Changes in Myocardial Repolarization and Connexin-43 are Involved in Susceptibility of Hypertrophic Heart to
Low K⁺-Induced Ventricular Fibrillation

Narcis Tribulova^a, Ludka Okruhlicova^a, Nobue Hirosawa^b, K Ogawa^c, and Issei Imanaga^b

^aInstitute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovakia
^bDepartment of Physiology and ^cDepartment of Anatomy, Fukuoka University, Fukuoka, Japan

Correspondence: N Tribulova, Institute for Heart Research, SAS, Dubravska cesta 9, P.O. Box 104, 840 05 Bratislava, Slovakia.
E-mail: usrdtri@savba.sk, phone +421 2 54774405, fax +421 2 54776637

1.  Introduction

Alterations in myocardial electrical coupling at the gap junctions play an important role in arrhythmogenesis. Our previous studies indicate that age-, NO-deficient hypertension- or diabetes-related gap junction remodeling (Okruhlicova et al., 2002; Tribulova et al. 2002, 2002a) and acute impairment of the intercellular coupling due to Ca²⁺ overload (Imanaga et al. 2002; Tribulova et al. 2001) facilitate occurrence of sustained ventricular or atrial fibrillations. We, as well as others (Peters et al. 1977) have found that the ability of the heart to ensure myocardial synchronization and electrical stability is decreased if the number of gap junctions is reduced and their distribution is abnormal or locally absent. Remodeling of gap junctions appears to create anatomic substrate of arrhythmias (Safitz 1999), while acute disorders of intracellular free Ca²⁺ and intercellular coupling are crucial in the initiation and maintenance of malignant arrhythmias. The aim of this study was to investigate possible arrhythmogenic factors in spontaneously hypertensive rats (SHR) as well as their susceptibility to low K⁺-induced arrhythmias.

2.  Material and Methods

The experiments were performed on isolated hearts of 13 week-old SHR and age-matched Wistar Kyoto rats (WKY). Equilibration of the heart by Langendorff perfusion with oxygenated, 37º C warm, standard Krebs solution at a constant pressure was followed by perfusion with low K⁺ for 60 min unless sustained ventricular fibrillation (SVF) occurred earlier. Electrocardiogram and epicardial monophasic action potentials (MAPs) in two points were continuously monitored. Myocardial tissue was taken for ultrastructural analysis as well as for immunodetection and expression of the main gap junction protein, connexin-43 (Cx43).

3.  Results

A	B

Figure 1. A Susceptibility of WKY and SHR hearts to low K⁺-induced arrhythmias. VPBs – ventricular premature beats; BG – bigeminy; VT – ventricular tachycardia; TVF – transient and SVF – sustained ventricular fibrillations. *significant difference (p<0.05) between SHR and WKY rats; B WB data showing a significantly lower amount of the phosphorylated (P1) form of connexin-43 in SHR vs. WKY hearts under control conditions (white columns), as well as a decrease of P1/P0 ratio due to low K⁺ perfusion (black columns, *P<0.001).

Besides, much frequent side-to-side than end-to-end gap junctions were found in these hearts. Low K⁺ perfusion itself results in increase of MAP₉₀ to 221.5+9.4ms in WKY and 319.2+19.7ms in SHR. In parallel with MAP prolongation there was an increase in incidence of triggered electrical activity manifested by early afterdepolarizations (EADs) linked with the occurrence of premature beats (that occurred earlier in SHR than WKY hearts). Low K⁺-induced impairment of Cx43 was characterized by decrease in phosphorylated isoform (Fig 1B) as well as by localized loss of both immunostaining and integrity of gap junctions. Consequently the incidence of ventricular tachycardia (70% vs. 50%) and both transient (50% vs. 25%) and sustained (60% vs. 25%) ventricular fibrillation was higher in SHR than WKY (Fig 1A).

4.  Discussion

The main finding of this study is that both prolongation of MAP duration and decrease in connexin-43 phosphorylation likely contribute to higher susceptibility of SHR hearts to low K⁺-induced arrhythmias. Moreover, we have demonstrated a close relationship between the prolongation of MAPs and the occurrence of EADs (linked with VPBs) in the whole heart preparation.

It has been reported that hypertrophic hearts of various etiologies may develop Ca²⁺ oscillations and triggered activity (Balke and Shorofsky 1998, McIntosh et al. 1998). Therefore, it can be expected that the hypertrophic heart of SHR, which exhibits prolongation of APD (Hojo et al.,1992) and/or MAP (shown in this study) will be prone to develop Ca²⁺ disturbances. Hypokalemia itself prolongs repolarization and contributes to Ca²⁺ oscillations resulting in EADs and VPBs (Tribulova et al. 2002, January and Riddle 1989). The higher vulnerability to Ca²⁺ overload and Ca²⁺-related disorders of SHR hearts may result also from abnormal Ca²⁺ handling, i.e., from increased expression of Na⁺/Ca²⁺ exchanger and suppression of the sarcoplasmic reticulum (Hojo et al 1992). The increased susceptibility of SHR hearts to low K⁺-related Ca²⁺ disturbances may account not only for the earlier incidence of premature beats that initiate transient arrhythmias, but also for the significantly higher incidence of VF compared with WKY.

Triggered activity is known to precede ventricular tachycardia and fibrillation, whereby re-entry is considered as the underlying mechanism (Peters et al. 1997). Re-entry was reported to be closely associated with both dispersion of myocardial repolarization and decreased intercellular gap junctional coupling. Coordinated contraction of the heart depends on the rapid, orderly propagation of action potentials via specialized channels clustered in gap junctions (Tribulova et al., 2001, Safitz 1999). Since connexin-43 is a phosphoprotein, changes in its phosphorylation state could modulate the degree of cell-to-cell coupling (Duthe et al., 2000, Imanaga et al., 2002). Abnormal phosphorylation due to low K+ perfusion as well as localized loss of immunostaining and integrity of gap junctions suggest further impairment of myocardial electrical coupling. These changes may promote re-entry arrhythmias.

In conclusion, these results indicate that hypertension-related electrical and gap junction remodeling are important pro-arrhythmic factors facilitating initiation and persistence of ventricular fibrillation in the setting of acute Ca²⁺ disorders due to hypokalemia.

Acknowledgements

This work was partially supported by grant-in-aid of The vehicle racing commemorative foundation to I.I. and VEGA grants 2/3124/23, 2/2064/22, 2/2065/22.

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