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

Abstract

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.

Keywords: Piezo 2; TRPC6; blood pressure; exercise mechanoreflex; exercise pressor reflex; sympathetic nervous system.

Figure 1. Effects of serial injections of SAR7334 and GsMTx‐4 on the pressor, tension, renal sympathetic, blood flow and vascular conductance responses to static contraction of the triceps surae muscles

Data are presented as the mean ± SD changes over time in blood pressure, tension, RSNA, blood flow and vascular conductance induced by static contraction. The averaged time course includes 2 s of baseline, 30 s of contraction and 2 s after the end of contraction. Contractions were evoked before, 10 min after the first injection, and 10 min after the second injection. Figures in the left panels represent the experiments during which SAR7334 (51 ± 8 µm in 100 µl) was injected first and GsMTx‐4 (51 ± 8 µm in 100 µl) was injected second (n = 8). Figures in the right panels represent the experiments during which GsMTx‐4 (51 ± 8 µm in 100 µl) was injected first and SAR7334 (51 ± 8 µm in 100 µl) was injected second (n = 9). Abbreviations: MAP, mean arterial pressure; RSNA, renal sympathetic nerve activity. †P < 0.05 between pre‐blockade and first blockade. ‡P < 0.05 between pre‐blockade and second blockade.

Figure 2. Effects of serial injections of SAR7334 and GsMTx‐4 on the integrated pressor, tension and renal sympathetic nerve activity responses to static contraction of the triceps surae muscles

Data are presented as individual (males as open circles and females as closed circles) and group means (open bars) for the integrated changes evoked by static contraction. Contractions were evoked before, 10 min after the first injection, and 10 min after the second injection. Figures in the left panel represent the experiments during which SAR7334 (51 ± 8 µm in 100 µl) was injected first and GsMTx‐4 (51 ± 8 µm in 100 µl) was injected second (n = 8). Figures in the right panel represent the experiments during which GsMTx‐4 (51 ± 8 µm in 100 µl) was injected first and SAR7334 (51 ± 8 µm in 100 µl) was injected second (n = 9). Abbreviations: BPi, blood pressure index calculated as the integrated blood pressure response to contraction; TTi, tension time index calculated as the integrated tension response to contraction; RSNAi, integrated renal sympathetic nerve activity. ∗P < 0.05, ∗∗ P < 0.01, and ∗∗∗P < 0.001 between the corresponding data points.

Figure 3. Effects of serial injections of SAR7334 and GsMTx‐4 on the pressor, tension, renal sympathetic, blood flow and vascular conductance responses to passive stretch of the triceps surae muscles

Data are presented as the mean ± SD changes over time in blood pressure, tension and RSNA induced by passive stretch. The averaged time course includes 2 s of baseline, 30 s of contraction and 2 s after the end of stretch. Stretches were evoked before, 10 min after the first injection, and 10 min after the second injection. Figures in the left panels represent the experiments during which SAR7334 (51 ± 8 µm in 100 µl) was injected first and GsMTx‐4 (51 ± 8 µm in 100 µl) was injected second (n = 8). Figures in the right panels represent the experiments during which GsMTx‐4 (51 ± 8 µm in 100 µl) was injected first and SAR7334 (51 ± 8 µm in 100 µl) was injected second (n = 9). Abbreviations: MAP, mean arterial pressure; RSNA, renal sympathetic nerve activity. †P < 0.05 between pre‐blockade and first blockade. ‡P < 0.05 between pre‐blockade and second blockade. # P < 0.05 between the first and second blockade.

Figure 4. Effects of serial injections of SAR7334 and GsMTx‐4 on the integrated pressor, tension and renal sympathetic nerve activity responses to passive stretch of the triceps surae muscles

Data are presented as individual (males as open circles and females as closed circles) and group means (open bars) for the integrated changes evoked by passive stretch. Stretches were evoked before, 10 min after the first injection, and 10 min after the second injection. Figures in the left panel represent the experiments during which SAR7334 (51 ± 8 µm in 100 µl) was injected first and GsMTx‐4 (51 ± 8 µm in 100 µl) was injected second (n = 8). Figures in the right panel represent the experiments during which GsMTx‐4 (51 ± 8 µm in 100 µl) was injected first and SAR7334 (51 ± 8 µm in 100 µl) was injected second (n = 9). Abbreviations: BPi, blood pressure index calculated as the integrated blood pressure response to contraction; TTi, tension time index calculated as the integrated tension response to stretch; RSNAi, integrated renal sympathetic nerve activity. ∗P < 0.05, ∗∗ P < 0.01, and ∗∗∗P < 0.001 between the corresponding data points.

Figure 5. Percentage change from baseline of the integrated blood pressure response to static contraction and passive stretch following injection of SAR7334 and/or GsMTx‐4

Data are presented as individual (males as open circles and females as closed circles) and group means (open bars) for the percentage changes from baseline in the integrated blood pressure evoked by static contraction and passive stretch. Stretches and contractions were evoked before, 10 min after the first injection, and 10 min after the second injection. Figure in the left panel represents the effects of SAR7334 (51 ± 8 µm in 100 µl) alone and the effects of SAR7334 when injected after GsMTx‐4 (51 ± 8 µm in 100 µl). Figure in the right panel represents the effects of GsMTx‐4 (51 ± 8 µm in 100 µl) alone and the effects of GsMTx‐4 when injected after SAR7334 (51 ± 8 µm in 100 µl). Abbreviations: BPi, blood pressure index calculated as the integrated blood pressure response to contraction; ∗P < 0.05 between the corresponding data points.

Figure 6. Representative traces and individual effects of 1 µm and 4 µm of SAR7334 on the inward current of heterologous cells overexpressing Piezo 2 in response to membrane stretch

Data are presented as individual (open dots) and group means (open bars) for the peak inward current evoked by membrane stretch. Membrane stretch was evoked by exposing the cells to hypoosmotic solution (Hypo‐Osm). A more negative value indicates higher inward current. Two concentrations of SAR7334 were used, 1 µm and 4 µm, which corresponded to the calculated concentration of drugs reached in the interstium of myocytes following intra‐arterial injections.