Effect of oxidants on Na,K,ATPase and its reversal

Summary Ischemia-reperfusion heart cell injury may be mediated, at least in part, through the generation of oxy radicals. Therefore, mechanisms of action of two oxidants on a membrane model, partially purified Na,K,ATPase, were investigated. Effects of H₂O₂, an oxygen intermediate postulated to play a primary role in reperfusion injury, on the function of the enzyme were time-dependent and potentiated by Fe ions. The inhibition of enzyme activity was prevented by chelators, but not by hydroxyl radical scavengers. The results support the view that the possible mode of enzyme modification involves H₂O₂-derived, Fe ion-catalyzed, localized (site-specific) hydroxyl radical formation. The action of hypochlorous acid (HOCl), a powerful oxidant postulated to be produced by activated neutrophils, was quantitatively similar to that of H₂O₂ plus Fe ions in causing enzyme dysfunction. This is partly because relatively large doses of oxidants were required, due to the presence of physiological anti-oxidant defense mechanisms in the membrane. Although a combination of deferoxamine (Fe ion chelator) and dithiothreitol (DTT) (sulfhydryl reducing agent) was most effective in preventing the enzyme modification, once enzyme inactivation by oxidants is in progress, deferoxamine plus DTT could only arrest further deterioration of the enzyme function. Therefore, the oxidant-induced change in membrane dysfunction advances with time; the advance can be stalled, but the enzyme activity cannot be restored to normal.     

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     

Age-dependent production of mitochondrial hydrogen peroxide,lipid peroxides and fluorescent pigments in the rat heart

Summary Mitochondria were prepared from hearts of 3-, 14-, 18-, and 24-month-old male Wistar rats. Respiratory control ratio (RCR) values did not change with age in the glutamate or succinate-induced respiration except at 24 months in which RCR values significantly increased with both the substrates. Using still glutamate or succinate as substrates the production of H₂O₂ was measured in the presence of antimycin. A 70% and 25% increase in H₂O₂ formation was observed at 14 and 18 months of age, respectively, in comparison to the youngest group. Only in the presence of succinate was a 25% elevation in H₂O₂ found at 24 months of age. These observations parallel with the decrease of the ratio between tissue levels of reduced and oxidized glutathione that was observed at 14 and 18 months of age. The concentration of myocardial malondialdehyde, a secondary product of lipid peroxidation, remained the same at all ages measured, most probably because it is readly metabolized in vivo. On the contrary the myocardial level of lipofuscin, which is not degraded by the cell, progressively increased beginning from 18 months of age.     

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.     

Glutathione alters calcium responsiveness of cardiac skinned fibers

Summary The glutathione status of cardiac muscle, that is the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) changes in certain forms of cardiomyopathy and during reperfusion of ischemic heart. Here we show that it also affects the sensitivity of contractile proteins to calcium. GSH (4 mM) increased the pCa₅₀ for force development in skinned fibers by 0.2 pCa units and increased force ba 44%±5.4% at pCa 5.6 whereas GSSG (4 mM) decreased it by 54%±17.8% at pCa 5.6. Half maximal activations and inhibitions were seen with 4 mM GSH or GSSG, respectively.In contrast to GSH, the reducing agent dithiotreitol at 5 mM had no activating effect. Our results suggest that the loss of contractility observed after a reperfusion of the ischemic heart my, at least in part, be due to a decreased responsiveness of the contractile proteins due to an altered glutathione status.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 320).     

Cytokines induce stress protein formation in cultured cardiac myocytes

Summary Diseases accompanied by severe cardiac impairment like sepsis and chronic uremia are frequently linked to an increase in cytokine release. In order to investigate possible toxic effects of the immune mediators on myocardial cells, we studied the contractility of cardiac myocytes and the de novo formation of stress proteins in cultured heart cells under cytokine exposition. All cytokines investigated induce, concentration-dependently, arrhythmias and cessation of spontaneous contractions. Interleukin(IL)-2, IL-3, IL-6, and tumor necrosis factor (TNF) stimulate the synthesis of a 30 kD stress protein in heart cells, whereas IL-1 additionally evokes two proteins of the 70 kD family. These findings confirm a direct interference of the interleukins and TNF with myocytes and, especially, myocardial protein formation. As the induction of stress proteins makes cells more resistant towards a subsequent challenge, the cytokines are possibly involved in the activation of cell protecting mechanisms in cardiac myocytes.     

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     

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.     

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.     

