L-type Ca2+ channel alpha(1c) subunit isoform switching in failing human ventricular myocardium

The objectives of this study were to determine the relative abundance of th e L-type Ca channel alpha(1c) IVS3 isoforms that result from alternative sp licing in normal human ventricular myocytes and to measure the changes in i soform expression in end stage heart failure. Methods: mRNA was isolated fr om left ventricular tissue and myocytes From non-failing and failing human hearts. RT-PCR with isoform-specific primers was used to obtain cDNAs that were then mutated for use in competitive PCR reactions. An RNase protection assay was also used to confirm the presence of one of the novel isoforms. Results: Four different alpha(1c) IVS3 isoforms were found in non-failing h uman ventricular myocytes using RT-PCR. Two isoforms contained exon 31 (ter med IVS3A isoforms) and two isoforms contained exon 32 (termed IVS3B isofor ms). One of these isoforms has not been observed previously and contains ex on 31 and all but the last six base pairs of exon 32. in non-failing human ventricular myocytes the IVS3A isoform is 2.5 times more abundant than the IVS3B isoform. There were significant changes in the relative abundance of these isoforms in failing hearts, with the IVS3B isoform being twice as abu ndant as the IVS3A isoform. All isoforms were confirmed by RNase protection analysis. Conclusions: These experiments show that there are at least four L-type Ca channel mRNA isoforms in the normal human heart and that the rel ative abundance of these isoforms changes significantly in heart failure. T hese alpha(1c) isoform changes in heart failure are associated with dysfunc tional electromechanical disturbances, but the specific physiological role of each L-type Ca channel isoform in normal and failing hearts. (C) 2000 Ac ademic Press.