-LC and restores the specific Ca2(+)-binding site but not regulation of the actin-activated Mg2+ ATPase. A second mutation was produced by substituting the last 11 residues of the COOH terminus with 15 different residues. This mutant restores the specific Ca2(+)-binding site, but does not restore Ca2+ regulation to the actin-activated ATPase activity. Several other point mutations do not alter light chain function. The experiments directly establish that the divalent cation-binding site of domain I is functionally distinct from the specific Ca2(+)-binding site. The results indicate that an intact domain I and the COOH terminus are required to suppress the myosin ATPase activity. The fact that the domain I mutation and the COOH-terminal mutation disrupt regulation but do not affect Ca2(+)-binding indicates that these two aspects of regulation are separable and, therefore, the R-LC has distinct functional regions.
J Mol Biol 1990 Nov 05
PMID:Regulation of scallop myosin by mutant regulatory light chains. 214 99

Alterations of cardiac contractility caused by thiamine deficiency were studied on three groups of 2 month old male Wistar rats: B1, fed a thiamine deficient diet, PF pair fed, which received an amount of thiamine free diet determined on the daily consumption of B1 animals, supplemented with appropriate thiamine supply, C ad libitum fed controls. The animals were studied after 35 days of dietary treatment. Force-velocity curves were determined in right ventricle papillary muscles. Shortening velocity was significantly lower in B1 and PF than in C muscles and in B1 than in PF muscles. The ability to develop tension was not altered. Myosin ATPase activity was assayed in preparations of myofibrils and in preparations of purified myosin. Both Ca-Mg activated myofibrillar ATPase activity and Ca-activated myosin ATPase activity were significantly reduced in B1 and PF compared to C myocardium. Furthermore Ca-activated ATPase activity was lower in B1 than in PF myocardium. Myosin isoenzyme distribution was determined by pyrophosphate gel electrophoresis of purified myosin preparations. When compared to C animals both B1 and PF animals showed a myosin electrophoretic pattern shifted towards the slow isoform V3; such a shift was more pronounced in B1 animals. Information concerning excitation-contraction coupling was obtained by determining the steady state and transient force-interval relation and by recording transmembrane action potential. B1 and PF myocardium exhibited, when compared to C, a less sensitivity to a reduction of the interval of stimulation, a faster mechanical restitution, a prolonged action potential duration. Such alterations were generally more pronounced in B1 than in PF myocardium. The results support the view that in the rat cardiac contractility is deeply affected by thiamine deficiency. The alterations of cardiac contractility seem to be caused by adaptive mechanisms rather than by cardiac failure and seem to be attributable for a big part to the reduction of food supply.
J Mol Cell Cardiol 1990 Oct
PMID:Altered contractile properties of rat cardiac muscle during experimental thiamine deficiency and food deprivation. 215 36

We previously reported setting up an in vitro system for the observation of actin filament sliding along myosin filaments. The system involved a minute amount of fluorescently labelled F-actin, and its movement was monitored by fluorescence microscopy. Here, we report observations of the Ca2+-dependent movement of F-actin complex with tropomyosin plus troponin (regulated actin) added to the movement system in place of pure F-actin. In a wide range of pCa (-log10[Ca2+]) between 3 and 5.5 at 30 degrees C, regulated actin filaments moved rapidly, and the average velocity depended little on the Ca2+ concentration (about 7.5 microns/s). However, when the Ca2+ concentration was decreased to pCa = 5.8 or lower, the filaments suddenly stopped moving. In striking contrast to these observations, unregulated actin moved rapidly within the whole pCa range examined, the average velocity (about 7.5 microns/s) being essentially Ca2+-independent. These observations indicate that (1) tropomyosin-troponin actually gave Ca2+-sensitivity to F-actin, and (2) the movement system was regulated by Ca2+ in an on-off fashion within a narrow range of Ca2+ concentration. In a pCa range between 5.8 and 6.0, regulated actin filaments did not exhibit thermal motion; instead, they had fixed positions in the specimen, possibly because they remained associated with myosin filaments in the background, without sliding past each other. Although regulated actin moved fast in the presence of 1 mM-CaCl2 (pCa = 3) at 30 degrees C, it became entirely non-motile as the temperature was decreased to 25 degrees C or lower. Such a sharp movement/temperature relation was never found for unregulated actin. We assayed regulated actin-activated myosin ATPase in the same conditions as used for microscopy, and found that the ATPase activity depended both on pCa and on the temperature considerably less than the movement of regulated actin. The results suggest that the sliding velocity in the in vitro system would not be proportional to the rate of actin-activated ATPase.
J Mol Biol 1989 Feb 20
PMID:Calcium-triggered movement of regulated actin in vitro. A fluorescence microscopy study. 252 55

