E. Erdmann

Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure.

BackgroundExperiments were performed in human ventricular myocytes to investigate properties of excitation-contraction coupling in patients with terminal heart failure. Myocytes were isolated from left ventricular myocardium of patients with cardiac failure caused by dilated or ischemic cardiomyopathy undergoing transplantation. These results were compared with those obtained from cells of healthy donor hearts that for technical reasons were not suitable for transplantation. Methods and Results[Ca2+]i, transients and Ca2+ currents were recorded from isolated cells under voltage clamp perfused internally with the Ca2+ indicator fura 2. In cells that were stimulated externally, the cell-permeant form of the indicator, fura 2-AM, was used. When action potentials were to be recorded, cells were stimulated in current clamp mode. Unstimulated Ca2+ current densities were not significantly different in myopathic and control cells. In diseased myocytes, resting [Ca2+]i levels were 165±61 nmol/l, compared with 95±47 nmol/l in normal cells. With 5 mmol/l Na+ in the pipette, peak [Ca2+]i transients were 367±109 and 746±249 nmol/l, respectively. The decline of [Ca2+]i during diastole was significantly slower in myopathic cells than in control cells. This was a result of a prolongation of the action potential and of a reduced Ca2+ sequestration by the sarcoplasmic reticulum. ConclusionsThese results may partly explain the alterations of contractility in vivo in patients with heart failure.

Alterations of K+ currents in isolated human ventricular myocytes from patients with terminal heart failure.

Prolongation of the action potential has been postulated to be a major reason for the altered diastolic relaxation of the heart in patients with severe heart failure. To investigate the electrophysiological basis for this action potential prolongation in terminal heart failure, K+ currents were recorded in single ventricular myocytes isolated from 16 explanted hearts of patients undergoing transplantation. Results from diseased hearts were compared with ventricular cells isolated from six undiseased donor hearts. Action potential duration was significantly prolonged in cells from patients with heart failure. A delayed rectifier K+ current was hardly detectable in most cells, and if it could be recorded, it was very small in both diseased and undiseased cells. When currents were normalized for cell surface area, the average current density of the inward rectifier K+ current was significantly reduced in diseased cells when compared with normal control cells (hyperpolarization at -100 mV, -15.9 +/- 2.2 vs -9.0 +/- 1.2 microA/cm2; P < .01). In addition, a large transient outward K+ current could be recorded in human myocytes. The average current density of the time-dependent component of this transient outward K+ current was significantly reduced in heart failure (depolarization at +40 mV, 9.1 +/- 1.0 vs 5.8 +/- 0.64 microA/cm2; P < .01). Action potential prolongation in severe heart failure may partially be explained by a reduction in current densities of the inward rectifier K+ current and of the transient outward K+ current. These alterations may thereby have a significant effect on cardiac relaxation.

Characteristics of transient outward current in human ventricular myocytes from patients with terminal heart failure.

A variety of outward currents exists in ventricular myocardium of different species influencing action potential duration and electrical activity. Transient outward currents have been reported in ventricular tissue of some animals but are small or absent in others. This study was conducted to investigate whether a transient outward current exists in human ventricular myocardium and to characterize its basic electrophysiological properties. Currents were recorded from enzymatically isolated human ventricular myocytes obtained from explanted hearts of 22 patients with terminal heart failure. In almost all cells studied, a transient outward current could be recorded on depolarization to between -20 and +80 mV. The size of the transient outward current was usually large enough to mask the Ca2+ current. It could be recorded under conditions in which Ca2+ influx and intracellular Ca2+ transients were suppressed. Basic current characteristics were similar to transient outward currents observed in other species. Inactivation of the transient outward current was monoexponential, with a time constant of 54.8 +/- 3.7 milliseconds at +40 mV. Half-maximal activation occurred at 16.7 +/- 1.6 mV; half-maximal steady-state inactivation occurred at -34.5 +/- 2.3 mV. Frequency-dependent reduction of peak transient outward current was 29.8 +/- 1.4% at 2 Hz compared with resting conditions. Recovery from inactivation was voltage dependent and had a biexponential time course; the faster time constant (41.0 +/- 6.5 milliseconds at -80 mV) accounted for 86.0 +/- 5.2% of total current. The transient outward current was sensitive to 4-aminopyridine (IC50, 1.15 mM). These results indicate that a large Ca(2+)-independent transient outward K+ current is present in human ventricular myocytes that might be regulated by physiological or pathological events and is a potential site for pharmacological intervention.

