Martin Morad

Excitation-contraction coupling in heart muscle: Membrane control of development of tension☆

III. EXCITATION-CONTRACT/ON COUPLING PROCESSES IN THE MAMMALIAN VENTRICLE A. Structure 1. Transverse-tubular system 2. Sarcoplasmic reticulum B. Functional Implications of the Structure of the Mammalian Myocardium C. Action Potential Control of Contraction D. Membrane Potential and Development of Tension in Mammalian Ventricle E. Potassium Chloride Contracture Studies F. Ionic and Drug Dependence of the Two Components of Tension in Mammalian Heart 1. The phasic response 2. The tonic response G. The Mechanism of Development of the Tonic Tension H. The Mechanism of the Triggered Release of the Phasic Store 1. Secondary inward current and activation of contraction 2. Other observations concerning the secondary inward current I. Replenishment of the Phasic Store 1. Depletion of the activator store 2. Beat-dependent kinetics 3. Quantity of activator in the phasic store and the concept of recirculation 4. An estimate of the magnitude of the recirculated fraction of activator 5. Delay in availability of activator 6. Anatomical correlates 7, Plateau and replenishment J. A Model of the E.-C Coupling Mechanism in Mammalian Heart Muscle K. Events Leading to a Normal Contraction L. A Mechanism for the Altered Inotropic State (the Interval Strength Relation) 1. Post-extrasystolic potentiation 2. Treppe 3. Other theories (a) Inotropic mediator hypothesis (b) Sodium-lag hypothesis

ROLE OF CA2+ CHANNEL IN CARDIAC EXCITATION-CONTRACTION COUPLING IN THE RAT: EVIDENCE FROM CA2+ TRANSIENTS AND CONTRACTION

1. Optical methods were used to measure simultaneously unloaded cell shortening and intracellular Ca2+ transients in whole‐cell voltage clamped rat ventricular myocytes. Red light (greater than 670 nm) was used to measure cell shortening with a linear photodiode array. The dyes Fura‐2 (Kd = 140 nM) and Mag‐Fura‐2 (Kd = 44 microM) were used as Ca2+ indicators with fluorescence excitation at 340 and 410 nm and emission at 510 nm. 2. Repeated measurements at 6 s intervals as 0.4 mM‐Fura‐2 diffused into the cell from the tip of the voltage clamp pipette showed no decrease in the rate of rise and peak value of the intracellular Ca2+ transient and only a small suppression of cell shortening, suggesting that the molecular mechanisms regulating the Ca2+ release were not significantly altered by the buffering capacity of the Fura‐2. 3. Experiments in which the sarcoplasmic reticulum (SR) was depleted of Ca2+ either by exposure to caffeine or by repeated brief (20 ms) voltage clamp depolarizations confirm that the SR is the major source of activator Ca2+. 4. Mag‐Fura‐2 (1 or 5 mM) was used to register the initial rapid development of the [Ca2+]i transient but the later time course of the Ca2+ transients measured with this dye was obscured by motion artifacts resulting from cell shortening. 5. Both Fura‐2 and Mag‐Fura‐2 showed that depolarization to 0 mV from a holding potential of ‐80 mV resulted in a [Ca2+]i transient which developed with a delay of 3‐9 ms and approached its peak value in an additional 8‐19 ms. Both Ca2+ indicators also showed that the Ca2+ transient approached its peak value more slowly as the clamped membrane potential was made increasingly more positive. 6. The voltage dependencies of the Ca2+ signal (Fura‐2) and cell shortening were both bell‐shaped and were qualitatively similar to the voltage dependence of Ca2+ current simultaneously measured. This was observed with holding potentials of both ‐40 and ‐80 mV. 7. Comparison of the temporal relation of the Ca2+ current, ICa, and intracellular Ca2+ transient (Fura‐2) and cell shortening at different membrane potentials showed that Ca2+ transient measured 25 ms into the depolarization correlated closely to the integral of the Ca2+ current measured prior to this time. Cell shortening, on the other hand, peaked about 100 ms later and correlated with measurements of the Ca2+ activity at the later time.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro Modeling of Ryanodine Receptor 2 Dysfunction Using Human Induced Pluripotent Stem Cells

Background/Aims: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. Methods: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. Results: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca2+ release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca2+-induced Ca2+-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. Conclusion: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

Relaxing effects of catecholamines on mammalian heart

1. The effect of catecholamines on the time course and amplitude of contraction and on KCl‐induced contractures has been studied in mammalian hearts.

