Connexin 43 Dephosphorylation: A Potential Underlying Mechanisms Involved in Exercise-Induced Cardiac Fatigue?

Research Article

Austin Biol. 2016; 1(4): 1018.

Connexin 43 Dephosphorylation: A Potential Underlying Mechanisms Involved in Exercise-Induced Cardiac Fatigue?

Vitiello D1,2,3,4*

¹Institute for Biomedical and Epidemiological Research in Sport, France

²EA 7329, Paris Descartes University, Sorbonne Paris Cité, Paris, France

³National Institute of Sport, Expertise and Performance – INSEP, Paris, France

4Avignon University, LAPEC EA4278, F-84000 Avignon, France

*Corresponding author: Vitiello D, School of Sport Sciences, Paris Descartes University, 1 Rue Lacretelle, 75015, Paris, France

Received: November 09, 2016; Accepted: December 05, 2016; Published: December 07, 2016


Background: Endurance exercise races have grown in popularity over the last several years. Even if regular exercise is beneficial for cardiovascular health, many studies also demonstrated that running a marathon or a longdistance triathlon induces transient Left Ventricle (LV) dysfunctions associated in some athletes with myocardial damages. The underlying mechanisms of these ventricular dysfunctions are not fully elucidated.

Methods: Adult male Wistar rats were randomly assigned to the following 4 groups (8 rats per group): Control (Ctrl), Exercised Group (4 hours run on treadmill) (Ex) and 2 groups in which NADPH oxidase enzyme was inhibited with apocynin treatment (Ctrl-APO and Ex-APO, respectively). Cardiac function was evaluated ex vivo during basal conditions using the isolated perfused Langendorff heart model. Connexin 43 (Cx43) dephosphorylation was assessed in LV myocardium by western blotting.

Results: The prolonged exercise of 4 hours on treadmill induced a decrease in LV intrinsic myocardial contractility and relaxation and a dephosphorylation of Cx43 in the LV myocardium. Interestingly, the apocynin treatment, specific inhibitor of the NADPH enzyme, reversed LV dysfunctions and prevented Cx43 dephosphorylation.

Conclusion: The present findings show a potential implication of the NADPH oxidase enzyme in myocardial connexin 43 dephosphorylation which can be involved in LV dysfunctions following a prolonged exercise in rat. This new underlying mechanism insight of exercise-induced cardiac fatigue may help to determine the long-term effects of prolonged exercises on cardiac risk in athletes.

Keywords: Cardiac fatigue; Connexin 43; Oxidative stress; Prolonged exercise


cTnI: Cardiac Troponin I; Ctrl: Control Group; Ctrl-APO: Control Group Treated with apocynin; Cx43: Connexin 43; dP/ dtmax: Left Ventricle Intrinsic Myocardial Contractility; dP/dtmin: Relaxation; Ex: Exercised Group; Ex-APO: Exercised Group Treated with apocynin; LV: Left Ventricle; NADPH: Nicotinamide Adenine Dinucleotide Phosphate Oxidase Enzyme; PSE: Prolonged Strenuous Exercise


The popularity of endurance exercise races has increased worldwide over the last thirty years. However, a growing number of studies also demonstrated that running a long-distance race induces transient ventricular dysfunctions associated in some subjects with an increase in Cardiac Troponin I (cTnI), a biomarker of myocardial damages [1,2]. Oxidative stress has been proposed to explain this endurance exercise-induced cardiac fatigue [3,4]. Among the potential sources of oxidative stress like xanthine oxidase, uncoupled nitric oxide synthases and the mitochondrial electron transport chain, the Nicotinamide Adenine Dinucleotide Phosphate Oxidase Enzyme (NADPH) is considered as major sources of ROS in the myocardium [5,6]. Our research team has previously reported a significant link between NADPH oxidase-dependent oxidative stress and myocardial dysfunction after a Prolonged Strenuous Exercise (PSE) on treadmill in rat [3]. Despite some advances in the explanation of exerciseinduced cardiac fatigue, it was recently mentioned that mechanisms producing the decrease in cardiac function after exercise still not clear because of the multiplicity of factors which may contribute to this phenomenon [7]. Gap junctions are essential for the heart function notably by their important role in impulse conduction. They are low resistance connections between adjacent myocardial cells and are predominantly composed by the connexin 43 (Cx43) [8]. In addition, the phosphorylation of Cx43 is essential for gap junction plaque forming and thus for intercellular communication [9-11]. Previous studies in animals have demonstrated that pathological conditions like heart failure and cardiac ischemia induce Cx43 protein dephosphorylation which regulate connexin function [12- 14]. Furthermore, Cx43 dephosphorylation can be induced by oxidative stress generated by the NADPH oxidase enzyme which can exhert arrhythmogenic effect [12]. Therefore, among the potential mechanisms, gap junctions remodeling might be involved in PSEinduced LV dysfunctions.

