Calciumregulation in Kardiomyozyten haploinsuffizienter Cacna1c+/- -Ratten

Das CACNA1C-Gen codiert für die porenbildende α1C-Untereinheit der L-Typ-Calcium-Kanal (LTCC)-Isoform Cav1.2, welche im Gehirn, in Glattmuskelzellen und im Herzen exprimiert wird. Im Gehirn und in dessen Entwicklung ist die Cav1.2-Isoform beteiligt an der Regulation der synaptischen Plastizität, der...

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Bibliographic Details
Main Author: Fender, Hauke Ansgar
Contributors: Kockskämper, Jens (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2021
Online Access:PDF Full Text
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The CACNA1C gene encodes the pore-forming α1C-subunit of the L-type calcium channel (LTCC) isoform Cav1.2, which is expressed in the brain, smooth muscle and the heart. In the brain and in its development, the Cav1.2 isoform is involved in the regulation of synaptic plasticity, neuronal activity and thus also in learning processes and behaviour. In the heart, Cav1.2 is responsible for the sarcolemmal calcium (Ca) influx during the action potential and the Ca-induced Ca-release from the sarcoplasmic reticulum (SR) which leads to contraction of the cardiomyocyte. In genome-wide association studies, polymorphisms in the CACNA1C gene have been linked to the occurrence of psychiatric disorders (depression, bipolar disorder, schizophrenia, anxiety disorders & autism), but variants of the gene can also manifest in heart disease (Brugada syndrome, Timothy syndrome or long-QT syndrome). On the one hand, psyche and heart function are linked via the CACNA1C gene; on the other hand, psychiatric diseases also increase the risk of developing cardiac diseases and vice versa. The heterozygous Cacna1c+/- knockout in the rat/mouse model leads to the development of a behavioural phenotype associated with deficits in social communication, increased anxiety and autism-like behaviours - confirming the important role of the gene at the behavioural level. This study presents the first characterisation of the Cacna1c+/- rat model at the cardiac level. For this purpose, Ca-homeostasis of ventricular myocytes as well as expression and phosphorylation of Ca-handling proteins in the heart of these animals were investigated and compared to their wild-type (WT) littermates. In a basal characterisation, the Cacna1c+/- animals showed unchanged heart rates but decreased blood pressure values without evidence of the development of cardiac hyper- or hypotrophy. Electrically stimulated Ca-transients (CaT) were examined in isolated cardiomyocytes at the confocal microscope using the Ca dye Fluo-4. CaT, SR Ca content, fractional SR Ca release and sarcolemmal Ca influx in Cacna1c+/- myocytes under basal conditions were comparable to WT myocytes, despite a 30% reduction in Cav1.2 expression in Cacna1c+/- myocardium. However, remodelling of Ca-handling proteins accompanied by increased expression of SR-Ca-ATPase (SERCA) and Na-Ca exchanger (NCX) as well as basally increased protein kinase A (PKA) dependent phosphorylation at Cav1.2 (Ser1928) and ryanodine receptor type-2 (RyR2) (Ser2808) could be detected, which can be regarded as a kind of compensatory mechanism. Since the Cacna1c+/- rat model has a behavioural phenotype and psychological stress is also associated with increased sympathetic activity, the second part of the work simulated sympathetic stimulation in which ventricular myocytes were treated with the β-adrenergic agonist isoprenaline (ISO). Under ISO (100 nM), the amplitude of CaT was increased and the decline in CaT accelerated. Both WT and Cacna1c+/- myocytes showed a corresponding response to ISO treatment, although the increase in CaT amplitude was significantly weaker in Cacna1c+/- myocytes. After ISO administration, fractional SR Ca release increased from 60% to 90% in WT myocytes, whereas the increase from 60% to 75% was also weaker in Cacna1c+/- myocytes. Accordingly, using an ISO concentration-response curve, a significantly higher EC50 of 13 nM was determined in Cacna1c+/--myocytes compared to WT (EC50: 4 nM). Furthermore, after blocking the SR, the sarcolemmal Ca-influx was decreased under ISO in Cacna1c+/- myocytes. In Langendorff-perfused hearts, the ISO-mediated increase in phosphorylation of key Ca-handling proteins was investigated. The increase in phosphorylation of RyR2 at Ser2808 was significantly weaker in Cacna1c+/- hearts than in WT. Since in the Cacna1c+/- myocardium this site was already more strongly phosphorylated basally, the ISO-mediated increase could not be as pronounced as in the WT. This reduced phosphorylation reserve in Cacna1c+/- myocytes is presumably responsible for the impaired ability to stimulate SR-Ca release and increase CaT to the same extent as the WT. Compensatory mechanisms take place in Cacna1c+/- myocytes that allow maintenance of normal CaT under basal conditions. However, under stressed conditions, these mechanisms reach their limits, revealing an impaired response of Cacna1c+/- myocytes to sympathetic stress. This study thus provides the first evidence that a heterozygous knockout of the Cacna1c+/- gene, in addition to the development of a behavioural phenotype, also results in a phenotype at the cardiac level.