TRPC channels are necessary mediators of pathologic cardiac hypertrophy

Abstract

Pathologic hypertrophy of the heart is regulated through membrane-bound receptors and intracellular signaling pathways that function, in part, by altering Ca²⁺ handling and Ca²⁺-dependent signaling effectors. Transient receptor potential canonical (TRPC) channels are important mediators of Ca²⁺-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 Ca²⁺ 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 Ca²⁺-dependent hypertrophy-inducing pathway. Thus, TRPC channels are necessary mediators of pathologic cardiac hypertrophy, in part through a calcineurin–NFAT signaling pathway.

Pathologic cardiac hypertrophy, an enlargement of the adult heart caused by disease-inducing stimuli, can cause sudden death and is a leading predictor for the development of heart failure (1). The growth of individual myocytes is programmed by neuroendocrine factors that signal through membrane-bound receptors leading to activation of signal-transduction pathways and alterations in gene expression (2). Augmentation in intracellular Ca²⁺ is thought to be critically involved in signaling cardiac hypertrophy, in part through the Ca²⁺-activated protein phosphatase calcineurin, which leads to activation of the transcription factor, nuclear factor of activated T cells (NFAT), which induces hypertrophic response genes (3). Transient receptor potential canonical (TRPC) channels are cation-selective influx channels that can initiate cardiac hypertrophy when overexpressed, in part because of increased Ca²⁺ influx and calcineurin activation (4–7). Functional TRPC channels are comprised of homo- or heterotetramers between either TRPC1/4/5 or TRPC3/6/7 subfamily members, although overexpression of any one subunit alone can produce enhanced currents (8). In general, TRPC3/6/7 are activated by diacylglycerol (DAG) generated by G protein-coupled receptors (GPCR)/Gαq/phospholipase C signaling, and TRPC1/4/5 can be activated by depletion of intracellular Ca²⁺ stores (store-operated Ca²⁺ entry) or by stretch (8, 9). Once activated, these channels induce signal transduction through cytoplasmic elevations in Ca²⁺ and Na⁺ or through refilling of endoplasmic reticulum Ca²⁺ stores to ensure prolonged signaling events (8, 9).

Overexpression of TRPC3 or -6 was reported to induce cardiac hypertrophy through calcineurin/NFAT signaling in transgenic (TG) mice (4, 5). More recently, some data have emerged suggesting that TRPC channels are required for cardiac hypertrophy, because Trpc1^−/− mice and use of the TRPC3 inhibitory compound Pyr3 showed reduced pressure-overload growth (10, 11). However, it remains unknown whether endogenous TRPC channels and associated Ca²⁺ influx are altered in pathologic cardiac hypertrophy and which subfamily might be required for pathologic growth. Here we generated TG mice with inhibition of the TRPC3/6/7 and TRPC1/4/5 subfamilies, which equivalently inhibited membrane “leak” of Ca²⁺ in pathologic hypertrophy and inhibited growth following agonist stimulation and pressure overload.

Results

Myocytes from Hypertrophic Hearts Have Increased Membrane Ca²⁺ Influx.

Depletion of intracellular Ca²⁺ in most cell types leads to activation of store-operated Ca²⁺ influx through defined channel complexes that include Orai and possibly TRPCs in the plasma membrane (12). Even if TRPC channels are not bona fide regulators of store-operated Ca²⁺ entry, Ca²⁺ depletion conditions in conjunction with GPCR stimulation is often used as a surrogate for assessing their activity. Here, adult cardiac myocytes from WT mouse hearts were bathed in Ca²⁺-free buffer with the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) and then immediately were switched to buffer containing 1 mM Ca²⁺ to monitor intracellular Ca²⁺ levels by Indo-1 fluorescence. WT myocytes showed no Ca²⁺ influx under these conditions (Fig. 1A and Fig. S1). However, myocytes isolated from hypertrophic mouse hearts after transverse aortic constriction (TAC) showed substantial Ca²⁺ influx (Fig. 1 B and C and Fig S1). Most of the myocytes (≈80%) showed modest Ca²⁺ influx (Fig. 1C), but ≈20% of the myocytes showed robust Ca²⁺ influx activity (Fig. 1B and Fig. S1). Induction of Ca²⁺ influx observed in hypertrophied adult myocytes was not inhibited with the L-type Ca²⁺ channel inhibitor verapamil or the Na⁺/Ca²⁺ exchanger (NCX) inhibitor KB-R7943 (Fig. 1 D and E) but was completely inhibited with SKF-96265, a known TRPC-channel inhibitor (Fig.1F).

