Abstract 5332: Overexpression of Connexin43 Reverses Loss of Conduction Velocity in an In Vitro, Anisotropic Model of Interstitial Cardiac Fibrosis
Interstitial cardiac fibrosis is a pathological condition found in aging, hypertrophy, atrial fibrillation, heart failure, and myocardial infarction. Fibrosis reduces the electrical coupling between cardiomyocytes because of fibroblast-deposited collagenous septa that electrically isolate cardiac cells. In this study, we characterized the therapeutic benefits of connexin 43 (Cx43) overexpression in fibrotic monolayers of neonatal rat ventricular myocytes (NRVMs).
Methods: Anisotropic monolayers were obtained by growing cells on parallel, 20 μm-wide fibronectin lines formed by microcontact printing. Fibrosis was obtained by treating the predominately NRVM monolayer with 1 μM angiotension II or TGF-β1 for 48 hours to enhance myofibroblast proliferation and collagen production. Lentivirus encoding Cx43 (LV-Cx43) was then added to a subset of fibrotic monolayers. Four days later, non-treated and LV-Cx43 treated monolayers were analyzed for collagen content and Cx43, and optically mapped with voltage-sensitive dye, di-4-ANEPPS. For comparison, non-fibrotic (control) monolayers were also treated with LV-Cx43.
Results: Increase in extracellular matrix in fibrotic monolayers was confirmed by picrosirius red stain (n=5) and by collagen type I immunostaining (n=5). Western blots confirmed the effectiveness of LV-Cx43 transduction with a 5-fold upregulation of Cx43 in control monolayers and a 3.5-fold upregulation in fibrotic monolayers. Longitudinal (LCV) and transverse conduction velocity (TCV) decreased as expected in fibrotic monolayers (18±1 and 5±0.5 cm/s, respectively; n=3) compared with control monolayers (28±4 and 13±5 cm/s, respectively; n=3). Cx43 overexpression in fibrotic monolayers boosted LCV and TCV (to 29±6 and 8±2cm/s, respectively; n=5). Unexpectedly, LCV and TCV decreased in control monolayers subjected to LV-Cx43 treatment (18±3 and 7±3cm/s, respectively; n=4), possibly as a result of supranormal electrical loading.
Conclusion: A novel in vitro model of cardiac fibrosis is presented that features increased interstitial collagen and anisotropically slowed conduction. Lentiviral overexpression of Cx43 reverses the loss of conduction velocity in this model.