Published online before print January 2, 2009, doi: 10.1161/CIRCRESAHA.108.184051
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© 2009 American Heart Association, Inc.
Cellular Biology |
The CaV3.2 T-Type Ca2+ Channel Is Required for Pressure Overload–Induced Cardiac Hypertrophy in Mice
From the Institute of Biomedical Sciences (C.-S.C., C.-H.H., H.C., Y.-T.C., D.C., J.-J.C., Y.-C.C.,Y.-H.C., C.-C.C.), Academia Sinica, Taipei, Taiwan; Graduate Institute of Life Sciences (C.-H.H., C.-C.C.), National Defense Medical Center, Taipei, Taiwan; Center for Neural Science (H.-S.S.), Korea Institute of Science and Technology, Seoul, Korea; and Department of Physiology and Biophysics and Department of Neurology (K.P.C.), Howard Hughes Medical Institute, University of Iowa, Iowa City.
Correspondence to Chien-Chang Chen, Institute of Biomedical Sciences, Academia Sinica, 128 Academia Rd Sec 2, Nankang, Taipei 11529, Taiwan. E-mail ccchen{at}ibms.sinica.edu.tw
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Abstract |
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Voltage-gated T-type Ca2+ channels (T-channels) are normally expressed during embryonic development in ventricular myocytes but are undetectable in adult ventricular myocytes. Interestingly, T-channels are reexpressed in hypertrophied or failing hearts. It is unclear whether T-channels play a role in the pathogenesis of cardiomyopathy and what the mechanism might be. Here we show that the
1H voltage-gated T-type Ca2+ channel (Cav3.2) is involved in the pathogenesis of cardiac hypertrophy via the activation of calcineurin/nuclear factor of activated T cells (NFAT) pathway. Specifically, pressure overload–induced hypertrophy was severely suppressed in mice deficient for Cav3.2 (Cav3.2–/–) but not in mice deficient for Cav3.1 (Cav3.1–/–). Angiotensin II–induced cardiac hypertrophy was also suppressed in Cav3.2–/– mice. Consistent with these findings, cultured neonatal myocytes isolated from Cav3.2–/– mice fail to respond hypertrophic stimulation by treatment with angiotensin II. Together, these results demonstrate the importance of Cav3.2 in the development of cardiac hypertrophy both in vitro and in vivo. To test whether Cav3.2 mediates the hypertrophic response through the calcineurin/NFAT pathway, we generated Cav3.2–/–, NFAT-luciferase reporter mice and showed that NFAT-luciferase reporter activity failed to increase after pressure overload in the Cav3.2–/–/NFAT-Luc mice. Our results provide strong genetic evidence that Cav3.2 indeed plays a pivotal role in the induction of calcineurin/NFAT hypertrophic signaling and is crucial for the activation of pathological cardiac hypertrophy.
Key Words: hypertrophy • cardiomyopathy • T-type Ca2+ channel
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Introduction |
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Hypertrophic growth and remodeling of the adult heart begin as normal compensatory responses to a variety of physiological and pathological stimuli including exercise, pregnancy, pressure overload, hypertension, myocardial infarction, and primary genetic abnormalities.1–3 Prolonged hypertrophic response may eventually lead to heart failure and death. Pathological hypertrophy is often associated with structural and functional remodeling of the heart. Profound changes in gene expression during pathological cardiac hypertrophy are common, particularly in the case of genes that code for proteins involved in regulating contraction ion channels, for example.4
Alterations of intracellular Ca2+ handling could lead to abnormal Ca2+ signaling cascades, a phenomenon that has been shown to contribute to the pathogenesis of cardiac hypertrophy and heart failure.5,6 However, the detailed mechanism of how the cardiac myocytes distinguish Ca2+ transients that occur at every heartbeat from those meant to trigger intracellular hypertrophic signaling remains largely unknown. Intracellular Ca2+ levels could be altered because of either changes in Ca2+ release from intracellular organelles or the influx of extracellular Ca2+, and in both cases, this could be attributable to the activities of either ligand- or voltage-activated Ca2+ channels. Indeed, ligand-activated Ca2+ channels, such as store-operated Ca2+ channels, have been implicated in the signaling pathway that leads to cardiac hypertrophy.7 Candidate proteins for store-operated Ca2+ channels, such as transient receptor potential proteins (including TRPC1, TRPC3, and TRPC6), have been shown to act as positive regulators of calcineurin/NFAT-mediated signaling, which drive cardiac hypertrophy both in vitro and in vivo.8–11
