Myosin P-light chain isoenzymes in the human heart: evidence for diphosphorylation of the atrial P-LC form

Summary We studied myosin light chains (LC) of human atrium and ventricle of normal and diseased individuals by a high-resolution 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique. Atrial LCs (ALC-1, ALC-2 (=P-LC)) revealed both higher molecular weights and lower isoelectric points (IEP) than their ventricular counterparts (VLC-1, VLC-2 (=P-LC)). Different P-LC forms with their distinct myosin isoenzymes have been designated as P-LC-polymorphism and myosin P-LC isoenzymes, respectively. In the dephosphorylated state two VLC-2 forms (VLC-2 and VLC-2*) with the same MW and different IEP, but only one ALC-2 form, were found. In the partially phosphorylated state ALC-2 appeared to be single- and double-phosphorylated (three spots in the 2D-PAGE), whereas the two VLC-2 forms appeared to be single-phosphorylated each (four spots in the 2D-PAGE). Phosphoryl-transfer from ATP to the P-LC forms was studied using skinned fibers incubated with MLCK (myosin light chain kinase) and (-³²P)ATP. Ventricular myosin P-LC isoenzyme pattern was usually the same in normal and diseased patients: the VLC-2 to VLC-2* ratio was approx. 70/30, but in one patient with valvular heart disease (VHD) the relation was 55/45 (shift to the VLC-2* form). In hypertrophied atria of VHD patients a shift of the myosin P-LC isoenzyme pattern to the VLC-2* form occurred, too.     

Further studies on the effects of myosin P-light chain phosphorylation on contractile properties of skinned cardiac fibres

Summary We investigated the influence of myosin P-light chain phosphorylation by Ca²⁺-calmodulin dependent myosin light chain kinase (MLCK) on the sensitivity of the tension-pCa relation and maximum unloaded shortening velocity (v _max) of chemically skinned heart fibres of the pig.Submaximum Ca²⁺ stimulation (pCa 5.5) induced 20±5% of the isometric tension achieved at maximum Ca²⁺ activation (pCa 4.3).MLCK-induced myosin P-light chain phosphorylation increased the isometric force development at pCa 5.5 by 40% whereas maximum tension at pCa 4.3 was not affected.Unloaded shortening velocity (v _max) was not altered by myosin P-light chain phosphorylation either at maximum or at submaximum Ca²⁺ concentration, being c. 1.2 muscle length/s at pCa 5.5 and 2.2 muscle length/s at pCa 4.3.The MLCK-induced increase of the myosin P-light chain phosphorylation level was evaluated by determination of³²P-incorporation. Two phosphorylatable myosin P-light chains could be demonstrated.     

Atrial and ventricular myosins from human hearts. I. Isoenzyme distribution during development and in the adult

Summary Two distinct types of native isomyosins, referred to as human fetal HF and human ventricular myosin HV-3, have been identified in the human fetal heart during two different periods of gestation and in the neonatal state, whose relative parts change with development. In the adult ventricle, only HV-3 was found. Two myosin isoforms designated as HA-3 and HA-1 occur in the atrial myocardium of the normal human heart, which are electrophoretically distinct from the fetal isoenzymes.In fetal tissue, the myosin light chain complement is composed of atrial and ventricular light chains. In support of recent results, we also found identical spots from the atrial ALC-1 and the fetal light chain FLC, suggesting a homology between them. Apart from the light chains typical for this tissue, the atrial myocardium also contains ventricular light chains. Therefore we hypothesize that atrial myosin consists of two atrial isoenzymes and presumably of a ventricular type, too.Differences between atrial and ventricular myosin from human hearts were demonstrated by measuring the temperature dependence of the Ca²⁺-ATPase.     

