R01HL155377
Project Grant
Overview
Grant Description
Investigating Cardiac Ion Channels by Novel Methods
Our long-term overall goals are to discover physiologic homeostatic mechanisms underlying regulation of Cav1.2 channels in the heart and to identify novel therapeutic targets for heart failure and arrhythmias.
Cav1.2, the L-type Ca2+ channel that plays a key role in cardiac excitation-contraction coupling, is an important target of the sympathetic nervous system and several signaling pathways. Increased cardiac contractility during fight-or-flight response is caused by β-adrenergic augmentation of Cav1.2 channels. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant Cav1.2 A1C and β subunits, this regulation persists, implying involvement of extra-channel factors.
Recently, we identified the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca2+ channels. We expressed A1C or β2B subunits conjugated to ascorbate-peroxidase in mouse hearts and used multiplexed, quantitative proteomics to track hundreds of proteins in close proximity to Cav1.2. We observed that the Ca2+ channel inhibitor RAD, a monomeric G-protein, is enriched in the Cav1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific serine residues on RAD decreases its affinity for auxiliary β-subunits and relieves constitutive inhibition of Cav1.2 observed as an increase in channel open probability.
We propose three aims:
(1) Using knock-in mice with the four PKA phosphorylation sites of RAD mutated to alanine, and mice with cardiac-specific expression of a mutant Cavβ subunit that cannot bind RAD, we will determine in cardiomyocytes the role of RAD phosphorylation in regulating cardiac contractility in vivo.
(2) Having successfully applied proximity labeling, we now also propose to identify the A-kinase anchoring proteins (AKAPs) that facilitate β-adrenergic regulation of Cav1.2 in cardiomyocytes. The identity of the AKAP that facilitates β-adrenergic regulation of Cav1.2 in cardiomyocytes is unknown.
(3) PKG activation by cGMP inhibits Cav1.2 and counteracts β-adrenergic stimulation of Ca2+ current in cardiomyocytes. Strategic PKG activation could therefore serve as a targeted suppressor of adrenergic stimulation of Cav1.2 and concomitant arrhythmias. We hypothesize that PKG signaling blocks β-adrenergic-induced stimulation of Cav1.2 by at least one of several mechanisms: i) by direct PKG phosphorylation of A1C or β2B; ii) by preventing the recruitment of PKA to the Cav1.2 complex; iii) by preventing the dissociation of RAD from the Cav1.2 complex in the heart.
To assess whether PKG phosphorylation of A1C or β2B is required, we will utilize our fully phospho-mutant A1C and β2B transgenic mice that have normal β-adrenergic stimulation of Cav1.2. To dissect the upstream signaling pathways, we will utilize proximity proteomics.
The three aims, which will provide key new understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding the molecular mechanisms responsible for the modulation of cardiac contractility and arrhythmogenesis.
Our long-term overall goals are to discover physiologic homeostatic mechanisms underlying regulation of Cav1.2 channels in the heart and to identify novel therapeutic targets for heart failure and arrhythmias.
Cav1.2, the L-type Ca2+ channel that plays a key role in cardiac excitation-contraction coupling, is an important target of the sympathetic nervous system and several signaling pathways. Increased cardiac contractility during fight-or-flight response is caused by β-adrenergic augmentation of Cav1.2 channels. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant Cav1.2 A1C and β subunits, this regulation persists, implying involvement of extra-channel factors.
Recently, we identified the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca2+ channels. We expressed A1C or β2B subunits conjugated to ascorbate-peroxidase in mouse hearts and used multiplexed, quantitative proteomics to track hundreds of proteins in close proximity to Cav1.2. We observed that the Ca2+ channel inhibitor RAD, a monomeric G-protein, is enriched in the Cav1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific serine residues on RAD decreases its affinity for auxiliary β-subunits and relieves constitutive inhibition of Cav1.2 observed as an increase in channel open probability.
We propose three aims:
(1) Using knock-in mice with the four PKA phosphorylation sites of RAD mutated to alanine, and mice with cardiac-specific expression of a mutant Cavβ subunit that cannot bind RAD, we will determine in cardiomyocytes the role of RAD phosphorylation in regulating cardiac contractility in vivo.