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. (D–F) 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.

Generation of Dominant-Negative TRPC3 TG Mice.

To determine if TRPC channels might underlie the observed induction of sarcolemmal Ca²⁺ influx in myocytes from hypertrophic hearts, we generated TG mice expressing dominant-negative (dn) TRPC3 with the α-myosin heavy chain (αMHC) promoter. Two independent lines were generated that each showed abundant overexpression in the heart without affecting endogenous TRPC3 levels (Fig. 2A). Importantly, the overexpressed dnTRPC3 protein was localized properly to the sarcolemma and the T-tubular network of adult myocytes from these hearts, coincident with NCX1 (Fig. 2B). To investigate the effectiveness of the dnTRPC3 mutant in vivo, we crossed line 6.6 dnTRPC3 TG mice with mice expressing WT TRPC3 that we described previously (5). Protein levels were not altered in dn TG mice, indicating no promoter competition (Fig. 2C). Adult myocytes isolated from WT TRPC3 overexpressors showed a robust Ca²⁺ influx activity under store-depleted conditions, as described previously (ref. 5 and Fig. 2D). However, this TRPC3-dependent Ca²⁺ influx activity was blocked completely by the presence of the dnTRPC3 transgene (Fig. 2E). These data also were quantified from multiple myocytes to show the extent of Ca²⁺ entry with TRPC3 and its complete inhibition with the dnTRPC3 transgene (Fig. S1).

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.

Adult myocytes from hearts of unstressed sham mice showed no Ca²⁺ influx activity in more than 60 myocytes examined from WT or dnTRPC3/6 TG mice (Fig. 2F and Fig. S1). More importantly, adult myocytes isolated from hearts of dnTRPC3 mice subjected to TAC showed a nearly complete loss of Ca²⁺ influx activity (90% of all myocytes showed no activity), although ≈10% of myocytes showed a minor Ca²⁺ influx activity (Fig. 2 G and H and Fig. S1). These results suggest that expression of the dnTRPC3 transgene in the heart blocks induction of most aberrant sarcolemmal Ca²⁺ influx activity caused by pathological cardiac hypertrophy.

dnTRPC3 TG Mice Have Reduced Pathologic Cardiac Hypertrophy.

We hypothesized that the TRPC-dependent Ca²⁺ influx activity observed in hypertrophic hearts initiated reactive growth signaling. To examine this hypothesis, we subjected adult dnTRPC3 TG mice to coinfusion of phenylephrine (PE) and angiotensin II (AngII) to model a neuroendocrine-GPCR–stimulated hypertrophy response. Remarkably, dnTRPC3 TG mice showed significantly less cardiac hypertrophy than WT control mice of the same strain infused with PE/AngII for 2 weeks (Fig. 3A). To extend these results, two lines of adult dnTRPC3 TG mice also were subjected to pressure-overload stimulation by TAC; both lines showed a significant reduction in cardiac hypertrophy compared with WT controls over 2 weeks of stimulation (Fig. 3B and Fig. S2A). During this 2-week time course, neither WT nor TG groups showed a reduction in fractional shortening from echocardiograms (Fig. S2B). Moreover, dnTRPC3 TG mice continued to show less cardiac hypertrophy than WT control mice after 8 weeks of TAC (Fig. S2C). dnTRPC3 TG mice also showed less induction of hypertrophic marker gene expression after TAC than WT mice (Fig. 3 C and D). Consistent with this reduction in the hypertrophic program, dnTRPC3 TG mice showed less of a reduction in fractional shortening after 8 weeks of TAC than WT controls, were protected from lung edema that characterizes heart failure, and showed less ventricular fibrosis than WT controls (Fig. 3 E–G). However, dnTRPC3 TG mice hypertrophied normally following physiologic exercise stimulation with 21 days of swimming (Fig. 3H). These results suggest that blockade of the TRPC3/6/7 subfamily antagonizes neuroendocrine-like and pressure-overload–induced pathologic cardiac hypertrophy in vivo, as well as transition to heart failure, but has no involvement in physiologic hypertrophy.

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.

dnTRPC6 Transgenic Mice Show Reduced Pathologic Cardiac Hypertrophy.

