1Philipps-Universität Marburg Pharmakologie und Klinische Pharmazie Marburg, Deutschland; 2Philipps-Universität Marburg Behavioral Neuroscience Marburg, Deutschland
Introduction
CACNA1C codes for Cav1.2, the L-type calcium channel, found in neurons, cardiac myocytes and smooth muscle cells. Cacna1c haploinsufficient rats are a model for altered social behaviour. Recently, it was shown that these rats also exhibit a cardiac phenotype including altered expression and phosphorylation of calcium handling proteins in left ventricular (LV) myocardium and an attenuated response of calcium transients and sarcomere shortening to beta-adrenergic stimulation by isoprenaline (ISO) in ventricular myocytes. We hypothesized that this altered cytosolic calcium regulation would also affect mitochondrial calcium regulation in Cacna1c+/- myocytes.
Methods and Results
Ventricular myocytes were isolated from adult female Cacna1c haploinsufficient rats (Cacna1c+/-) and their wild-type littermates (WT, +/+). Myocytes were loaded with X-Rhod-1 followed by cobalt chloride quenching of cytosolic fluorescence in order to measure mitochondrial calcium by means of confocal linescan imaging. Under these conditions, ventricular myocytes exhibited a striped pattern of X-Rhod-1 fluorescence, which has been shown previously to coincide with mitochondria. Electrical stimulation (1 Hz) of the cells resulted in mitochondrial calcium transients (mitoCaTs). In WT myocytes (n=26), mitoCaTs displayed amplitudes of 0.18±0.01 dF/Frest, rise times of 72±7 ms and time constants tau of Ca decay of 106±4 ms. Similar values were obtained for mitoCaTs in ventricular myocytes from Cacna1c+/- (n=20) indicating that, under baseline conditions, mitoCaTs did not differ between WT and Cacna1c+/-. In line with these results, we found that the expression of major mitochondrial calcium handling proteins in LV myocardium, i.e. the mitochondrial calcium uniporter (MCU), the mitochondrial sodium/calcium/lithium exchanger (NCLX) and the putative mitochondrial hydrogen/calcium exchanger (LETM1) did not differ between WT and Cacna1c+/- (N=8 hearts for each genotype). In ventricular myocytes from both WT and Cacna1c+/- stimulated at 1 Hz, beta-adrenergic stimulation with ISO (30 nM) increased the amplitude and accelerated the decay of mitoCaTs. When, in the presence of ISO, stimulation frequency was increased from 1 Hz to 2 Hz and 4 Hz, there was a progressive acceleration of mitoCaT decay evident as a decrease of the time constant tau of decay. In WT myocytes, tau of decay decreased from 78±2 ms at 1 Hz to 37±2 ms at 4 Hz, whereas in Cacna1c+/- myocytes the respective values were 76±3 ms at 1 Hz and 32±2 ms at 4 Hz. Amplitudes of mitoCaTs amounted to 0.29±0.02 dF/Frest at 1 Hz and 0.30±0.03 dF/Frest at 4 Hz in WT versus 0.35±0.05 dF/Frest at 1 Hz and 0.40±0.05 dF/Frest at 4 Hz in Cacna1c+/- myocytes (P<0.05 vs WT).
Conclusions
MitoCaTs do not differ between WT and Cacna1c+/- ventricular myocytes under baseline conditions and expression of MCU, NCLX and LETM1 is comparable in LV myocardium from WT and Cacna1c+/- rats. ISO increased and accelerated mitoCaTs in both WT and Cacna1c+/- myocytes. Despite an attenuated ISO effect on cytosolic CaTs, however, Cacna1c+/- myocytes exhibited an augmented ISO effect on mitochondrial CaTs.