The effect of coronary reperfusion on the uptake of cardiac glycosides by ischemic myocardium was studied in 17 open chested dogs undergoing anterior wall infarction produced by snaring confluent branches of the left coronary system. Epicardial electrograms delineated ischemic zones of S-T elevation, border, and nonischemic zones. Animals were reperfused by snare release 1, 2, and 6 hr after occlusion. After 15 min of reperfusion, 1.0 mg of [3H] digoxin was given intravenously, and 2 hr later the hearts were excised and endocardial (endo) and epicardial (epi) samples from each zone were analyzed for [3H] digoxin concentration. In five dogs occluded for 1 hr and reperfused, [3H] digoxin uptake was comparable in endo and epi layers of all three zones. In six dogs reperfused after 2 hr of occlusion, mean (+/-S.E.) [3H] digoxin concentrations (nanograms per gm) were significantly reduced by 54 percent in endo (111 +/-18) and 35 percent in epi (151 +/- 23) layers of the ischemic zone as compared with the mean nonischemic concentration (endo249 +/- 34; epi 239 +/- 34). Border zone endo and epi [3H] digoxin uptake was reduced by 21 and 17 percent, respectively. In six dogs reperfused after 6 hr of occlusion, [3H] digoxin uptake in the ischemic zone was markedly reduced by 85 percent in endo (34 +/- 4) and 60 percent in epi (86 +/- 12) layers as compared with the nonischemic concentration (endo 232 +/- 19; epi 217 +/- 15). Border zone uptake was decreased by 54 percent in endo and 38 percent in epi regions. We conclude that coronary reperfusion between 2 and 6 hr of coronary occlusion is associated with markedly reduced myocardial digoxin uptake, more pronounced in subendocardial regions of ischemic tissue. This alteration in digoxin binding by reperfused ischemic myocardium is consistent with ischemia-induced structural or functional alterations in the putative digitalis receptor, (Na++K+)-ATPase.
The influences of hypoxia and of the interactions of hypoxia with digoxin and ouabain on myocardial stiffness were studied at two extracellular calcium concentrations (2.5 mM and 4.0 mM) in isolated, isometrically contracting cat papillary muscles. Stiffness (delta T/delta L), defined as a change in tension (delta T) in response to an imposed length change (delta L), was measured during contraction and during rest by use of a sinusoidal forcing function. Neither digoxin, ouabain, nor increased extracellular calcium altered contraction or resting stiffness in the well-oxygenated environment. Resting stiffness was increased at the end of hypoxia only in the presence of digoxin in both 2.5 mM and 4.0 mM Ca. Contraction stiffness was increased in 2.5 mM Ca by hypoxia alone and by hypoxia in the presence of digoxin or ouabain, but was not increased in experiments carried out in 4.0 mM Ca. Thus it appears that hypoxia per se increases contraction stiffness, but increasing extracellular calcium from 2.5 mM to 4.0 mM prevents the increase elicited by hypoxia; resting stiffness, however, is increased by hypoxia only in the presence of digoxin, and this occurs in both 2.5 mM and 4.0 mM Ca.
Characterization of the energy metabolism pattern of the specialized heart muscle of bovine heart was studied in comparison with that of the ordinary heart muscle. Mitochondrial oxygen consumption of the specialized heart muscle was significantly lower than that of the ordinary heart muscle with succinate as the substrate. On the other hand, there was no significant difference in oxygen consumption between both heart muscles with glutamate + malate as the substrates. The activity levels of succinate dehydrogenase and lactate dehydrogenase were much lower than those of the ordinary heart muscle. The isozyme pattern of LDH of the specialized heart muscle consisted of one major component of LDH-1 (H4) and that of the ordinary heart muscle consisted of two major components of LDH-1 (H4) and LDH-2 (H3M). The ratio of NADH to NAD of the specialized heart muscle was remarkably lower than that of the ordinary heart muscle. These results indicate that the specialized heart muscle depends not only upon anaerobic metabolism but also upon aerobic metabolism for its energy supply.
