Rest- and stimulation-dependent changes in exchangeable calcium content in rabbit ventricular myocardium

Summary Ca²⁺ shifts in the isolated, perfused ventricular muscle of rabbit hearts were investigated with the aid of⁴⁵Ca under the conditions of complete equilibration of preparations with⁴⁵Ca containing solution. The cellular⁴⁵Ca content was calculated by subtraction of⁴⁵Ca²⁺ dissolved in the free water of extracellular space from the total tissue⁴⁵Ca²⁺ content. The cellular content of⁴⁵Ca in stimulated (60 per min) preparation was 0.887±0.067 mmol/kg wet weight (w.w.). Six minutes of rest resulted in the drop of this content to 0.503±0.054 mmol/kg w.w. despite continued perfusion with⁴⁵Ca containing solution. Contractile force (CF) decreased at that time to 23% of control. The first post-rest contraction (RSC) resulted in a gain of 0.073 mmol⁴⁵Ca/kg w.w. Both the content of⁴⁵Ca and CF returned to the pre-rest values when stimulation was resumed.The difference between the⁴⁵Ca content of post-rest stimulated and rested preparations (0.384 mmol/kg w.w.) is equivalent to Ca₂ fraction (Ca₂), previously described in guinea pig ventricular myocardium (17, 12). However, the volume of rabbit Ca₂ is only about 42% of that in guinea pig. Caffeine in concentration 12.5 mM, which did not displace Ca₂ from guinea pig ventricular muscle, decreased Ca₂ in the rabbit ventricle by 44%. CCCP, a protonofore destroying the mitochondrial protone gradient essential for Ca²⁺ uptake and maintainability, displaced Ca₂ completely from rabbit ventricles. These results, although far from conclusive, do suggest that both the mitochondrial and sarcoplasmic reticulum might be the site of the rate-dependent Ca₂ fraction. The physiological meaning of differences in Ca₂ content between rabbit, guinea pig, and rat ventricular myocardium is discussed.This study was supported by the grant CPBR, 11.6     

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.     

Epinephrine-stimulated contractile and metabolic reserve in postischemic rat myocardium

Summary Recovery of contractilc function and of fatty acid oxidation may be delayed in viable postischemic myocardium. To determine whether a metabolic reserve is preserved after reperfusion of reversibly injured myocardium, we studied the effect of epinephrine on myocardial fatty acid oxidation in isolated rat hearts perfused retrogradely with crythrocyte enriched buffer containing albumin 0.4 mM, palmitate 0.4 mM, and glucose 11 mM. Hearts were subjected to 60 min of low-flow ischemia (5% of control flow) followed by 60 min of reperfusion. Five minutes following the onset of reperfusion, developed left ventricular pressure (DLVP) and oxidation of palmitate were reduced to 53% (p<0.01) and 46% (p<0.01), respectively, of values measured in nonischemic control hearts. Subsequently, DLVP and oxidation of palmitate gradually recovered to 78% (NS) and 91% (NS) by 60 min of reperfusion. Epinephrine 5·10^–4M elicited an immediate stimulation of both contractile function and palmitate oxidation. Early after reperfusion stimulated DLVP and palmitate oxidation were still lower compared to values measured in control hearts exposed to the same concentration of epinephrine. Later than 15 min after the onset of reperfusion the response of DLVP and of palmitate oxidation to epinephrine no longer differed between control and reperfused hearts.These results indicate that viable postischemic myocardium exhibits a remarkable oxidative metabolic reserve. The observation provides further evidence for the view that impairment of myocardial energy production is not responsible for contractilc dysfunction early after reperfusion.Supported by the Swiss National Science Foundation grant # 3.917-0.87     

Net transsarcolemmal Ca2+ shifts versus Ca/Ca exchange in guinea pig ventricular muscle

