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* Department of Pediatrics, University of Cincinnati, Children’s Hospital Medical Center, Cincinnati, Ohio, USA; and
Department of Surgery, University of Cincinnati, Cincinnati, Ohio, USA
1Correspondence: Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229, USA. E-mail: jeff.molkentin{at}cchmc.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: signaling • calcium • store-operated • NFAT
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. CnB contains four EF hand Ca2+ binding motifs and it tightly binds CnA at submicromolar concentrations of Ca2+. At low Ca2+ concentrations, CaM is not bound to the complex, and an autoinhibitory domain within the C terminus of CnA sterically blocks the active site. As Ca2+ concentration elevates, it binds the EF hand motifs of CaM, facilitating greater interaction with CnA and a conformational shift that displaces the autoinhibitory domain, permitting calcineurin activation (1)
. Once activated, calcineurin regulates development and stress adaptation of multiple organ systems in mammals (2
3
4
5)
. While higher organisms have a number of confirmed targets for this phosphatase, many of the biological effects of calcineurin are mediated through two classes of transcriptional effectors: nuclear factor of activated T cells (NFAT) and myocyte enhancer factor 2. For example, in the heart calcineurin functions as a potent regulator of cardiac hypertrophy through activation of NFAT transcription factors to mediate the growth response (6)
.
Activation of calcineurin-NFAT requires a unique mechanism of Ca2+ alteration within most cells such that intracellular Ca2+ concentration is dramatically increased over a significant period (7
8
9
10)
. For example, engagement of immune cell receptors leads to sustained Ca2+ entry in the form of ICRAC, a type of store-operated Ca2+ entry, and this sustained Ca2+ entry mechanism is necessary for NFAT nuclear localization/activation and proper cytokine expression (7
8
9
10
11
12
13)
. The current known as ICRAC is hypothesized to mediate store-operated Ca2+ entry in part through canonical members of the transient receptor proteins (TRP) of the C class, or TRPC. Drosophila melanogaster carrying trp mutations generate only a transient photoreceptor depolarization in response to sustained light signals, suggesting a function as a store-operated Ca2+ entry channel (14)
. Since this initial description, a large superfamily of TRP homologous channels has been elucidated in mammalian species (15)
, some of which are prominently expressed in the heart (16
, 17)
. In response to agonist stimulation resulting in the generation of diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3), Ca2+ is released and eventually depleted from the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR). One hypothesis is that TRPC activity is directly activated after IP3 and DAG signals in the sensing of ER/SR Ca2+ depletion, thus inducing Ca2+ entry to replete internal stores (18)
. That such a mechanism exists in cardiomyocytes is uncertain given that essentially all Ca2+ exchange during each contractile cycle can be attributed to other channels and pumps associated with excitation-contraction coupling (19)
.
Recent studies have demonstrated the existence of a store depletion-sensitive Ca2+ entry mechanism in both neonatal and adult rat cardiomyocytes. Treatment of adult cells with IP3 or IP3-generating agonists and agents that block ER Ca2+ reuptake resulted in store depletion and induced extracellular Ca2+ influx sensitive to pharmacologic inhibitors of store-operated Ca2+ entry, but not L-type Ca2+ current (20
, 21)
. In addition, store-operated Ca2+ entry in neonatal cardiomyocytes was associated with NFAT nuclear localization and hypertrophy, while L-type channel inhibitors had no effect on these processes (21)
. More recently, store-operated Ca2+ entry was shown to partially regulate SR Ca2+ homeostasis in neonatal rabbit ventricular myocytes (22)
. Consistent with these observations, store-operated Ca2+ entry also plays an important role in maintaining internal contractile Ca2+ levels in smooth muscle cells (23)
. Here we generated lines of cardiac-specific TRPC3 expressing transgenic mice to evaluate the potential association between store-operated Ca2+ entry and calcineurin-NFAT activation and cardiac hypertrophy in vivo. TRPC3 transgenic mice showed a prominent increase in cardiac NFAT transcriptional activity that was associated with, but preceded by, mild cardiac hypertrophy or cardiomyopathy. Moreover, TRPC3 transgenic mice showed a dosage-dependent enhancement in cardiac hypertrophy after neuroendocrine as well as enhanced hypertrophy after pressure overload stimulation, which was ameliorated by calcineurin Aß gene targeting. Our results are the first to suggest that TRPC proteins have the ability to regulate calcineurin signaling and the hypertrophic growth of the myocardium in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-myosin heavy chain (-MHC) promoter expression vector (gift from Jeffrey Robbins, Children’s Hospital, Cincinnati, OH, USA). The -MHC-TRPC3 transgene was injected into newly fertilized oocytes to generate transgenic mice (FVB/N background). NFAT-luciferase reporter transgenic mice and calcineurin Aß gene-targeted mice were each described previously (24
, 25)
. Experiments involving animals were approved by the Institutional Animal Care and Use Committee.
