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TRPC Channels Are Necessary Mediators of Pathologic Cardiac Hypertrophy

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TRPC Channels Are Necessary Mediators of Pathologic Cardiac Hypertrophy

Xu Wu et al. Proc Natl Acad Sci U S A.

Abstract

Pathologic hypertrophy of the heart is regulated through membrane-bound receptors and intracellular signaling pathways that function, in part, by altering Ca(2+) handling and Ca(2+)-dependent signaling effectors. Transient receptor potential canonical (TRPC) channels are important mediators of Ca(2+)-dependent signal transduction that can sense stretch or activation of membrane-bound receptors. Here we generated cardiac-specific transgenic mice that express dominant-negative (dn) TRPC3, dnTRPC6, or dnTRPC4 toward blocking the activity of the TRPC3/6/7 or TRPC1/4/5 subfamily of channels in the heart. Remarkably, all three dn transgenic strategies attenuated the cardiac hypertrophic response following either neuroendocrine agonist infusion or pressure-overload stimulation. dnTRPC transgenic mice also were partially protected from loss of cardiac functional performance following long-term pressure-overload stimulation. Importantly, adult myocytes isolated from hypertrophic WT hearts showed a unique Ca(2+) influx activity under store-depleted conditions that was not observed in myocytes from hypertrophied dnTRPC3, dnTRPC6, or dnTRPC4 hearts. Moreover, dnTRPC4 inhibited the activity of the TRPC3/6/7 subfamily in the heart, suggesting that these two subfamilies function in coordinated complexes. Mechanistically, inhibition of TRPC channels in transgenic mice or in cultured neonatal myocytes significantly reduced activity in the calcineurin-nuclear factor of activated T cells (NFAT), a known Ca(2+)-dependent hypertrophy-inducing pathway. Thus, TRPC channels are necessary mediators of pathologic cardiac hypertrophy, in part through a calcineurin-NFAT signaling pathway.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Pressure overload induces sarcolemmal Ca2+ entry in ventricular myocytes. (A) Ca2+ trace for store repletion from an adult cardiac myocyte isolated from a WT sham-operated mouse. (B and C) WT mice subjected to TAC stimulation showed robust Ca2+ entry in 20% of isolated cardiac myocytes and modest Ca2+ entry in the remaining 80% of myocytes. (DF) Verapamil and KB-R7943 did not reduce the Ca2+ entry in 20% of myocytes after TAC stimulation, but SKF-96265 eliminated all Ca2+ entry in all myocytes. CPA was given throughout to inhibit SR reloading of Ca2+. All data were collected in multiple myocytes from three to six mice.
Fig. 2.
Fig. 2.
Overexpression of dnTRPC3 in the hearts of TG mice inhibits TAC-induced Ca2+ entry. (A) Western blots for the dnTRPC3 truncation protein and endogenous TRPC3 protein from hearts of WT and two dnTRPC3 TG lines. (GAPDH was used as a loading control.) (B) Immunocytochemistry from a dnTRPC3 TG myocyte reacted with an anti-TRPC3 antibody (green) and NCX1 (red). (C) Western blots for endogenous TRPC3, overexpressed dnTRPC3, and GAPDH from hearts of WT, TRPC3 TG, dnTRPC3 TG, and double transgenic (DTG) mice. (D) Ca2+ influx tracing in an adult ventricular myocyte isolated from TRPC3 TG mice with PE addition (50 μM). (E) Ca2+ influx tracing in an adult ventricular myocyte isolated from TRPC3 × dnTRPC3 DTG mice with PE. (F) Ca2+ influx tracing in an adult ventricular myocyte isolated from dnTRPC3 TG mice subjected to a sham surgical procedure. (G and H) Ca2+ influx tracings in adult ventricular myocytes isolated from dnTRPC3 TG mice subjected to a TAC surgical procedure to induce hypertrophy. All data were collected in multiple myocytes from three to six mice.
Fig. 3.
Fig. 3.
