The relationship between intracellular [Ca(2+)] and Ca(2+) wave characteristics in permeabilised cardiomyocytes from the rabbit.
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Spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and propagated intracellular Ca(2+) waves are a consequence of cellular Ca(2+) overload in cardiomyocytes. We examined the relationship between average intracellular [Ca(2+)] and Ca(2+) wave characteristics. The amplitude, time course and propagation velocity of Ca(2+) waves were measured using line-scan confocal imaging of beta-escin-permeabilised cardiomyocytes perfused with 10 microM Fluo-3 or Fluo-5F. Spontaneous Ca(2+) waves were evident at cellular [Ca(2+)] > 200 nM. Peak [Ca(2+)] during a wave was 2.0-2.2 microM; the minimum [Ca(2+)] between waves was 120-160 nM; wave frequency was approximately 0.1 Hz. Raising mean cellular [Ca(2+)] caused increases in all three parameters, particularly Ca(2+) wave frequency. Increases in the rate of SR Ca(2+) release and Ca(2+) uptake were observed at higher cellular [Ca(2+)], indicating calcium-sensitive regulation of these processes. At extracellular [Ca(2+)] > 2 microM, the mean [Ca(2+)] inside the permeabilised cell did not increase above 2 microM. This extracellular-intracellular Ca(2+) gradient could be maintained for periods of up to 5 min before the cardiomyocyte developed a sustained and irreversible hypercontraction. Inclusion of mitochondrial inhibitors (2 microM carbonyl cyanide m-chlorophenylhydrazone and 2 microM oligomycin) while perfusing with > 2 microM Ca(2+) abolished the extracellular-intracellular Ca(2+) gradient through the generation of Ca(2+) waves with a higher peak [Ca(2+)] compared to control conditions. Under these conditions, cardiomyocytes rapidly (< 2 min) developed a sustained and irreversible contraction. These results suggest that mitochondrial Ca(2+) uptake acts to delay an increase in [Ca(2+)] by blunting the peak of the Ca(2+) wave. (+info)
Mechanisms underlying the frequency dependence of contraction and [Ca(2+)](i) transients in mouse ventricular myocytes.
(34/8946)
In most mammalian species force of contraction of cardiac muscle increases with increasing rate of stimulation, i.e. a positive force-frequency relationship. In single mouse ventricular cells, both positive and negative relationships have been described and little is known about the underlying mechanisms. We studied enzymatically isolated single ventricular mouse myocytes, at 30 degrees C. During field stimulation, amplitude of unloaded cell shortening increased with increasing frequency of stimulation (0.04 +/- 0.01 Delta L/L(0) at 1 Hz to 0.07 +/- 0.01 Delta L/L(0) at 4 Hz, n = 12, P < 0.05). During whole cell voltage clamp with 50 microM [K5-fluo-3](pip), both peak and baseline [Ca(2+)](i) increased at higher stimulation frequencies, but the net Delta[Ca(2+)](i) increased only modestly from 1.59 +/- 0.08 Delta F/F(0) at 1 Hz, to 1.71 +/- 0.11 Delta F/F(0) at 4 Hz (n = 17, P < 0.05). When a 1 s pause was interposed during stimulation at 2 and 4 Hz, [Ca(2+)](i) transients were significantly larger (at 4 Hz, peak F/F(0) increased by 78 +/- 2 %, n = 5). SR Ca(2+) content assessed during caffeine application, significantly increased from 91 +/- 24 micromol l(-1) at 1 Hz to 173 +/- 20 micromol l(-1) at 4 Hz (n = 5, P < 0.05). Peak I(Ca,L) decreased at higher frequencies (by 28 +/- 6 % at 2 Hz, and 45 +/- 8 % at 4 Hz), due to slow recovery from inactivation. This loss of I(Ca,L) resulted in reduced fractional release. Thus, in mouse ventricular myocytes the [Ca(2+)](i)-frequency response depends on a balance between the increase in SR content and the loss of trigger I(Ca,L). Small changes in this balance may contribute to variability in frequency-dependent behaviour. In addition, there may be a regulation of the contractile response downstream of [Ca(2+)](i). (+info)
Citalopram inhibits L-type calcium channel current in rat cardiomyocytes in culture.