Isolation of adult cardiomyocytes for Ca2+ measurements
Cardiomyocytes were isolated for analysis, and only Ca2+-tolerant cells with clear cross striations and without spontaneous contractions or significant granulation were selected for experimental studies. In brief, the heart was rapidly excised and placed in Tyrode solution containing (in mmol/l): 120 NaCl, 5.4 KCl, 1.2 NaH2PO4, 5.6 glucose, 20 NaHCO3, 1.6 MgCl2, 10 2,3-butanedione monoxime (BDM), and 5 taurine (buffer A), gassed with 95% O2-5% CO2. All solutions were filtered and equilibrated with 95% O2-5% CO2 for at least 20 min before use. The heart was retrogradely perfused with buffer A for 4–5 min, then with buffer A containing 1 mg/ml collagenase type II (Worthington, Freehold, NJ, USA) and 0.08 mg/ml protease type XIV (Sigma) at 37°C. After 2 min of enzyme perfusion, 50 µM Ca2+ was added to the enzyme solution. When the heart became "swollen and soft" after 
5 min of digestion, the enzyme was recirculated. The heart was perfused for an additional 8–12 min or until flow rate surpassed pre-enzyme flow rate. After perfusion, the ventricles were separated from the atria and minced. Myocytes were incubated with 2 µmol/l Fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR, USA) for 30–35 min in buffer C (buffer A with 1 mmol/l Ca2+ without BDM) at room temperature. After being loaded, the cells were washed and resuspended in buffer C. After three washes (40 min), the cell suspension was placed in a plexiglas chamber containing 500 µl of fresh buffer C. The Fura-2 fluorescence ratio was determined at room temperature using a Delta Scan dual-bean spectrofluorophotometer (Photon Technology, Birmingham, NJ, USA), operating at an emission wavelength of 510 nM with excitation wavelengths of 340 and 380 nM. The stimulating frequency for Ca2+ transient measurements was 0.5 Hz. Baseline amplitude (estimated by 340/380 nM ratio) of the Ca2+ signal was acquired and data were analyzed using software from Felix 1.1 Software and IonWizard (IonOptix). Adult myocytes were pretreated with 1 µM Ang II and the reversible sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor CPA (cyclopiazonic acid, 5 µM) for 9–10 min in Ca2+-free buffer to achieve more complete SR store depletion. Myocytes were then given buffer with an extracellular Ca2+ of 1.8 mM to assay for store-operated entry measured as a sustained increase in Fura-2 fluorescence (measured as the 340/380 ratio) 4–5 min after Ca2+ addition. In some experiments verapamil (10 µM) or KB-R7943 (5 µM) was added to the 1.8 mM solution to abolish [Ca2+]i spikes and L-type Ca2+ entry, or the possibility of Na+/Ca2+ activity, but this did not alter the sustained increase in the Fura-2 ratio in a small percentage of wild-type cells. SKF 96365 was used at 5 µM, which inhibits store-operated Ca2+ entry although its cross-reactivity with other channels has not been proven, so its true specificity is uncertain.