L-type Ca2+ channels are the main voltage-activated Ca2+ channels responsible for triggering Ca2+-induced Ca2+ release during excitation–contraction coupling. Their role in the development of cardiac hypertrophy remains controversial. In general, the current density of L-type Ca2+ channels is either unchanged or slightly elevated when hypertrophy is mild and slightly reduced when hypertrophy is severe.12–14 Although normally expressed during developmental stages and not expressed in adult cardiac myocytes, T-channels are reexpressed after development of pathological hypertrophy, in the postinfarction heart, and during stimulation with certain hormones.15–23 The pore-forming subunits of T-channels are encoded by 3 genes, Cav3.1, -3.2 and -3.3,24–26 and both Cav3.1 and Cav3.2 are present in cardiac tissue.27–29 Mice lacking Cav3.1 T-channels display abnormal sinoatrial node pacemaker activity and atrioventricular conduction,30 whereas mice lacking Cav3.2 exhibit recurrent coronary vasospasms.31 Cav3.1 and Cav3.2 have been reported to be upregulated in various animal models following cardiac hypertrophy.18,19,22 However, the physiological role of T-channel reexpression under these conditions is unclear.
In this study, we aimed to determine whether Cav3.2 T-channels are involved in the development of pathological cardiac hypertrophy and whether the calcineurin–NFAT pathway is downstream of Cav3.2 T-channels during cardiac hypertrophy. Using Cav3.2–/– and Cav3.1–/– mice, we show that Cav3.2 but not Cav3.1 T-channels are required for the development of cardiac hypertrophy induced either by pressure overload and angiotensin (Ang) II treatment in vivo and in vitro. We also discover that the T-channel blocker ethosuximide could prevent the development of pressure overload–induced cardiac hypertrophy in wild-type (WT) mice, and that the stimulation of NFAT signaling in response to pressure overload requires the presence of Cav3.2. Overall, our results demonstrate that Cav3.2 T-channels regulate pathogenic cardiac hypertrophy in vivo by activating the calcineurin–NFAT signaling cascade.
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Materials and Methods |
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Animals were kept under specific pathogen-free conditions, and all procedures performed were approved by the Institutional Animal Care and Use Committee of Academia Sinica, Taiwan. An expanded Materials and Methods section detailing the methods and protocols used in the present study is available in the online data supplement at http://circres.ahajournals.org.
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Results |
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Impaired Cardiac Hypertrophy in Cav3.2–/– but Not Cav3.1–/– Mice Following Transverse Aortic Banding
Although an association between T-channel reexpression and cardiac hypertrophy has been established, an in vivo loss-of-function approach to causality has not previously been undertaken. In this study, we compared the extent of cardiac hypertrophy in WT, Cav3.2–/–, and Cav3.1–/– mice following pressure overload to determine the role of T-channels in the development of cardiac hypertrophy. Measurement of the Doppler flow pressure gradient (in meters per second) following constriction of the transverse aorta (Table I in the online data supplement) indicated that WT, Cav3.2–/–, and Cav3.1–/– mice were subjected to a comparable pressure load. Notably, Cav3.2–/– mice or their sham-operated animals have a smaller body size relative to those age-matched WT mice (supplemental Table I), thus resulting in a higher ratio of heart weight/body weight (HW/BW) (in milligrams per gram) (Figure 1A and supplemental Table I). In WT and Cav3.1–/– mice, by 2 weeks following transverse aortic banding (TAB) treatment, the HW/BW ratio had increased significantly by 39.9% and 22.9%, respectively, relative to that in age-matched, sham-operated WT and Cav3.1–/– mice (P<0.001). In Cav3.2–/– mice, by contrast, the HW/BW ratio in the banded animals and their sham controls did not differ significantly (P=0.07) (Figure 1A). Measurement and comparison of another hypertrophy index, the HW/tibial length (in milligrams per millimeter) ratio, across groups yielded similar results (Figure 1B). We also measured the blood pressure of WT and Cav3.2–/– mice at both basal and 2 weeks after TAB. There is no significant difference in blood pressure between WT and Cav3.2–/– mice after TAB (109±3 for WT versus 109±2 mm Hg for Cav3.2–/–).