Atrial and ventricular myosins from human hearts II. Isoenzyme distribution after myocardial infarction

Summary Human atrial and ventricular myosins were prepared from autopsy specimens from subjects with coronary heart disease. Cardiac myosin light chain isotypes were resolved using twodimensional gel electrophoresis, whereas myosin isozymes were detected by pyrophosphate gel electrophoresis.Myocardial infarction and associated work overload cause a transition in the light chain complements of the myosins. Thus ventricular myosin light chains were found in pressure overloaded atria and atrial light chains have also been identified in the infarct ventricle of the human heart.Two molecular isoenzymes of the human atrial myosin, the relative proportions of which are changed after infarction, were separated under non-dissociating conditions by gel electrophoresis. A decrease in HA-3 and a corresponding increase in HA-1 were observed. Ventricular hypertrophy in patients with coronary insufficiency induces a second ventricle isomyosin, called HV-1, with the same electrophoretic mobility as HA-1. The relative part of this myosin type amounts to 20%. Comparative peptide mapping studies were carried out on myosin subfragment-1 preparations from normal and infarct ventricles. In the primary structures, the chymotrypsic digestions produced slight differences.These data demonstrate the heterogeneity of human atrial and ventricular myosins in patients with coronary heart disease.     

The idiopathic dilated cardiomyopathy in man. A biochemical and molecular study on myosin

Summary We studied subunit composition and Ca⁺⁺-activated ATPase activity of myosin isolated from atria and ventricles of hearts explanted from patients suffering from idiopathic dilated cardiomyopathy. At variance with previously published data, we have been unable to detect in the ventricular subendocardial layers a significant amount of myosin atrial-like light chain 1 (ALC1), which has been reported to be related to some hemodynamic features of the hypertrophied and failing heart. Such a subunit was not visible in the septum and in the subepicardial layers either. On the contrary, in both atria a ventricular-like light chain 2 (VLC2) was found. The nature of this additional light chain was confirmed on the basis of two-dimensional electrophoresis and immunoblotting techniques with polyclonal antibodies reacting with VLC2. In these patients we also observed a depressed Ca⁺⁺-activated ATPase activity, both in atrial and ventricular myosin. The explanation for this finding in ventricles still remains obscure since neither myosin light chains, nor myosin heavy chains showed any difference between patients with dilated cardiomyopathy and controls. On the contrary, in atria we clearly identified changes consistent with the expression of myosin heavy chains of ventricular type and VLC2, which can account for the depressed Ca⁺⁺-activated ATPase activity.     

Calcium sensitivity and myosin light chain pattern of atrial and ventricular skinned cardiac fibers from patients with various kinds of cardiac disease

In the present study, the Ca2(+)-sensitivity and myosin light chain patterns of skinned fibers of right atrium and left papillary muscles of 27 patients suffering from mitral valve disease (MVD, moderate heart failure), ischemic cardiomyopathy (ICM, severe heart failure), dilated cardiomyopathy (DCM, severe heart failure), and coronary heart disease (CHD, no heart failure, no atrial hypertrophy) were investigated. Myosin light chains of both chemically skinned and intact samples were studied by two-dimensional gel electrophoresis (2D-PAGE). Ca2(+)-sensitivity of ventricular fibers was about 0.14 pCa-units higher than that of atrial fibers in all groups except dilated cardiomyopathy where this difference was markedly diminished (only 0.06 pCa-units). Generally, Ca2(+)-sensitivity of skinned ventricular fibers was the same among the different heart diseases. Skinned atrial fibers from patients with dilated cardiomyopathy, however, were significantly (about 0.08 pCa-units) more sensitive for Ca2+ than those of the other groups (coronary heart disease, mitral valve disease or ischemic cardiomyopathy) which showed similar Ca2(+)-tension relationships. Ventricle-specific P-light chain forms could be observed in atrial samples from patients of all groups, whereas no atrium-specific light chain forms were detectable in any ventricular sample. It is concluded that there is no difference in Ca2(+)-sensitivity of the ventricular contractile elements of the human heart in different heart diseases. In atrial myocardium, there is an increased Ca2(+)-sensitivity of skinned fibers from hearts with dilated cardiomyopathy which is probably related to an elevation of right atrial pressure.     