Cardiac glycoside receptor, (Na+ + K+)ATPase activity and force of contraction in rat heart

Abstract In order to elucidate the role of (Na+ + K+)ATPase and of the ouabain binding sites in the pharmacological effects of ouabain in the digitalis insensitive species rat, specific [3H]ouabain binding and (Na+ + K+)ATPase activity were measured simultaneously in a rat heart cell membrane preparation. Specific [3H]ouabain binding, 86Rb+-uptake and force of contraction were also measured simultaneously in electrically stimulated contracting ventricular strips of rat heart. The following results were obtained: (1) Rat heart cell membranes exhibit two classes of [3H]ouabain binding sites with apparent dissociation constants (KD) of the [3H]ouabain-receptor complex 1–2.3 × 10−7 M and 2.8 × 10−5 M. (2) (Na+ + K+)ATPase activity of rat heart cell membranes is half-maximally inhibited by ouabain at a concentration of 4 × 10−5 M when assayed at the same conditions as [3H]ouabain binding. (3) Specific [3H]ouabain binding to electrically stimulated (1 Hz) contracting ventricular strips of rat heart exhibited only one class of receptors (KD = 3 × 10−7M). Force of contraction increased half-maximally at 3 × 10−7 M ouabain when measured simultaneously and 86Rb+uptake was inhibited half-maximally at 3 × 10−5 M ouabain. Thus, there is a serious discrepancy between the effect of ouabain on (Na+ + K+)ATPase activity and 86Rb+ uptake on one hand and on force of contraction on the other hand, whereas there is a good correlation between [3H]ouabain-receptor binding and increase in force of contraction. These results indicate that inhibition by ouabain of active cation transport is not a mandatory prerequisite of its positive inotropic effect, at least in the rat heart.

Contribution of cAMP-phosphodiesterase inhibition and sensitization of the contractile proteins for calcium to the inotropic effect of pimobendan in the failing human myocardium.

Previous studies have shown reduced effects of cAMP-dependent positive inotropic agents in the failing human myocardium; thus other cAMP-independent mechanisms of action may be useful to increase force of contraction in this condition. The purpose of this investigation was to determine whether a positive inotropic effect of the cAMP-phosphodiesterase (PDE) inhibitor pimobendan is observed in the failing human myocardium and to study whether other factors, such as an increase in the Ca2+ sensitivity of myofilaments, play a functional role in the increase in force of contraction. Pimobendan produced a positive inotropic effect in isolated preparations from nonfailing donor hearts; however, in moderately (New York Heart Association class II-III, NYHA II-III) and severely (NYHA IV) failing myocardium, this effect was reduced. In addition, in NYHA IV specimens pimobendan inhibited the crude cAMP-PDE (crude PDE) and the isoenzymes I-III (PDE I-III) in a concentration-dependent way. As judged from the IC50 values found in this tissue for the inhibition of PDE III and of crude PDE, the potency of the compound was 18.1 times greater on PDE III. Consistent with a cAMP-PDE-dependent mechanism of action, the positive inotropic effect was potentiated by isoproterenol and inhibited by adenosine in failing myocardium. In failing myocardium, pimobendan also increased the sensitivity of skinned cardiac fibers to Ca2+ and shifted the Ca(2+)-tension relation to the left. This sensitizing effect began at 0.01 mumol/l in NYHA II-III and NYHA IV and rose to about 200% at 300 mumol/l in both groups. In contrast, the demethylated metabolite UD-CG 212 Cl failed to produce positive inotropic effects in failing myocardium alone, but in the presence of isoproterenol, it exerted an increase in force of contraction. The potency of UD-CG 212 Cl for PDE III inhibition in NYHA IV was greater than that of pimobendan. The metabolite pronouncedly decreased the sensitivity of skinned cardiac fibers to Ca2+ at 30-300 mumol/l in NYHA II-III and NYHA IV. It is concluded that in the failing human heart pimobendan inhibited PDE III and sensitized contractile proteins for Ca2+. Both effects appear to be involved in the positive inotropic effect of the compound, because its metabolite, UD-CG 212 Cl, had no effect on force of contraction and on the Ca2+ sensitivity of skinned cardiac fibers but inhibited PDE III even more potently than pimobendan.(ABSTRACT TRUNCATED AT 400 WORDS)