Cardiac sarcoplasmic reticulum characteristics in hypertrophic hearts from spontaneously hypertensive rats

Summary Sarcoplasmic reticulum (SR) from left ventricles of rats that developed spontaneous hypertension was studied in vitro. Similar increases of left ventricular mass were found when grouping the animals into mild and severe hypertensives (average systolic arterial pressure of 168±4 and 202±6 mm Hg, respectively). The amount of SR protein (mg/g of left ventricle) was higher when obtained from hypertrophic ventricles of both hypertensive groups than from ventricles of the control group. The result agreed with the enhanced Ca²⁺ uptake exhibited by left ventricular homogenates of the hypertensive groups. Consequently, Ca²⁺ uptake in SR microsomes isolated per gram of left ventricle (nmol Ca²⁺/g muscle) was 51.62±10.06 and 64.99±12.84 in mildly and severely hypertensive groups vs. 17.37±5.79 in the control group (P<0.05). The SR microsomes obtained from ventricles of hypertensive rats showed an enhanced Ca²⁺ activated ATPase activity that was not accompanied by increased Ca²⁺ uptake at saturating calcium concentrations, but by increased affinity for calcium (K'_app of 1.09±0.28 and 2.67±0.16 M in SR microsomes of hypertrophic and control ventricles respectively; P<0.05). The rats of calcium loss, measured in SR vesicles passively loaded with⁴⁵Ca, were similar when assayed in SR obtained from ventricles of both hypertensive and normal rats. These results enable us to suggest that in hearts of rats presenting spontaneous hypertension, the function of the SR system could account for a normal handling of cytosolic calcium. They might support the absence of mechanical alterations described in hearts of young rats of the SHR strain.This work was supported by grants from the Consejo nacional de Investigaciones Científicas y Tecnicas de la República Argentina (CONICET).     

Hydrogen peroxide generation by mitochondria isolated from regionally ischemic and nonischemic dog myocardium

Summary We occluded the left anterior descending coronary artery of anesthetized, open-chest dogs, for 1 or 2 h. Some hearts were reperfused for 1 h after 1 h of ischemia. We isolated mitochondria from the central ischemic zone (CIZ) and a surrounding nonischemic zone (NIZ) of the left ventricle, and assayed H₂O₂ production using a horseradish peroxidase-dual wavelength spectrophotometric technique. Mitochondria, studied in the absence of exogenous respiratory chain inhibitors, generated H₂O₂ during State 4 respiration with succinate as the substrate. NIZ mitochondria in all groups produced ca. 1.5 nmols H₂O₂/min/mg protein (no significant differences between groups). The State 4 O₂ consumption rates of NIZ mitochondria from hearts subjected to 1 h ischemia plus reperfusion, or 2 h of ischemia (ca. 30 nmols/min/mg) were significantly higher than that of NIZ mitochondria of hearts subjected to only 1 h of ischemia (23 nmols/min/mg). Thus, the ratio between H₂O₂ produced and State 4 O₂ consumption fell from 6.5% to 5%. Mitochondria from all CIZ samples had State 4 O₂ consumption rates that were not different from corresponding NIZ values. However CIZ mitochondria of hearts subjected to 1 h ischemia without reperfusion produced less H₂O₂ (1.1±0.1 nmols/min/mg), and had a slightly reduced H₂O₂/O₂ ratio (4.4±0.7%), compared with their NIZ samples (1.5±0.1 nmols/min/mg; 5.3%). Reperfusion after 1 h of ischemia abolished these regional differences. The CIZ mitochondria from hearts subjected to 2 h ischemia produced only 0.75±0.22 nmols H₂O₂/min/mg (2.5% of State 4 O₂ consumption). These values were 50% of corresponding NIZ values, and were significantly less than for any other group or tissue region. If similar phenomena occur in conscious animals subjected to incomplete regional ischemia, especially of relatively brief duration or if accompanied by reduced intracellular defenses against oxidants such as H₂O₂, they suggest that mitochondria persist as H₂O₂ sources and so may contribute to the oxidant load and myocardial dysfunction.Supported by grants from the National Institutes of Health (HL-29499) and from the American Heart Association of Michigan     

Effects of in vivo manganese administration on calcium exchange and contractile force of rat ventricular myocardium