(2) Having successfully applied proximity labeling, we now also propose to identify the A-kinase anchoring proteins (AKAPs) that facilitate β-adrenergic regulation of Cav1.2 in cardiomyocytes. The identity of the AKAP that facilitates β-adrenergic regulation of Cav1.2 in cardiomyocytes is unknown.
(3) PKG activation by cGMP inhibits Cav1.2 and counteracts β-adrenergic stimulation of Ca2+ current in cardiomyocytes. Strategic PKG activation could therefore serve as a targeted suppressor of adrenergic stimulation of Cav1.2 and concomitant arrhythmias. We hypothesize that PKG signaling blocks β-adrenergic-induced stimulation of Cav1.2 by at least one of several mechanisms: i) by direct PKG phosphorylation of A1C or β2B; ii) by preventing the recruitment of PKA to the Cav1.2 complex; iii) by preventing the dissociation of RAD from the Cav1.2 complex in the heart.
To assess whether PKG phosphorylation of A1C or β2B is required, we will utilize our fully phospho-mutant A1C and β2B transgenic mice that have normal β-adrenergic stimulation of Cav1.2. To dissect the upstream signaling pathways, we will utilize proximity proteomics.
The three aims, which will provide key new understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding the molecular mechanisms responsible for the modulation of cardiac contractility and arrhythmogenesis.
Funding Goals
TO FOSTER HEART AND VASCULAR RESEARCH IN THE BASIC, TRANSLATIONAL, CLINICAL AND POPULATION SCIENCES, AND TO FOSTER TRAINING TO BUILD TALENTED YOUNG INVESTIGATORS IN THESE AREAS, FUNDED THROUGH COMPETITIVE RESEARCH TRAINING GRANTS. SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM: TO STIMULATE TECHNOLOGICAL INNOVATION, USE SMALL BUSINESS TO MEET FEDERAL RESEARCH AND DEVELOPMENT NEEDS, FOSTER AND ENCOURAGE PARTICIPATION IN INNOVATION AND ENTREPRENEURSHIP BY SOCIALLY AND ECONOMICALLY DISADVANTAGED PERSONS, AND INCREASE PRIVATE-SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT FUNDING. SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM: TO STIMULATE TECHNOLOGICAL INNOVATION, FOSTER TECHNOLOGY TRANSFER THROUGH COOPERATIVE R&D BETWEEN SMALL BUSINESSES AND RESEARCH INSTITUTIONS, AND INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL R&D.
Grant Program (CFDA)
Awarding / Funding Agency
Place of Performance
New York
United States
Geographic Scope
State-Wide
Related Opportunity
Analysis Notes
Amendment Since initial award the End Date has been extended from 05/31/25 to 05/31/29 and the total obligations have increased 482% from $528,708 to $3,076,196.
The Trustees Of Columbia University In The City Of New York was awarded
Cardiac Ion Channels Regulation for Heart Health
Project Grant R01HL155377
worth $3,076,196
from National Heart Lung and Blood Institute in June 2021 with work to be completed primarily in New York United States.
The grant
has a duration of 8 years and
was awarded through assistance program 93.837 Cardiovascular Diseases Research.
The Project Grant was awarded through grant opportunity NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed).
Status
(Ongoing)
Last Modified 8/20/25
Period of Performance
6/5/21
Start Date
5/31/29
End Date
Funding Split
$3.1M
Federal Obligation
$0.0
Non-Federal Obligation
$3.1M
Total Obligated
Activity Timeline
Transaction History
Modifications to R01HL155377
Additional Detail
Award ID FAIN
R01HL155377
SAI Number
R01HL155377-3830280112
Award ID URI
SAI UNAVAILABLE
Awardee Classifications
Private Institution Of Higher Education
Awarding Office
75NH00 NIH National Heart, Lung, and Blood Institute
Funding Office
75NH00 NIH National Heart, Lung, and Blood Institute
Awardee UEI
QHF5ZZ114M72
Awardee CAGE
3FHD3
Performance District
NY-90
Senators
Kirsten Gillibrand
Charles Schumer
Charles Schumer
Budget Funding
Federal Account | Budget Subfunction | Object Class | Total | Percentage |
---|---|---|---|---|
National Heart, Lung, and Blood Institute, National Institutes of Health, Health and Human Services (075-0872) | Health research and training | Grants, subsidies, and contributions (41.0) | $1,174,906 | 100% |
Modified: 8/20/25