(-)-Adrenaline caused concentration-dependent increases in cAMP levels and the rate of beating in eight-day-old heart cell cultures of newborn rats. Half-maximal increases in both parameters (5- and 0.2 -fold, respectively) occurred at about 10(-6)M. Following the addition of 3 X 10(-7) M adrenaline, the cellular cAMP level rose to a max imum in 30 sec. The rise was abolished by 5 X 10(-8) M (-)-propranolol and was greatly magnified by 10(-4) M 1-methyl-3-isobutylxanthine. In the presence of the latter compound, the average rate of accumulation of cAMP in thecultures during the first 10 seconds of exposure to 3 X 10(-7) M adrenaline was 8.78 pmol/mg of protein-sec, which is 230 times more rapid than the basal accumulation rate. These findings may be taken as evidence in support of the view that cAMP is involved in the positive chronotropic action of adrenaline on cardiac pacemaker cells.
Phase contrast microscopy of cultured embryonic heart cells showed the beating frequency decreased more rapidly and the regularity the rhythm of of the beating cells was lost sooner in heart cells from cardiomyopathic hamsters than from the control hamsters. Studies of cultured heart cells by differential interference contrast (with Nomarski's prism) and by electron microscopy revealed a significant impediment in the maturation of the sarcomeric units in the diseased animals compared to controls. The incorporation of [14C] leucine into acid-insoluble fractions was studied, and no significant difference in incorporation between the two groups was found. An analysis of polyacrylamide gel electrophoresis revealed the possible existence of a quantitative difference in one of the composing proteins of the erythrocyte membrane between the two groups. The protein kinase activity of ghosts from the control group was more sensitive to cAMP than that from the diseased animals. In addition, the binding of [3H] cAMP to the ghost was almost identical between the two. The morphological and biochemical observations lead one to the plausible supposition that there are some differences in the interaction of the so-called catalytic and regulatory subunits between the two groups and that there is an impairment of the higher arrangement of myofibrils from their building blocks in the diseased hamster. The significance of the existence of abundant corpuscles resembling neurosecretory granules was not established by this study. They may have an etiological significance or they may be related to a disturbed function in the cultured cells of the cardiomyopathic hamster.
Mitochondria from cardiomyopathic hamster hearts have elevated calcium levels, which may cause a defect of oxidative phosphorylation in a small fraction of them. Using a combination of dual labeling density-gradient centrifugation, it was not possible to isolate such an abnormal fraction. However, cardiomyopathic mitochondria are more susceptible to damage by calcium in vitro, suggesting that an abnormal fraction does exist nevertheless.
When isolated rat hearts are perfused with Ca2+-containing medium, after a brief Ca2+-free period, irreversible cell damage occurs (calcium paradox). This phenomenon is concomitant with a rapid consumption of myocardial high-energy phosphate stores, prior to the appearance of these compounds in the effluent perfusion medium. A possible mechanism for the origin of myocardial necrosis, caused by intracellular Ca2+ overload, is discussed.
A computer simulation indicates that intracellular sodium concentration within a space near the inner surface of sarcolemma fluctuates during a cycle of myocardial function. The sodium transient (a transient increase in sodium ion concentration associated with membrane excitation) is enhanced by the inhibition of (Na+, K+)-ATPase by ouabain, but an accumulation of myocardial sodium does not occur until the inhibition exceeds a critical point. The critical magnitude of sodium pump inhibition that causes a progressive sodium accumulation is dependent on the heart rate.
