Abstract 953: Ca2+-permeable TRP Channels In Human Cardiac Fibroblasts
In our laboratory, recent single cell electrophysiologic studies have demonstrated the absence of voltage-gated Ca2+ channels in human cardiac fibroblasts. The more positive membrane potential found in these cells suggests that Ca2+ entry occurs through a different mechanism. We hypothesized that non-voltage-gated Ca2+-permeable TRP channels are responsible for Ca2+ entry in human cardiac fibroblasts. With informed consent, right atrial biopsies were obtained from patients undergoing cardiac surgery (n=4:.3M, 1F; mean age 65±8 yrs, EF 63±5%, LVEDP 24±4 mm Hg). Fibroblasts were dissociated and cultured for 7 to 10 days. We found that TRPC1, TRPC4, TRPC6, TRPV4, TRPV5, TRPV6, TRPM4 and TRPM7 were detectable at message levels by RT-PCR. Functional expression of these channels was evaluated by patch-clamp technique. An outward rectifying current with typical I–V relation of TRPM7 was readily recorded in the fibroblasts. The averaged current density was 14.5±0.8 pA/pF (mean±SEM, n=60 from four patients). These currents exhibit similar properties to those of heterologues expressed TRPM7, including permeation to Ca2+ and Mg2+, single channel conductance, and inhibition by 2-APB. Moreover, the TRPM7-like current in human fibroblasts was activated by acidic pH conditions, suggesting that this channel function may be enhanced under ischemic conditions. To study whether this TRPM7-like current promotes fibroblast proliferation, we designed siRNA specifically targeting human TRPM7. Fibroblasts treated with TRPM7 siRNA for five days showed a 76.3% decrease in current density, indicating that transcription of trpm7 produces the message for the Ca2+-pemeable channel in human cardiac fibroblasts. Consistent with the decreased current amplitude, Ca2+ influx in siRNA treated fibroblasts was significantly smaller than those of control fibroblasts, further suggesting that TRPM7-like current contributes to calcium signaling in these cells. Taken together, our results indicate that TRPM7 provides the molecular basis of the Ca2+-permeable cation channel in human cardiac fibroblast, and that TRPM7 may play a crucial role in the fibrogenesis cascade in human heart.