(35/8946)
Selective serotonine reuptake inhibitors (SSRI) are believed to be less dangerous in the treatment of depressive disorder in comparison with tricyclic antidepressants (TCA) due to their relative lack of cardiotoxicity. Thus, we investigated the effect of citalopram (SSRI) on membrane electrophysiology in rat cardiomyocytes in tissue culture. The results were compared with those from amitriptyline (TCA). The whole-cell configuration patch-clamp technique was used. Both citalopram and amitriptyline exhibited the concentration-dependent inhibition of the L-type calcium channel current (ICa). Citalopram in concentrations of 3 microM and 10 microM inhibited peak calcium current by 2.7% and 8%, respectively. We demonstrated the same potency of citalopram and amitriptyline to inhibit ICa. These observations led us to conclude that citalopram and amitriptyline are drugs, which exhibit a similar potency for causing concentration-dependent inhibition of ICa. (+info)
Effects of sprint training on contractility and [Ca(2+)](i) transients in adult rat myocytes.
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The effects of 6-8 wk of high-intensity sprint training (HIST) on rat myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients were investigated. Compared with sedentary (Sed) myocytes, HIST induced a modest (5%) but significant (P < 0.0005) increase in cell length with no changes in cell width. In addition, the percentage of myosin heavy chain alpha-isoenzyme increased significantly (P < 0.02) from 0.566 +/- 0.077% in Sed rats to 0.871 +/- 0.006% in HIST rats. At all three (0.6, 1.8, and 5 mM) extracellular Ca(2+) concentrations ([Ca(2+)](o)) examined, maximal shortening amplitudes and maximal shortening velocities were significantly (P < 0.0001) lower and half-times of relaxation were significantly (P < 0.005) longer in HIST myocytes. HIST myocytes had significantly (P < 0.0001) higher diastolic [Ca(2+)](i) levels. Compared with Sed myocytes, systolic [Ca(2+)](i) levels in HIST myocytes were higher at 0.6 mM [Ca(2+)](o), similar at 1.8 mM [Ca(2+)](o), and lower at 5 mM [Ca(2+)](o). The amplitudes of [Ca(2+)](i) transients were significantly (P < 0.0001) lower in HIST myocytes. Half-times of [Ca(2+)](i) transient decline, an estimate of sarcoplasmic reticulum (SR) Ca(2+) uptake activity, were not different between Sed and HIST myocytes. Compared with Sed hearts, Western blots demonstrated a significant (P < 0.03) threefold decrease in Na(+)/Ca(2+) exchanger, but SR Ca(2+)-ATPase and calsequestrin protein levels were unchanged in HIST hearts. We conclude that HIST effected diminished myocyte contractile function and [Ca(2+)](i) transient amplitudes under the conditions studied. We speculate that downregulation of Na(+)/Ca(2+) exchanger may partly account for the decreased contractility in HIST myocytes. (+info)
Hypothesis: troponin degradation is one of the factors responsible for deterioration of left ventricular function in heart failure.
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A hypothesis is presented that explains one of the mechanisms by which a heart starts to fail. The hypothesis is that myocardial function of an overloaded or otherwise stressed heart may become impaired by cellular troponin degradation in vital cardiomyocytes. The troponins (I, T and C) regulate actin-myosin interaction, thereby controlling contraction and relaxation. Troponins have been shown to be targets of activated calpain I. This enzyme, that is activated by elevated intracellular Ca2+ concentrations, such as occurs during ischemia, degrades troponins, leading to impaired interaction between actin and myosin and, thereby, less contractile force. Several reports about troponin degradation in viable myocardium support this hypothesis. Also, results are discussed that demonstrate the presence of immunoreactive troponin fragments in plasma under conditions in which myocardial necrosis can be excluded or is unlikely. The hypothesis implicates that release of troponin and/or troponin degradation products is not specific for necrotic myocardium but may occur from viable myocardium as well. To test this hypothesis, several lines of research are suggested. If the hypothesis is not rejected in the near future, the concept that a positive troponin test reflects 'even microscopic zones of myocardial necrosis' as used by the Joint ESC/ACC Committee for the Redefinition of Myocardial Infarction [The Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction. Myocardial infarction redefined-A consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction. Eur Heart J 2000;21:1502-1513], should be withdrawn. (+info)
Involvement of cyclin D activity in left ventricle hypertrophy in vivo and in vitro.