Generation of purified adult cardiomyocytes for Western blot
Wild-type and TRPC3 transgenic adult mouse hearts (2 months of age) were dissected, washed in ice-cold cannulation buffer (10 mM 2,3-butanedione monoxime, 25 µM CaCl2 in MEM), and perfused with digestion media (1 mg/ml BSA, 90 U/ml collagenase, 10 mM 2,3-butanedione monoxime, 25 µM CaCl2 in MEM) until the myocardium began to visually dissolve. Perfused hearts were then dissociated in a dish with mechanical disruption and filtered through a 200 µM mesh into MEM containing 10 mM 2,3-butanedione monoxime and 10 µM CaCl2 until individual cells were generated. Cells were collected by centrifugation and differentially plated to remove any residual fibroblasts, after which the cardiac myocytes were collected for Western blot.
Western blot analysis
Ventricle samples from mouse hearts were frozen in liquid nitrogen and stored at –70°C. Protein samples for membrane-rich fractions were prepared from heart tissue using extraction buffer (250 mM sucrose, 10 mM Tris-HCl pH 7.5, 1 mM DTT, and protease inhibitors Complete EDTA-free, Roche, Nutley, NJ, USA) as described previously (26)
. In brief, heart samples were homogenized in extraction buffer, and homogenates were centrifuged at 3000 g for 5 min at 4°C, and the resultant supernatant was centrifuged at 28,000 g for 30 min at 4°C. The pellets were resuspended in extraction buffer and stored at –70°C until use. Western blot conditions have been described (24
, 25)
. Whole cell protein homogenates were extracted from adult cardiomyocytes in cell lysis buffer (20 mM sodium phosphate, pH 7.0, 150 mM NaCl, 2 mM MgCl2, 10 mM NaF, 0.1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM dithiothreitol, 1% Nonidet P-40, 10% glycerol, 10 µg of leupeptin per ml, 10 µg of aprotinin per ml, 10 µg of pepstatin per ml, 10 µg of N--tosylphenylalanyl chloromethyl ketone (TPCK) per ml, 10 µg of N--tosyllysyl chloromethyl ketone (TLCK) per ml). Forty micrograms of protein was loaded in each lane of all Western blots. Antibodies included TRPC1, 4, 5, 6, 3/6/7 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) TRPC3 (Alomone Labs, Jerusalem, Israel), and GAPDH (Research Diagnostics Inc., Flanders, NJ, USA). Isoform-specific calcineurin A and Aß rabbit polyclonal antibodies were custom synthesized in rabbits and affinity purified by Zymed Laboratories (San Francisco, CA, USA).
Echocardiography and surgical models
Mice from all genotypes or treatment groups were anesthetized with isoflurane and echocardiography was performed using a Hewlett Packard 5500 instrument with a 15 MHz microprobe. Echocardiographic measurements were taken on M-mode in triplicate from at least four separate mice per group. The investigator was blinded to the identity of the mice for analysis. Two-month-old nontransgenic and transgenic mice were subjected to transverse aortic constriction (TAC) as described (24)
. Alzet miniosmotic pumps (no. 2002; Alza Corp., Mountain View, CA, USA) containing a mixture of phenylephrine (PE) (50 mg/kg/day) and angiotensin II (Ang II) (432 µg/kg/day), isoproterenol (60 mg/kg/day), or PBS (vehicle control) were surgically inserted dorsally and subcutaneously in 2-month-old mice under isoflurane anesthesia. All mice were sacrificed 2 wk after the procedure.
Reporter assays in mouse hearts
Luciferase reporter assays from mouse hearts were performed as described previously (24)
. In brief, TRPC3 transgenic mice were crossbred with NFAT-luciferase reporter mice and ventricles were excised from double and single transgenic mice and stored at –70°C. The frozen hearts were homogenized in 1 ml luciferase assay buffer (100 mM KPO4, pH 7.8, 0.5% Nonidet P-40, and 1 mM DTT). Homogenates were centrifuged at 3000 g for 10 min at 4°C and the supernatants assayed for luciferase activity as described previously (24)
.
