Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart

Abstract

The manner in which Ca2+-sensitive signaling proteins are activated in contracting cardiomyocytes is an intriguing theoretical problem given that the cytoplasm is continually bathed with systolic Ca2+ concentrations that should maximally activate most Ca2+-sensitive signaling kinases and phosphatases. Store-operated Ca2+ entry, partially attributed to transient receptor potential (TRP) proteins, can mediate activation of the Ca2+-sensitive phosphatase calcineurin in nonexcitable cells. Here we investigated the gain-of-function phenotype associated with TRPC3 expression in the mouse heart using transgenesis to examine the potential role of store-operated Ca2+ entry in regulating cardiac calcineurin activation and ensuing hypertrophy/myopathy. Adult myocytes isolated from TRPC3 transgenic mice showed abundant store-operated Ca2+ entry that was inhibited with SKF96365 but not verapamil or KB-R7943. Associated with this induction in store-operated Ca2+ entry, TRPC3 transgenic mice showed increased calcineurin-nuclear factor of activated T cells (NFAT) activation in vivo, cardiomyopathy, and increased hypertrophy after neuroendocrine agonist or pressure overload stimulation. The cardiomyopathic phenotype and increased hypertrophy after pressure overload stimulation were blocked by targeted disruption of the calcineurin Abeta gene. Thus, enhanced store-operated Ca2+ entry in the heart can regulate calcineurin-NFAT signaling in vivo, which could secondarily impact the hypertrophic response and cardiomyopathy.

Figure 1

Generation of TPRC3 transgenic mice. (A) Schematic of the cardiac transgene with the cardiac-specific α-MHC promoter fused to the TRPC3 cDNA. (B) Western blot for TRPC3 and GAPDH (control) protein from the hearts of wildtype (Wt) and line 21 and line 23 TRPC3 transgenic mice. (C) Western blot for the indicated proteins from enriched adult ventricular cardiac mycoytes from wildtype mice and the two TRPC3 transgenic lines. Brain extract was used as a positive migration control (not shown).

Figure 2

Analysis of store-operated Ca²⁺ entry in adult myocytes isolated from wiltype mice. (A) Representative fura-2 ratio fluorescence tracing from a wildtype adult cardiomyocyte that essentially showed no store-operated entry (left panel) and (B) one that showed a minimal but detectable response (right panel). The arrow indicates the timing of Ca²⁺ addition (1.8 mM). The baseline fura-2 fluorescence was subtracted from the peak fluorescence. Approximately 75% of the myocytes showed no Ca²⁺ entry while 25% showed a minimal, albeit, detectable signal. (C) Addition of the Na⁺/Ca²⁺ inhibitor KB-R7943 did not appreciably alter the ability to identify store-operated Ca²⁺ entry in a minor population of myocytes.

Figure 3

Analysis of store-operated Ca²⁺ entry in adult myocytes isolated from TRPC3 transgenic mice. (A) Representative fura-2 ratio fluorescence tracing from a TRPC3 transgenic adult cardiomyocyte. The arrow indicates the timing of Ca²⁺ addition (1.8 mM). The baseline fura-2 fluorescence was subtracted from the peak fluorescence. (B) Representative fura-2 ratio fluorescence tracing from a TRPC3 transgenic adult cardiomyocyte treated with verapamil or (C) KB-R7943. The arrow indicates the timing of Ca²⁺ addition (1.8 mM). (D) Representative fura-2 ratio fluorescence tracing from a TRPC3 transgenic adult cardiomyocyte treated with SKF96365 at the indicated time point (arrow).

Figure 4

Cardiac phenotype of TRPC3 transgenic mice. (A) Heart-weight normalized to tibial-length in wildtype (Wt) and lines 21 or 23 transgenic mice at the indicated times. *P<0.05 versus wt. (B) Survival analysis of high expressing line 23 TRPC3 male transgenic mice (N=31) versus wildtype male mice (N=49) over time. (C) RT-PCR for ANF and L7 (control) from the hearts of Wt or the two TRPC3 transgenic lines at 2 months of age. Either 27 or 30 cycles of amplification is shown. (D) M-mode representation of cardiac echocardiography from wildtype and low expressing TRPC3 transgenic line at 12 months of age. (E) Quantitation of left ventricular end diastolic dimension by echocardiography in wildtype and low expressing TRPC3 transgenic mice at 12 months of age. *P<0.05 versus wt. (F) Quantitation of fractional shortening (FS) by echocardiography in wildtype and low expressing TRPC3 transgenic mice at 12 months of age. *P<0.05 versus wt.