Summary We investigated the net transsarcolemmal Ca²⁺ shifts and Ca/Ca exchange by means of⁴⁵Ca in isolated, perfused ventricles of guinea pig heart treated with vanadate to inhibit ATP-driven sarcolemmal Ca²⁺ pump. The heart was stimulated (at the rate of 60/min) and perfused with a solution containing⁴⁵Ca for 60 min. Thereafter stimulation was stopped and either perfusion with radioactive solution was continued or the solution was exchanged for a non-radioactive one. In the first case, tissue⁴⁵Ca content (equivalent to the exchangeable Ca²⁺ content) dropped from 1.960±0.120 mmol/kg of wet weight (w.w.) to 0.715±0.049 mmol/kg w.w. and stabilized at this level between 5th and 10th min. In the second case, decrease in⁴⁵Ca content continued and within 40 min attained 0.047±0.004 mmol/kg w.w., despite stabilizing of the total exchangeable Ca²⁺ content. Drop of⁴⁵Ca content in the rested heart perfused (until the end of experiments) with radioactive solution resulted from the net transsarcolemmal Ca²⁺ shift and it was strongly inhibited by removal of extracellular Na⁺. The continuing drop in⁴⁵Ca content in the heart perfused with non-radioactive solution while total Ca²⁺ content stabilized must have resulted from Ca/Ca exchange; it was stimulated by removal of extracellular Na⁺. These experiments separate two modes of⁴⁵Ca fluxes and suggest that a common route of these fluxes is the Na/Ca exchanger.This work was supported by a grant: C.P.B.R.11.6 No 58.     

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.     

Effects of ouabain and low-Na+ perfusion on rest-decay and post-rest recovery of cellular Ca content in ventricular muscle of guinea-pig heart

Summary Ca²⁺ shifts in isolated, perfused ventricular muscle of guinea-pig hearts were investigated with the aid of⁴⁵Ca under the conditions of complete equilibration of preparations with isotopecontaining solutions. The content of⁴⁵Ca in stimulated preparations (rate 60/min) was 1.30 ±0.12 mmol/kg of wet weight (w.w.). 6 min rest resulted in the drop of this content to 0.37 ±0.05 mmol/kg w.w. despite continued perfusion with⁴⁵Ca containing solution. The difference of 0.93 mmol/kg w.w. is equivalent to fraction Ca₂ (15) and is labelled accordingly. Ouabain (1 M) increased the⁴⁵Ca content to 1.53±0.15 mmol/kg w.w. in the stimulated and to 1.12±0.23 mmol/kg w.w. in the rested muscle. The respective values after low (50 mM) sodium perfusion were 1.70±0.11 and 1.07±0.13 mmol/kg w.w. The differences between the stimulated and rested preparations (Ca₂ fraction) were 0.41 and 0.63 mmol/kg, respectively. In the control experiments the force of the first post-rest beat dropped to 20±5% of the force of steady-state beats. During oubain and low-sodium perfusion, the force of the first contraction increased markedly and its peak was larger than that of the few subsequent beats. It is concluded that Na–Ca exchange is the important factor in the rate-dependent control of Ca₂ fraction content and of contractile force.This study was supported by the grant CPBR, 11.6     

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.     

Ultrastructural damage and Ca2+-shifts in the canine myocardium subjected to regional incomplete ischemia

Summary The role of Ca²⁺ in the pathogenesis leading to ischemic myocardial cell death is still controversial. To gain insight into this phenomenon a cytochemical procedure, the phosphate pyroantimonate method, was used to localize different subcellular Ca²⁺-pools at the ultrastructural level. After 45 min of left anterior descending coronary artery (LAD) occlusion, the coronary arteries were perfused with triphenyltetrazoliumchloride staining (TTC) to identify viable ischemic and infarcted tissue. In non-ischemic tissue, Ca²⁺-deposits were confined to the sarcolemma, sarcolemma-derived vesicles, transverse tubules, and intercalated disks. In infarcted tissue (TTC-negative), the sarcolemma lost its Ca²⁺-binding capacity and mitochondria were either overloaded with Ca²⁺-precipitate or they contained amorphous densities. In viable ischemic areas (determined with the TTC-technique) the sarcolemma was virtually devoid of Ca²⁺-deposits. Mitochondria in this area frequently showed clumping of the cristae, associated with an accumulation of Ca²⁺-precipitate in between the clustered cristae. The results of this study indicate that Ca²⁺-shifts occur in ischemic myocardial cells before the occurrence of other ultrastructural signs of irreversible injury which, therefore, narrows the possibility that Ca²⁺-overload is only a consequence of ischemic cell death.     

