All tissues examined express CaMKII, but the specific isoforms expressed in several tissues, including gastric and colonic smooth muscle, have not been identified. RT-PCR, real time PCR, and Northern blotting are used to determine the relative expression levels of the CaMKII isoform mRNAs. In situ mRNA hybridization histochemistry is being used to localize the isoforms within the different tissue layers. In skeletal muscle, CaN-dependent transcriptional pathways regulate slow or fast twitch muscle fiber expression.
Cardiac development requires activation of CaN-dependent transcriptional pathways.
Activation of CaN and dephosphorylation of NFAT initiates a transcriptional cascade that ultimately leads to certain forms of cardiac hypertrophy. NFAT and CaN are expressed in gastrointestinal smooth muscle, but the physiological roles and transcriptional cascades regulated by CaN-dependent NFAT activation are unknown. A major focus of this lab is to investigate the isoform expression of CaN and NFAT in fundus and proximal colon smooth muscle tissues. Immunohistochemistry, RT-PCR, Northern blotting, luciferase reporter assays, and in situ hybridization are being used to elucidate the mechanisms of NFAT activation by CaN in fundus and proximal colon smooth muscle tissue. The major goals of this project are to elucidate the mechanisms underlying the substrate selectivity and sub-cellular distribution of the calcineurin a and b catalytic subunit isoforms.