GsMTx-4 inhibits the exercise pressor reflex and the muscle mechanoreflex primarily through TRPC inhibition

Evidence suggests that Piezo 2 and TRPC6 channels play important roles in evoking the mechanical component of the exercise pressor reflex. However, the pharmacological tools used in previous studies, namely GsMTx-4 (Piezo 2) and SAR7334 (TRPC6), have potential overlapping effects. GsMTx-4, in particular, inhibits TRPC6 channels in addition to Piezo 2. Consequently, we determined in vivo the initial and combined effects of serial injection of GsMTx-4 and SAR7334 in evoking the mechanical component of the exercise pressor reflex in male and female decerebrated rats. In addition, in heterologous cells expressing Piezo 2 channels, we determined the effect of SAR7334 on the inward current evoked by membrane stretch. The exercise pressor reflex and the mechanoreflex were evoked by statically contracting and passively stretching the triceps surae muscles before and after injection of GsMTx-4 (51 ± 8µm) followed by SAR7334 (51 ± 8µm; n = 8-9) or after injection of SAR7334 (51 ± 8µm) followed by GsMTx-4 (51 ± 8µm; n = 8). In vivo, GsMTx-4 and SAR7334 inhibited the pressor and sympathetic nerve responses to passive stretch and static contraction when injected first. When GsMTx-4 was injected secondary to SAR7334, no reduction in blood pressure and sympathetic responses to static contraction or passive stretch was observed. In vitro, 1 µm of SAR7334 increased the inward current evoked by membrane stretch in heterologous cells expressing Piezo 2 (n = 6). In contrast, 4 µm increased the inward current in two cells, did not change the current in one cell and decreased the current in three cells. Our findings suggest that GsMTx-4 in our in vivo experiments antagonized TRPC as well as Piezo 2 channels. KEY POINTS: GsMTx-4 blocks both Piezo 2 channels and TRPC6 raising the possibility that its effects on the exercise pressor reflex and the mechanoreflex is mediated through TRPC6 inhibition. We determined the effects of GsMTx-4 alone, and after pre-treatment with a TRPC6 antagonist (SAR7334), to elucidate the potential overlapping effects of the drug on the exercise pressor reflex and the mechanoreflex. GsMTx-4 alone significantly inhibited the pressor and sympathetic responses to static contraction and passive stretch. However, when GsMTx-4 was injected after pre-treatment with a TRPC6 antagonist, the effects of GsMTx-4 were abolished. These results demonstrate that the effects of GsMTx-4 are mediated through TRPC6 channel inhibition and that the role played by Piezo 2 was previously overestimated.