The reciprocal protective effects of magnesium and calcium in hyperkalemic cardioplegic solutions on ischemic myocardium

Summary The cardioprotective effects of magnesium and calcium in hyperkalemic cardioplegic solutions were investigated in isolated rat hearts. Isolated rat hearts were arrested for 30 min at 37°C in cardioplegic solutions containing magnesium and calcium in varying concentrations. In St. Thomas' Hospital cardioplegic solution, the magnesium and calcium concentrations were varied as follows: Mg 0; Ca 0.1, 0.3, 0.6, 1.2 mmol/l (mM)./ Mg 4; Ca 0.1, 0.3, 0.6, 1.2 mM./ Mg 8; Ca 0.1, 0.3, 0.6, 0.9, 1.2 mM./ Mg 12; Ca 0.1, 0.6, 0.9, 1.2 mM./ Mg 16; Ca 0.1, 0.6, 1.2, 1.5 mM. For each magnesium concentration, the percentage recovery of aortic flow generated dose-response curves depending on calcium concentration. The maximum percentage recovery of aortic flow was 76.0%±2.7% (mean ±SEM) in the Mg0–Ca0.1 mM group, 77.1%±2.0% in the Mg4–Ca0.3 mM group, 78.5%±2.3% in the Mg8–Ca0.6 mM group, 79.8%±2.4% in the Mg12–Ca0.9 mM group and 80.0%±3.4% in the Mg16–Ca1.2 mM (ST solution) group. Significant difference in the recovery of aortic flow has not been observed among these groups, and furthermore, significant differences in the recovery of other parameters of cardiac function and Ck leakage have not been observed among these groups. These results suggest that the cardioprotective effects depend on the relative combination of magnesium and calcium concentrations, and that it is important to maintain an appropriate balance of magnesium and calcium in hyperkalemic cardioplegic solutions.     

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.     

The effect of calcium ions and the calcium ionophore A23187 on choline uptake and phosphatidylcholine biosynthesis in chick embryo hearts

Summary The effect of increases in extracellular calcium [Ca]₀ and the calcium ionophore A23187 on choline uptake and phosphatidylcholine biosynthesis was assessed in isolated cardiac myocytes. The cells were obtained from 7-day old chick embryos and were maintained in culture. Choline uptake was examined using [methyl 3H] choline. A23187 was found to increase choline uptake through the saturable choline uptake process. Pulse chase experiments using [methyl 3H] choline showed that after a 2 h incubation with choline, about 85% of the label was recovered in phosphocholine with most of the rest in phospholipid and a small amount in CDP-choline and glycerol phosphocholine. Increases in [Ca]₀ up to 10 mM did not affect the amount of label in phosphocholine or phospholipid, the rate of disappearance of label from phosphocholine, or the rate of appearance of labelled choline in phospholipid. In contrast, A23187, at concentrations up to 10^–4 M, was associated with a significant (p<0.05) increase in choline in the phosphocholine and phospholipid pool compared to control cells. The time course of the disappearance of choline from the phosphocholine pool and appearance in phospholipid pool was not significantly different between control cells and those treated with A23187. A23187 increased choline uptake via the specific uptake process. The effect on choline uptake may be attributed to the action of A23187 to facilitate the release of calcium from specific intracellular calcium storage sites rather than a nonspecific increase in [Ca]₁ that may have resulted from the increase in [Ca]₀.Supported in part by a grant from Canadian (British Columbia) Heart Foundation     