Familial Hypertrophic Cardiomyopathy-Linked Mutant Troponin T Causes Stress-Induced Ventricular Tachycardia and Ca2+-Dependent Action Potential Remodeling

Abstract— The cardiac troponin T (TnT) I79N mutation has been linked to familial hypertrophic cardiomyopathy and high incidence of sudden death, despite causing little or no cardiac hypertrophy in patients. Transgenic mice expressing mutant human TnT (I79N-Tg) have increased cardiac contractility, but no ventricular hypertrophy or fibrosis. Enhanced cardiac function has been associated with myofilament Ca2+ sensitization, suggesting altered cellular Ca2+ handling. In the present study, we compare cellular Ca2+ transients and electrophysiological parameters of 64 I79N-Tg and 106 control mice in isolated myocytes, isolated perfused hearts, and whole animals. Ventricular action potentials (APs) measured in isolated I79N-Tg hearts and myocytes were significantly shortened only at 70% repolarization. No significant differences were found either in L-type Ca2+ or transient outward K+ currents, but inward rectifier K+ current (IK1) was significantly decreased. More critically, Ca2+ transients of field-stimulated ventricular I79N-Tg myocytes were reduced and had slow decay kinetics, consistent with increased Ca2+ sensitivity of I79N mutant fibers. AP differences were abolished when myocytes were dialyzed with Ca2+ buffers or after the Na+-Ca2+ exchanger was blocked by Li+. At higher pacing rates or in presence of isoproterenol, diastolic Ca2+ became significantly elevated in I79N-Tg compared with control myocytes. Ventricular ectopy could be induced by isoproterenol-challenge in isolated I79N-Tg hearts and anesthetized I79N-Tg mice. Freely moving I79N-Tg mice had a higher incidence of nonsustained ventricular tachycardia (VT) during mental stress (warm air jets). We conclude that the TnT-I79N mutation causes stress-induced VT even in absence of hypertrophy and/or fibrosis, arising possibly from the combination of AP remodeling related to altered Ca2+ transients and suppression of IK1.

Species differences in the activity of the Na(+)-Ca2+ exchanger in mammalian cardiac myocytes.

1. Species differences in the activity of the exchanger were evaluated in isolated myocytes from rat, guinea‐pig, hamster ventricles and human atria. Fluorescence measurements using fura‐2 were carried out in conjunction with the whole‐cell patch‐clamp technique for simultaneous recording of membrane currents and intracellular Ca2+ concentration. 2. Ca2+ release from sarcoplasmic reticulum (SR) induced either by rapid application of caffeine or by Ca2+ current elicited inward Na(+)‐Ca2+ exchange currents (INa‐Ca). The magnitude of INa‐Ca was largest in hamster, smallest in rat, with guinea‐pig and human myocytes having intermediate values. The ratio of caffeine‐induced exchanger current densities, normalized with respect to the peak Ca2+ release, was 4:2:1.5:1 for hamster > guinea‐pig > or = human > or = rat myocytes. 3. The rates of Ca2+ removal in the presence of caffeine, which reflect primarily the Ca2+ extruding activity of the Na(+)‐Ca2+ exchanger, followed the same order of hamster > guinea‐pig > or = human > or = rat. 4. The kinetics of INa‐Ca vs. Ca2+ transients were different among species. In rat myocytes, the kinetics of the INa‐Ca and the Ca2+ transients were similar, with INa‐Ca linearly proportional to intracellular Ca2+ concentration ([Ca2+]i). In hamster myocytes, the time course of INa‐Ca tracked only the declining phase of the Ca2+ transient with INa‐Ca having faster kinetics during the Ca2+ release. These findings suggest that the Ca2+ concentrations in the vicinity of the exchanger were significantly higher than those of the cytosol during Ca2+ release in hamster myocytes. 5. We concluded that there are significant species differences in the exchanger activity of cardiac myocytes, arising from differences in exchanger densities, their modulation and/or their spatial distribution with respect to the ryanodine receptors of cardiac myocytes.

Remodelling of ionic currents in hypertrophied and failing hearts of transgenic mice overexpressing calsequestrin

1 Overexpression of cardiac calsequestrin (CSQ) impairs Ca2+ signalling in murine myocytes, leading to marked cardiac hypertrophy. Here we report on contractile, histological and electrophysiological changes accompanying the development of cardiac hypertrophy and failure in CSQ‐overexpressing mice. 2 CSQ mice developed contractile dysfunction after 60 days of age, with only 40% survival at 6 months. Four‐ to 6‐month‐old CSQ mice revealed biventricular dilatation, cardiomyocyte hypertrophy, patchy interstitial fibrosis and tissue calcifications. 3 Cardiac hypertrophy of CSQ mice was accompanied by progressive P‐R and Q‐T interval prolongation, conduction blocks, 2‐fold prolongation of the ventricular action potential and increased cellular membrane capacitance. 4 Remodelling of ionic currents included marked reduction of both density and absolute magnitude of transient outward (Ito) and inward rectifying (IK1) K+ currents. The density, but not the absolute magnitude, of basal and isoproterenol (isoprenaline)‐stimulated Ca2+ current (ICa) was decreased by 42% and the inactivation kinetics of ICa were significantly slowed. Na+ current density was suppressed by 50%, but its steady‐state activation and inactivation were shifted to more positive potentials. The density of Na+‐Ca2+ exchange current was increased by 35%. 5 In CSQ but not in control myocytes dialysed with cAMP, isoproterenol continued to enhance ICa. This apparent lower responsiveness of ICa to cAMP could be reversed by the non‐hydrolysable cAMP analogue 8‐Br‐cAMP or the phosphodiesterase inhibitor IBMX, suggesting high phosphodiesterase activity of CSQ myocytes. 6 In young CSQ mice (< 60 days) with compensated cardiac hypertrophy, only Ito was significantly suppressed. All other currents remained relatively intact. 7 An increase in cardiac Ca2+‐storage capability by overexpression of CSQ results in a dilated cardiomyopathy with tissue fibrosis, calcifications, impaired β‐adrenergic signalling and progressive remodelling of ionic currents. The extent of the changes in ionic currents was age dependent.

