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Calcium Current in Human Heart. Introduction: Significant species-, issue-, and age-dependent differences have been described for the L-type calcium current (ICa). Therefore, extrapolation data obtained from the many animal models to human cardiac physiology is difficult. In this study, we have characterized the voltage-dependent properties of ICa from pediatric and adult, atrial and ventricular human heart tissue. Methods and Results: ICa, was measured in single human heart muscle cells using the “whole cell,” voltage clamp method. Single myocytes were isolated from myocardial specimens obtained intraoperatively from both pediatric and adult patients (ages 3 months to 75 years) undergoing cardiac surgery. Cells obtained for these experiments appeared to be healthy; the resting potential was between -80 and -85 mV. The action potential shape and duration and current-voltage relationship for 1Ca were similar to that reported by others for human heart cells. The steady-state activation variable, dx was found to be similar in both pediatric atrial and ventricular cells but shifted approximately 5 mV negative in the adult atrial and ventricular cells. I, of all cells displayed biex-ponential inactivation and steady-state inactivation was incomplete at positive potentials (steady-state inactivation curves turned up at positive potentials) consistent with inactivation arising from voltage-dependent and calcium-dependent processes as reported in heart cells from many species. The potential of maximal inactivation was more negative for adult cells (around -10 mV) than pediatric cells (around 0 mV). Estimates of the calcium “window” current, using a modified Hodgkin-Huxlcy model, could explain measured differences in action potential shape and duration. Conclusion: Human cardiac I, can be investigated using whole cell, voltage clamp methods and a modified Hodgkin-Huxley model. Quantitative characterization of many of the properties of ICa in human heart tissue suggests that important species differences do exist and that further investigations are required to characterize the dependence of inactivation on [Ca2+]i in human heart cells. Since the array of characteristics of ICa in different species varies, the study of human myocardial cells per se continues to be important when examining human cardiac physiology.

ATP-Sensitive Potassium Channels in Human Heart. Introduction: The purpose of this study was to examine the electrophysiologic derangements that underlie contractile failure in single human heart muscle cells exposed to metabolic inhibition. Methods and Results: Single myocytes were isolated from right atrial appendage specimens obtained intraoperatively from patients undergoing routine cardiac surgery. On exposure to lO-mM 2-deoxyglucose (to inhibit glycolysis) and 2-mM cyanide (to inhibit oxidative phosphorylation), twitch shortening decreased to undetectable levels over 5–6 minutes. The action potential duration declined in parallel with the contractile failure. Using voltage clamp depolarizations of a fixed duration the twitch was maintained in metabolic blockade until the development of maintained (rigor) contracture. At this time a large increase in K+ conductance, which can be attributed to the activation of ATP-sensitive K + channels (K ATP channels), was measured. In isolated inside-out membrane patches, the ATP dependence of KaTP channel activity was described by a sigmoid curve with Ki,ATP (ATP concentration required for half-maximal inhibition of K ATPchannel activity) = 8 μM and Hill coefficient (nH) = 1.2. The single channel current-voltage relationship reversed close to the K + equilibrium potential and the conductance was approximately linear (g = 29 pS) over the voltage range included in the action potential (-60 m V to +20 mV). Conclusion: In human atrial cardiac myocytes subjected to complete metabolic inhibition, contractile failure is caused by action potential shortening resulting from an increase in K + conductance presumably through the activation of KATP channels. (J Cardiovasc Electrophysiol, Vol. 3, pp. 56–63, February 1992)

Experimental data do not support the idea that excitation-contraction coupling in heart muscle can be explained by a simple calcium-induced calcium release mechanism alone or a simple voltage-dependent calcium release mechanism alone. Our data on excitation-contraction coupling combined with the results of others suggest the need to either develop more complex and more sophisticated single-mechanism models, or to establish dual-control models.

Benign metastasizing leiomyoma is a rare clinical entity that has been described in several previous reports. Although the exact pathophysiology of the disease is unknown, two predominant theories exist: (1) metastasis from ah existing leiomyoma (cornmonly seen with uterine leiomyoma) or (2) multicentric leiomyomatous growths rather than actual metastases. We present an interesting case in which several elements of the patient′s history complicated the differential diagnosis.