Sarcolemmal Ca++-ATPase, Mg++-ATPase, and (Na+-K+)-ATPase activities were increased in late stages of heart failure in myopathic hamsters (BIO 14.6) without any changes in the adenylate cyclase activity. On the other hand, these hamsters at early and moderate stages of heart failure showed depressions in mitochondrial calcium binding and uptake and microsomal calcium binding. Sarcolemmal (Na+-K+)-ATPase was decreased in failing hearts because of substrate lack, oxygen lack, and perfusion with Ca++-free, Na+-free, or K+-free medium. Both Mg++-ATPase and Ca++-ATPase activities of sarcolemma did not change on perfusing the hearts with substrate-free, hypoxic, Na+-free, or K+-free medium. Adenylate cyclase activity decreased on substrate-free or Ca++-free perfusion. Intracellular calcium overload produced by perfusing the hearts with medium containing calcium after Ca++-free perfusion was associated with decrease in all the sarcolemmal-bound enzyme activities. All types of failing hearts employed in this study showed a dramatic shift in the electrolyte composition. Failure of the cardiac muscle to generate contractile force on treatment with trypsin was associated with defects in the functions of sarcolemma, mitochondria, and sarcoplasmic reticulum, whereas such an effect on treatment with phospholipase C was limited to alterations in the activities of sarcolemma. The data suggest that abnormality at the level of sarcolemma plays an important role in the pathogenesis of heart dysfunction; however, the degree and direction of alterations in the sarcolemmal functions seem to be dependent upon the type of heart failure.
Based on the assumption that circulating acetaldehyde (AcH) is cardiotoxic, D-penicillamine was administered to dogs given alcohol orally, or given AcH intravenously. Paralleling the increase in plamsa norepinephrine (NE) and epinephrine (E) induced by AcH infusion, hemodynamic measurements showed a positive inotropic response with increase in pulse, blood pressure, left ventricular contractility, and cardiac output. Infusion of D-penicillamine abruptly lowered circulating levels of AcH and catecholamines, which was accompanied by an appropriate hemodynamic response.
Phosphate-supported calcium uptake by guinea pig cardiac sarcoplasmic reticulum has been shown to exhibit a pattern different from that of oxalate-supported calcium uptake. Ionophore-induced calcium release has been demonstrated following phosphate-supported calcium uptake. Rat cardiac sarcoplasmic reticulum has been shown to differ from the guinea pig in its sensitivity to ionophore.
The binding of [3H]atropine by the primary subcellular fractions and plasma membrane-enriched fractions from atria and ventricles of various species was measured by the Millipore filtration technique. Although all of the primary particulate fractions exhibited binding activities, the bulk of the total homogenate binding activity was associated with the washed particles sedimenting at the lower gravitational forces; this was observed with either atria or ventricles of dog, guinea pig, rabbit, hamster, and rat. Plasma membrane-enriched fractions isolated from the right atrium of guinea pig exhibited atropine binding activities with characteristics similar to dog atrial membranes; binding activity was moderately enriched in these membranes with respect to the starting material.
Following two and four hours of left ventricular ischemia, 20 minutes of blood reflow cannot produce a complete return to normal oxidative metabolism in ischemic canine myocardium. Glycolysis remains increased and aerobic metabolism is most extensively depressed in the subendocardium.
Action potential durations in premature excitations showed paradoxical prolongation at the shorter coupling intervals; this was abolished by manganous ions. Voltage-clamp experiments also disclosed a transient increase of slow inward current in premature excitations. These results indicate that prolongation of action potential durations was mainly brought about by changes in slow inward current, especially in its characteristics of recovery from inactivation.
Excitation-contraction coupling in heart muscle cells depends upon an inward displacement of Ca2+ from the extra- to the intracellular phase. Some of the Ca2+ which is displaced inwards is probably derived from superficially located binding sites. Drugs which increase the peak tension developed during contraction, e.g., catecholamines and the cardiac glycosides, may act by increasing the amount of Ca2+ which becomes available for participation in the events associated with excitation-contraction coupling. The catecholamines increase in the amount of Ca2+ which is displaced inward from the extracellular phase, whereas the cardiac glycosides apparently act by increasing the exchangeability of the Ca2+ which is stored at binding sites located at the plasma membrane. Other drugs, e.g., verapamil, act in the reverse manner, decreasing the amount of Ca2+ which is available for displacement from the superficially located binding sites. These observations will be discussed in terms of an hypothesis that the plasma membrane represents an important site of drug action, and that many drugs which alter the contractile performance of the heart do so because of their ability to modify the ability of the plasma membrane to accumulate and exchange Ca2+.
