Abstract 111: High Activity Gating of Caveolae-Targeted L-Type Ca2+ Channels Can Initiate Pathological Hypertrophy
Introduction: The source of Ca2+ that activates pathological cardiac hypertrophy is not clearly defined. We hypothesize that high activity gating of L-type Ca2+ channels (LTCCs) localized in caveolae signaling microdomains stabilized by caveolin-3 (Cav-3) provides the Ca2+ influx that locally activates calcineurin (Cn)-mediated nuclear factor of activated T-cells (NFAT) to induce hypertrophic gene expression. We generated novel reagents that specifically activate or inhibit the gating of LTCCs in caveolae for analysis of the hypertrophic program, as well as any potential effect on ICa and contraction.
Methods and Results: We targeted the known LTCC inhibitory protein Rem or an LTCC subunit (β2a) that promotes high activity gating specifically to caveolae by fusing them to a caveolin-binding domain peptide (termed Cav-Rem and Cav-β2a). We infected adult feline left ventricular myocytes (AFLVMs) with adenoviruses containing Cav-Rem or Cav-β2a for membrane localization and functional studies. NFAT nuclear translocation was determined by co-infecting AFLVMs with ad-NFAT-GFP and either ad-Cav-Rem or ad-Cav-β2a and pacing cells to induce Ca2+ influx mediated nuclear NFAT-GFP translocation. Membrane fractionation experiments showed that Cav-3 membrane domains contain 26.2 +/- 12.7% of membrane targeted LTCCs and blocking these with Cav-Rem eliminated a small fraction of the LTCC current (<15%) and almost all Ca2+ influx induced NFAT nuclear translocation (>90%), but did not reduce myocyte contractility. Conversely, selective enhancement of LTCC activity within caveolae with Cav-β2a caused a significant increase in NFAT nuclear translocation (>70%) but had no significant effect on contractility.
Conclusions: We provide proof of concept that specific Cav-targeted reagents can be used to enhance or inhibit LTCC activity within caveolae microdomains to amplify or block the hypertrophic response. Our results suggest that high activity gating of LTCCs, which is known to be present in the hypertrophied failing human heart, can activate signaling pathways linked to cardiac hypertrophy. Selectively inhibiting these caveolae localized LTCCs could be a novel mechanism to block pathological hypertrophy without reducing cardiac contractility.
- © 2012 by American Heart Association, Inc.