Susanne Hoischen

The enhanced contractility in phospholamban deficient mouse hearts is not associated with alterations in (Ca2+)-sensitivity or myosin ATPase-activity of the contractile proteins.

Abstract Work performing heart preparations from hypercontractile, phospholamban deficient mouse hearts showed no change in parameters of contraction or relaxation in response to isoproterenol stimulation. Thus, the aim of the present study was to investigate whether or not changes at the level of the contractile apparatus occur in addition to the altered expression of Ca2+-regulating proteins observed in these mouse models, e. g., phospholamban, ryanodine receptors. Triton-X skinned fiber preparations from phospholamban deficient (n = 9) and wild-type (n = 10) mice were used and the Ca2+-activated force as well as the myosin ATPase-activity were simultaneously measured. The tension dependent ATPase-activity was unchanged in phospholamban deficient animals when compared to controls. The SERCA 2a-inhibitor cyclopiazonic acid did not affect myosin ATPase-activity in this system. The Ca2+-sensitivity of Ca2+-activated force and myosin ATPase were unchanged as well. Comparison of the concentrations needed to achieve half maximal activation of the myosin ATPase-activity and force demonstrated that the Ca2+-sensitivity of the myosin ATPase was higher compared to the Ca2+-sentivity of tension development. This holds true for phospholamban deficient mice (EC50 ATPase: 0.9 ± 0.2 μmol/l; tension: 1.7 ± 0.3 μmol/l; p < 0.001) and wild-type controls (1.1 ± 0.01 μmol/l; 2.2 ± 0.4 μmol/l; p < 0.01). The myosin ATPase-activity and force were correlated to each other in both, phospholamban deficient mice and controls and did not change at submaximal Ca2+ concentrations. The ATPase/force-ratio, as a parameter of tension cost, was similar in either phospholamban deficient mice or controls. Thus, the present study provides evidence that at the level of the contractile proteins regulation of Ca2+-activated force and energy demand of force development are not altered in phospholamban deficient mice with enhanced myocardial performance. At the level of the regulation of crossbridge interaction, no adaptive or compensatory mechanisms have been initiated by ablation of phospholamban.

Potent vasodilatory with minor cardiodepressant actions of mibefradil in human cardiac tissue

1 The present study compared the cardiovascular effects of mibefradil (MIB), a novel Ca2+‐channel antagonist with high selectivity for T‐type Ca2+‐channels to the effect of the L‐type Ca2+‐channel‐antagonists nifedipine (NIF) and diltiazem (DIL) in left ventricular myocardium and coronary arteries of hearts obtained from patients suffering from dilated cardiomyopathy (NYHA IV). Right atrial myocardium from patients undergoing aortocoronary bypass surgery without signs of cardiac failure was studied as well. 2 NIF and DIL (100 μmol l−1) completely depressed force of contraction (FOC) in electrically driven left ventricular myocardium (NIF 6.5±1.4% and DIL 7.1±1.2% of control), whereas a similar concentration of MIB only reduced force of contraction to 55.1±4.0% of the basal FOC. The negative inotropic potency as measured by the concentration needed to reduce basal FOC for 25% was NIF (0.0095 μmol l−1)>DIL (0.041 μmol l−1)>MIB (9.47 μmol l−1). 3 All three Ca2+‐channel antagonists were more potent in human atrial compared to human left ventricular myocardium to reduce FOC. 4 The rank order of Ca2+‐antagonistic moiety as measured by the decrease of the intracellular Ca2+‐transient (fura‐2 ratio method) was NIF>DIL>MIB. 5 All Ca2+‐channel antagonists completely relaxed human coronary arteries (% of papaverine effect: MIB 81.7±5.5%, DIL 91.3±0.9%, NIF 96.4±3.7%) precontracted with PGF2α (0.3 μmol l−1). The rank order of vasodilatory potency was NIF (EC50; 0.02 μmol l−1)>DIL (0.13 μmol l−1)>MIB (2.05 μmol l−1). 6 The vasoselectivity measured by the ratio of the concentration needed to achieve a 25% decrease in force and the concentration needed for 25% vasodilatation was 316 for MIB, 1.5 for NIF and 1.0 for DIL. 7 The present study provides evidence that blockade of T‐type Ca2+‐channels (e.g. mibefradil) results in potent vasodilatory properties with only minor cardiodepressant effects.