Summary The purpose of this study was to investigate the effect of prolonged (14 days) intragastric administration of Mn²⁺ (0.25 mmol/kg daily) on Ca²⁺ exchange and contractility of rat ventricular myocardium. Left-ventricular pressure and its first derivative (dP/dt) were recorded by means of a balloon catheter inserted via the left atrium into the left ventricle of the rat heart perfused by Langendorff method. Ca²⁺ exchange in the stimulated and rested ventricular myocardium was investigated with the aid of⁴⁵Ca under the conditions of complete equilibration of preparations with a solution containing⁴⁵Ca²⁺. The cellular⁴⁵Ca²⁺ content was calculated by subtraction of⁴⁵Ca²⁺ dissolved in the free water of extracellular space from the total tissue⁴⁵Ca²⁺ content. The cellular⁴⁵Ca²⁺ content in the stimulated (60/min) ventricles of control rats (without Mn²⁺) was 0.83 ± 0.09 mmol/kg wet weight (w.w.). Ten minutes of rest resulted in a gain of 0.06 mmol⁴⁵Ca/kg w.w. (not statistically significant). Fourteen days' exposure to Mn²⁺ resulted in an increase of the mean⁴⁵Ca content to 1.61 ± 0.09 mmol/kg w.w. in the stimulated preparations and to 1.35 ± 0.06 mmol/kg w.w. in the rested ones (p < 0.001). Thus, the control rest preparations did not change their Ca²⁺ content, while in the rats treated with Mn²⁺ the rest resulted in an increase at exchangeable Ca by 52 %. The maximal ventricular developed pressure (P_max) after 14 days of Mn²⁺ administration was increased by 35 % and dP/dt_max was 228 % of the value in the control group. The mean time from the maximal value of the first derivative to P_max (t-dP/dt_max) was reduced by one-half and mean time to peak developed pressure (TPT) was shortened to one-third of these in the control group. These results, although far from conclusive, do suggest that the long-lasting exposition to Mn²⁺ resulted in increased myocardial contractility caused, most probably, by the inhibition of Ca²⁺ efflux from the cell. Perhaps this is a compensation for the inhibitory effect of Mn²⁺ on the slow calcium channels.     

Calcium metabolism and depressed contractility in isolated human and porcine heart muscle

Summary Contractility is often depressed in isolated heart muscle. To analyze this phenomenon, we measured the derivative of left ventricular pressure (dP/dt) in intact and in isolated, blood perfused pig hearts, and peak force (F) or stress (F/mm²) in ventricular trabeculae of man and pig. When the heart was in the steady state at a priming frequency of 2 Hz an extrasystolic interval of 0.3 s was interposed, followed by four postextrasystolic intervals of 0.8 s. In the case of isolated trabeculae the priming frequency was 0.2 Hz, the extra interval 0.4 s, and the post-extrasystolic intervals were 5 s. The exponential decay of potentiation is characterized by the constant D: a low value of D indicates a rapid decay of potentiation. DP/dt was about 1000 mm Hg/s in the intact hearts, but within 1 h after isolation dP/dt decreased to about 700 mm Hg/s, and this was associated with a decrease in D from 0.63 to 0.40. Developed stress in the isolated trabeculac was about 2 mN/mm² and D was about 0.20 under standard, in vitro conditions (a.o. 1.5 mM Ca²⁺, 0.2 Hz stimulus frequency). This stress is only 10% of the calculated stress in the intact heart. An increase of priming frequency, or of [Ca²⁺], or addition of 30 nM isoproterenol to the perfusate caused a marked increase in F and D. Properties of human and porcine trabeculae were quantitatively similar. The strong correlation between dP/dt, or F, and D suggests a causal relationship. This is consistent with the current model of e-c coupling in heart muscle, in which the activity of the Ca²⁺ pump of the sarcoplasmic reticulum determines the decay of potentiation and the amount of releasable Ca²⁺ in the reticulum determines force of contraction. Since isoproterenol stimulates the Ca²⁺ pump in the reticulum, the increase in D and F induced by this drug is consistent with the model. We conclude, that the decreased dP/dt, F, and D in isolated preparations was due to impaired sarcoplasmic reticulum function. The role of this phenomenon in the stunned heart syndrome, species differences and possible causes are discussed.     

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.     

Mitochondrial respiration in myocardial biopsy samples as a criterion of postischemic recovery of the cardiac contractility

Summary Isolated perfused guinea pig hearts were arrested by a high K⁺ cardioplegic solution containing (PG group) or lacking (control group) 10 mM phosphocreatine +15 mM glutamate. Total normothermic ischemia lasted 45 min followed by 30 min reperfusion. Mitochondrial respiration in the absence and presence of different concentrations of ADP and creatine was studied in biopsy samples (6–8 mg) after saponin treatment. The samples were taken before and after ischemia, as well as after the reperfusion period. A slightly better relative recovery of developed pressure (RRDP) in PG group was associated with higher mitochondrial acceptor control ratio after reperfusion (5.74±0.32 vs. 4.54±0.21 in PG and control groups, resp., p<0.01). When the results obtained in both groups were treated together, tight correlations between the pre- or postischemic mitochondrial state and RRDP were revealed. Higher values of RRDP were found for the hearts with lower preischemic values of (low ADP+creatine)-stimulation of mitochondrial respiration (r=–0.57, p<0.01). Relative changes in this mitochondrial parameter during ischemic period were in a good correlation with the RRDP (r=0.82, p<0.001). The data suggest that the study of the mitochondrial function in myocardial biopsy samples before ischemia and reperfusion could provide a useful information for the prognosis of cardiac function recovery.     

