Irregular Pacing of Ventricular Cardiomyocytes Induces Pro-Fibrotic Signalling Involving Paracrine Effects of Transforming Growth Factor Beta and Connective Tissue Growth Factor

AIMS: Atrial fibrillation is the most prevalent sustained arrhythmia associated with arrhythmic ventricular contractions, incident heart failure, increased morbidity and mortality. The relationship between arrhythmic contractions and ventricular remodelling is incompletely understood. The aim of this study was to characterize the influence of irregular contractions on pro-fibrotic signalling in neonatal rat ventricular cardiomyocytes (NRVM). METHODS AND RESULTS: Neonatal rat ventricular cardiomyocytes were paced via field stimulation at 3 Hz for 24 h. Irregularity was created by pseudorandomized variation of stimulation intervals and compared to regular pacing. Treatment of neonatal cardiac fibroblasts (NCF) with medium of irregularly paced NRVM increased protein expression of collagen I (206 ± 62%, P = 0.0121) and collagen III (51 ± 37%, P = 0.0119). To identify the underlying mechanism, expression of pro-fibrotic connective tissue growth factor (CTGF) and transforming growth factor beta (TGF-β) was assessed. In irregularly paced NRVM, increased protein expression of CTGF (80 ± 22%, P = 0.0035) and TGF-β (122 ± 31%, P = 0.0022) was associated with enhanced excretion of both proteins into the medium. Electron paramagnetic resonance spectroscopy revealed an increased production of reactive oxygen species (46 ± 21%, P = 0.0352) after irregular pacing accompanied by increased 8-hydroxydeoxyguanosine staining (214 ± 53%, P = 0.0011). Irregular pacing was associated with elevated mRNA levels of anti-oxidative superoxide dismutase 1 (25 ± 7%, P = 0.0175), superoxide dismutase 3 (20 ± 7%, P = 0.0309), and catalase (20 ± 7%, P = 0.046). CONCLUSION: These data demonstrate that irregular pacing is an important inductor of pro-fibrotic signalling in NRVM involving paracrine effects of CTGF and TGF-β as well as increased oxidative stress. Thus, irregularity of the heart beat might directly be involved in the progression of maladaptive remodelling processes in atrial fibrillation.