T- and L-Type Ca2+-Channel Antagonists Reduce Contractility in Guinea Pig Cardiac Myocytes

The aim of this study was to investigate the influence of L- and T-type Ca2+-channel blockade on myocardial contractility in guinea pig cardiomyocytes. Left ventricular myocardium from guinea pig contains both L- and T-type Ca2+ channels. The T-type Ca2+ influx was inhibited with mibefradil (1-100 microM), a novel compound with a threefold higher affinity for T- compared with L-type Ca2+ channels. In comparison, L-type Ca2+ influx was reduced by the benzodiazepine diltiazem (1-100 microM). The effect of mibefradil and diltiazem on electrically driven (0.5 Hz) isolated cardiomyocytes (n = 12) was studied in a concentration-dependent manner. The change of the contraction amplitude (percentage of cell shortening) was continuously recorded with an one-dimensional high-speed camera. Both mibefradil and diltiazem concentration-dependently reduced (p < 0.05 vs. control) the contraction amplitude in isolated myocytes from guinea pig. The concentration at which the contraction amplitude of guinea pig cardiomyocytes was reduced by 50% (EC50) was 31.6 microM for diltiazem and 6.3 microM for mibefradil, indicating that the T-type Ca2+-channel blocker mibefradil is more potent in reducing contractility in guinea pig cardiac myocytes in comparison with the L-type Ca2+-channel antagonist diltiazem. Mean values for cell shortening in percentage +/- SEM for mibefradil (0, 1, 10, 100 microM) were 100%, 78 +/- 9.2%, 36 +/- 5.4%, and 24 +/- 3.6%. The corresponding values for diltiazem were 100%, 92 +/- 12.5%, 79 +/- 8.9%, and 35 +/- 2.6%. In contrast, the increase of the extracellular Ca2+ concentration (2-7.5 mM) resulted in a significant increase of the contraction amplitude (+213 +/- 14%). Therefore, blockade of the Ca2+ influx through voltage-dependent T- or L-type Ca2+ channels decreases contraction in isolated cardiac myocytes from guinea pigs containing L- and T-type Ca2+ channels.

Die Ca2+-Sensitizer CGP 48506 und EMD 57033, nicht aber der Na+-Kanal-Modulator BDF 9148, verlängern die Relaxation an isolierten Kardiomyocyten des Meerschweinchens

The sodium channel modulator DPI 201-106 has been described to posses Ca2+-sensitizing properties. Therefore, the present study investigated the inotropic effect of the Na+-channel modulator BDF 9148 (1 μM), a congener of DPI 201-106, in comparison with the Ca2+-sensitizers CGP 48506 (1–50 μmol/l) and EMD 57033 (1–30 μmol/l) in electrically driven left ventricular cardiomyocytes isolated from guinea pigs. The changes of the contraction amplitude in comparsion to the basal cell shortening (cell shortening in μm and %) were continuously recorded with a one-dimensional high speed camera. BDF 9148, CGP 48506, and EMD 57033 exerted a significant increase in the contraction amplitude (p<0.05 vs. control). The maximal positive inotropic effects of CGP 48506 (50 μmol/l) and EMD 57033 (30 μmol/l) were +249±30% and +226±28%, respectively. The corresponding value for BDF 9148 (1 μmol/l) was +176±16%. However, only the Ca2+-sensitizers CGP 48506 and EMD 57033, but not BDF 9148, prolonged the contractile twitch. Especially in patients with an already enhanced intracellular myocardial Ca2+-concentration, Ca2+-sensitizers, which impair relaxation, may be disadvantageous for therapeutial use despite their positive inotropic effect. Ca2+-Sensitizer sind Substanzen, die die Kontraktionskraft am Myokard Ca2+- und Energie-unabhängig steigern können. Auch Natriumkanalmodulatoren wird eine Ca2+-sensibilisierende Eigenschaft zugeschrieben. Deshalb wurde in der vorliegenden Untersuchung der inotrope Effekt des Na+-Kanalmodulators BDF 9148 (1 μM) im Vergleich zu den Ca2+-Sensitizern CGP 48506 (1–50 μmol/l) und EMD 57033 (1–30 μmol/l) an isolierten, elektrisch stimulierten Kardiomyocyten des Meerschweinchens untersucht. Die Änderung der Zellverkürzung wurde kontinuierlich mit einer eindimensionalen High Speed-Kamera aufgenommen. BDF 9148, CGP 48506 und EMD 57033 bewirkten eine signifikante Zunahme der Zellverkürzung (CGP 48506 (50μmol/l): +249±30%, EMD 57033 (30 μmol/l): +226±28%, BDF 9148 (1 μmol/l): +176±16%). Nur die Ca2+-Sensitizer CGP 48506 und EMD 57033 verlängerten die Relaxation. Besonders bei Patienten mit bereits erhöhten intrazellulären myokardialen Ca2+-Konzentrationen, wie dies bei der dilatativen Kardiomyopathie beschrieben ist, sind Ca2+-Sensitizer, die neben der positiv inotropen Wirkung zusätzlich die Relaxation verzögern, möglicherweise ungünstig.