Hyperkalemic depolarized cardiac arrest has been the cornerstone of myocardial protection during cardiac surgery for more than 30 years. Many of the advances in myocardial protection seek to minimize the cellular damage and to reduce the ongoing metabolic processes occurring as a direct consequence of the depolarized state. Ideally, cardiac arrest at hyperpolarized cellular membrane potentials--the natural resting state of the heart--will meet all the requirements of modern cardioplegia, namely, electromechanical asystole and cardiac relaxation, while preserving the vital integrity of the heart itself.To determine whether activation of adenosine triphosphate-sensitive potassium channels by pharmacologic agents could produce hyperpolarized cardiac arrest, we tested the ability of aprikalim, a known adenosine triphosphate-sensitive potassium channel opener, to arrest the intact beating heart. In a normothermic (37 degrees C) isolated rabbit heart preparation, aprikalim was found to rapidly shorten the action potential duration and produce cardiac asystole that was maintained during 20 minutes of "no-flow" global ischemia without a rise in end-diastolic pressure. Cardiac rhythm and function were fully restored by reperfusion alone (developed pressure was 100.6% +/- 7.9% of prearrest value after 30 minutes of reperfusion). In contrast, 20 minutes of unprotected normothermic global ischemia resulted in a 2.7 +/- 0.55 mmHg rise in end-diastolic pressure and only 58.2% +/- 3.8% recovery of developed pressure after 30 minutes of reperfusion. By way of comparison, 20 minutes of standard hyperkalemic depolarized normothermic rest was accompanied by a 1.2 +/- 0.6 mmHg rise in end-diastolic pressure and only 80.8% +/- 2.6% recovery of developed pressure after 30 minutes of reperfusion. To directly compare hyperkalemic depolarized cardiac arrest to hyperpolarized cardiac arrest induced by potassium channel openers and to better define the characteristics of such hyperpolarized arrest, we studied a fixed (4 mmHg rise in end-diastolic pressure--contracture) ischemic injury model. The time to development of the contracture was prolonged by hyperkalemic arrest (35.8 +/- 1.7 minutes) and significantly more so by hyperpolarized arrest (47.0 +/- 3.3 minutes) when compared with that of unprotected hearts (24.0 +/- 1.2 minutes). Moreover, aprikalim resulted in significantly better postischemic recovery of function (developed pressure was 69.0% +/- 6.7% of prearrest value after 30 minutes of reperfusion) than after no cardioplegia (45.4% +/- 7.5%) or standard hyperkalemic cardioplegia (44.3% +/- 5.7%).Pharmacologic activation of adenosine triphosphate-sensitive potassium channels can result in predictable and sustainable hyperpolarized cardiac arrest that is reversible by reperfusion. This method of myocardial protection was found to fully preserve cardiac electromechanical function after a 20-minute period of global normothermic ischemia. Furthermore, hyperpolarized arrest induced by potassium channel openers significantly prolonged the period to the development of contracture and afforded a significantly better postischemic recovery of function than obtained in either hearts protected with hyperkalemic depolarized arrest or those not protected by any form of cardioplegia.

The British Regional Heart Study seeks to define risk factors for cardiovascular disease, to examine their interrelationships, and to explain the geographic variations in cardiovascular disease in Britain. A clinical survey of men aged 40-59 in 24 British towns was carried out and preliminary data from the survey analysed. On a town basis cardiovascular mortality was associated with mean systolic blood pressure and the prevalence of heavy cigarette smoking and heavy alcohol consumption. No such association was seen for body mass index or mean serum total cholesterol or high-density-lipoprotein cholesterol concentration. Cigarette smoking and alcohol intake and, to a less degree, systolic blood pressure were related to the social class (percentage of manual workers) of a town, and these factors may determine to some extent the increased risk of cardiovascular disease in manual workers. Blood pressure in individual subjects was affected predominantly by age, body mass index, and alcohol intake. Body mass index appeared to affect blood pressure to a greater extent than alcohol intake and did so with a consistent and positive linear trend. Nevertheless, the differences between towns in mean blood pressure readings appeared to be more closely associated with variations in the prevalence of heavy drinking than with variations in body mass index. Alcohol intake and body mass index explained only a part of the striking differences between towns in mean blood pressure readings, and some important "town"factors remained unexplained.

