Gene Transfer of TRPC6DN (Dominant Negative) Restores Erectile Function in Diabetic Rats

Abstract

Introduction. Transient receptor potential (TRP) channels play an important role in modulating intracellular Ca2+ ([Ca2+](i) ) levels. Aim. We examined the hypothesis that overexpression of TRPC6DN (dominant negative) may contribute to decreased [Ca2+](i) levels in corporal smooth muscle (CSM). We also investigated whether gene transfer of TRPC6DN could restore erectile function in diabetic rats. Methods. For the in vitro study, the K(Ca), K(ATP), and TRPC6DN channel genes were transferred using cDNA, into cultured human CSM cells and human embryonic kidney cells. For the in vivo study, young adult rats were divided into three groups: normal controls; diabetic controls transfected with vector only; and a diabetic group transfected with pcDNA of the TRPC6DN gene. Main Outcome Measures. After gene transfer, the effects of reducing [Ca2+](i) levels were assessed by Fura-2-based imaging analysis. The intracavernosal pressure (ICP) response to cavernosal nerve stimulation was assessed after intracorporal injection of TRPC6DN pcDNA. The transgene expression of the TRPC6DN was examined by reverse transcription polymerase chain reaction (RT-PCR) in rats transfected with TRPC6DN pcDNA. Results. Gene transfer of ion channels effectively reduced [Ca2+](i) . Among these channels, transfer of the TRPC6DN gene resulted in the greatest reduction of [Ca2+](i) in human CSM. The mean (+/- standard error of the mean) ratio of ICP to mean arterial pressure (BP) in the gene-transfer rats was 79.4 +/- 2.4% (N = 8). This was significantly higher than that in control rats (55.6 +/- 3.7% [N = 8]), and similar to that in the young control rats (83 +/- 2.2% [N = 12]). The RT-PCR showed expression of TRPC6DN genes in the transfected rats. Conclusion. Gene transfer of TRPC6DN not only reduced [Ca2+](i) in human CSM but also restored erectile function in diabetic rats. These results suggest that pcDNA transfer of TRPC6DN may represent a promising new form of therapy for the treatment of male erectile dysfunction in the future. Jung JH, Kim BJ, Chae MR, Kam SC, Jeon J-H, So I, Chung KH, and Lee SW. Gene transfer of TRPC6DN (dominant negative) restores erectile function in diabetic rats. J Sex Med 2010;7:1126-1138.