Figure 5

NFAT activation in TRPC3 transgenic mice. (A) Fold activation of luciferase activity from the hearts of NFAT-luciferase transgenic mice crossed with wildtype or low expressing TRPC3 transgenic mice. Hearts were collected at 2 weeks, 2 months, or 8 months. The number of mice analyzed in each group is shown in the figure. *P<0.05 versus corresponding wt. (B) Fold activation of luciferase activity from the hearts of NFAT-luciferase transgenic mice crossed with wildtype, low, or high expressing TRPC3 transgenic mice at baseline or after 14 days of PE + Ang II infusion. Hearts were collected at 2 months of age. The number of mice analyzed in each group is shown in the figure. *P<0.05 versus wt with PBS. #P<0.05 versus Wt with agonist. (C) Relative luciferase activity from hearts of NFAT-luciferase transgenic mice crossed with wildtype, low, or high expressing TRPC3 transgenic mice at baseline or after 14 days of isoproterenol (Iso) infusion. Hearts were collected at 2 months of age. The number of mice analyzed in each group is shown in the figure. *P<0.05 versus wt with PBS. #P<0.05 versus no Iso within each group. (D) Western blot for calcineurin Aα and Aβ from the hearts of the indicated mice, or brain as a control.

Figure 6

Analysis of propensity towards cardiac hypertrophy in TRPC3 transgenic mice. (A) Heart-weight normalized to tibial-length in wildtype and TRPC3 transgenic mice at 2 months of age subjected to 14 days of PE + Ang II infusion. The number of mice used in each cohort is shown in the bars. *P<0.05 versus no agonist, #P<0.05 versus wt with agonist. (B) Heart-weight normalized to tibial-length in 2 month-old wildtype or low expressing TRPC3 transgenic subjected to TAC or sham procedure for 14 days. The number of mice used in each cohort is shown in the bars. *P<0.05 versus Wt sham, #P<0.05 versus wt TAC. (C) Histological analysis of cardiomyocyte cross-sectional areas in 2 month-old wildtype or low expressing TRPC3 transgenic subjected to TAC or sham procedure for 14 days. The number of mice used in each cohort is shown in the bars. *P<0.05 versus Wt sham, #P<0.05 versus wt TAC. (D) Fractional shortening (FS) in 2 month-old wildtype or low expressing TRPC3 transgenic subjected to TAC or sham procedure for 14 days. The number of mice used in each cohort is shown in the bars. *P<0.05 versus Wt TAC. (E) Lung-weight normalized to tibial-length in 2 month-old wildtype or low expressing TRPC3 transgenic subjected to TAC or sham procedure for 14 days. The number of mice used in each cohort is shown in the bars. *P<0.05 versus Wt TAC.

Figure 7

Analysis of calcineurin-NFAT association with TRPC3-mediated hypertrophy. (A) Fold activation of luciferase activity from the hearts of NFAT-luciferase transgenic mice crossed with wildtype or low expressing TRPC3 transgenic mice that were subjected to a sham or TAC procedure for 14 days. The number of mice analyzed in each group is shown in the figure. *P<0.05 versus wt sham, #P<0.05 versus wt TAC. (B) Heart-weight normalized to tibial-length in 2 month-old wildtype or low expressing TRPC3 transgenic crossed into the calcineurin Aβ wildtype or null background. Mice were then subjected to TAC or sham procedure for 14 days. The number of mice used in each cohort is shown in the bars. *P<0.05 versus Wt CnAβ+/+ sham, #P<0.05 versus wt CnAβ+/+ TAC. (C) Lung-weight normalized to tibial-length in 2 month-old wildtype or low expressing TRPC3 transgenic crossed into the calcineurin Aβ wildtype or null background. Mice were then subjected to TAC or sham procedure for 14 days. The number of mice used in each cohort is shown in the bars. *P<0.05 versus Wt CnAβ+/+ TAC. (D) Fractional shortening (FS) in 2 month-old wildtype or low expressing TRPC3 transgenic crossed into the calcineurin Aβ wildtype or null background. Mice were then subjected to TAC or sham procedure for 14 days. The number of mice used in each cohort is shown in the bars. *P<0.05 versus Wt CnAβ+/+ TAC. (E) Control western blot for TRPC3 transgenic protein expression in the hearts of wildtype and calcineurin Aβ−/− mice.