Histological and hypertrophic marker gene analyses
Hearts were collected at the indicated times, fixed in 10% formalin containing PBS, and embedded in paraffin. Serial 7 µm heart sections from each group were analyzed. Samples were stained with H&E, Masson’s trichrome, or wheat germ agglutinin-TRITC conjugate at 50 µg/ml to accurately identify sarcolemmal membranes so that myofiber diameter could be quantified. Reverse transcriptase-polymerase chain reaction (RT-PCR) for atrial natriuretic factor (ANF) and control L7 mRNA was performed as described previously (27)
.
Statistical analysis
All results are presented as means ± SE. Statistical analysis between two experimental groups was performed by Student’s t test using InStat 3.0 software (GraphPad software for Science Inc., San Diego, CA, USA). Data analysis for more than three groups was performed using 1- or 2-way ANOVA, with post hoc Tukey’s test or Dunnett’s test by Prism 3.0 software (GraphPad software for Science Inc.). Probability values < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MHC promoter to generate cardiac-specific transgenic mice (Fig 1
A). Two lines were generated that were characterized by low and high levels of expression by Western blot from total heart protein extracts from 2-month-old mice (Fig 1B
). Since the heart is comprised of many cell types, it was also of interest to survey which TRPC family members were expressed in adult ventricular myocytes, as well as to examine whether overexpression of TRPC3 in cardiac myocytes might alter expression of other TRPC isoforms. To this end, ventricular myocytes were enzymatically disassociated and enriched from wild-type, line 21, and line 23 TRPC3 transgenic mice at 2 months of age for Western blot. TRPC1, 3, 4, and 5 were detectable and not altered in the TRPC3 transgenic line (expect for TRPC3 itself), while TRPC6 and 7 were not detectable (Fig 1C
).
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Analysis of store-operated Ca2+ entry in wild-type and TRPC3 mice
To evaluate the potential physiological relevance of TRPC3 overexpression in the mouse heart, store-operated Ca2+ entry was analyzed from individual adult myocytes isolated from the hearts of 3- to 4-month-old, high expressing transgenic and nontransgenic mice. While myocytes from adult rat hearts have been shown to have store-operated Ca2+ entry (20)
, adult mouse myocytes were reported to be devoid of this activity (28)
. Fura-2 loaded adult myocytes from wild-type mice were prepared in Ca2+-free buffer and stimulated with Ang II in the presence of CPA, a SERCA inhibitor to deplete internal Ca2+ stores. Ca2+ was then returned to the media and entry into the cell was monitored by a change in the Fura-2 fluorescence absorbance ratio. Under these conditions, 75% of adult mouse myocytes showed no store-operated Ca2+ entry, although 25% of myocytes showed a very subtle but significant entry that could be blocked with SKF96365 (21
, 22)
, an inhibitor of store-operated Ca2+ entry (Fig 2
A, B, and data not shown). This very subtle Ca2+ signal in a fraction of the myocytes was not blocked with the L-type Ca2+ channel inhibitor verapamil or the Na+/Ca2+ exchanger inhibitor KB-R7943 (Fig 2C
, and data not shown). A potential qualification of these data is the sole reliance on SKF96365 to implicate a TRPC-dependent mechanism, since this compound can act somewhat nonselectively toward Ca2+ currents in general.
