c-Src kinase inhibition reduces arrhythmia inducibility and connexin43 dysregulation after myocardial infarction

Abstract

Objectives: The aim of this study was to evaluate the role of tyrosine kinase cellular-Src (c-Src) inhibition on connexin43 (Cx43) regulation in a mouse model of myocardial infarction (MI).

Background: MI is associated with decreased expression of Cx43, the principal gap junction protein responsible for propagating current in ventricles. Activated c-Src has been linked to Cx43 dysregulation.

Methods: MI was induced in 12-week-old mice by coronary artery occlusion. MI mice were treated with c-Src inhibitors (PP1 or AZD0530), PP3 (an inactive analogue of PP1), or saline. Treated hearts were compared to sham mice by echocardiography, optical mapping, telemetry electrocardiographic monitoring, and inducibility studies. Tissues were collected for immunoblotting, quantitative polymerase chain reaction, and immunohistochemistry.

Results: Active c-Src was elevated in PP3-treated MI mice compared to sham at the scar border (280%, p = 0.003) and distal ventricle (346%, p = 0.013). PP1 treatment restored active c-Src to sham levels at the scar border (86%, p = 0.95) and distal ventricle (94%, p = 1.0). PP1 raised Cx43 expression by 69% in the scar border (p = 0.048) and by 73% in the distal ventricle (p = 0.043) compared with PP3 mice. PP1-treated mice had restored conduction velocity at the scar border (PP3: 32 cm/s, PP1: 41 cm/s, p < 0.05) and lower arrhythmic inducibility (PP3: 71%, PP1: 35%, p < 0.05) than PP3 mice. PP1 did not change infarct size, electrocardiographic pattern, or cardiac function. AZD0530 treatment demonstrated restoration of Cx43 comparable to PP1.

Conclusions: c-Src inhibition improved Cx43 levels and conduction velocity and lowered arrhythmia inducibility after MI, suggesting a new approach for arrhythmia reduction following MI.

Keywords: Src; connexin43; gap junctions; myocardial infarction; sudden death.

Figure 1. Wall motion defects and evidence of ROS production after MI

(A) Representative trichrome staining and echocardiography images of sham and MI mice, demonstrating scar formation and decreased anterior wall movement in the MI model. (B). Representative images of fluorescent nitrotyrosine stains indicative of increased ROS in the distal ventricle and scar border of PP3 and PP1-treated post-MI mice. Quantification of relative fluorescent units (RFUs) demonstrates significantly elevated ROS in the distal ventricle and scar border of both PP3 and PP1 groups compared to sham. There were no differences between treatment groups.

Figure 2. c-Src inhibition with PP1 improves Cx43 levels after MI

(A) Representative images of p-Src, Cx43, and GAPDH Western blots. Cx43 blots are subcategorized into P0, P1, or P2 bands reflecting increasing levels of phsophorylation. (B) Quantification of band intensity of p-Src and Cx43 normalized to GAPDH expression and relative to sham expression levels. (C) Distribution of Cx43 phosphorylation states according to treatment group.

Figure 3. c-Src inhibition improves conduction velocity after MI

(A) Representative images of activation maps of sham, PP1 and PP3-treated mice. (B) Conduction velocities of PP1, PP3, and sham mice at the distal ventricle and scar border. Sham mice could not be consistently captured at cycle lengths faster than 140 ms.

Figure 4. c-Src inhibition reduces arrhythmia inducibility after MI

(A) Representative ECG at the initiation of stimulus protocol. Stimulus artifacts are labeled as “Stim.” (B) Representative ECG patterns with stimulus pacing. (C) The number of mice in each treatment arm that were inducible or not. NSVT, PMVT, and SMVT stand for nonsustained VT, polymorphic VT, and sustained, monomorphic VT, respectively.