Characteristics of calcium-current in isolated human ventricular myocytes from patients with terminal heart failure

The Ca(2+)-current plays a prominent role in triggering excitation-contraction coupling in the mammalian heart. It is also a target of clinically important drugs such as catecholamines or Ca(2+)-channel blockers. Until now studies of Ca(2+)-channels in human ventricular myocardium have been hampered by the fact that adequate voltage control cannot be obtained in multicellular preparations. To characterize the properties of human myocardial Ca(2+)-currents, ventricular myocytes were isolated from explanted hearts of patients with end-stage heart failure undergoing cardiac transplantation. The current-voltage relation and voltage-dependent inactivation of L-type currents were similar to those in non-diseased guinea-pig myocardium. Currents could be stimulated with isoprenaline in a dose-dependent manner. When cells were superfused with a Na(+)-free solution in the presence of Tetrodotoxin, Cs+ and Tetraethylammonium to block interfering Na+ and K(+)-currents, depolarization from a holding potential of -90 mV to -80-(-)50 mV did not elicit any time-dependent inward-current. Changing the holding potential from -90 to -45 mV did not alter the current-voltage relation. We conclude that T-type Ca(2+)-currents do not seem to make a detectable contribution to the transmembrane Ca(2+)-influx and that L-type currents in human ventricular myocytes of patients with severe heart failure have characteristics that are similar to those in other mammalian species.

Ca2+ -currents and intracellular [Ca2+]i-transients in single ventricular myocytes isolated from terminally failing human myocardium

The purpose of the present study was to test the hypothesis that steps between the excitation of the cell membrane and contraction are altered in cardiac failure. Ca(2+)-currents and [Ca2+]i-transients were measured in single ventricular myocytes isolated from explanted hearts of patients with terminal heart failure undergoing transplantation, or from donors whose organs could not be used for technical reasons. Peak Ca(2+)-current densities were unchanged, as was the current-voltage relation. However, in myocytes isolated from severely failing hearts resting [Ca2+]i-levels were elevated, peak [Ca2+]i-transients were significantly smaller, and the diastolic decline of [Ca2+]i was markedly slowed. As the trigger for the release of Ca2+ from the sarcoplasmic reticulum is unchanged and the systolic [Ca2+]i-transient is reduced, severe heart failure can be described as partial electromechanical uncoupling.

Characterization of A1 adenosine receptors in atrial and ventricular myocardium from diseased human hearts.