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Robust store-operated Ca2+ entry was easily detectable in 70% of adult myocytes isolated from high expressing TRPC3 transgenic mice (Fig 3
A). The peak change in Fura-2 fluorescence (340/380 ratio) averaged 0.401 ± 0.042 in adult myocytes from TRPC3 transgenic mice (n=34 cells from 7 mice) and 0.065 ± 0.010 in wild-type myocytes (n=44 cells from 8 mice). This store-operated Ca2+ entry observed in TRPC3 transgenic myocytes was not blocked with the L-type Ca2+ channel blocker verapamil (Fig 3B
) or the Na+/Ca2+ exchanger inhibitor KB-R7943 (Fig 3C
), but was completely inhibited with SKF96365 (Fig 3D
). These data indicate that TRPC3 overexpression in the mouse heart substantially enhances store-operated Ca2+ entry. Finally, the potential effect of TRPC3 overexpression on the Ca2+ transient itself was also assessed in adult ventricular myocytes isolated from wild-type and TRPC3 transgenic mice. Remarkably, the peak amplitude of the Ca2+ transient was significantly increased in TRPC3 transgenic myocytes compared with wild-type myocytes, although baseline Fura-2 readings, the time to 70% decay in the Ca2+ transient, and the time constant were not altered (Table 1
, and data not shown). These readings were generated from younger animals prior to the onset of heart failure. Even in nonfailing mice, this increase in Ca2+ transient amplitude was unexpected, but may be related to greater sarcolemmal Ca2+ influx or "membrane leak," causing enhanced SR loading, consistent with a recent description suggesting that store-operated Ca2+ entry could influence SR Ca2+ content in neonatal rabbit ventricular myocytes (22)
.
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TRPC3 transgenic mice display cardiomyopathy
Low and high expressing TRPC3 transgenic mice were examined for signs of cardiac dysfunction or alterations in overall phenotype. Low expressing mice were essentially normal at 2 and 8 months of age with respect to overt hypertrophy as assessed by measurement of heart weight normalized to tibial length (Fig 4
A). However, by 12 months of age low expressing TRPC3 transgenic mice showed cardiac hypertrophy. High expressing TRPC3 transgenic mice also showed almost no cardiac enlargement at 2 and 8 months of age, but nearly all succumbed to lethality by 12 months of age (Fig 4A, B
). While the cause of lethality is unknown, we speculate that disturbed Ca2+ homeostasis induced by increased Ca2+ influx could cause arrhythmia and sudden death. Indeed, high expressing male transgenic mice showed nearly 50% lethality by 50 days of age (Fig 4B
), while 9/34 female transgenic mice died by 8 wk of age, suggesting a sex-dependent difference in lethality associated with TRPC3 overexpression. While both high and low TRPC3 transgenic mice had no hypertrophic enlargement at 2 and 8 months of age, cardiac ANF expression was significantly induced at 2 months of age, suggesting that the cardiac stress response was evoked and preceded an overt hypertrophic phenotype (Fig 4C
). To more carefully evaluate the affect of TRPC3 overexpression, echocardiography was performed in the low transgenic line at 12 months of age (Fig 4D-F
). These TRPC3 transgenic mice showed a significant dilation of the left ventricle (Fig 4E
) and a loss of ventricular performance as assessed by measurement of fractional shortening (Fig 4F
). Thus, overexpression of TRPC3 in the heart generally leads to cardiomyopathy as mice age, but hypertrophy is only seen at later stages.