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OBJECTIVE: Cardiac hypertrophy is induced by a number of stimuli and can lead to cardiomyopathy and heart failure. Present knowledge suggests that cell-cycle regulatory proteins take part in hypertrophy. We have investigated if the D-type cyclins are involved in cardiac hypertrophy. METHODS: The expression and activity of the D-type cyclins and associated kinases in cardiomyocytes were studied during angiotensin II- and pressure overload-induced hypertrophy in rats (Rattus norvegicus) and in isolated, neonatal cardiomyocytes. Expression of the D-type cyclins was manipulated pharmacologically and genetically in neonatal myocytes. RESULTS: In the left ventricle, there was a low, constitutive expression of the D-type cyclins, which may have a biological role in normal, adult myocytes. The protein level and the associated kinase activity of the D-type cyclins were up-regulated during hypertrophic growth. The increase in cyclin D expression could be mimicked in vitro in neonatal cardiac myocytes. Interestingly, the cyclin Ds were up-regulated by hypertrophic elicitors that stimulate different signalling pathways, suggesting that cyclin D expression is an inherent part of cardiac hypertrophy. Treatment of myocytes with the compound differentiation inducing factor 1 inhibited expression of the D-type cyclins and impaired hypertrophic growth induced by angiotensin II, phenylephrine and serum. The response to hypertrophic elicitors could be restored in differentiation inducing factor 1-treated myocytes by expressing cyclin D2 from a heterologous promoter. CONCLUSION: Our results point to the D-type cyclins as important regulators of cardiac hypertrophy. This supports the notion that cell-cycle regulatory proteins regulate hypertrophic growth. (+info)
C-Reactive protein augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes.
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OBJECTIVE: Nitric oxide (NO) production by inducible NO synthase (iNOS) can exert negative inotropic and cytotoxic effects on cardiac myocytes and may play an important role in the pathogenesis of cardiac dysfunction and remodeling. An elevated serum level of C-reactive protein (CRP) is an important predictive factor for cardiac disorders including acute myocardial infarction and dilated cardiomyopathy. The basic mechanisms responsible for this association are not clear; CRP may merely be a marker of inflammation with no specific role in the pathogenesis of cardiac disease or may directly modulate the disease process. We investigated the effects of CRP on iNOS expression and subsequent NO synthesis in rat cardiac myocytes, and the mechanism by which CRP exerts its effects. METHODS: NO production by culture neonatal rat cardiac myocytes was determined by measurement of nitrite contents in the culture medium. The expression of iNOS mRNA and protein were measured by reverse transcription-polymerase chain reaction and Western blotting, respectively. The levels of nuclear factor (NF)-kappaB proteins were analyzed by a gel retardation assay. RESULTS: Incubation of cardiac myocytes with interleukin-1beta (IL-1beta; 10 ng/ml) caused a significant increase in nitrite production. CRP significantly increased the IL-1beta-induced nitrite production in a dose-dependent manner (10-100 microg/ml). Incubation with IL-1beta induced the expression of iNOS mRNA and protein in cardiac myocytes, and CRP enhanced their expression. Addition of IL-1beta activated NF-kappaB in cardiac myocytes, while CRP did not affect IL-1beta-induced NF-kappaB activation. CONCLUSIONS: These results indicated that CRP directly enhances NO synthesis in IL-1beta-stimulated cardiac myocytes through an NF-kappaB-independent mechanism. (+info)
Interaction of different potassium channels in cardiac repolarization in dog ventricular preparations: role of repolarization reserve.
(40/8946)
1 The aim of this study was to investigate the possible role of the interaction of different potassium channels in dog ventricular muscle, by applying the conventional microelectrode and whole cell patch-clamp techniques at 37 degrees C. 2 Complete block of I(Kr) by 1 micro M dofetilide lengthened action potential duration (APD) by 45.6+/-3.6% at 0.2 Hz (n=13). Chromanol 293B applied alone at 10 micro M (a concentration which selectively blocks I(Ks)) did not markedly lengthen APD (<7%), but when repolarization had already been prolonged by complete I(Kr) block with 1 micro M dofetilide, inhibition of I(Ks) with 10 micro M chromanol 293B substantially delayed repolarization by 38.5+/-8.2% at 0.2 Hz (n=6). 3 BaCl(2), at a concentration of 10 micro M which blocks I(Kl) without affecting other currents, lengthened APD by 33.0+/-3.1% (n=11), but when I(Kr) was blocked with 1 micro M dofetilide, 10 micro M BaCl(2) produced a more excessive rate dependent lengthening in APD, frequently (in three out of seven preparations) initiating early afterdepolarizations. 4 These findings indicate that if only one type of potassium channels is inhibited in dog ventricular muscle, excessive APD lengthening is not likely to occur. Dog ventricular myocytes seem to repolarize with a strong safety margin ('repolarization reserve'). However, when this normal 'repolarization reserve' is attenuated, otherwise minimal or moderate potassium current inhibition can result in excessive and potentially proarrhythmic prolongation of the ventricular APD. Therefore, application of drugs which are able to block more than one type of potassium channel is probably more hazardous than the use of a specific inhibitor of one given sort of potassium channel, and when simultaneous blockade of several kinds of potassium channel may be presumed, a detailed study is needed to define the determinants of 'repolarization reserve'. (+info)