Different phosphorylation patterns of cardiac myosin light chains using ATP and ATP gamma S as substrates

Controversial views have been reported regarding the role of myosin light chain phosphorylation in the regulation of cardiac contractility (for review see. In the past, adenosine 5'-(-thio)triphosphate) (ATP gamma S) instead of ATP has frequently been used to study mechanical and biochemical consequences of myosin P-light chain (P-LC, LC-2) phosphorylation since thiophosphorylated sites are not significantly attacked by phosphatases. Unlike thiophosphorylation phosphorylation of myosin by myosin light chain kinase did neither decrease maximal (unloaded) shortening velocity of cardiac skinned fibres nor ATPase activity of cardiac myofibrils. We have accordingly investigated the phosphorylation pattern of purified cardiac myosin light chains using radioactive labeled ATP gamma S and ATP. We found that both the 28 kDa myosin light chain (LC-1) and the 18 kDa myosin light chain (LC-2, P-LC) were phosphorylated when ATP gamma S was present. In the presence of ATP, however, only LC-2 was found to be phosphorylated.     

Phosphorylation and thiophosphorylation by myosin light chain kinase: different effects on mechanical properties of chemically skinned ventricular fibers from the pig

The influence of myosin light chain phosphorylation (treatment with myosin light chain kinase = MLCK, calmodulin and ATP) and thiophosphorylation (incubation with MLCK, calmodulin and ATP gamma S) on the maximal shortening velocity (Vmax) and Ca2+ sensitivity of chemically-skinned ventricular fibers from the pig has been studied. Vmax was determined by the slack-test method and by extrapolation of the force-velocity relation by the isotonic quick release method. Vmax was 1.53 muscle length/s (L/s) and 1.94 L/s using the force-velocity relation and the slack-test, respectively. Phosphorylation increased the Ca2+ sensitivity for isometric force development of skinned fibers but had no influence on Vmax. Thiophosphorylation decreased Vmax but had no influence on Ca2+ sensitivity. Phosphorylation pattern of the myosin light chains of the skinned fibers was studied using [gamma-32P]ATP or [gamma-P35S]ATP (250 muCi each) and autoradiography. Incubation of skinned fibers with labeled ATP led to a phosphate incorporation into the 18-kDa myosin light chain (MPLC or regulatory light chain) while incubation with labeled ATP gamma S led to an incorporation of thiophosphate into the 28-kDa myosin light chain (alkali light chain) and tropomyosin. We suggest that the difference in mechanical behavior between phosphorylated and thiophosphorylated skinned fibers are due to differences in the phosphorylation profiles of myofibrillar regulatory proteins.     

Tension-pCa relations of saponin-skinned rabbit and human heart muscle

We tested if interaction between the thick and thin filaments, as revealed by the rate of cross-bridge cycling or the pattern of calcium activation, influences the relation between tension and free calcium concentration (pCa) of cardiac muscle. Bundles of rabbit ventricular cells which contained either fast-cycling RV1 myosin or slowly-cycling RV3 myosin and bundles of human atrial cells were chemically skinned by exposure to saponin. Skinned bundles were rapidly activated with calcium using the method of Moisescu and Thieleczek (1978). Our results suggest that the relation between pCa and tension is not different for skinned bundles of rabbit ventricular cells that contain either fast or slowly cycling myosin cross-bridges. The pattern of calcium activation, i.e. maximal to submaximal or submaximal to maximal, was also found not to influence the relation between tension and pCa for either rabbit ventricular or human atrial muscle. The tension-pCa relation of cardiac muscle was not altered by conditions that have been shown to alter the tension-pCa relation of skeletal muscle. These results suggest that the mechanisms responsible for regulating force development at the level of the thick and thin filaments that have been reported for skeletal muscle, are absent in cardiac muscle.     