Effect of D-600 on ischemic and reperfused rabbit myocardium: relation with timing and modality of administration

Summary In this study we have investigated the possibility that D-600, a phenylalkylamine calcium antagonist, protects the isolated rabbit heart against ischemia and reperfusion-induced damage.D-600 was either subcutaneously injected (2 mg/kg, twice daily for 5 to 6 days) in the rabbit before isolation of the heart, or delivered to the isolated hearts in the perfusate (10^–7 M), either at the onset of ischemia and during reperfusion, or only during post-ischemic reperfusion.Ischemia (90 min) was induced by reducing coronary flow from 25 to 1 ml/min, followed by 30 min of reperfusion. Myocardial damage was determined in terms of mechanical function, release of creatine phosphokinase (CPK) and noradrenaline, mitochondrial function, calcium homeostasis, and endogenous stores of ATP and creatine phosphate (CP). Administration of D-600 to the rabbits or to the isolated hearts at the time of ischemia exerted protection. There are four groups of evidence in support of this conclusion: 1) the rise in diastolic pressure during ischemia was diminished with greater recovery of developed pressure during reperfusion; 2) CPK and noradrenaline release during reperfusion were reduced; 3) the oxygen consumption and ATP generating capacities of mitochondria were better maintained; and 4) associated with this preservation of mitochondrial function was the maintenance of near normal calcium homcostasis and of endogenous ATP and CP stores. The two different modalities of administration did not produce substantially different results.When administered to the isolated hearts after the ischemic period, D-600 failed to improve mechanical recovery and release of endogenous substances. However, it reduced mitochondrial calcium overload and improved ATP production. The mechanism of the protective effect of D-600 seems to be multiple: energy-sparing effect, reduction of the toxicity mediated by endogenous catecholamines, and direct inhibition of mitochondrial calcium transport.     

The relationship between regional blood flow and contractile function in normal,ischemic, and reperfused myocardium

Summary The prevailing paradigm of coronary physiology and pathophysiology is that a balance between blood flow (i.e., supply) and function (i.e., demand) exists under normal conditions and that an imbalance between supply and demand occurs during ischemia. However, this paradigm is derived largely from studies relating changes in total coronary inflow to global ventricular function. The present article examines the relationship between myocardial blood flow and function on a regional level and proposes that a change may be needed in the current paradigm of coronary pathophysiology. In normal myocardium, considerable heterogeneity of regional blood flow exists, indicating either similar heterogeneity of metabolic demand and function or questioning the precision of metabolic coupling between flow and function. After the onset of ischemia, a transient imbalance between the reduced blood flow and function may exist. However, myocardial function rapidly declines and during early steady-state ischemia regional myocardial blood flow and function are once again evenly matched. Such supply-demand balance may persist over prolonged periods of ischemia enabling the myocardium to remain viable through reduction of energy expenditure for contractile function, i.e., to hibernate. Whereas in hibernating ischemic myocardium, regional myocardial blood flow and function are both reduced but appropriately matched to one another, flow and function appear to be largely uncoupled in reperfused stunned myocardium. The clinical identification of viable but ischemic (hibernating) and postischemic (stunned) myocardium is of utmost importance in patients undergoing reperfusion procedures. A new paradigm of coronary and myocardial pathophysiology, encompassing a regional as well as a global view of perfusion and function, will have to include explanations for phenomena such as myocardial hibernation and myocardial stunning.     

