TRPC6 fulfills a calcineurin signaling circuit during pathologic cardiac remodeling
Koichiro Kuwahara, Yanggan Wang, John McAnally, James A. Richardson, Rhonda Bassel-Duby, Joseph A. Hill, Eric N. Olson
Koichiro Kuwahara, Yanggan Wang, John McAnally, James A. Richardson, Rhonda Bassel-Duby, Joseph A. Hill, Eric N. Olson
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Research Article Cardiology

TRPC6 fulfills a calcineurin signaling circuit during pathologic cardiac remodeling

Abstract

The heart responds to injury and chronic pressure overload by pathologic growth and remodeling, which frequently result in heart failure and sudden death. Calcium-dependent signaling pathways promote cardiac growth and associated changes in gene expression in response to stress. The calcium/calmodulin-dependent phosphatase calcineurin, which signals to nuclear factor of activated T cells (NFAT) transcription factors, serves as a transducer of calcium signals and is sufficient and necessary for pathologic cardiac hypertrophy and remodeling. Transient receptor potential (TRP) proteins regulate cation entry into cells in response to a variety of signals, and in skeletal muscle, expression of TRP cation channel, subfamily C, member 3 (TRPC3) is increased in response to neurostimulation and calcineurin signaling. Here we show that TRPC6 was upregulated in mouse hearts in response to activated calcineurin and pressure overload, as well as in failing human hearts. Two conserved NFAT consensus sites in the promoter of the TRPC6 gene conferred responsiveness to cardiac stress. Cardiac-specific overexpression of TRPC6 in transgenic mice resulted in heightened sensitivity to stress, a propensity for lethal cardiac growth and heart failure, and an increase in NFAT-dependent expression of β–myosin heavy chain, a sensitive marker for pathologic hypertrophy. These findings implicate TRPC6 as a positive regulator of calcineurin-NFAT signaling and a key component of a calcium-dependent regulatory loop that drives pathologic cardiac remodeling.

Authors

Koichiro Kuwahara, Yanggan Wang, John McAnally, James A. Richardson, Rhonda Bassel-Duby, Joseph A. Hill, Eric N. Olson

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Figure 3

TRPC6 activates NFAT-dependent transcription in ventricular myocytes.

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TRPC6 activates NFAT-dependent transcription in ventricular myocytes. (A...
(A) COS-1 cells were cotransfected with the expression vectors for NFATc4-GFP (green) and TRPC6 or empty vector, fixed, permeablized, and immunostained with anti-TRPC6 (red). Nuclei are stained blue (DAPI). Magnification, ×400. (B) Effects of TRPC6 on NFATc4 subcellular distribution were quantified with microscopic examination of greater than 100 cells per condition. N, NFATc4-GFP localized exclusively in nucleus; N>C, nuclear NFATc4-GFP localization exceeds cytoplasmic; N≤C, cytoplasmic NFATc4-GFP localization equals or exceeds nuclear; C, NFATc4-GFP localized exclusively in cytoplasm. (C) COS-1 cells were cotransfected with RCAN1-luciferase and TRPC6 and RCAN1 expression vectors. Fold activation over RCAN1-luciferase without expression plasmids is shown. (D) Myocytes were cotransfected with RCAN1-luciferase reporter plasmid and a plasmid expressing rat TRPC6 at various doses in the presence or absence of ET-1. Fold activation over RCAN1-luciferase alone is shown. *P < 0.05 versus control; †P < 0.05 versus control, ET-1, and TRPC6 (200 ng). (E) Myocytes were cotransfected with RCAN1-luciferase and plasmids expressing rat TRPC6 and RCAN1. Fold activation over RCAN1-luciferase without expression plasmids is shown. *P < 0.05 versus TRPC6 alone. (F) Rat smooth muscle cells transfected with rat TRPC6 siRNA. Quantitative RT-PCR for TRPC1–TRPC7 was performed on RNA. Trpc5 and -7 mRNA were not detectable. Percent change of Trpc gene expression in cells transfected with TRPC6 versus control siRNA is shown. (G) Myocytes were cotransfected with RCAN1-luciferase and TRPC6 siRNA alone or with ET-1 or PE. Fold activation over RCAN1-luciferase with vehicle alone is shown. Control values were assigned as 1.0.