A genetic abnormality of cardiac myocytes from the blind mutant (RC) chick heart: Abnormalities of cardiac structure and choline transport

Summary A new genetic cardiomyopathy was identified in a blind mutant avian strain. Cardiac myocytes were cultured from 7-day-old chick embryos from Rhode Island Red chickens and from another strain of this species that has been identified to have several abnormalities, the most striking of which is blindness. Cardiac myocytes were maintained in tissue culture. Morphologically, in culture, the cardiac myocyte from the blind mutant strain assumed a spherical shape and showed abnormalities of sarcolemmal membrane compared to control myocytes from heterozygous animals. Choline uptake and metabolism were examined, using [methyl³ H] choline, because it is a sarcolemmal transport process and choline is metabolised to phosphatidylcholine, an important phospholipid for cellular structure and function. Choline uptake through the active transport process was markedly and significantly reduced in the mutant cells compared to control cells, while choline metabolism to phosphatidylcholine was not significantly altered. These results demonstrate a new abnormality of cardiac myocytes, a cardiomyopathy that can be studied in cell culture and one with abnormalities of cellular choline transport.Supported in part by a grant from the Canadian (British Columbia) Heart Foundation     

Adaptation to stress exposure prevents arrhythmogenic and contractural effects of the excess of Ca2+ on the heart by the increased activity of sarcoplasmic reticulum

Summary Adaptation of rats to repcated short-term stress exposure prevents, to a considerable extent, contractural and arrhythmogenic effects of high concentrations of extracellular Ca²⁺ on isolated heart. Increased efficiency of SR Ca²⁺-pump functioning and a significant increase in Ca²⁺ pump resistance to autolysis are proved to play the main role in this effect.     

Excitation-contraction coupling in voltage-clamped cardiac myocytes

Summary Myocytes isolated from guinea pig ventricles were voltage-clamped using patch pipettes in the whole-cell configuration. For proper voltage control fast Na⁺ current was blocked by TTX or inactivated by an appropriate prepulse. Zero-load cell shortening was monitored by a photoelectric device. The mechanical response to a short depolarizing clamp was mainly a phasic (transient) contraction. Long-lasting depolarizations caused a tonic (sustained) shortening of a cell. Different clamp patterns were used to study the mode of activation of phasic contraction. 1) With a constant Ca²⁺ preload established by a train of conditioning pulses, the shortening-voltage relation measured with test pulses of varying height was a bell-shaped curve reflecting the slow inward current (I_Ca)-voltage relation. The test pulse had a striking influence on the first contraction of the following conditioning series, resulting in an S-shaped relation between post-test contraction and test potential. 2) With series of identical clamps of varying height, steady-state contraction was maximal around 40 mV and not in proportion to I_Ca. In these measurements Ca²⁺ preload was likely to increase with increasing potential. It is concluded that I_Ca initiates phasic contraction by inducing a release of Ca²⁺ from internal stores while replenishment of the stores is largely determined by an electrogenic transsarcolemmal Na⁺–Ca²⁺ exchange. The data suggest that Na⁺–Ca²⁺ exchange is not only involved in long-term changes of cardiac contractility but also in beat-to-beat regulation.     

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.     

Capillary neoformation in the rat heart-stereological studies on papillary muscles in hypertrophy and physiologic growth

Summary Stereological investigations on myocardial capillaries provided evidence that the common estimator of capillarity, the capillary density (i.c., number of capillary profiles per unit transverse sectional area), underestimates the true capillary supply since the capillary axes are not oriented perfectly in parallel to the myofiber axes. Recently, we studied the true capillarity, i.c., the length density of capillaries (L_v=capillary length per capillary volume), in some experimental models of cardiac hypertrophy which have been published elsewhere. It has been shown that L_v decreases in renovascular hypertension, but is maintained in physical exercise and after chronic thyroxin application. However, the growth pattern of capillaries in hypertrophic hearts has not yet been analyzed. In the present paper it is demonstrated that important information on the capillary network can be derived from the two-dimensional capillary-to-fiber ratios (2D CFR: capillary profiles per myofiber profiles in transverse sections) and from the three-dimensional capillary-to-fiber ratios (3D CFR: capillary length per unit myofiber length). Increase in both suggests neoformation of additional capillary branches in parallel connection. Retrospective analysis of the quantitative data indicates that in hypertrophy induced by physical exercise or by chronic thyroxin application capillary neoformation in parallel connection counterbalances increase of oxygen diffusion distance due to myofiber enlargement. In renovascular hypertension, capillary neoformation in parallel connection does not occur. Studies on normal growth indicated both a slight decrease of L_v of capillaries, as well as a continuous neoformation of additional capillary branches.