To substantiate further our conclusion that the TRPC3/6/7 subclass is necessary for mediating cardiac hypertrophy, we generated two lines of cardiac-specific dnTRPC6 TG mice (Fig. 4C). These mice showed a significant reduction in TAC-induced Ca²⁺ influx across the sarcolemma in adult myocytes, similar to dnTRPC3 TG mice (Fig. 4 A and B and Fig. S1). The dnTRPC6 protein is not a truncation like the dnTRPC3 protein but instead contains mutations that disable pore functionality (13). Compared with WT mice, dnTRPC6 TG mice showed complete inhibition of PE/AngII-induced cardiac hypertrophy after 2 weeks of infusion (Fig. 4D) and a significant inhibition of hypertrophy following 2 weeks of TAC stimulation (Fig. 4E). Even after 8 weeks of TAC stimulation, dnTRPC6 TG mice continued to show less cardiac hypertrophy than WT mice (Fig. 4F). Interestingly, compared with WT mice, dnTRPC6 TG mice showed hyperfunctionality at baseline and were partially protected from a loss of cardiac ventricular performance after 8 weeks of TAC stimulation (Fig. 4G). Consistent with these results, dnTRPC6 TG mice showed less ventricular fibrosis than WT mice after 8 weeks of TAC (Fig. 4H). These results further indicate that the TRPC3/6/7 subclass is necessary for mediating the full extent of pathologic cardiac hypertrophy and transition to failure.

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.

dnTRPC4 Transgenic Mice Show Reduced Pathologic Cardiac Hypertrophy.

Although both dnTRPC3 and dnTRPC6 attenuated cardiac hypertrophy, it was uncertain if the TRPC1/4/5 subfamily was similarly involved in the hypertrophic response. Therefore we generated dnTRPC4 TG mice with cardiac-specific expression. The dnTPRC4 truncated protein was overexpressed robustly in the heart without altering endogenous TRPC4 expression (Fig. 5A). Immunocytochemistry showed that, similar to dnTRPC3, overexpressed dnTRPC4 and NCX1 colocalized to the sarcolemma and T-tubules in isolated adult myocytes (Fig. 5B). Importantly, the induction of Ca²⁺ influx that occurs in adult myocytes from hypertrophied WT hearts was significantly reduced in dnTRPC4 TG hearts subjected to TAC (Fig. 5C, WT TAC: 0.7 ± 0.14, dnTRPC4 TAC: 0.58 ± 0.13, P < 0.05). Associated with this inhibition of Ca²⁺ influx activity, dnTRPC4 TG mice also showed less cardiac hypertrophy after 2 weeks of TAC, similar to dnTRPC3 and dnTRPC6 TG mice (Fig. 5D). dnTRPC4 TG mice also showed less induction of hypertrophic marker gene expression and less fibrosis after 6 weeks of TAC than did WT TAC mice (Fig. S3 A and B). These results suggest that the TRPC1/4/5 subfamily also is involved in regulating cardiac hypertrophic signaling.

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.

Although TRPC3/6/7 and TRPC1/4/5 subfamily members generally prefer self-oligomerization, examples of cross-oligomerization between the subfamilies have been observed (14–17). Thus we crossed dnTRPC4 mice with WT TRPC3 TG mice to assess Ca²⁺ influx in isolated myocytes, which showed significant inhibition of TRPC3-dependent Ca²⁺ influx under store-depleted conditions (Fig. 5E). Moreover, immunoprecipitation of TRPC3 protein from double transgenic (DTG) hearts identified the dnTRPC4 protein, suggesting that these two channels could coassociate across these two subfamilies (Fig. 5F). However, this “promiscuity” was not complete, because crossing the dnTRPC4 and dnTRPC3 transgenes together resulted in a 100% inhibition of all TAC-induced Ca²⁺ entry in isolated myocytes (Fig. 5G). As a control, we showed that the dnTRPC4 and dnTRPC3 truncation proteins could immunoprecipitate with their respective homotypic, full-length counterparts in neonatal myocytes and even that dnTRPC6 could interact with TRPC3 (Fig. S4 A–C). Taken together, these results suggest that TRPC channels can form complexes across the subfamilies in cardiac myocytes so that overexpression of any dnTRPC family member renders many potential TRPC tetrameric channel assemblies inactive.

dnTRPC Inhibits Calcineurin–NFAT Signaling.

