The sarcolemma (plasma membrane) of myocardial cells has high levels of Na+-Ca2+ exchange activity. Na+-Ca 2+ exchange is an important mechanism for regulating myocardial Ca 2+ and thereby also regulates contractility. This laboratory has recently cloned the Na+-Ca 2+ exchanger allowing molecular analysis of the exchanger to begin. The long-term objective of this project is to understand the distribution, regulation, and gene structure of the exchanger. Towards this goal, the specific aims of the project are as follows: 1. Isoforms and distribution. cDNA clones representing isoforms of the Na+-Ca2+ exchanger will be isolated, sequenced and expressed. Isoforms function and distribution will be assessed. 2. Regulation. The effects of thyroid hormone, chronic digitalis treatment, and ischemia on exchanger protein and RNA levels will be determined. These three interventions all affect Na+-Ca2+ exchange activity, and the proposed experiments will analyze these effects at the molecular level. 3. Gene structure. Transcriptional regulatory elements and intron/exon boundaries will be analyzed. Characterization of upstream regulatory elements will aid in understanding the regulated expression of different exchanger gene products. Intron/exon maps are useful in understanding evolutionary and protein domain relationships. 4. Antisense oligonucleotides. Oligonucleotides with sequences antisense to mRNA for the Na+-Ca2+ exchanger will be introduced into cells. This should block synthesis of exchanger protein and the response of excitation-contraction coupling pathways will be analyzed. The Na+-Ca 2+ exchanger is an important regulator of contractility. Further molecular knowledge of this essential cardiac transporter will aid in understanding the workings of both normal and diseased hearts.