Evaluation of parameters for the assessment of regional myocardial contractile function during asynchronous left ventricular contraction

Summary The primary purpose of this study was to evaluate parameters used for the measurement of regional myocardial contractile function in the setting of left ventricular (LV) asynchrony. Secondarily, we tested whether the peak negative value of left ventricular dP/dt (-dP/dt) can be used to estimate global LV end-systole during asynchrony. In seven anesthetized (Isoflurane) swine the left anterior descending coronary artery was cannulated and perfused at constant blood flow rates. To produce LV asynchrony, dobutamine (D) was infused into the perfusion system. This was repcated later during coronary hypoperfusion (HYPO) sufficient to produce regional contractile dysfunction. The amount of LV wall thickening during systole (% WT, sonomicrometry) was calculated using either - dP/dt or the closure of the aortic valve (AO, electromagnctic flow probc) for estimating the timing of global LV end-systole. % WT was compared to other paramcters which are not dependent upon the timing of global LV end-systole, including the amplitude of the first harmonic of the Fourier transform (AMP) and regional myocardial work (WI) estimated from the left ventricular pressure-wall thickness relationship. A close correlation between global LV end-systole defined by the AO or - dP/dt existed during control. D or HYPO. During HYPO+D no such relationship was found (r=.22, NS), and % WT calculated using - dP/dt as an estimate of end-systole was underestimated when comparcd to % WT calculated by use of the AO to estimate end-systole (2.9±6.8% vs 6.3±6.6%, p<.05). % WT, AMP, and WI showed similar results during control, D and HYPO. However, D during HYPO increased the AMP from .59±.23 mm to .76±.32 mm and WI from 67±20 mm Hg*mm to 95±24 mm Hg*mm (p<.05), respectively. This increase in regional myocardial function, however, was not detected by % WT (10.5±6.4% vs 6.3±6.6%). Thus, during left ventricular asynchrony, the measurement of LV-dP/dt to estimate the timing of global LV end-systole is inappropriate and can lcad to inaccuracies in the measurement of regional contractile function. Parameters such as AMP or WI are advantageous since global LV end-systole docs not need to be accurately defined.Supported in part by the American Heart Association California Affiliate grant-in-aid #86-S105, and by the German Research Foundation (He 1320/3-2). Dr. Guth is the recipient of a Research Fellowship from the Alexander von Humboldt-Stiftung, Jean-Paul-Straße 12, D-5300 Bonn 2     

Effects of caffeine on energy output of rabbit heart muscle

Summary The effects of caffeine (1 mmol·1^–1) on mechanical and energetic parameters in the arterially perfused interventricular rabbit septa were examined at various frequencies of stimulation. Even though 1 mmol^–1 caffeine induced a negative inotropic effect only at stimulation rates higher than 0.33 Hz. relaxation was impaired at all frequencies tested. The ratio between maximum rate of relaxation and developed tension was consistently lowered by caffeine, indicating a more marked effect on relaxation over contraction. In addition, while time-to-peak tension was unaffected by caffeine at the dose used, the last part of the relaxation (i.e., of the contractile event) was prolonged at all frequencies in the presence of the drug. Resting heat production (H _t ) was increased in the presence of caffeine (1.6±0.6 mW·g^–1). The ratios between active heat production and either developed tension (H_a/T) or tension time integral (H_a/TtI), increased at all frequencies examined (53.3±8.5 J·mN^–1·g^–1 and 68.2±9.9 J·mN^–1·s^–1·g^–1, respectively), indicating a lowered economy of the contractile process. This is consistent with the lower ATP/Ca ratio reported for the sarcoreticular Ca pump (i.e., one ATP hydrolyzed/2 Ca transported) with respect to the sarcolemmal mechanisms such as Na–Ca exchanger or the sarcolemmal Ca pump, with an ATP/Ca ratio of 1 to 1. Thus, inhibition of the SR-Ca pump by caffeine would induce a higher rate of ATP hydrolysis with the consequent increase in the H_a/T ratio. As a result of the increase in both H_a/T ratio and H_r induced by caffeine, the ratio between total heat production and developed tension (H_t/T) also increased. Therefore, the contractile process appeared to be more efficient in the presence of an active SR, since it is energetically less costly to generate a given level of isometric tension.     