Ventricular myosin pattern of spontaneously hypertensive turkeys is unaffected by labetalol treatment

Summary In most animal species, left ventricular hypertrophy due to pressure overload is associated with an advantageous increase of the slow V₃ isomyosin. In contrast, in spontaneously hypertensive turkeys, the development of left ventricular hypertrophy is associated with the synthesis of a fast V₁-like isomyosin, with high incidence of cardiac failure. This could be related to the high catecholamine levels found in these animals. This is why we studied the ventricular myosin pattern after lowering of blood pressure and regression of cardiac hypertrophy obtained by means of labetalol, an - and -blocking drug which inhibits the effects of catecholamines.From the 2nd to the 32nd week of age, 22 turkeys were treated with increasing doses of p.o. labetalol (from 20 to 35 mg/kg body weight daily) and 16 other turkeys were given daily p.o. placebo. Blood pressure and heart rate were periodically measured by an indirect method. After sacrifice, the degree of cardiac hypertrophy was evaluated by the biventricular weight to body weight ratio, ventricular myosin was purified, Ca⁺⁺-activated ATPase activity assessed, and ventricular myosin pattern was determined by two-dimensional gel electrophoresis of myosin heavy chains. Plasma and cardiac catecholamines were measured by high performance liquid chromatography.Throughout the study period, blood pressure and heart rate were significantly reduced in the labetalol-treated animals as compared to the untreated ones. At the end of the study period, the ventricular mass was significantly lower in the labetalol group. Nevertheless, no differences were obscrved in ventricular myosin pattern and Ca⁺⁺-activated ATPase activity levels between the two groups. In the labetalol group, an increase in plasma catecholamines and only a slight, but not significant, increase in cardiac catecholamines was found.These data indicate that in spontancously hypertensive turkeys, the synthesis of the fast V₁-like isomyosin is not influenced by known pathophysiological stimuli like blood pressure, cardiac hypertrophy and catecholamines.     

Contractile proteins in globally “stunned” rabbit myocardium

Summary The isolated working rabbit heart preparation was used to study whether the contractile machinery remains unchanged in globally stunned myocardium. The function of the heart has been measured in nonischemic and postischemic conditions. The effect of isoprenaline or calcium chloride administration in both conditions was also studied. Myocardial contractile function was significantly depressed after 20-min global ischemia and returned to normal after CaCl₂ and supranormal values after isoprenaline administration. From hearts used in experiments myofibrils were prepared and their ATPase activity was determined. It was observed that myofibrils prepared from stunned myocardium showed about 50 % increase in ATPase activity in the presence of CaCl₂. Subjection of the heart to ischemia caused a decrease in calcium sensitivity of the myofibrillar ATPase. Myofibrils obtained from ischemic hearts but subjected to isoprenaline or CaCl₂ administration exhibited increased calcium sensitivity over that of control heart. These effects were accompanied by changes in the extent of phosphorylation of troponin I (TNI) and myosin light chains. The modification of contractile apparatus in the postischemic period described in this paper may contribute to the overall mechanism of myocardial stunning.     

Skinned fibers of human atrium and ventricle: myosin isoenzymes and contractility

Different myosin isoenzymes of pig and human atrium and ventricle and rat ventricle were characterized by two approaches: pyrophosphate polyacrylamide gel electrophoresis (PP-PAGE) and analysis of the myosin P light chains by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We further investigated the relation between atrial and ventricular myosin isoenzymes of human, pig, and rat, and the maximum (unloaded) shortening velocity (Vmax) and the Ca2+ sensitivity of chemically skinned fibers of the same species. The myosin isoenzymes of both human and pig atrium comigrated in the PP-PAGE with rat V2 isomyosin, whereas the ventricle of human and pig comigrated with rat V3. In both human and pig ventricle, a myosin P light chain polymorphism exists (two phosphorylatable P light chains with the same molecular weight but different isoelectric points). In contrast, we found no P light chain polymorphism in the atrium of human and pig and in the ventricle of rat (one phosphorylatable P light chain only). A correlation exists between Vmax, Ca2+ sensitivity, and atrium- and ventricle-specific myosin isoenzymes of human and pig. Vmax was determined by the slack-test method. Plots of delta l versus delta t of atrial and ventricular skinned fibers were well fitted by a single straight line up to delta l = 15% and delta l = 13%, respectively. Vmax of skinned ventricular fibers was lower than Vmax of skinned atrial fibers in both human and pig. Ca2+ sensitivity of skinned fibers of ventricle, however, was higher than Ca2+ sensitivity of atrial skinned fibers in both human and pig.     