However, to date, the impact of PSE on NADPH oxidasedependent oxidative stress on Cx43 phosphorylation status associated with LV myocardial function assessment have not been previously described. Thus, the hypothesis of the present study is that NADPH oxidase-dependent redox status modulation induces Cx43 dephosphorylation which represents one of the underlying mechanisms involved in LV myocardial dysfunctions after a PSE.


All procedures were performed in agreement with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85-23, revised 1996) and approved by the French ministry of agriculture. Adult Wistar male rats were randomly assigned to the following groups (8 rats per group): one Control Group (Ctrl) and one Exercised Group (Ex). Two other groups of 8 rats were treated with apocynin, (Sigma Aldrich, France) a bio-absorbable specific inhibitor of the NADPH oxidase enzyme, in drinking water (1.5 mM) as previously described [15] and were assigned to a Ctrl-APO and Ex-APO groups. All exercise sessions were performed on a motorized rodent treadmill. All exercised animals were first familiarized with the treadmill over 5 days then performed an incremental test to determine their maximal running speed and finally performed a Prolonged Strenuous Bout of Exercise (PSE) at 60-65% of their maximal running speed for 4 hours.

All the Ctrl, Ctrl-APO (n = 8 / group) and Ex, Ex-APO (n = 8 / group) animals were killed within 30 min following exercise for isolated heart experiments as previously described [3]. Another set of animals (Ctrl, Ctrl-APO and Ex, Ex-APO; n = 4 / group) were killed within 30 min following exercise for western blots analyses of the nonphosphorylated form (i.e. P0 form) of the Cx43 protein (monoclonal Cx43 antibody (13-8300), Thermo Fisher Scientific, France) [16].

Statistical Analysis

Data were analyzed using one-way or two-way ANOVA among groups. When significant interactions were found, a Bonferroni t-test was applied with adjusted P>0.05 (Stat view 2.20; Adept Scientific, Letch worth, UK). Data are presented as means ± S.E.M.


The cardiac function of Ex animals was altered by the PSE compared to Ctrl animals (Figure 1). The LV intrinsic myocardial contractility (dP/dtmax) and relaxation (dP/dtmin) were respectively significantly decreased by 37% (P>0.05) and 34% (P>0.05) in Ex rats compared to Ctrl rats. The specific inhibition of NADPH oxidase by the apocynin treatment improved myocardial function in Ex-APO rats to the level of Ctrl and Ctrl-APO rats (Figure 1). Similarly, LV dP/dtmax (Figure 1, upper graph), dP/dtmin (Figure 1, lower graph) were normalized in Ex-APO rats to the level found in Ctrl and Ctrl- APO rats. The PSE induced a 41% increase of the non-phosphorylated status of Cx43 in Ex animals compared to Ctrl animals (P>0.05, Figure 2). Interestingly, the apocynin treatment normalized Cx43 phosphorylation status in Ex-APO rats to the level found in Ctrl and Ctrl-APO rats (Figure 2).

Citation: Vitiello D. Connexin 43 Dephosphorylation: A Potential Underlying Mechanisms Involved in Exercise- Induced Cardiac Fatigue?. Austin Biol. 2016; 1(4): 1018.