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Histological examination of hearts also showed a significant increase in ventricular wall thickness in the banded WT mice but less in the banded Cav3.2–/– mice (Figure 2A). One hallmark of cardiac hypertrophy is the enlargement of individual myocytes, and thus cardiomyocyte hypertrophy was also assessed at the cellular level. TAB treatment was found to lead to a significant increase in the cardiac myocyte cross-sectional diameter (in microns) by 23% in banded WT mice compared with sham-operated controls (P<0.001) (Figure 2B). In contrast, there was no significant change in the myocyte cross-sectional diameter from the banded Cav3.2–/– mice compared with the sham-operated controls (P=0.1). The progression to the cardiomyopathy is accompanied by increased fibrosis. Cav3.2–/– mice have been shown to display focal myocardial fibrosis caused by abnormal coronary function.31 Indeed, there was more fibrosis in sham-operated Cav3.2–/– mice than those in WT mice (Figure 2C). After TAB treatment, there was a marked increase in myocardial fibrosis in both WT and Cav3.2–/– mice, and no significant difference in myocardial fibrosis between WT and Cav3.2–/– mice was observed after TAB (Figure 2A and 2C).
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Because cardiac hypertrophy is frequently accompanied by a reexpression of the cardiac fetal genes, we examined the expression of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), -myosin heavy chain (-MHC), β-myosin heavy chain (β-MHC), and skeletal -actin (-SA) in the banded WT and Cav3.2–/– hearts using real-time quantitative PCR. As expected, ANF, BNP, β-MHC, and -SA were significantly upregulated in the banded WT heart, whereas -MHC was downregulated (Figure 2D). Notably, despite the attenuation of hypertrophy in the Cav3.2–/– heart, the expression of ANF, BNP, β-MHC, and -SA were also upregulated and the expression of -MHC was downregulated under these conditions (P<0.05), suggesting that Cav3.2 channels are not involved in the reexpression of these fetal genes during pathological hypertrophy.
We also performed serial echocardiography to characterize the change in cardiac structure and function after pressure overload. Left ventricle mass (LVM) and the LVM/BW ratio had increased significantly in the banded WT and Cav3.1–/– mice at 2 and 4 weeks after banding relative to animals at basal condition. In Cav3.2–/– mice, however, LVM and the LVM/BW ratio did not increase at all in 2-week banded Cav3.2–/– mice, although a modest but significant increase in LVM/BW was observed at 4 weeks after banding (Figure 3A and 3B and supplemental Table I). The hypertrophic response in banded Cav3.2–/– mice was markedly reduced compared to that in banded WT animals for the same period, whereas hypertrophic response in banded Cav3.1–/– mice was comparable to that in banded WT animals (Figure 3B). Cardiac function, as indicated by the percentage of fractional shortening, did not show significant difference in WT at basal or after 2- and 4-week TAB, nor did it show a difference in Cav3.2–/– or Cav3.1–/– mice. Fractional shortening was comparable among WT, Cav3.2–/–, and Cav3.1–/– mice when subjected to the same period of TAB (Figure 3C).
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Compromised Ang II–Induced Cardiac Hypertrophy in Cav3.2–/– Mice
Pressure overload–induced cardiac hypertrophy is mediated in part via the mechanical stress–dependent and neuroendocrine factor–dependent pathways, such as those mediated by Ang II and endothelin 1.1,5 To test whether Ang II can induce cardiac hypertrophy in Cav3.2–/– mice, Ang II was continuously infused into WT and Cav3.2–/– mice for 2 weeks. Ang II infusion markedly increased systemic blood pressure in both WT and Cav3.2–/– mice, suggesting that Cav3.2–/– mice, although having a compromised vascular function, remain functionally intact in response to Ang II–induced hypertension (Figure 4A). As in the case of the TAB results, Ang II treatment led to a significant increase in the LVM/BW in WT but not in Cav3.2–/– mice (Figure 4B). After 2 weeks of Ang II treatment, the ratio of LVM/BW increased significantly by 33.6% in WT mice (P<0.01), whereas the ratio did not change significantly in Cav3.2–/– mice.