The aim of this study was to determine whether variations of isomyosin expression occurred during doxorubicin-induced cardiomyopathy. A suitable experimental model in which pure delayed cardiotoxic effects could be easily studied was adopted. Young adult female Sprague Dawley rats received 9 mg/kg of doxorubicin (DXR) i.v. divided into three subdoses of 3 mg/kg every third day. Control animals received equal volumes of saline. The animals were examined 9 weeks after treatment. At this time the animals treated with DXR showed ECG alterations, reduction of body weight and a marked decrease of both atrial and ventricular mass, but were still fully hemodynamically compensated. Loss of myofibrillar material could be documented by the reduced recovery of myofibril and myosin. The contractile response of papillary muscles isolated from the right ventricle of treated animals was markedly impaired. Ca-Mg-activated and Mg-activated myofibrillar ATPase activity and Ca-activated myosin ATPase activity were determined on ventricular myocardium of control and treated animals. Both myofibrillar and myosin ATPase activities were found to be significantly reduced. Pyrophosphate gel electrophoresis of purified myosin was carried out. The isomyosin pattern of DXR-treated animals showed a pronounced shift towards V3, the percent of alpha heavy chains being 54.6% in treated rats (80.5% in control rats). This isomyosin shift can explain the reduced myofibrillar and myosin ATPase activity found in treated animals.
J Mol Cell Cardiol 1989 Jan
PMID:Reduction of myofibrillar ATPase activity and isomyosin shift in delayed doxorubicin cardiotoxicity. 252 75

Corticosteroids have been shown to produce a myopathy of peripheral skeletal muscle, characterized predominantly by Type II fiber atrophy. To determine if similar histologic and histochemical changes occur in the diaphragm and whether the in vitro contractile properties of this muscle are adversely affected by steroids, we studied two groups of hamsters. The experimental group received triamcinolone while a control group received saline, both given daily for 3 wk as i.m. injections. Soleus (Sol) and extensor digitorum longus (EDL) muscles and costal diaphragm muscle sections were stained for histologic (hematoxylin and eosin, modified Gomori trichrome) and histochemical (myosin ATPase, succinate dehydrogenase [SDH]) analysis. Muscle fiber proportions and cross-sectional areas (CSA) were measured from myosin ATPase sections. In vitro studies of isometric contractions were carried out on small strips of costal diaphragm, measuring maximal isometric twitch (Pt) and tetanus (Po) tensions, time to peak tension (TTP), half relaxation time (1/2 RT), force-frequency relationship, and fatigue characteristics (60 Hz tetani; duty cycle, 0.5). Triamcinolone treatment resulted in no change in muscle fiber proportions. There was no effect on Type I fiber CSA; however, there was Type IIa (Sol, EDL) and Type IIb (diaphragm, EDL) fiber atrophy in triamcinolone-treated animals. Pt and Po (normalized for weight) of diaphragm strips were not different. There was a prolongation in TTP and 1/2 RT, a left shift in the force-frequency curve, and a reduced fatiguability of triamcinolone-treated diaphragm (P less than 0.05). We conclude that a steroid myopathy could be explained by a loss of muscle mass (Type IIb fiber atrophy) rather than an intrinsic impairment in contractile function.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1989 Sep
PMID:Pathologic changes and contractile properties of the diaphragm in corticosteroid myopathy in hamsters: comparison to peripheral muscle. 262 59


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