Because calcineurin–NFAT has been implicated in mediating TRPC-dependent hypertrophy, we evaluated the ability of our dnTPRC proteins to affect this signaling pathway. First, TG mice containing an NFAT-luciferase reporter transgene (18) were crossed with dnTRPC3 TG mice and subjected to TAC stimulation. NFAT-luciferase TG mice alone showed a 6-fold induction in NFAT activity in the heart following 2 weeks of TAC, but this activation was inhibited by ≈50% in DTG mice that also contained the dnTRPC3 transgene (Fig. 6A). Similar results also were obtained in cultured neonatal rat ventricular myocytes (NRVM). NRVMs were infected with an NFAT-luciferase reporter adenovirus and stimulated with PE, which produced robust NFAT activity that was reduced by ≈60% with an adenovirus encoding dnTRPC6 (Fig. 6B). Similarly, AdTRPC3- or AdTRPC4-mediated overexpression significantly enhanced PE-induced NFAT activity in NRVMs (Fig. 6C). Calcineurin association with calmodulin, which indicates the fraction of calcineurin in the activated state, also was reduced with dnTRPC6 overexpression in PE-stimulated myocytes (Fig. 6 D and E). Finally, we also showed that dnTRPC3 and dnTRPC4 overexpression could reduce PE-enhanced NFAT activity caused by TRPC3 or TRPC4 overexpression in NRVMs (Fig. S5). These results indicate that prohypertrophic GPCR signaling utilizes a TRPC-dependent Ca²⁺ signal in activating the calcineurin-NFAT circuit.

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.

Discussion

Ca²⁺-dependent signaling effectors are present in cardiac myocytes, where they influence the cardiac hypertrophic response (2, 3), although, given the excitable nature of this cell type and the dynamic fluxing of Ca²⁺ that bathes the entire cytoplasm during each contractile cycle, specifically how these effectors might be activated remains a mystery. One hypothesis is that Ca²⁺-activated signaling effectors are compartmentalized in membrane microdomains in direct proximity or even attached to Ca²⁺ influx channels (3). For example, calmodulin-dependent protein kinase II is regulated, in part, by a perinuclear Ca²⁺ pool associated with the inositol triphosphate receptor that controls translocation of histone deacetylase 5 out of the nucleus, presumably to permit hypertrophic gene expression (19). T-type Ca²⁺ channels, which are induced in hypertrophic hearts, provide a local Ca²⁺ signal to NOS3 to generate an antihypertrophic and protective effect through cGMP-dependent protein kinase type I (20). Similarly, induction of TRPC channel activity during cardiac hypertrophy is hypothesized to generate a distinct Ca²⁺ signaling microdomain that can impact calcineurin signaling and the hypertrophic response directly (21).

Simple overexpression of TRPC3 or TRPC6 in the mouse heart was sufficient to induce Ca²⁺ entry and enhance the cardiac hypertrophic response (4, 5). Mechanistically, we and others have shown that TRPC channels engage calcineurin–NFAT signaling in the heart, a well-known prohypertrophic pathway that is both necessary and sufficient for growth (4–7). Indeed, deletion of calcineurin Aβ reduced hypertrophic inducibility by TRPC3 overexpression in the heart (5). Although TRPC channels can induce pathologic cardiac growth when overexpressed, the necessity of these channels was not investigated until very recently. For example, the TRPC3 chemical inhibitor Pyr3 administered to mice at 0.1 mg/kg/d was shown to attenuate the cardiac hypertrophic response following pressure-overload stimulation (11). Moreover, Trpc1^−/− mice were shown to develop less cardiac hypertrophy in response to 8 weeks of pressure-overload stimulation or 4 weeks of AngII infusion (10). Although our study is in agreement, we identified several concepts that further establish an overarching Ca²⁺ regulatory paradigm in the heart. First, we observed that the sarcolemma from hypertrophic cardiac myocytes is altered profoundly and effectively is “leaky” to Ca²⁺ through non–L-type or non–T-type channels (non-NCX1). Second, this enhanced Ca²⁺ influx profile observed under store-depleted conditions was inhibited with either dnTRPC3, dnTRPC6, or dnTRPC4 overexpression in vivo. Third, the TRPC3/6/7 and TRPC1/4/5 subclasses appeared to function interdependently in the heart to mediate pathologic hypertrophy.

