Abstract 170: CAPON Interacts With Neuronal Nitric Oxide Synthase To Modulate L-type Calcium Current And Action Potential Duration In Ventricular Cardiomyocytes
Background: We have previously demonstrated expression of endogenous CAPON in heart and overexpression of CAPON resulting in shortening of action potential duration (APD) via reduction of ICa,L and increase of IKr. We hypothesized that the effects of CAPON overexpression are due to modifications of the NOS-NO pathways in the heart. Therefore, we sought to explore how CAPON interacts with NOS in ventricular myocytes (VM) and to dissect the mechanisms of CAPON overexpression-mediated changes in cellular electrophysiology.
Methods and Results: To probe protein-protein interaction of CAPON and NOS, normal guinea pig VM lysates were immunoprecipitated with CAPON antibody and probed by CAPON, nNOS and eNOS antibodies, respectively. We found that CAPON-nNOS, but not eNOS, exists as a physiological complex in VM. To explore how CAPON overexpression affects NOS, freshly isolated VM were in vitro transduced with an adenoviral vector (AdCAPON-GFP), or the reporter only AdGFP, or not infected with any virus. After 40.3 ± 1.9 hours of cell culture, nNOS became hardly detectable in control VM with and without AdGFP transduction by western blot, whereas nNOS was preserved in the AdCAPON-GFP transduced VM (n=3). We further imaged intracellular NO using DAR-4M AM in living VM isolated 3–5 days after in vivo gene transfer of CAPON. With 2 mM L-arginine, the NO fluorescence was increased in CAPON-overexpressed VM (1421 ± 28.2 au, n=43) compared to that of the control VM (1329 ± 19.1 au, n=166; p<0.05). In patch-clamp studies, in CAPON-overexpressed VM, the peak ICa,L density was smaller (−5.1 ± 0.9 pA/pF, n=14 vs. −6.5 ± 0.8 pA/pF, n=14; p<0.001) and the APD90 was shorter (221.6 ± 18.2 ms, n=6 vs. 333.7 ± 18.7 ms, n=7; p<0.01) than those of the control VM. Pretreatment with 1 mM L-NAME reversed the peak ICa,L density reduction (−6.2 ± 1.4 pA/pF, n=14 vs. −6.6 ± 1.3 pA/pF, n=14; p=NS) and the APD90 abbreviation (348.3 ± 44.2 ms, n=7 vs. 378.5 ± 44.8 ms, n=5; p=NS) in CAPON-overexpressed VM.
Conclusions: We demonstrate CAPON-nNOS protein-protein interaction in VM and find that overexpression of CAPON may upregulate the nNOS-NO pathway, thereby modulating ICa,L and APD. The findings provide a rationale for the association of CAPON gene variants with extremes of the QT interval in human populations.