Sensitivity to oxidants of mitochondrial and sarcoplasmic reticular calcium uptake in saponin-treated cardiac myocytes

Summary Calcium transport functions of mitochondria and sarcoplasmic reticulum (SR) were studied without prior extraction using isolated rat heart myocytes permeabilized with saponin. Calcium uptake by SR was rapid and its affinity was high in comparison to calcium uptake by mitochondria, which had a higher capacity. The sensitivity of uptake to two oxidants, H₂O₂ and HOCl (hypochlorous acid), depended on the cytosolic calcium concentration; when this was similar to the concentration in diastole (180 nM), HOCl inhibited calcium uptake by mitochondria and SR, whereas when the calcium concentration was 750 nM, mitochondrial calcium uptake showed relatively high resistance, although SR uptake was still markedly inhibited by HOCl. Calcium uptake of both mitochondria and SR was less sensitive to the action of H₂O₂ than to HOCl, and the H₂O₂ effect was less dependent on the cytosolic calcium concentration. Therefore, HOCl, when produced by activated leukocytes and supplied to the heart cells, may seriously impair the excitation-contraction coupling function of SR, whereas H₂O₂, possibly generated directly by mitochondria or generated from superoxide anions, may be tolerated relatively well by heart SR and mitochondria.Preliminary accounts of this work were presented as FASEB 1988. Dr. Kaminishi was a Canadian Heart Foundation Post-Doctoral fellow; his present address is Yamagata University, Japan.     

Mechanical catecholamine responsiveness and myosin isoenzyme pattern of pressure-overloaded rat ventricular myocardium

Summary Pressure-overloaded cardiac hypertrophy was induced by abdominal aortic constriction in 10-week-old male Wistar rats. 24–26 weeks after aortic constriction, the hearts were excised and a myocardial mechanical study was performed using isolated left ventricular papillary muscles. There was no significant difference in isometric developed tension (T) between sham-operated control and aortic constriction (AC) rats (control vs AC rats=2.9±0.6 vs 2.7±0.7 g/mm²). dT/dt_max of AC rats, on the other hand, was significantly lower than that of controls (controls vs AC rats=32.8±7.5 vs 26.3±6.1 g/mm²sp<0.05). Myocardial mechanical responses to isoproterenol (10^–7 mol/l) were depressed in the group with aortic constriction compared with the control group (T:18.5±6.7 vs 12.1±4.9%,p<0.05, dT/dt: 25.2±6.2 vs 17.5±5.8%,p<0.02). Responses of the parameters to dibutyryl cyclic AMP (10^–5 mol/l) were also smaller in the AC group than in the control group (T: 18.0±5.6 vs 13.3±4.0%,p<0.05, dT/dt: 20.4±6.9 vs 14.7±4.1%,p<0.05). Left ventricular myosin isoenzyme pattern, revealed by pyrophosphate gel electrophoresis, shifted towards VM-3 under pressure overload. The present study demonstrates that post-membrane processes may be mainly responsible for the decreased myocardial mechanical catecholamine responsiveness in pressure-overloaded cardiac hypertrophy.Supported by Tanaka Memorial Medical Research Fund     

The effect of calcium concentration on spontaneous ventricular defibrillation and VF threshold

Summary In order to investigate the influence of the effective refractory period on spontaneous ventricular defibrillation, isolated rat hearts were perfused with Krebs-Henseleit solution containing 0.5, 2.7 and 5.1 mM calcium. After measuring the fibrillation threshold at spontaneous rate (SR), ventricular fibrillation (VF) was induced during basic ventricular pacing of 110% SR, or the highest rate permitting 11 electromechanical coupling. The VF threshold was significantly reduced from 13.6±3.5 to 7.9±5.3 and 5.1±3.4 mA at 0.5, 2.7 and 5.1 mM Ca⁺⁺ concentrations, respectively. The incidence of spontaneous recovery from VF, induced during basic pacing, was 100%, 83% and 50% at calcium concentrations of 0.5, 2.7 and 5.1 mM, respectively, (p<0.01 for the incidences at 0.5 mM versus 5.1 mM Ca⁺⁺). The incidence of spontancous defibrillation decreased when the hearts were driven rapidly, with spontaneous recovery rates of 92%, 58% and 0% (p<0.0001)) for corresponding increases in Ca⁺⁺ concentration. Induced ventricular fibrillation of fine morphology was frequently observed at 5.1 mM Ca⁺⁺. It appears that progressive impairment of spontaneous defibrillation is caused by an increase in calcium concentration, this effect being more pronounced at high ventricular rates. Variations in the effective refractory period, caused by alterations in extracellular calcium concentration and differences in intracellular Ca⁺⁺ accumulation, may account for the above results.     