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To determine whether the reduced cardiac hypertrophy observed in Cav3.2–/– mice was attributable to the lack of Cav3.2 channels in cardiac myocytes, we isolated and cultured neonatal cardiac myocytes from WT and Cav3.2–/– mice and tested their hypertrophic responses to Ang II in vitro. Ang II significantly increased the surface area of only the WT cardiac myocytes (by 17.8%, P<0.001) but not that of the Cav3.2–/– cardiac myocytes (Figure 5A and 5B). RT-PCR analysis indicated that neonatal WT mouse myocytes express all 3 Cav3 channel isoforms (Figure 5C). On Ang II stimulation, only Cav3.2 mRNA but not Cav3.1 or Cav3.3 mRNA was markedly increased in neonatal WT myocytes (Figure 5C and 5D), whereas the level of Cav3.1 and Cav3.3 mRNA expression remained unchanged in neonatal Cav3.2–/– myocytes. Our in vivo and in vitro studies suggest the activation of Cav3.2 channels is important to the development of cardiac hypertrophy.
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Inhibition of T-type Ca2+ Channels Blunts Cardiac Hypertrophy
Because pressure overload– and Ang II–induced cardiac hypertrophy was blunted in Cav3.2–/– mice, we tested whether cardiac hypertrophy could be inhibited by blocking T-channels. Because no specific T-channel blocker is available commercially, we chose ethosuximide, which has been used to block T-channels in vitro and in vivo,32–34 to test our hypothesis. WT mice were subjected to TAB and concurrently infused with ethosuximide for 2 weeks. As shown in Figure 6A, TAB-induced cardiac hypertrophy (as assessed by the LVM/BW ratio) was significantly attenuated in the ethosuximide-treated groups by 15.6% (P<0.01), when compared to the corresponding vehicle-treated group. We also found that ethosuximide can reduce Ang II–mediated increase of LVM/BW ratio by 19% (P<0.05) (Figure 6B). These data demonstrate that blocking T-channels in WT mice can reduce cardiac hypertrophy induced by either pressure overload or treatment with Ang II.
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Electrophysiological Examinations of Ca2+ Currents From Acutely Isolated Left Ventricular Myocytes
Reappearance of the T-current has been observed in hypertrophied ventricular cells in the association with pressure overload in feline16 and rat.18,19 Using the whole-cell patch-clamp technique, we examined whether T-type Ca2+ current (T-current) reappeared in the left ventricular myocytes from 2-week TAB WT mice. Small but detectable T-currents were recorded in WT myocytes after TAB, whereas they are not detectable or very small in WT myocytes following sham operation (Figure 7) (at –40 mV, –0.64±0.17 pA/pF for TAB [n=5] and –0.11±0.02 pA/pF for sham [n=12]; P<0.05). The T-current recorded in WT myocytes after TAB was peaked at approximately –40 mV, and bath application of 100 µmol/L Ni2+ caused a rapid reduction of T-current amplitude to 
75% of control, indicating relatively high sensitivity of the current to Ni2+ (Figure 7B, inset). The L-type Ca2+ current (L-current) density was similar in sham-operated WT myocytes compared with sham-operated Cav3.2–/– myocytes (peak inward at 10 mV, –7.83±0.49 pA/pF for WT sham [n=14] and –8.09±0.48 pA/pF for knockout sham [n=15]). After TAB, there is no significant change in the L-current density in both WT and Cav3.2–/– myocytes (peak inward at 10 mV, –8.23±0.52 pA/pF for WT TAB [n=15] and –7.98±0.41 pA/pF for knockout TAB [n=15]) (Figure 7C). It has been reported that the reduction of the L-current by continuous infusion of L-channel blocker35 or the knockdown of L-channel accessory β subunit36 alleviates hypertrophic response. Our data rule out the possibility that blunted hypertrophy response in banded Cav3.2–/– mice results from the downregulation of L-type Ca2+ currents.