1. Calcium current (ICa) was recorded in single rat heart cells at two periods during development: (1) at 2-7 days post-partum (neonatal), and (2) at 6-8 weeks (adult). 2. We measured both transient and steady-state components of ICa and could describe ICa in terms of the steady-state activation (d infinity) and inactivation (f infinity) parameters, the channel reversal potential (Echannel) and a relative conductance parameter, gr. 3. In adult single cells, the application of ryanodine (10 microM), an agent known to alter the function of the sarcoplasmic reticulum (SR), abolished contraction rapidly and increased ICa. Ryanodine also produced a 13 mV shift in f infinity towards more positive potentials and altered its slope, while producing a small increase in gr but no effect on d infinity. In neonatal single cells, ryanodine (10 microM) had no significant effect on contraction, ICa, d infinity, f infinity, or gr. Caffeine (10 mM), a less specific agent widely used to investigate sarcoplasmic reticulum function, had actions similar to those of ryanodine. 4. In adult myocytes, when EGTA (10 or 20 mM) or bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 10 mM) were included in the pipette solution, contractions were rapidly abolished, while a small (4 mV) shift of f infinity to more positive potentials was seen. A large additional shift of f infinity was observed when ryanodine (10 microM) was added to the superfusion solution in the continued presence of EGTA or BAPTA. The alterations of ICa in EGTA (or BAPTA) plus ryanodine were the same as those seen in ryanodine alone. In neonatal cells, in contrast, when EGTA or BAPTA were included in the pipette solution we observed only a small effect on f infinity and the application of ryanodine had no effect. 5. Electron micrographs of our preparations show that the dissociated adult cells have sharp sarcolemmal borders, fully developed sarcomeres with T-tubules and sarcoplasmic reticulum membranes. In contrast, the neonatal cells that we use have few of these intracellular structures. Our observations in these preparations are consistent with the work of others (e.g. Penefsky, 1974; Hirakow & Gotoh, 1975; Ishikawa & Yamada, 1975; Legato, 1975; Hoerter, Mazet & Vassort, 1981). 6. Our data suggest that fully developed sarcoplasmic reticulum in rat heart cells can affect ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of increased protein kinase C activity were studied in neonatal rat myocytes grown in primary culture. The changes in mechanical and electrical behavior, as well as protein phosphorylation, that followed the apparent activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined. As spontaneous beating frequency was increased minimally by 10 nM TPA and by 100% with 85 nM TPA, shortening amplitude, shortening velocity, and relaxation velocity decreased concomitantly. In contrast, 4-alpha-phorbol-12,13-didecanoate (alpha-PDD), which does not activate protein kinase C, had no effect on beating behavior at 800 nM. In voltage-clamped single myocytes, both steady-state and transient components of the cadmium-sensitive calcium current were increased by the addition of TPA (65 nM). Neither the time constant for the inactivation of the transient component of this calcium current nor the reversal potential was altered by TPA. The phosphorylation state of a discrete set of proteins, with apparent molecular weights of 32 and 83 kDa, was enhanced when TPA was added to intact myocytes. Angiotensin II enhances the phosphorylation state of the same set of proteins as observed with TPA. We conclude that activation of protein kinase C can modify mechanical behavior and increase L-type Ca2+ channel activity in cultured neonatal rat ventricular myocytes. The remarkable similarity in mechanical, electrical, and protein phosphorylation responses of cultured neonatal myocytes following TPA or angiotensin II application indicate that protein kinase C may mediate the action of angiotensin II.