Overexpression of dnTRPC3 inhibits pathological cardiac hypertrophy. (A) Ratio of heart weight to body weight (HW/BW) in WT and dnTRPC3 TG mice after 2 weeks of PE/Ang II infusion versus vehicle treatment with PBS. *, P < 0.05 vs. vehicle; #, P < 0.05 vs. WT PE/AngII. (B) HW/BW ratio in WT and line 6.6 dnTRPC3 TG mice after 2 weeks of TAC stimulation. *, P < 0.05 vs. sham; #, P < 0.05 vs. WT TAC. (C and D) RT-PCR for relative B-type natriuretic peptide (BNP) and β-myosin heavy chain (βMHC) mRNA levels from hearts of the indicated groups. *, P < 0.05 vs. sham; #, P < 0.05 vs. WT TAC. (E) Fractional shortening (FS) by echocardiography in WT and dnTRPC3 TG mice after 8 weeks of TAC or sham treatment. (F) Lung weight to body weight (LW/BW) ratio in WT and dnTRPC3 TG mice after 8 weeks of TAC stimulation. *, P < 0.05 vs. sham. (G) Ventricular fibrosis after 8 weeks of TAC in the indicated groups, measured from histologically stained sections. *, P < 0.05 vs. sham; #, P < 0.05 vs. WT TAC. (H) HW/BW ratios in WT and dnTRPC3 TG mice after 21 days of swimming exercise. *, P < 0.05 vs. sham. The number of mice analyzed in each group is shown in the bars.
Fig. 4.
Fig. 4.
dnTRPC6 inhibits pathological cardiac hypertrophy and heart failure. (A and B) Ca2+ influx tracings in adult ventricular myocytes isolated from dnTRPC6 TG mice subjected to TAC. (C) Western blots of TRPC6 and GAPDH protein in hearts from WT and dnTRPC6 TG mice. (D) HW/BW ratio in WT and dnTRPC6 TG mice after PE/AngII infusion for 2 weeks. *, P < 0.05 vs. vehicle; #, P < 0.05 vs. WT PE/AngII. (E and F) HW/BW in WT and dnTRPC6 TG mice after 2 and 8 weeks of TAC stimulation. *, P < 0.05 vs. sham; #, P < 0.05 vs. WT TAC. (G) FS in WT and dnTRPC6 TG mice after 8 weeks of TAC stimulation. *, P < 0.05 vs. sham; #, P < 0.05 vs. WT TAC. (H) Ventricular fibrosis after 8 weeks of TAC in the indicated groups, measured from histologically stained sections. *, P < 0.05 vs. sham; #, P < 0.05 vs. WT TAC. The number of mice analyzed in each group is shown in the bars.
Fig. 5.
Fig. 5.
dnTRPC4 inhibits pathologic cardiac hypertrophy in TG mice. (A) Western blot for endogenous TRPC4 and the dn deletion mutant of TRPC4 from WT and dnTRPC4 TG mouse heart protein extracts. (GAPDH was used as a loading control.) (B) Immunocytochemistry of a myocyte from a dnTRPC4 TG heart reacted with an anti-TRPC4 (green) or NCX1 (red) antibody. (C) Ca2+ influx tracing in an adult ventricular myocyte isolated from a dnTRPC4 TG mouse subjected to TAC. (D) Heart weight to tibia length (HW/TL) ratios in WT and dnTRPC4 TG mice after 2 weeks of TAC stimulation. *, P < 0.05 vs. sham; #, P < 0.05 vs. WT TAC. The number of animals examined is shown in the bars of the graph. (E) Ca2+ influx tracing in an adult ventricular myocyte treated with PE isolated from TRPC3 × dnTRPC4 DTG mice. (F) Western blot for TRPC3 and dnTRPC4 after immunoprecipitation of TRPC3 (IgG was used as a control) from TRPC3 × dnTRPC4 DTG mouse hearts. (G) Ca2+ influx tracing in an adult ventricular myocyte from dnTRPC3 × dnTRPC4 DTG mice after TAC stimulation.
Fig. 6.
Fig. 6.
dnTRPCs attenuate calcineurin–NFAT signaling in cardiac myocytes. (A) NFAT-luciferase activity from hearts of NFAT-Luc single TG mice versus NFAT-Luc x dnTRPC3 DTG mice subjected to TAC. *, P < 0.05 vs. sham; #, P < 0.05 vs. Luc-TG TAC. Number of mice analyzed is shown in the bars of the graph. (B) NFAT luciferase activity in Adßgal and Ad-dnTRPC6 coinfected NRVM with or without PE treatment for 48 h. *, P < 0.05 vs. vehicle; #, P < 0.05 vs. Adßgal PE. Number of plates of myocytes used to sum the results is shown in the bars of the graph. (C) NFAT luciferase activity in NRVM with or without PE, infected with the indicated viruses. *, P < 0.05 vs. vehicle; #, P < 0.05 vs. Adßgal PE. (D and E) Western blots (D) and quantitation after immunoprecipitation of calmodulin (CaM) (E) to pull down calcineurin B (CnB) or calcineurin A (CnA) from NRVMs infected with control or Ad-dnTRPC6, with or without PE treatment for 48 h.

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