Grants and Contracts Details
In the heart, the L-type calcium channel (LTCe) is essential for excitation-contraction coupling (ECC), regulating calcium stores, and signaling to the nucleus via its carboxyl terminus (CCT). Pilot data shows that in excised embryonic day (E) 10 mouse hearts, chronic pharmacological inhibition of LTCCs perturbs heart development. Therefore, the goal of this project is to discover the mechanism that governs this phenomenon. To this end, there are two possibilities: 1) LTCC regulation of calcium dynamics controls heart development and 2) CCT signaling to the nucleus controls heart development. We postulate that LTCC nuclear signaling via the CCT is the primary means by which the LTCC regulates heart development. To test this, dispersed E10 mouse cardiomyocytes chronically treated with pharmacological LTCC block (LTCCS) will be used as a model system in the following specific aims: Specific Aim 1 will test the hypothesis that signaling induced by sustained LTCCS is directly transduced by the LTCC itself; sustained LTCCS does not necessarily alter ECC or sarcoplasmic reticulum (SR) calcium load. Spark frequency, an index of ECC, and SR load will be measured using calcium imaging. To confirm that LTCCS is not affecting LTCC current (ICa,L), a contributor to ECC and SR load, ICa,L will be measured using perforated patch. Specific Aim 2 will test two hypotheses. The first hypothesis is that the LTCC is a signaling molecule; therefore LTCCS will increase nuclear localization of CCT. To test this, immunocytochemistry of overexpressed epitope-tagged CCT as well as endogenous CCT will be performed to confirm that the CCT localizes to the nucleus and that this localization is regulated by LTCCS. The second hypothesis is that active Cav1.2 channels, the primary LTCC subtype in the heart, anchor CCT. For these experiments E10 cardiac fibroblasts (CFs), which can be harvested co-currently with E10 cardiomyocytes, will be used because they show increased CCT nuclear localization. Presumably, this is due to a lack of LTCCs; therefore, LTCC mRNA will first be measured using qPCR to confirm this. To test that active Cav1.2 anchors CCT, Cav1.2 will be overexpressed in CFs and CCT nuclear localization will be measured using immunocytochemistry.
|Effective start/end date||7/1/07 → 6/30/09|
- American Heart Association Ohio Valley Affiliate: $42,000.00
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