TRPC channels remodeling in pulmonary arterial hypertension contributes to pulmonary arterial smooth muscle cells dysfunctions - 17/02/23

Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca²⁺) signaling contributes to abnormalities in human PA smooth muscle cells (hPASMCs), including aberrant proliferation, exacerbated migration, apoptosis resistance and arterial contractility. The transient receptor potential canonical (TRPC, six members in humans, TRPC1, -C3, -C4, -C5, -C6, -C7) channels constitute calcium-permeable nonselective cation channels involved in Ca²⁺ homeostasis in smooth muscle cells. However, their implications in Ca²⁺ signaling, properties and signaling pathways are unclear in PAH-hPASMCs.

Using a combination of approaches, we investigated the roles of TRPC channels in PA remodeling in PAH. In vitro, we studied the impact of TRPC knockdown (KD) by siRNA on control and PAH-hPASMCs using Ca²⁺ imaging, molecular biology, proliferation, apoptosis and migration assays. We also determined the in vivo consequences of pharmacological TRPC3 inhibition using the experimental model of pulmonary hypertension (PH) induced by monocrotaline (MCT)-exposure.

In PAH-hPASMCs, we showed unchanged TRPC1 and -C4 expression, overexpression of TRPC3 and -C6 while TRPC5 and -C7 were not detected at mRNA level (in control and PAH). We found that the KD of TRPC1, -C3, -C4 or -C6 reduced store-operated Ca²⁺ entry (SOCE) and proliferation of PAH-hPASMCs. In addition, only TRPC1 KD decreased the PAH-hPASMCs migration capacity. TRPC1 or -C6 KD reduced the expression of the cell cycle inhibitor protein P21 and the apoptosis inhibitor survivin, while the KD of TRPC3 or -C4 increased P21 and survivin expression. Moreover, TRPC6 KD increased the phosphorylation of Akt (p308), the Caspase9/procaspase9 ratio, and reduced ERK1/2 phosphorylation. To further evaluate the putative role of TRPC channels in the apoptosis resistance of PAH-hPASMCs, we exposed cells to staurosporine (apoptosis inducer). In staurosporine-exposed PAH-hPASMCs, the KD of TRPC1, -C3 or -C6 increased the Caspase9/procaspase9 ratio. TRPC1 or -C4 KD increased Akt phosphorylation, while TRPC1 KD reduced the survivin expression. Finally, in vivo “curative” administration of the TRPC3 inhibitor PYR3 attenuated the PH development in rats MCT-PH model.

In human PAH-hPASMCs, the expression of TRPC3 and -C6 is increased compared to control. We also show stronger involvement of TRPC channels in PAH-hPASMCs phenotypes (proliferation, migration and apoptosis resistance). These preliminary results suggest that all expressed TRPC channels contribute to cellular dysfunctions occurring in PAH-hPASMCs and could be innovative therapeutic targets in PAH.