Molecular Determinants of L-type Ca2+ Channel Inactivation SEGMENT EXCHANGE ANALYSIS OF THE CARBOXYL-TERMINAL CYTOPLASMIC MOTIF ENCODED BY EXONS 40–42 OF THE HUMAN α1CSUBUNIT GENE

Recently we have described a splice variant of the L-type Ca2+ channel (α1C,86) in which 80 amino acids (1572–1651) of the conventional α1C,77 were substituted by another 81 amino acids due to alternative splicing of exons 40–42. Ba2+ current (IBa ) through α1C,86 exhibited faster inactivation kinetics, was strongly voltage-dependent, and had no Ca2+-dependent inactivation. An oligonucleotide-directed segment substitution and expression of the mutated channels in Xenopus oocytes were used to study the molecular determinants for gating of the channel within the 80-amino acid domain. Replacement of segments 1572–1598 or 1595–1652 of the “slow” α1C,77 channel with the respective segments of the “fast” α1C,86 gave rise to rapidly inactivating α1C,86-like channel isoforms. We found that replacement of either motifs 1572IKTEG1576 or1600LLDQV1604 of α1C,77 with the respective sequences of α1C,86 caused strong but partial acceleration of I Ba inactivation. Replacement of both sequences produced an α1C,86-like fast channel which had no Ca2+-dependent inactivation. These results support the hypothesis that motifs 1572–1576 and 1600–1604 of α1C,77 contribute cooperatively to inactivation kinetics of α1C and are critical for Ca2+-dependent inactivation of the channel.

Does the “pacemaker current” generate the diastolic depolarization in the rabbit SA node cells?

Small preparations of spontaneously beating rabbit sino-atrial node (SA node) were voltage clamped with the two-microelectrode technique. The effects of 0.25–5 mM Cs+ on the spontaneous pacing rate and the time-dependent inward “pacemaker” current,ih, were studied. In the presence of 2 mM Cs+, the spontaneous pacing rate decreased only slightly even thoughih was strongly depressed at potentials negative to −60 mV Cs+ had little or no effect on other time-dependent currents observed with clamp pulses less negative than −50 mV. Since no voltage-dependence to the Cs+ effect onih could be measured (between −90 mV and −20 mV), it was considered unlikely that the lack of Cs+ effect on the rate of diastolic depolarization results from a voltage-dependent effect of Cs+ on theih channel.Adrenaline produced a marked positive chronotropic effect in Cs+-treated SA node cells. This effect was accompanied by marked enhancement of the slow inward current (isi) with no change in the Cs+-blockedih current. These results are consistent with the idea thatih plays a minor role in generation of pacemaker depolarization, and suggest a more prominent role ofisi in the generation of diastolic depolarization in SA nodal cells.

Enhanced Na(+)-Ca2+ exchange activity in cardiomyopathic Syrian hamster.

The signaling of contraction by Ca2+ in the Syrian hamster (BIO 14.6) heart in the late stage of the cardiomyopathy (220 to 300 days old) was compared with that in age-matched healthy hamster hearts. Membrane current and cell shortening or intracellular Ca2+ transients were measured simultaneously in isolated whole-cell-clamped myocytes. The density of the L-type Ca2+ current was smaller in myopathic than in normal myocytes (2.13 +/- 0.3 versus 3.21 +/- 0.4 pA/pF at 0 mV, P < .05). In both control and myopathic myocytes, the L-type Ca2+ current gated the release of Ca2+ and activation of contraction. In myopathic myocytes, activation of contraction also activated a slowly inactivating inward current of 1.73 +/- 0.2 pA/pF. The Na(+)-Ca2+ exchanger generated this current (INa-Ca), because it was suppressed by rapid replacement of Na+ with Li+ and depletion of the intracellular Ca2+ pool by caffeine. INa-Ca, activated by rapid application of caffeine, was not significantly different in both groups (3.7 +/- 0.5 pA/pF in control hearts versus 3.9 +/- 0.5 pA/pF in cardiomyopathic hearts). The activation of the inward exchanger current in myopathic myocytes coincided with a significant prolongation of contraction and the intracellular Ca2+ transient and a delay in the onset of relaxation. These results suggest that the enhanced activity of the Na(+)-Ca2+ exchanger may be related to compromised sequestration of Ca2+ in these animals.