Myosin Light Chain Kinase Is Involved in the Mechanism of Gastrointestinal Dysfunction in Diabetic Rats

Background  

It is well established that smooth muscle contractility is regulated by an elevation of cytosolic Ca²⁺ via myosin light chain phosphorylation, which is activated by myosin light chain kinase (MLCK). Recently, MLCK has been demonstrated to play an important role in smooth muscle contraction and normal gastrointestinal motility.     

Improvement of relaxation velocity parameters by calcium channel blockers in the aging rabbit myocardium

Summary Normal aging in man is known to be associated with a reduction in left-ventricular diastolic function, including the rates of relaxation and filling. Calcium channel blockers have been reported to improve left-ventricular diastolic function in patients with various forms of heart disease. Clinically, the action of calcium channel blockers may be related to either a direct myocardial effect or may be secondary to the peripheral or coronary vasodilation effects. The purpose of this study is to investigate a possible direct effect of calcium channel blockers on modulation of the reported age-related reduction in myocardial relaxation.The direct effects on myocardial relaxation of the dihydropyridine calcium channel blocker, nifedipine, were studied in isolated, perfused interventricular septa and left-ventricular wall from eight young (ages 9 to 18 months) and 14 old (ages 3 to 5 years) rabbits. Septa were perfused with oxygenated Ringer's solution and paced at 48 beats/min. Maximum relaxation velocity per unit of developed tension [–dT/dt]/T, and relaxation time per unit of developed tension t_r/T were continuously measured before and after infusion of calcium channel blockers. In absence of drugs, the older rabbits demonstrated a mean [–dT/dt]/T which was 32% lower (p<0.003) and a mean t_r/T which was 45% higher (p<0.005) than the younger rabbits. When nifedipine was introduced at concentrations>10^–8 M equivalent to doses above the therapeutic free-plasma concentration in humans, all contraction and relaxation parameters were depressed. However, at lower doses, equivalent to doses in the clinical therapeutic range, [–dT/dt]/T was increased in the older rabbit septa by 18% in the presence of nifedipine. t_r/T was shortened in the older rabbit septa by 17% in the presence of nifedipine. Myocardial relaxation in older rabbits after drug infusion approximated these parameters in the younger rabbits prior to drug infusion (P=NS). Calcium channel blockers had similar beneficial effects on the relaxation properties of the myocardium in younger rabbits. All beneficial effects were observed at concentrations of calcium channel blockers which were within and below the clinically therapeutic range of plasma free drug concentration, i.e., 5×10^–9 to 4×10^–8 M.Potential differences in relaxation effects related to different segments of the myocardium and different mechanical recording vectors were evaluated. Isolated left ventricle preparations from aging rabbits demonstrated improvements in t_r/T and [–dT/dt]/T similar to those observed in the septum. Furthermore, improvement in mechanical function along the y-axis and x-axis vectors of the septum was similar. The data suggest that, in this rabbit model, calcium channel blockers may improve or reverse the age-related reduction in myocardial relaxation.     

