TY - JOUR
T1 - TRPC6 N338S is a gain-of-function mutant identified in patient with doxorubicin-induced cardiotoxicity
AU - Lu, Tong
AU - Sun, Xiaojing
AU - Necela, Brian M.
AU - Lee, Hon Chi
AU - Norton, Nadine
N1 - Publisher Copyright:
© 2022 Elsevier B.V.
PY - 2022/11/1
Y1 - 2022/11/1
N2 - The canonical transient receptor potential 6 gene, TRPC6, has been implicated as a putative risk gene for chemotherapy-induced congestive heart failure, but knowledge of specific risk variants is lacking. Following our genome-wide association study and subsequent fine-mapping, a rare missense mutant of TRPC6 N338S, was identified in a breast cancer patient who received anthracycline-containing chemotherapy regiments and developed congestive heart failure. However, the function of N338S mutant has not been examined. Using intracellular Ca2+ imaging, patch clamp recording and molecular docking techniques, we assessed the function of N338S mutant heterologously expressed in HEK293 cells and HL-1 cardiac cells. We found that expression of TRPC6 N338S significantly increased intracellular Ca2+ levels ([Ca2+]i) and current densities in response to 50 μM 1-oleoyl 2-acetyl-sn-glycerol (OAG), an activator of TRPC6 channels, compared to those of TRPC6 WT. A 24-h pretreatment with 0.5 μM doxorubicin (DOX) further potentiated the OAG effects on TRPC6 N338S current densities and [Ca2+]i, and these effects were abolished by 1 μM BI-749327, a highly selective TRPC6 inhibitor. Moreover, DOX treatment significantly upregulated the mRNA and protein expressions of TRPC6 N338S, compared to those of TRPC6 WT. Molecular docking and dynamics simulation showed that OAG binds to the pocket constituted by the pore-helix, S5 and S6 domains of TRPC6. However, the N338S mutation strengthened the interaction with OAG, therefore stabilizing the OAG-TRPC6 N338S complex and enhancing OAG binding affinity. Our results indicate that TRPC6 N338S is a gain-of-function mutant that may contribute to DOX-induced cardiotoxicity by increasing Ca2+ influx and [Ca2+]i in cardiomyocytes.
AB - The canonical transient receptor potential 6 gene, TRPC6, has been implicated as a putative risk gene for chemotherapy-induced congestive heart failure, but knowledge of specific risk variants is lacking. Following our genome-wide association study and subsequent fine-mapping, a rare missense mutant of TRPC6 N338S, was identified in a breast cancer patient who received anthracycline-containing chemotherapy regiments and developed congestive heart failure. However, the function of N338S mutant has not been examined. Using intracellular Ca2+ imaging, patch clamp recording and molecular docking techniques, we assessed the function of N338S mutant heterologously expressed in HEK293 cells and HL-1 cardiac cells. We found that expression of TRPC6 N338S significantly increased intracellular Ca2+ levels ([Ca2+]i) and current densities in response to 50 μM 1-oleoyl 2-acetyl-sn-glycerol (OAG), an activator of TRPC6 channels, compared to those of TRPC6 WT. A 24-h pretreatment with 0.5 μM doxorubicin (DOX) further potentiated the OAG effects on TRPC6 N338S current densities and [Ca2+]i, and these effects were abolished by 1 μM BI-749327, a highly selective TRPC6 inhibitor. Moreover, DOX treatment significantly upregulated the mRNA and protein expressions of TRPC6 N338S, compared to those of TRPC6 WT. Molecular docking and dynamics simulation showed that OAG binds to the pocket constituted by the pore-helix, S5 and S6 domains of TRPC6. However, the N338S mutation strengthened the interaction with OAG, therefore stabilizing the OAG-TRPC6 N338S complex and enhancing OAG binding affinity. Our results indicate that TRPC6 N338S is a gain-of-function mutant that may contribute to DOX-induced cardiotoxicity by increasing Ca2+ influx and [Ca2+]i in cardiomyocytes.
KW - Cardiotoxicity
KW - Diacylglycerol analog
KW - Doxorubicin
KW - Gain-of-function mutation
KW - TRPC6
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U2 - 10.1016/j.bbadis.2022.166505
DO - 10.1016/j.bbadis.2022.166505
M3 - Article
C2 - 35882306
AN - SCOPUS:85134915901
SN - 0925-4439
VL - 1868
JO - Biochimica et Biophysica Acta - Molecular Basis of Disease
JF - Biochimica et Biophysica Acta - Molecular Basis of Disease
IS - 11
M1 - 166505
ER -