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TRPC3 transgenic mice show activation of NFAT in the heart
In nonexcitable cells, store-operated Ca2+ entry has been shown to regulate calcineurin-NFAT signaling, providing the necessary long-term increase in intracellular Ca2+ (7
8
9
10
11
12
13)
. Moreover, the source or microdomain of Ca2+ that regulates calcineurin-NFAT in the heart has not been determined, although store-operated entry has been suggested as a source (21)
. Here we investigated whether TRPC3 overexpression would enhance calcineurin-NFAT signaling in the heart using NFAT-luciferase reporter transgenic mice (24)
. Low expressing TRPC3 transgenic mice showed a roughly 2-fold increase in cardiac NFAT-luciferase activity at 2 wk, 2 months, and 8 months of age (Fig 5
A). A similar profile of increased NFAT-luciferase activity was observed in high expressing TRPC3 transgenic mice. A dose-dependent increase in NFAT-luciferase reporter activity was likely not seen between the high and low TRPC3 transgenic lines due to the relative insensitivity of the reporter transgene and/or the possibility that even low TRPC3 overexpression gives a near-maximal response toward calcineurin-NFAT signaling. That NFAT-luciferase activity is increased as early as 2 wk and 2 months of age, which significantly precedes any signs of cardiac pathology, suggests that TRPC3 overexpression proximally regulates NFAT activity. However, TRPC proteins are typically activated by depletion in stored Ca2+ or by phospholipase C (PLC) -inducing stimuli. Thus, we also investigated the ability of the G-protein-coupled PLC agonists PE + Ang II to further up-regulate NFAT-luciferase activity in the hearts of TRPC3 transgenic mice (Fig 5B
). Indeed, both low and high lines of TRPC3 transgenic mice showed a greater increase in cardiac NFAT-luciferase transgene activity after 14 days of agonist stimulation in 2-month-old mice (Fig 5B
). These results are consistent with the hypothesis that G-protein-coupled receptor agonists that promote PLC activation and formation of IP3 and DAG in turn promote greater TRPC channel activity and hence more NFAT activation. As a control, isoproterenol was also infused in TRPC3 transgenic mice, which functions as a ß-adrenergic receptor agonist that signals independent of PLC. Remarkably, isoproterenol infusion did not augment NFAT-luciferase activity in the hearts of wild-type or TRPC3 transgenic mice (high or low line), but instead led to significant inhibition (Fig 5C
). This inhibition may be due to activation of coregulated pathways, such as p38 mitogen-activated protein kinases or c-Jun N-terminal kinase that can directly phosphorylate and inactivate NFAT transcription factors (5)
. As a final control, we also measured total calcineurin A and Aß protein levels in the hearts of wild-type and TRPC3 transgenic mice, which showed no difference, suggesting that the alteration in NFAT-luciferase activity is not due to a secondary change in calcineurin A protein levels (Fig 5D
).
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Agonist and pressure overload stimulation promote greater cardiac hypertrophy in TRPC3 transgenic mice
While baseline hypertrophy was only minimally altered by constitutive TRPC3 expression in the heart, it is possible that full store-operated Ca2+ entry through the overexpressed TRPC3 protein requires stimulation. Indeed, significantly greater NFAT-luciferase activity was observed in TRPC3 transgenic mice infused with PE + Ang II (Fig 5B
). Associated with this greater increase in NFAT-luciferase activity was a corresponding augmentation in cardiac hypertrophy. Two-month-old wild-type male mice showed a characteristic 15% increase in cardiac hypertrophy after agonist infusion (Fig 6
A). However, low and high TRPC3 male transgenic mice showed significantly greater cardiac hypertrophy after 14 days of agonist infusion (Fig 6A
). Thus, TRPC3 overexpression predisposed the heart to greater hypertrophy, in association with augmented NFAT activity, suggesting a role for store-operated Ca2+ entry in this response.
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In addition to agonist infusion for 14 days, wild-type male and TPRC3 male transgenic mice were subjected to pressure overload stimulation to further assess the hypertrophic phenotype and its modulation by TRPC3 overexpression. Consistent with the agonist infusion studies, TRPC3 transgenic mice developed significantly greater cardiac hypertrophy compared with wild-type mice after 2 wk of TAC stimulation (Fig 6B
). Analysis of myocytes from heart ventricular histological sections also showed a significantly greater increase in cardiomyocyte hypertrophy in low expressing TRPC3 transgenic mice compared with wild-type mice after TAC stimulation (Fig 6C
). Associated with this greater increase in cardiac hypertrophy observed in TRPC3 transgenic mice was an enhanced propensity toward heart failure. Specifically, TRPC3 transgenic mice showed a dramatic reduction in fractional shortening after 2 wk of TAC stimulation, while wild-type mice were fully compensated during this time (Fig 6D
). There was also a trend toward increased left ventricular end diastolic dimension in the transgenic mice after TAC, although it was not significant (data not shown). Low expressing TRPC3 transgenic mice showed increased lung weight normalized to tibial length after 2 wk of TAC, suggesting pulmonary edema, while wild-type mice did not (Fig 6E
). Thus, TRPC3 transgenic mice are predisposed to greater cardiac hypertrophy and failure after pressure overload stimulation, associated with greater calcineurin-NFAT activation. Indeed, TAC stimulation produced a synergistic increase in cardiac NFAT-luciferase activity in the hearts of low expressing TRPC3 transgenic mice (Fig 7
A).