Inorganic phosphate inhibits contractility and ATPase activity in skinned fibers from human myocardium

Summary During hypoxic heart failure, inorganic phosphate (Pi) accumulates. We report the effects of Pi on force development and on myofibrillar ATPase-activity of human skinned atrial fibers, both at normal and at reduced levels of Mg-ATP. Pi (10 mM) depressed force production at maximal calcium activation (pCa 4.3) by about 40%. At higher pCa values (pCa 5.6), foree inhibition was even more pronounced, but at low concentrations of Mg-ATP (10 M), Pi was less effective. In contrast to contractile force, myofibrillar ATPase was only inhibited by about 10% at pCa 4.3, whereas it could be inhibited by 40–50% at submaximal calcium activation (pCa 5.6). As Pi inhibited contractile force more than ATPase activity, the ratio of ATPase-activity to foree (tension cost) was increased by inorganic phosphate. ATPase-activity and tension cost were significantly reduced by loweing Mg-ATP concentration to 10 M, whereas contractile force was less affected. Pi did not affect ATPase under these conditions at 10 mM Mg-ATP. Pi also shifted the calcium-force relationship towards higher Ca⁺⁺ concentrations, that is, it decreased calcium sensitivity. In contrast, the calcium sensitivity of myofibrillar ATPase was less affected. These findings suggest that inorganic phosphate may affect the myocardium by altering crossbridge kinetics rather than the calcium affinity of troponin-C. Because of its inhibitory effect on myofibrillar ATPase, inorganic phosphate may be partly cardioprotective in the hypoxic myocardium. However, this energy sparing effect is probably offset by the greater tension cost that decreases the efficiency of tension maintenance in the presence of inorganic phosphate.     

Binding of bepridil to isolated rat heart mitochondria

Summary We investigated mass action of isolated rat heart mitochondria with the calcium antagonist bepridil. At pH 7.20 bepridil in basic form b associates rapidly with the mitochondrial membrane but the amount fixed is higher in non-energized mitochondria than in mitochondria energized by succinate or ATP Mg²⁺. This effect is related to the dissociation state of the drug since conditions favoring the acidic form bH⁺ suppress this difference. Tritiated bepridil bound to mitochondrial membrane is only partially displaced by high concentrations of unlabeled drug (5 10^–5 M). No membrane energization effect is noted on this displacement. Binding values of bepridil to mitochondrial membrane (K_D 1.7 10^–5 M; B_max 23.8 nmol·mg^–1 protein) show only low affinity receptor sites. Bepridil binding to the lipid part of the inner membrane surface is postulated. This interaction is used to explain some of the in vitro effects of this calcium antagonist on membrane bound enzyme activities.     

Effects of physical training on the myocardium of streptozotocin-induced diabetic rats

Summary Effects of endurance swimming training on myocardial contractility and left ventricular myosin isoenzymes were examined in diabetic rats. A diabetic condition was induced in 15-weck-old male Wistar rats, by intravenous injection of streptozotocin (50 mg/kg). Swimming training was carried out for five to six weeks (90 min/day, 6 days/week). In order to estimate myocardial contractility, the isometric developed tension of the isolated left ventricular papillary muscle was measured. Myosin isoenzymes were obtained by pyrophosphate gel electrophoresis. Fasting blood glucose of the trained group was significantly lower than that of the sedentary group (sedentary vs. trained=409.6±25.9 vs. 266.3±20.5 mg/dl, p<0.001). There was no significant difference in isometric developed tension (T) between the two groups, and the dT/dt_max of the trained group showed a tendency to increase (sedentary vs. trained, T: 2.8±0.8 vs. 2.9±0.8 g/mm², dT/dt_max: 23.1±3.6 vs. 26.2±3.5 g/mm² · 2, p<0.1). Myocardial mechanical responses to isoproterenol and dibutyryl cAMP were increased in the trained group. Left ventricular myosin isoenzyme pattern was shifted towards VM-1 by endurance swimming (sedentary vs. trained, VM-1: 5.6±4.5 vs. 19.6±8.8%, p<0.001, VM-3: 75.1±10.0 vs. 54.9±14.7%, p<0.001). These results indicate that endurance swimming can improve disordered glucose metabolism and also influence myocardial contractility, myocardial catecholamine responsiveness, and energetics in myocardial contraction.     

