In the heart, calcium influx via Cav1.2 channels has a key role in excitation-contraction coupling, and in determining the plateau phase of the action potential. Although CaV1.2 channels are known to associate with proteins that regulate channel trafficking, localization, turnover, and function, the components of these protein complexes have not yet been fully identified. Physiologic b-adrenergic activation of PKA during the sympathetic ?fight or flight? response increases calcium influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. In pathological conditions, increased CaV1.2 currents can trigger electrical instability, early after- depolarizations, arrhythmias, and sudden death, frequently in the setting of adrenergic stimulation or decreased repolarizing currents. The molecular mechanisms of b-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but up-regulation of CaV1.2 mediated by activation of PKA is required for this process. Based upon the failure to identify the regulatory sites in the heart, the difficulties in reconstituting the regulation using heterologous expression, and the challenges in creating knock-in mice or using adenoviral-based expression, the Marx laboratory developed the straightforward but rigorous approach of using doxycycline- inducible, tissue-specific, transgenic-mice-expressing FLAG-epitope-tagged, dihydropyridine (DHP)-resistant pore-forming a1C subunits. Recent data suggest that b-adrenergic regulation of CaV1.2 does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse a1C subunits. b-adrenergic regulation of a1C may require, however, a unique combination of species-specific phospho-regulatory sites in a1C. To test this hypothesis, I generated transgenic mice with doxycycline-inducible expression of rabbit a1C with alanine-substitutions of all conserved and non-conserved potential PKA sites in the intracellular regions (N-terminal, intracellular loops, and C-terminal regions). If b-adrenergic regulation is preserved in these mice, I will cross these mice with mice expressing a mutant b2b subunit in which consensus PKA phosphorylation sites are substituted with alanines. This study will test whether prior failures to identify a mechanism is because of redundancy between the a and b subunits and will provide the definitive answer about whether PKA phosphorylation of any a1C or b2 residues is necessary. Since it appears that the a1C and b2 subunits may not the primary functional PKA targets and due to the inability to reconstitute adrenergic regulation when the primary subunits are heterologously expressed, I further hypothesize that additional proteins expressed in cardiomyocytes may be required for adrenergic regulation of CaV1.2.
In Aim 2, I seek to identify and test these novel regulators of CaV1.2 in the heart. The two Aims will provide new insights the mechanisms responsible for b-adrenergic regulation of Ca2+ influx in cardiomyocytes.

Public Health Relevance

In the heart Ca2+ influx via Cav1.2 has a key role in excitation-contraction coupling and in determining the plateau phase of the action potential. Abnormal regulation of Cav1.2 is associated electrical instability, early after- depolarizations (EADs), arrhythmias, and sudden death frequently in the setting of adrenergic stimulation or decreased repolarizing currents. My overall goal is to identify mechanisms responsible for b-adrenergic regulation of CaV1.2 channels in the heart.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31HL142178-03
Application #
10063904
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Huang, Li-Shin
Project Start
2018-12-20
Project End
2021-12-19
Budget Start
2020-12-20
Budget End
2021-12-19
Support Year
3
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032