The observation that both dnTRPC4 and dnTRPC3/6 could inhibit pathologic cardiac hypertrophy equally when overexpressed in the mouse heart was unexpected, because they are from different functional and oligomerization subfamilies (8, 9). However, TRPC1 and TRPC3 were reported to coassemble in generating a DAG-sensitive Ca²⁺ channel in HEK293 cells (14). In the brain, TRPC1/4/5 also were identified in complexes with TRPC3 and TRPC6, and a dnTRPC5 mutant was capable of quenching TRPC3 currents when TRPC1 was co-overexpressed, but not TRPC3 alone (15). Moreover, TRPC3 and TRPC4 were shown to coassemble in forming a redox-sensitive cation channel in endothelial cells and HEK293 cells by FRET analysis, immunoprecipitation, and direct measurement of channel current (22). More provocatively, TRPC channels probably coexist with TRPM and TRPP subclasses of channels in even larger channel complexes (16, 17). Because all TRPC family members have been detected in the heart, some of which are up-regulated by hypertrophy (4–7, 23), it is likely that many combinations of tetramers are possible and can generate unique cation influx properties. However, combined inhibition of both subfamilies in dnTRPC4, dnTRPC3 DTG mice completely eliminated all TAC-associated Ca²⁺ entry, suggesting that not all TRPC complexes show cross-oligomerization in the heart.

The cardiac myocyte is perhaps one of the few cell types in the body that does not require store-operated Ca²⁺ entry to reload the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) compartment. Indeed, Ca²⁺ loading of the ER/SR occurs during each contractile cycle and can be explained fully by the action and equilibrium between the L-type Ca²⁺ channel, the NCX, the ryanodine receptor, and SERCA2 (24). Thus, the relatively high prevalence of TRPC channel expression in cardiac myocytes probably mediates other modes of Ca²⁺ entry for putative signaling functions. The results of our study, in conjunction with the evolving literature in this area, suggest that pharmacologic inhibitors of TRPC channels might be a strategy for attenuating local Ca²⁺ signals involved in pathologic cardiac hypertrophy or failure.

Methods

Myocytes Isolation and Ca²⁺ Measurements.

Adult myocytes from WT, dnTRPC, and TRPC3 TG mice were isolated as previously described (25). Myocytes were loaded with Indo-1 AM (10 μM) for 12–45 min in normal Tyrode solution (in mmol/L: CaCl₂ 1, NaCl 140, KCl 4, MgCl₂ 1, Hepes 5, glucose 10; pH 7.4) or MEM at room temperature. Cells were bathed in Ca²⁺-free normal Tyrode solution in the presence of 10 μM CPA for 30 min to deplete intracellular Ca²⁺ stores. The solution then was switched to 1 mM Ca²⁺ normal Tyrode solution with CPA to evoke store-operated Ca²⁺ entry at baseline or in the presence of PE (50 μM). Other inhibitors included verapamil (10 μM), KB-R7943 (5 μM), and SKF-96365 (5 μM). NRVMs for NFAT activity assays were isolated and cultured as described previously (18).

Animal Models and Procedures.

Animal procedures were approved by the Cincinnati Children's Hospital Institutional Animal Care and Use Committee. NFAT-luciferase and heart-specific TRPC3 TG mice were described previously (5, 18). TG mice were generated with the αMHC promoter to drive expression of dnTRPC3 (26), dnTRPC4 (27), and dnTRPC6 (13).

Echocardiography and Mouse Procedures.

Echocardiography under 2% isoflurane has been described previously (28). Pathologic hypertrophy was induced by TAC as described previously (18). Doppler echocardiography was used to confirm equal pressure gradients across the aortic constrictions in all TAC procedures. Alzet miniosmotic pumps (model 1002; Durect Corp.) containing a mixture of PE (100 mg/kg/d) and AngII (432 μg/kg/d), or PBS (vehicle control) were surgically inserted s.c. dorsally in mice under isoflurane anesthesia. Swimming exercise to induce hypertrophy has been described previously (18).

Western Blotting, Immunoprecipitation, and Immunocytochemistry.

Western blotting and immunoprecipitation were done as described previously (28). The antibodies used in this study were anti-TRPC3 polyclonal antibody (Abcam), anti-TRPC6 and anti-TRPC3 polyclonal antibodies (Alomone Labs), anti-TRPC4 (gift from M. Zhu, Center of Molecular Neurobiology, Ohio State University), anti-GAPDH antibody (Fitzgerald), anti-calcineurin A and B (Sigma-Aldrich), and anti-calmodulin (Zymed Laboratories). Immunocytochemistry was performed as described previously, and primary antibodies were used at 1:100 (19).

Statistics.

All data are expressed as mean ± SEM. Differences between experimental groups were evaluated for statistical significance using the Student's t test for unpaired data or for multiple groups using both two-tailed t test and two-way ANOVA. The data were distributed normally in all cases. P < 0.05 was considered statistically significant.