Effect of sarcomere length and filament lattice spacing on force development in skinned cardiac and skeletal muscle preparations from the rabbit

Summary Skinned cardiac and skeletal muscle freeze-dried preparations were activated in solutions strongly buffered for Ca²⁺. The response of single skeletal muscle fibres or thin strips of papillary muscle was investigated in relation to changes in Ca content of the perfusate. Sarcomere length was set and controlled during the experiments. The relation between the negative logarithm of the Ca concentration, the pCa, and the normalized developed force proved to be sigmoidal. The exact position of these curves proved to be dependent upon both sarcomere length and the distance between the filaments. The latter was shown by means of osmotic compression of the fibres using dextran. As a consequence of these observations. it was concluded that the length-tension relation is dependent upon the actual Ca concentration. The results are discussed in terms of cross-bridge interaction.     

Voltage-dependent calcium release in guinea-pig cardiac ventricular muscle as antagonized by magnesium and calcium

Summary An increase in extracellular potassium concentration from 4 to 16 mmol/l caused a decrease in membrane potential from –92 to –59 mV and selectively diminished the earlier of two contraction components of guinea-pig papillary muscles at 0.2 Hz stimulation frequency in the presence of noradrenaline. The influence on the early contraction component had a threshold of 8 mmol/l K⁺, corresponding to a membrane potential of –77 mV. However, test contractions elicited 800 ms after the 5 s stimulation interval exhibited an unimpaired early component. Since the activator calcium responsible for the early contraction component is derived, in mammalian ventricular muscle, from the junctional sarcoplasmic reticulum (20), it is assumed that the release site of the reticulum was filled with calcium shortly (800 ms) after a regular contraction, and lost its calcium at 16 mmol/l extracellular K⁺ during the 5 s stimulation interval. The potassium-induced depolarization determined the rate of calcium leakage during rest from the intracellular store. The depolarization-induced decline of the early contraction component was equally well antagonized by Mg²⁺ or Ca²⁺ without influencing the measured transmembrane potential. Both divalent cations shifted the relation between potassium concentration or membrane potential and the strength of the early contraction component to less negative membrane potentials. In order to reduce the early contraction component by 25% in the presence of 9.6 instead of 1.2 mmol/l Mg²⁺, the potassium concentration had to be increased from 9.6 to 22.0 mmol/l, with a respective decrease in resting membrane potential from –72.6 to –51.1 mV. The antagonistic effect of both divalent cations is thought to result from the neutralization of negative charges outside the sarcolemma with a respective decrease in the outside surface potential.     

The role of calcium in the enhanced myocardial contractility of the hyperthyroid rat heart

Summary The hyperthyroid rat myocardium exhibits enhanced contractility. There is evidence that altered calcium handling by the myocardium may be responsible for this enhanced state. To investigate this, isolated hyperthyroid and cuthyroid hearts were perfused in the working mode and exposed to alterations in external calcium concentration. Heart rate was not significantly different in either group of hearts, nor was it altered by the change in calcium. The concentration of calcium needed to elicit half-maximal contractility (dP/dt_max) was lower in the hyperthyroid (0.81±0.07 mM) than in the cuthyroid hearts (1.12±0.09 mM, p<0.05). This increase in calcium sensitivity was unlikely to be at the site of the sarcolemma as verapamil exerted equal negative inotropic effects on both groups of hearts. Dantrolene, which blocks calcium release from the sarcoplasmic reticulum, exerted a significantly greater (p<0.01) depression in dP/dt_max after 12 min in the hyperthyroid (50±7%) than in the cuthyroid heart (15±2%). We conclude from our results that the enhanced contractile state of the hyperthyroid rat heart is likely to involve an altered mechanical response to calcium which is possibly at the level of enhanced calcium release from the sarcoplasmic reticulum.