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Loss of calcineurin Aß rescues TRPC3-dependent hypertrophy and heart failure
The data presented above suggest that TRPC3 overexpression leads to cardiomyopathy and augmented stimulus-induced hypertrophy in association with greater NFAT activity. To more carefully evaluate this association, we reasoned that loss of calcineurin Aß might "short-circuit" the signaling process from TRPC3-to-Ca2+-to-calcineurin activation in the heart. Thus, low expressing TRPC3 transgenic mice were crossed into the calcineurin Aß null background or controls were generated from the same backcross that were wild-type for calcineurin Aß (Fig 7B-D
). As previously observed, calcineurin Aß null mice were largely resistant to hypertrophy after 2 wk of pressure overload stimulation (25)
(Fig 7B
). In contrast, wild-type mice showed a 25% increase in heart weight normalized to tibial length, and wild-type mice with the TRPC3 transgene showed a synergistic increase in hypertrophy (Fig 7B
). However, loss of calcineurin Aß blocked pressure overload induced hypertrophy and, more important, essentially blocked the affect of the TRPC3 transgene on the hypertrophic response (Fig 7B
). Loss of calcineurin Aß also obviated heart failure in TPRC3 transgenic mice after 2 wk of TAC, as assessed by measurement of lung weight normalized to tibia length and measurement of ventricular fractional shortening (Fig 7C, D
). As a control, we also verified that loss of calcineurin Aß did not alter the level of TRPC3 overexpression in the heart (Fig 7E
). In conclusion, these results suggest that calcineurin could be involved in facilitating TRPC3-dependent hypertrophy and heart failure, although loss of calcineurin could also more globally disrupt the hypertrophic potential of the heart independent of a store-operated Ca2+ entry mechanism.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Despite this observation, contractile Ca2+ can regulate calcineruin-NFATc1 activation and CaM-dependent protein kinase (CaMKII) -histone deacetylase 4 (HDAC4) signaling in skeletal muscle myotubes, at least establishing the possibility that such a mechanism could also occur in the heart (30
, 31)
. This last point notwithstanding, specialized pools of Ca2+, which are region specific or buffered from cytoplasmic Ca2+, could still be required to regulate select Ca2+-sensitive signaling proteins in the heart. However, demonstrating microdomains or region-specific buffering of Ca2+ has largely been beyond routine Ca2+ imaging techniques and thus has yet to be definitively described in cardiac myocytes. Despite the general lack of supportive data, Brochet et al. recently described region specific depletion of Ca2+ in the junctional cisternae of the SR within nanometer-sized stores (32)
. Such region-specific depletion may require local mechanisms for store-operated Ca2+ influx, such as through TRPC proteins, which could explain the relatively large array of expressed TRPC proteins found in the heart (Fig 1C
, and ref 33
). These results are also of interest given the recent description of store-operated Ca2+ entry serving to regulate SR Ca2+ homeostasis in neonatal rabbit ventricular cardiac myocytes (22)
. Alternatively, expression of TRPC proteins in the heart could also regulate local pools of Ca2+ within lipid-raft domains that are coupled with PLC-dependent signaling circuits, as is characteristic of most other nonexcitable cell types (18)
. Finally, in agreement with the general concept of region-specific control of Ca2+, Wu et al. recently inferred a mechanism whereby changes in nuclear envelope Ca2+, as mediated by the IP3 receptor, serves as a means of activating CaMK-HDAC5 in ventricular cardiac myocytes, although no alterations in global Ca2+ were observed (34)
.