Morphologically intact plasma membranes from guinea pig ventricles were obtained by exposing isolated cell segments to osmotic shock, followed by extraction of actomyosin in 1 M KC1. These preparations contained approximately 1/6 of the protein and 5-10 percent of the mitochondrial markers present in the original cell preparation. Both adenylate cyclase and (Na++K+)-activated ATPase activities were enriched 3-4 fold. The receptor for epinephrine stimulation of adenylate cyclase was retained. The "basal" ATPase activity of 5-6 mumoles of Pi/mg/hr, measured in 120 mM NaC1 or KC1, was approximately doubled in 100 mM NaC1+20 mM KC1. This increment, the (Na++K+)-activated ATPase, was abolished by 10(-5) M ouabain, the Ki for ouabain being approximately 3x10(-7) M. Adenylate cyclase, which had a basal activity of approximately 0.33 nmole of cyclic AMP produced/min/mg of protein, was significantly stimulated by both l-epinephrine and NaF. Half-maximal stimulation was seen at approximately 5x10(-6) M l-epinephrine. Increasing Ca2+ in the range between 10(-7) and 10(-3) M inhibited basal, l-epinephrine-, and NaF-stimulated adenylate cyclase activities. Basal rates of cyclic AMP production were more sensitive to Ca2+ than was l-epinephrine-stimulated adenylate cyclase activity, so that l-epinephrine stimulation was increased from approximately 60 percent in 0.5 mM ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid to approximately 150 percent in 10(-7)M Ca2+ and 400 percent in 10(-5) M Ca2+. The inhibitory effect of Ca2+ on adenylate cyclase activity may represent a negative feedback mechanism by which eelevation of intracellular Ca2+ concentration lowers cellular levels of cyclic AMP and thus reduces Ca2+ influx into the myocardium.
The origin and possible regulatory mechanism of tonic tension (ICa-independent component of active contractile activity) were investigated in frog antrial muscle under voltage-clamp conditions. Replacement of NaCl by LiCl resulted in a fast decrease in tonic tension; a similar fast decrease of this contractile component was induced by Ca-free solution. When low Na Ringer's solution was applied, tonic tension increased transiently and then decreased to a steady amplitude; at return to normal Ringer's, a further, substantial decrease in tonic tension occurred before the original level was reached. Similar behavior of tonic tension was observed when both [Na] o and [Ca]o were lowered, but the ratio [Ca]o/[Na]o2 remained constant; the transient changes were prevented by using low Ca and Na solutions and keeping the ratio of [Ca]o/[Na]o5 constant. The significance of Na-Ca exchange in regulating tonic tension and the possibility that this exchange may be electrogenic are discussed.
The Km value for the dog heart (Na+-K+)-ATPase was 0.31 mM (MgATP), whereas the values for the concentrations of K+ and Na+ varied from 1.2 to 2.7 mM and 12 to 20 mM for half-maximal activation, respectively. The concentrations of ouabain and calcium for 50 percent inhibition of (Na+-K+)-ATPase activity varied from 2.4 to 3.2 muM and 0.5 to 1.2 mM, respectively, the inhibitory effects of these agents were pH dependent. This preparation bound about 50 nmoles of 1-anilino-8-napthaline sulfonate (ANS)/mg of protein and exhibited fluorescence attributable to the ANS-enzyme complex. Cations such as Na+,K+,Ca++, and Mg++ increased ANS-enzyme fluorescence intensity and the number of ANS binding sites but decreased the apparent ANS binding constant. The enzyme activity, ANS binding, and ANS-enzyme fluorescence were decreased by phospholipase A, phospholipase C, and trypsin treatments. Although ouabain inhibited enzyme activity and ANS-enzyme fluorescence markedly, it caused only a slight depression in ANS binding. These results extend support for the allosteric nature of the cardiac (Na+-K+)-ATPase and provide evidence for conformational changes during its activation by Na+ and K+.