1. Calcium currents (ICa) from neonatal rat ventricular heart muscle cells grown in primary culture were examined using the 'whole-cell' voltage-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Examination of ICa was limited to one calcium channel type, 'L' type (Nilius, Hess, Lansman & Tsien, 1985), by appropriate voltage protocols. 2. We measured transient and steady-state components of ICa, and could generally describe ICa in terms of the steady-state activation (d infinity) and inactivation (f infinity) parameters. 3. We observed that the reduction of ICa by the calcium channel antagonist D600 can be explained by both a shift of d infinity to more positive potentials as well as a slight reduction of ICa conductance. D600 did not significantly alter either the rate of inactivation of ICa or the voltage dependence of f infinity. 4. The calcium channel modulator BAY K8644 shifted both d infinity and f infinity to more negative potentials. Additionally, BAY K8644 increased the rate of inactivation at potentials between +5 and +55 mV. Furthermore, BAY K8644 also increased ICa conductance, a change consistent with a promotion of 'mode 2' calcium channel activity (Hess, Lansman & Tsien, 1984). 5. We conclude that, as predicted by d infinity and f infinity, there is a significant steady-state component of ICa ('window current') at plateau potentials in neonatal rat heart cells. Modulation of the steady-state and transient components of ICa by various agents can be attributed both to specific alterations in d infinity and f infinity and to more complicated alterations in the mode of calcium channel activity.

The effect of angiotensin II on cultured neonatal rat heart myocytes was studied by measuring changes in cell length, the magnitude and kinetics of the calcium current, and changes in cyclic adenosine 3',5'-monophosphate (cAMP) and phosphoinositide metabolism. Spontaneous beating frequency of multicellular networks was increased by angiotensin II with a maximal increase of 100% above control values at concentrations of 5 nM or greater. The half-maximal response occurred at 0.6 nM angiotensin II. Shortening amplitude, shortening velocity, and relaxation velocity decreased concomitantly with the increasing contractile rate. In voltage-clamped single myocytes, both steady-state and transient components of the calcium current were increased by the addition of angiotensin II. Angiotensin II had no effect on either control or isoproterenol-stimulated adenylate cyclase activity in myocyte membranes. Neither the basal levels nor the isoproterenol-stimulated cAMP accumulation in intact cells was affected by addition of hormone. In myocytes labeled with [3H]inositol, angiotensin II stimulated the formation of [3H]inositol phosphates. One minute after addition of 5 nM angiotensin II, inositol monophosphate and inositol bisphosphate levels were increased to 73% and 99%, respectively, above control values and remained elevated at 10 minutes. Inositol trisphosphate levels were not significantly different from control values at either time point. Nifedipine (10 microM) had no effect on angiotensin II-induced increases in [3H]inositol phosphates. We conclude that the increases in both spontaneous beating rate and calcium current in angiotensin II-stimulated cultured neonatal heart cells are not dependent on cAMP or inositol trisphosphate levels but may involve sustained phosphoinositide hydrolysis.

We have investigated the links between electrical excitation and contraction in mammalian heart muscle. Using isolated single cells from adult rat ventricle, a whole-cell voltage-clamp technique and quantitative fluorescence microscopy, we have measured simultaneously calcium current (Ica) and [Ca2+]i (with fura-2). We find that the voltage-dependence of Ica and the [Ca 2+]i-transient and the dependence of [Ca2+]i-transient on depolarization-duration cannot both be readily explained by a simple calcium-induced Ca-release (‘CICR’) mechanism. Additionally, we find that when [Ca2+]i and [Na+]i are at their diastolic levels, activation of the Na-Ca exchange mechanism by depolarization does not measurably trigger the release of Ca2+i. Finally, measuring Ica in adult and neonatal rat heart cells and using the alkaloid ryanodine, we have carried out complementary experiments. These experiments show that there may be an action of ryanodine on Ica that is independent of [Ca2+]i and independent of a direct action of the alkaloid on the calcium channel itself. Along with experiments of others showing that ryanodine binds to the sarcoplasmic reticulum calcium-release channel/spanning protein complex, our data suggests a model to explain our findings. The model links the calcium channels responsible for Ica to the sarcoplasmic reticulum by means of one or more of the spanning protein(s). Information from the calcium channel can be communitated to the sarcoplasmic reticulum by this route and, presumably, information can move in the opposite direction from the sarcoplasmic reticulum to the calcium channel.