The purpose of the present study was to characterize adenosine receptors in human atrial and ventricular myocardium. In isolated electrically driven preparations, adenosine produced „direct” negative inotropic effects in atrial myocardium (AT). In ventricular myocardium (VE), it only had negative Inotropic properties when force of contraction had been stimulated with isoprenaline (“indirect” effect), but it has no inotropic effect alone. The adenosine receptor antagonist 8-phenyltheophylline antagonized the „direct” and „indirect” effects; these findings indicated that both effects were mediated by adenosine receptors. In cardiac membranes from human AT and VE, adenosine receptors were characterized with [3H]-8-cyclopentyl-l,3-dipropylxanthine (DPCPX) binding. The effects of agonists. R-(-)-N6-phenylisopropyladenosinc (R-PIA), S-(+)-N6-phenylisopropyladenosuie (S-PIA), and 5′-(N-ethylcarboxamido)adenosine (NECA) and the effects of guanine nucleotides [Gpp(NH)p] were studied also. The antagonist affinities as judged from the apparent affinity, Kd, of [3H] DPCPX were similar in AT (2.2 nmol/1; 95% confidence limits, 1.4–3.7) and VE (1.8 nmol/1; 95% confidence limits, 1.0–3.0). The number of adenosine receptors was 1.7 times greater in AT (26.9 ± 2.33 fmol/mg protein; n =5) than in VE (16.2 ± 23 fmol/mg protein; n =5). High and low affinity states of adenosine receptors evaluated with the influence of Gpp(NH)p on agonist competition with R-PIA were similar in AT or VE. The rank orders of potency for agonists (R-PIA>S-PIA>NECA) and antagonists (DPCPX>8-phenyltheophylline>theophylline) were characteristic for the A, receptor subtype. It is concluded that A, adenosine receptors exist in the human myocardium. Since binding properties were similar in AT and VE, the same A1 adenosine receptor probably couples to different effectors in a similar guanine nucleotide-dependent way. [3H] DPCPX is the first radiolabeled antagonist ligand that allows detection of A, adenosine receptors and their coupling in the human myocardium.

Inotropic and lusitropic dysfunction in myocardium from patients with dilated cardiomyopathy

Isometric force of contraction (DT), peak rate of tension increase (+T), peak rate of tension decrease (-T), time to peak tension (TPT), and time to half-relaxation (T 1/2 T) were measured in electrically driven human papillary muscle strips (New York Heart Association [NYHA] class IV heart transplants, dilated cardiomyopathy; nonfailing (NF) donor hearts, brain dead) (1 Hz, 37 degrees C) under basal conditions (1.8 mmol/L Ca2+) and after stimulation with isoprenaline, ouabain, and Ca2+. There was no difference in the isometric contraction (+T, -T, TPT, and T 1/2 T) between NYHA IV hearts and NF hearts under basal conditions. Inotropic stimulation above 300% of basal DT increased -T significantly more in NF hearts (p less than 0.05) compared with NYHA IV hearts. The effectiveness of ouabain and Ca2+ to increase DT was not significantly changed in NYHA IV hearts compared with NF hearts. The isoprenaline-mediated increase in DT was reduced (p less than 0.05) in NYHA IV hearts to a similar extent (70%) as beta-adrenoceptors were downregulated. When the rate of stimulation was increased to 3 Hz (force-frequency relationship), force of contraction increased only in NF preparations, whereas it decreased in NYHA IV myocardium (p less than 0.05). It was concluded that the contractile apparatus in terminally failing human myocardium is sufficient to maximally increase DT. During inotropic stimulation, abnormalities in diastolic rather than systolic contraction become evident. This may indicate abnormal intracellular Ca2+ handling.

Force-frequency relationship and inotropic stimulation in the nonfailing and failing human myocardium: implications for the medical treatment of heart failure

SummaryIn isolated papillary muscle strips from nonfailing donor hearts (NF) and from the hearts of patients with dilated cardiomyopathy with severe heart failure (NYHA IV), the force-frequency relationship was studied. Experiments were performed under basal conditions and in the presence of 0.01 μM or 0.1 μM isoprenaline and 0.02 μM ouabain. In NF, there was a positive inotropic effect following an increase of the stimulating frequency, whereas in NYHA IV, the force gradually declined under these conditions. Low concentrations (0.01) μM of isoprenaline prevented the negative inotropic effect in NYHA IV, whereas at 0.1 μM the mechanical function deteriorated in NF and NYHA IV. Ouabain had no effect on the force-frequency relationship compared to basal conditions. It is concluded that a reduction of high frequencies does improve the contractility in the failing myocardium. It is not unreasonable to speculate that this mechanism might be involved in the beneficial effects of drugs which reduce the heart rate, such as β-adrenoceptor antagonists and cardiac glycosides, in the condition of congestive heart failure in which the sympathetic tone is high.