Effects of captopril on left ventricular structure and function in SHR with established hypertension

Summary While antihypertensive therapy is considered to be an important clinical intervention in hypertensive patients, its effects on cardiac structure and function have not been intensely evaluated. In this study we tested the hypotheses that lowering blood pressure (BP) with the angiotensin I-converting enzyme inhibitor captopril, would: 1) normalize left ventricular mass and increase the cardiocyte mitochondria/myofibrils volume (V_mito/V_myo) ratio; and 2) not compromise peak ventricular performance. We treated 16-week-old SHR and WKY with captopril (40–80 mg/kg) and hydrochlorothiazide (500 mg/l) via their drinking water. After six weeks of treatment peak cardiac performance was measured during rapid volume overload. Tissue samples from the left ventricular wall were analyzed by electron microscopy and stereology. Captopril lowered BP in SHR and WKY but had no affect on the left ventricular/body weight ratio. The only intracellular change in treated SHR was an increase in sarcoplasmic volume density. Treated WKY exhibited decreased midmyocardial mitochondrial volume density. At peak cardiac output, acceleration of flow and cardiac index were not affected by treatment. Stroke work at peak cardiac output was decreased in the treated groups due to a decrease in mean arterial pressure. In addition, captopril treatment resulted in a shift of the cardiac output (CO)-left ventricular end diastolic pressure (LVEDP) curves, such that LVEDP at peak cardiac output was approximately 50% less in the treated groups compared to their respective control groups. Although captopril was efficacious in lowering BP, it is suggested that lowering BP with this agent does not, at least within six weeks, lead to a reversal of hypertrophy or to a significant alteration in the volume densities of myofibrils and mitochondria. However, an important effect of this antihypertensive drug which may be of clinical significance, is that it leads to a leftward shift of the CO-LVEDP curve in both hypertensive and normotensive rats.     

Determination of rat heart morphology and function in vivo in two models of cardiac hypertrophy by means of magnetic resonance imaging

Summary Quantitative magnetic resonance imaging (MRI) was applied to assess structural and functional parameters of the rat heart in vivo. Using ECG and respiratory triggering, MR images were obtained at different time points during the cardiac cycle. This allowed accurate determinations of the left ventricular (LV) mass, wall thickness, LV end-systolic and end-diastolic volumes, stroke volume, and ejection fraction. LV mass determined by MRI showed an excellent linear correlation with post mortem gravimetric determination of LV weight. MRI was then used to examine the pathophysiological changes in two models of LV hypertrophy. In one group of animals the aortic arch was banded to an outer diameter of 1.0 mm to elicit a pressure overload on the LV. A second group was subjected to a volume overload due to graded disruption of the aortic valve. Although both models exhibited a similar degree of LV hypertrophy as shown by the LV weight/body weight ratio, important functional and structural differences were revealed by MRI. Aortic stenosis resulted in an increase in wall thickness, whereas stroke volume and ejection fraction did not differ compared to control animals. In contrast, aortic valve insufficiency did not affect LV wall thickness, however, LV chamber volume as well as stroke volume were markedly increased. Ejection fraction was significantly reduced in these animals. In conclusion, MRI allows the reliable in vivo determination of important structural and functional parameters of hearts in small rodents.