Anti-arrhythmic Cardiac Phenotype Elicited by Chronic Intermittent Hypoxia Is Associated With Alterations in Connexin-43 Expression, Phosphorylation, and Distribution

Abstract

Remodeling of the cellular distribution of gap junctions formed mainly by connexin-43 (Cx43) can be related to the increased incidence of cardiac arrhythmias. It has been shown that adaptation to chronic intermittent hypobaric hypoxia (IHH) attenuates the incidence and severity of ischemic and reperfusion ventricular arrhythmias and increases the proportion of anti-arrhythmic n-3 polyunsaturated fatty acids (n-3 PUFA) in heart phospholipids. Wistar rats were exposed to simulated IHH (7,000 m, 8-h/day, 35 exposures) and compared with normoxic controls (N). Cx43 expression, phosphorylation, localization and n-3 PUFA proportion were analyzed in left ventricular myocardium. Compared to N, IHH led to higher expression of total Cx43, its variant phosphorylated at Ser368 [p-Cx43(Ser368)], which maintains "end to end" communication, as well as p-Cx43(Ser364/365), which facilitates conductivity. By contrast, expression of non-phosphorylated Cx43 and p-Cx43(Ser278/289), attenuating intercellular communication, was lower in IHH than in N. IHH also resulted in increased expression of protein kinase A and protein kinase G while casein kinase 1 did not change compared to N. In IHH group, which exhibited reduced incidence of ischemic ventricular arrhythmias, Cx43 and p-Cx43(Ser368) were more abundant at "end to end" gap junctions than in N group and this difference was preserved after acute regional ischemia (10 min). We further confirmed higher n-3 PUFA proportion in heart phospholipids after adaptation to IHH, which was even further increased by ischemia. Our results suggest that adaptation to IHH alters expression, phosphorylation and distribution of Cx43 as well as cardioprotective n-3PUFA proportion suggesting that the anti-arrhythmic phenotype elicited by IHH can be at least partly related to the stabilization of the "end to end" conductivity between cardiomyocytes during brief ischemia.

Keywords: arrhythmia; brief ischemia; chronic hypoxia; connexin-43; heart; n-3 PUFA.

Figure 1

Effect of chronic intermittent hypobaric hypoxia (IHH, black columns) on expression of total Cx43 (B) and its high-phosphorylated forms (P1+P2 Cx43) (C) compared with normoxic group (N, open columns). The representative image of western blotting is shown (A). Values are mean ± SEM, (n = 5 in each group), ^**p < 0.01.

Figure 2

Effect of chronic intermittent hypobaric hypoxia (IHH, black columns) on protein level of non-phosphorylated Cx43 (A), phosphorylated Cx43 at Ser368 (B), phosphorylated Cx43 at Ser279/282 (C), and phosphorylated Cx43 at Tyr265 (D) assessed by western blotting in the left ventricular myocardium. N, normoxic group (open columns). Values are mean ± SEM, (n = 5 in each group), ^**P < 0.01.

Figure 3

Effect of chronic intermittent hypobaric hypoxia (IHH, black columns) on phosphorylation of Cx43 at Ser364 (A), Ser365 (B), and Ser368 (C) assessed by Mass Spectrometry in left ventricular myocardium. N, normoxic group (open columns). Values are mean ± SEM, (n = 5 in each group), ^**P < 0.01.

Figure 4

Effect of chronic intermittent hypobaric hypoxia (IHH, black columns) on protein expression of Cx43 upstream protein kinases: protein kinase A (PKA) (A), protein kinase G (PKG) (B), and casein-kinase 1 (CK1) (C) assessed by western blotting in left ventricular myocardium. N, normoxic group (open columns). Values are mean ± SEM, (n = 5 in each group), ^**P < 0.01.

Figure 5

Effect of chronic intermittent hypobaric hypoxia (IHH) on distribution of total Cx43. The representative micrograph of longitudinal cryosections demonstrates distribution of t-Cx43 (A) and p-Cx43(Ser368) (C) after 10-min regional ischemia in normoxic (N Isch) and hypoxic (IHH Isch) left ventricular myocardium compared to control groups not subjected to ischemia (N, IHH). The green color corresponds to specific Cx43 staining, red color represents sarcolemma (counterstained by WGA) and the blue color indicates nuclei DAPI staining. Note the positive Cx43 staining (arrows) located predominantly at the intercalated discs, i.e., “end to end” junctions of the cardiomyocytes. The proportion of t-Cx43 and p-Cx43(Ser368) at “end to end” junctions in normoxic (N) and hypoxic (IHH) groups under control conditions and after ischemia (Isch) (B,D) is expressed as a percentage of total junctions. Values are mean ± SEM, (n = 6 in each group), ^* vs. corresponding normoxic group; ^t vs. corresponding non-ischemic group; ^tP ≤ 0.05, ^**/^ttP ≤ 0.01, ^***/^tttP ≤ 0.001. Scale bar represents 20 μm.

Figure 6

Effect of chronic intermittent hypobaric hypoxia on main polyunsaturated fatty acids [linoleic acid (18:2n−6), arachidonic acid (20:4n−6), docosahexaeonic acid (22:6n−3)], total n−6 PUFA and total n−3 PUFA proportion in total phospholipids in the left ventricular myocardium of normoxic (N) and hypoxic (H) rats under control (non-ischemic) conditions and after 10-min regional ischemia (NI and HI, respectively. Values are mean ± SEM, (n = 7 in each group), *P < 0.05 vs. normoxia, †P < 0.05 vs. corresponding control group.

Figure 7

Effect of chronic intermittent hypobaric hypoxia (IHH, black columns) on the incidence of ventricular tachyarrhythmias (tachycardia and fibrillation) (A), total duration of ventricular tachyarrhythmias (B) and total number of premature ventricular complexes (PVCs) occurring as singles, salvos and tachycardia (C) during 10-min regional ischemia. N, normoxic group (open columns). Values are mean ± SEM, (n = 13 in each group), ^*P < 0.05.