Einfluß einer veränderten extrazellulären K+- und Mg2+-Konzentration auf die intrazelluläre Ca2+-Homöostase, die Kontraktionskopplung und die Kraft-Frequenz-Beziehung am menschlichen Myokard

The intracellular Ca(2+)-homeostasis may be affected by changes of the extracellular K(+)- and/or Mg(2+)-concentrations. Mg2+ reduces the Ca(2+)-influx via L-type Ca(2+)-channels, facilitates Ca(2+)-uptake into the sarcoplasmic reticulum, modulates the Ca(2+)-induced Ca(2+)-release and the Ca(2+)-binding to troponin C. The extracellular K+ activates the Na+/K(+)-ATPase and changes the membrane potential thereby affecting the mode of action of the Na+/Ca(2+)-exchanger. Especially when intracellular Ca2+ regulation is altered, for example in heart failure, Mg2+ and K+ exert beneficial effects on the frequency-dependent force-generation in human myocardium. Thus, extracellular Mg2+ and K+ influence contraction coupling in the human myocardium.ZusammenfassungVeränderungen der extrazellulären K+- und Mg2+-Konzentration beeinflussen die intrazelluläre Ca2+-Homöostase und damit die Kontraktionskopplung am menschlichen Myokard. Mg2+ vermindert den Ca2+-Einstrom über L-Typ-Ca2+-Kanäle, beschleunigt die Ca2+-Aufnahme in das sarkoplasmatische Retikulum und vermindert die Ca2+-Sensitivität von Troponin C. Hohe extrazelluläre K+-Konzentrationen stimulieren die sarkolemmale Na+/K+-ATPase. Dies und das veränderte Ruhepotential nach Steigerung der K+-Konzentration modulieren die Pumprichtung des Na+/Ca2+-Austauschersystems. So können das extrazelluläre K+ und Mg2+ direkt die intrazelluläre Ca2+-Homöostase und damit die Kontraktionskraftentwicklung und die Relaxation beeinflussen. Dies ist besonders am Myokard von herzinsuffizienten Patienten mit gestörter intrazellulärer Ca2+-Homöostase zu beachten.SummaryThe intracellular Ca2+-homeostasis may be affected by changes of the extracellular K+- and/or Mg2+-concentrations. Mg2+ reduces the Ca2+-influx via L-type Ca2+-channels, facilitates Ca2+-uptake into the sarcoplasmic reticulum, modulates the Ca2+-induced Ca2+-release and the Ca2+-binding to troponin C. The extracellular K+ activates the Na+/K+-ATPase and changes the membrane potential thereby affecting the mode of action of the Na+/Ca2+-exchanger. Especially when intracellular Ca2+ regulation is altered, for example in heart failure. Mg2+ and K+ exert beneficial effects on the frequency-dependent force-generation in human myocardium. Thus, extracellular Mg2+ and K+ influence contraction coupling in the human myocardium.