Here we showed that graded overexpression of TRPC3 in the heart produced a dosage-dependent manifestation of cardiomyopathy. The rationale for overexpressing TRPC3 was simply to examine the gain-of-function phenotype associated with TRPC proteins in the heart and to examine the potential downstream signaling mechanisms. TRPC3 was selected for overexpression in the heart since TRPC3 is the most highly characterized and is regulated by both store depletion and by PLC-coupled signaling events (18)
. There is also good evidence that TRPC3 is functional on its own when overexpressed whereas TRPC1 appears to be unstable due to aberrant intracellular targeting (18)
. A final consideration is that the function of TRPC proteins is not entirely consistent with biophysical measures of store-operated Ca2+ entry in whole-cell configuration (18)
. While TRPC3 can elicit a store-operated Ca2+ entry event in most cells assayed, it is likely that other components are required to generate a properly regulated and store-operated channel with the correct conductance characteristics. Despite this concern, overexpression of TRPC3 in our transgenic mice did evoke a characteristic store-operated Ca2+ entry event in isolated adult myocytes, although it remains possible that TRPC3-dependent influx is somewhat distinct from an endogenous store-operated Ca2+ entry mechanism in the duration of a response, the localization of the channel to select microdomains, its activation by select agonists, or its regulation by cofactors.
Previous work has established that both neonatal and adult rat cardiomyocytes are capable of store-operated Ca2+ entry once depleted with PLC-dependent agonists (20
, 21)
, as well as neonatal rabbit ventricular myocytes (22)
. Moreover, Marchase and colleagues showed that general inhibitors of store-operated Ca2+ entry could reduce NFAT activation in cardiomyocytes, further supporting the hypothesis that specialized pools of Ca2+ are uniquely coupled to Ca2+-sensitive signaling proteins such as calcineurin (21)
. Indeed, in nonmyocytes, calcineurin-NFAT is prominently regulated by store-operated Ca2+ entry (7
8
9
10
11
12
13)
. In support of such a mechanism, Uehara et al. similarly showed that embryonic and neonatal mouse cardiomyocytes displayed store-operated Ca2+ entry using a similar experimental approach (28)
. However, Uehara et al. failed to identify store-operated Ca2+ entry in adult mouse cardiomyocytes in culture, consistent with our inability to detect such entry in 
75% of nontransgenic adult mouse cardiomyocytes (Fig 2A
).
In conclusion, it remains uncertain whether store-operated Ca2+ entry plays a physiological role in regulating reactive signaling in the adult mouse heart. Targeted deletion of specific TRPC family members may provide significant insight into this important issue. However, the observation that overexpression of TRPC3 in the heart promoted greater NFAT activation, cardiomyopathy, and enhanced stimulus-induced cardiac hypertrophy at least suggests that store-operated Ca2+ entry can serve in such a capacity, although definitive proof of the requirement of this signaling circuit as a required mediator of hypertrophy and/or myopathy awaits an in vivo loss-of-function approach.
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ACKNOWLEDGMENTS |
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Received for publication December 1, 2005. Accepted for publication March 31, 2006.
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Y.-K. Ju, Y. Chu, H. Chaulet, D. Lai, O. L. Gervasio, R. M. Graham, M. B. Cannell, and D. G. Allen Store-Operated Ca2+ Influx and Expression of TRPC Genes in Mouse Sinoatrial Node Circ. Res., June 8, 2007; 100(11): 1605 - 1614. [Abstract] [Full Text] [PDF]
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S. de Frutos, R. Spangler, D. Alo, and L. V. G. Bosc NFATc3 Mediates Chronic Hypoxia-induced Pulmonary Arterial Remodeling with {alpha}-Actin Up-regulation J. Biol. Chem., May 18, 2007; 282(20): 15081 - 15089. [Abstract] [Full Text] [PDF]
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