Molecular and functional characterization of cardiac Cav3.2 T-type calcium channels

T-type calcium (Ca²⁺) channels contribute to the normal development of the heart and are also implicated in pathophysiological states such as cardiac hypertrophy. Functionally distinct Cav3 T-type Ca²⁺ channel isoforms can be generated by alternative splicing from each of three different Cav3 genes (Cav3.1, Cav3.2 and Cav3.3), although it remains to be described whether specific splice variants are associated with developmental stages and pathological conditions. Using full length cDNA generated from rat cardiac tissues, this study identified ten major regions of alternative splicing and systematically identified alternative splice variants of cardiac Cav3.2 channels. Quantitative real-time PCR analysis on the mRNA expression of the most common variants revealed preferential expression of Cav3.2(-25) splice variant channels in the newborn rat heart, whereas in the adult heart approximately equal levels of expression of both (+25) and (-25) exon variants was observed. In the adult stage of hypertensive rats, an increase in overall Cav3.2 mRNA expression and a shift towards the expression of Cav3.2(+25) containing channels as the predominant form was observed. This is the first evidence to show that cardiac Cav3.2 is subject to considerable splicing. Moreover, this thesis is also the first study to show developmental and pathological changes in expression of specific splice variants of the Cav3.2 channels. The biophysical characteristics of cloned Cav3.2 splice variants and T-type Ca²⁺ currents from dissociated cultured newborn ventricular myocytes were investigated using whole cell patch clamp analysis. This study showed variant-specific voltage-dependent facilitation (VDF) of Cav3.2 channels attributed to the exclusion of exon 25 in Cav3.2 transcripts. Lastly, this thesis is the first to provide evidence on VDF of T-type currents in rat ventricular myocytes.Medicine, Faculty ofGraduat