CAPON modulates cardiac repolarization via neuronal nitric oxide synthase signaling in the heart

Abstract

Congenital long- or short-QT syndrome may lead to life-threatening ventricular tachycardia and sudden cardiac death. Apart from the rare disease-causing mutations, common genetic variants in CAPON, a neuronal nitric oxide synthase (NOS1) regulator, have recently been associated with QT interval variations in a human whole-genome association study. CAPON had been unsuspected of playing a role in cardiac repolarization; indeed, its physiological role in the heart (if any) is unknown. To define the biological effects of CAPON in the heart, we investigated endogenous CAPON protein expression and protein-protein interactions in the heart and performed electrophysiological studies in isolated ventricular myocytes with and without CAPON overexpression. We find that CAPON protein is expressed in the heart and interacts with NOS1 to accelerate cardiac repolarization by inhibition of L-type calcium channel. Our findings provide a rationale for the association of CAPON gene variants with extremes of the QT interval in human populations.

Fig. 1.

Identification of endogenous CAPON protein in the heart. (A) Tissue homogenates from normal guinea pig ventricular myocardium (VM), brain, and lung, and HEK293 cells with (T) and without (NT) in vitro transduction with AdCAPON-GFP were subjected to SDS/PAGE Western blotting. Both full-length (CAPON-L) and short-form of CAPON (CAPON-S) are expressed in VM. The higher bands (arrowhead) could be caused by either posttranslational modification of CAPON or nonspecific cross-reactivity. (B) Immunostaining of CAPON in AdCAPON-GFP-transduced (3) and nontransduced (2) ventricular myocytes. Negative controls (secondary antibody only) are depicted in 1.

Fig. 2.

Electrophysiological effects of CAPON overexpression in guinea pig ventricular myocytes. (A and B) Adenovirus alone does not affect electrophysiology. For details, see SI Results. (C–H) CAPON overexpression abbreviates the action potential duration and reduces I_Ca,L. (C) APD was markedly shortened in a representative AdCAPON-GFP-transduced myocyte compared with a representative control myocyte. (D) Significant abbreviation of APD can be seen spanning from APD₁₀ to APD₉₀ at 1-Hz stimulation in CAPON-overexpressing myocytes (CAPON, n = 9; control, n = 13). (E) The actual APD₅₀ and APD₉₀ measured from individual myocyte comprising each group reveal a consistent reduction of APD in CAPON-overexpressing myocytes. (F) Representative I_Ca,L recordings elicited by depolarizing voltage steps (500 ms) from −40 to +60 mV in 10-mV increments after a prepulse from −80 mV to −40 mV show reduction of I_Ca,L in a AdCAPON-GFP-transduced myocyte compared with a control myocyte. (G) The peak I_Ca,L density in CAPON-overexpressing myocytes was smaller than in control myocytes. (H) Averaged peak current–voltage relationships demonstrate attenuation of I_Ca,L at multiple depolarizing pulses in CAPON-overexpressing myocytes (CAPON, n = 8; control, n = 12). The number inside each bar graph indicates the number of cells studied.

Fig. 3.

Electrophysiological effects of CAPON overexpression in guinea pig ventricular myocytes. (A and B) CAPON overexpression does not affect sodium current. For more details, see SI Results. (C–E) CAPON overexpression enhances I_Kr. (C) Delayed rectifier K⁺ tail current was measured in response to a depolarizing pulse to +40 mV for 5 s followed by repolarization to −40 mV. I_Kr was recorded after steady-state suppression of I_Ks by chromanol 293B. Representative recordings show a larger I_Kr tail current in a CAPON-overexpressing myocyte compared with a control myocyte. (D) Neither peak I_K nor I_Ks tail current densities were significantly different between CAPON-overexpressing and control myocytes. However, peak I_Kr tail current density was modestly enhanced in CAPON-overexpressing myocytes. (E) Instantaneous I_K1 current density elicited by ramp protocol from −100 to + 70 mV (5 s) was not different between CAPON-overexpressing (n = 10) and control myocytes (n = 9).

Fig. 4.

CAPON interacts with NOS1, but not NOS3, in ventricular myocytes. Normal guinea pig ventricular tissue homogenates were immunoprecipitated overnight with anti-CAPON-bound protein G complex, separated by SDS/PAGE, and probed by anti-NOS1 (A), anti-CAPON (B and D), and anti-NOS3 (C), respectively. To include a negative control, the ventricular tissue homogenates were also incubated overnight with anti-CAPON-free protein G complex (beads). As a result, neither NOS1 nor CAPON could be detected in the eluted precipitates. WB, Western blotting.

Fig. 5.

CAPON overexpression stabilizes NOS1 in ventricular myocytes. (A) Freshly isolated ventricular myocytes were transduced in vitro with AdCAPON-GFP, or AdGFP, or not transduced with either virus. BF, bright-field microscopic images. (B) The in vitro AdCAPON-GFP transduction caused a 1.9-fold increase of the CAPON level. (C) At 0 h of culture, the NOS1 level was not different between AdCAPON-GFP-transduced and nontransduced myocytes (n = 3 animals). (D) After 40.3 ± 2.3 h of cell culture, NOS1 became down-regulated in both ventricular myocytes transduced with AdGFP and in nontransduced myocytes, whereas NOS1 was well preserved in the AdCAPON-GFP-transduced ventricular myocytes. The mean NOS1 level from nontransduced and AdGFP-transduced myocytes was lower than that of AdCAPON-GFP-transduced myocytes (*, P < 0.05 vs. control, n = 3 animals). Noncultured ventricular tissue homogenates (VM) served as positive control for NOS1 detection. The signal intensities of CAPON and NOS1 are normalized against the GAPDH signals.

Fig. 6.

Enhanced intracellular NO production in CAPON-overexpressing myocytes. (A) The specificity of DAR-4M AM was verified by a differential fluorescent intensity with the addition of SNP or l-NAME. (B) The NO fluorescence was enhanced after l-arginine stimulation. In GFP (+), CAPON-overexpressing myocytes, the NO fluorescence was stronger than in control myocytes. (C) Representative high-powered images illustrate a modest enhancement of the NO fluorescent marker in a CAPON-overexpressing myocyte. (D) The baseline fluorescent intensity was equivalent in CAPON-overexpressing (n = 30) and control myocytes (n = 90), whereas with the addition of l-arginine, the fluorescence intensity increased disproportionately in CAPON-overexpressing myocytes (CAPON-l-arg) (n = 43) compared with controls (Control-l-arg) (n = 166).

Fig. 7.

l-NAME reverses CAPON overexpression-mediated APD abbreviation and I_Ca,L reduction. (A) Representative action potential recordings show reversal of APD abbreviation with l-NAME in a CAPON-overexpressing myocyte. (B) Pretreatment with l-NAME significantly increased APD₉₀ and reversed APD abbreviation in CAPON-overexpressing ventricular myocytes, whereas the APD90 was not significantly changed with l-NAME in control myocytes. (C) Representative I_Ca,L recordings show reversal of I_Ca,L suppression with l-NAME in a CAPON-overexpressing myocyte. (D) Averaged peak current–voltage relationships reveal significant increase of peak I_Ca,L densities with l-NAME in CAPON-overexpressing myocytes but not in control myocytes. The number of cells studied in each group is indicated in bar graphs in E. (E) Pretreatment with l-NAME significantly increased peak I_Ca,L density and rescued I_Ca,L suppression in CAPON-overexpressing myocytes, whereas the peak I_Ca,L density was not significantly changed with l-NAME in control myocytes.