Abstract 4126333: <i>TRPC6 A404V</i> is a gain-of-function variant associated with doxorubicin-induced cardiotoxicity in patients

Background: Gain-of-function mutations in the canonical transient receptor potential 6 (TRPC6) channel contribute to heart failure (HF) and focal segmental glomerulosclerosis in humans. Our GWAS results indicated that the TRPC6 A404V variant, having a minor allele frequency of 12% in the population, is associated with doxorubicin-induced cardiotoxicity. However, the underlying ionic mechanisms of this variant have not been fully examined. Methods: Combining patch clamp recording, site-directed mutagenesis, and in-silico analysis, we evaluated the TRPC6 A404V variant function. Results: Whole-cell TRPC6 currents were elicited in HEK293 cells using a voltage ramp protocol from -100 mV to +100 mV with a holding potential of -60 mV, 48 hours after transfection with TRPC6 WT or A404V cDNAs. The inward-current densities at -100 mV and the outward-current densities at +100 mV for TRPC6 WT and A404V variant were similar at baseline. However, after exposure to 50 μM 1-oleoyl acetyl-sn-glycerol (OAG, a TRPC6 activator), these current densities were significantly increased by 2.6-fold and 4.5-fold respectively in the WT (n=20 cells), and by 8.4-fold and 8.9-fold respectively in the A404V variant (n=12 cells, p&lt;0.05 vs. WT). The OAG effects were abolished by superfusion with 1 μM BI-749327 (a specific TRPC6 inhibitor). Moreover, a 24-hour treatment with 0.5 μM doxorubicin (DOX) further amplified the effects of OAG, increasing the total inward- and outward-current densities by 7.2-fold and 9.5-fold respectively in TRPC6 WT (n=11 cells), and by 13.9-fold and 14.7-fold respectively in the A404V variant (n=13 cells, p&lt;0.05 vs. WT). In comparison, the OAG effects on TRPC6 WT and A404V channels were not potentiated by treatment with 0.5 μM doxorubicinol (DOXol, a metabolite of DOX) for 24 h (n=10-12 cells, p=N.S.). Molecular docking studies showed that OAG had a lower binding energy (-5.60 kcal/mol) and a smaller apparent equilibrium constant (Kd) of 0.079 mM with the A404V variant, compared to those of -4.49 kcal/mol and 0.511 mM respectively with TTRPC6 WT. Conclusion: TRPC6 A404V is a gain-of-function variant with a higher binding affinity to OAG. Treatment with DOX but not DOXol greatly enhances TRPC6 WT and A404V channel activation by OAG. Thus, cancer patients carrying the TRPC6 A404V variant may be more vulnerable to develop HF upon treatment with anthracycline.