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Calcineurin–NFAT Signaling Is Not Activated in Cav3.2–/– Mice After Pressure Overload
The calcineurin–NFAT signaling pathway is important in regulating the development of cardiac hypertrophy and the associated changes in gene expression. It is thus possible that the reexpression of Cav3.2 channels can activate the calcineurin–NFAT signaling pathway during cardiac hypertrophy. To test this possibility, we first examined whether the expression of Cav3.2 could enhance the NFAT–luciferase activity in HEK293 cells. It has been shown that increased extracellular Ca2+ concentration to 10 mmol/L could induce Ca2+ influx via the Cav3.2 T-channels that open at the resting membrane potential of a HEK293 cell (ie, window current).37,38 As shown in Figure 8A, 10 mmol/L Ca2+ significantly increased the NFAT–luciferase reporter activity relative to that of cells transfected with control vehicle and T-channel blocker mibefradil effectively blocked NFAT–luciferase activity triggered by calcium. We further investigated whether calcineurin–NFAT signaling is intact in the Cav3.2–/– heart by crossing the Cav3.2–/– mice with a NFAT–luciferase (Luc) reporter transgenic mice.39 The basal level of NFAT-Luc reporter activity in the left ventricle was not significantly different between WT/NFAT-Luc and Cav3.2–/–/NFAT-Luc mice (Figure 8B). As expected, cardiac NFAT-Luc reporter activity increased significantly by 2.5-fold (P<0.001) 2 weeks after TAB in the WT/NFAT-Luc mice. In contrast, there was no significant change in luciferase reporter activity 2 weeks after TAB in the Cav3.2–/–/NFAT-Luc mice. We also measured the protein levels of total calcineurin A from the left ventricles of WT/NFAT-Luc and Cav3.2–/–/NFAT-Luc mice and found no difference (Figure 8C). These data suggest that Cav3.2 channels are important in the activation of calcineurin–NFAT signaling and that the lack of NFAT–luciferase activity in the Cav3.2–/–/NFAT-Luc mice is not attributable to a secondary change in the level of calcineurin A protein. To determine whether there could be any interaction between Cav3.2 T-channels and calcineurin, we performed coimmunoprecipitation experiments in vitro. We coexpressed vectors containing FLAG-Cav3.2 or FLAG-Cav3.1 and calcineurin in HEK293 cells. As shown in Figure 8D, calcineurin can be coimmunoprecipitated with FLAG-Cav3.2. In contrast, there is no detectable calcineurin in the FLAG vector control and a much weaker band of calcineurin coimmunoprecipitated with FLAG-Cav3.1 despite a larger amount of FLAG-Cav3.1 in the input lysates. The results suggest that the Cav3.2 T-channels can interact with calcineurin in HEK293 cells in vitro and could play a role in the hypertrophic signaling.
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Discussion |
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Although T-type Ca2+ channels have been previously implicated in the growth of cardiac tissues and in the pathogenesis associated with Ca2+ overload and cardiomyopathy-related arrhythmias,16–23 little is known about the mechanism whereby these channels act. Using Cav3.2–/– and Cav3.1–/– mice, we were able to demonstrate that Cav3.2 T-channels are required for hypertrophic response to mechanical stress and Ang II infusion.
Upregulation of Cav3.1 mRNA was observed in hypertrophied ventricles and failure-stage hearts in rats.19,22 Paradoxically, in the hypertrophied rat hearts, the increased T-type calcium currents from left ventricular myocytes showed relatively high nickel sensitivity, a feature peculiar to Cav3.2 channel–generated currents.18 A recent study reported that, in the ventricular septum of the aortic banded mouse heart, Cav3.1 mRNA expression decreased, whereas Cav3.2 mRNA expression was comparable to that in sham-operated control hearts.40 These conflicting results may reflect the different genetic backgrounds of experimental animals and the surgical protocols whereby cardiac hypertrophy was induced. Nevertheless, these studies failed to clarify whether the reexpression of T-channels is the consequence of cardiac remodeling in pathological states or whether the reexpressed T-channels, instead, initiate cardiac remodeling. Our demonstrations that nickel sensitive T-currents reappear in WT mice after TAB and T-channel blocker ethosuximide can blunt the development of cardiac hypertrophy induced by pressure overload or Ang II in WT animals suggest that Cav3.2 T-channels are indeed important in the initiation of pressure overload– and Ang II–induced cardiac hypertrophy in mice.
Pathological cardiac hypertrophy is commonly accompanied by myocyte enlargement, activation of a fetal program of cardiac gene expression, and fibrosis.5 Our findings that banded Cav3.2 –/– mice were resistant to cardiac hypertrophy but not fetal gene activation and fibrosis suggest that Cav3.2 is not necessary for these pathological features during cardiac remodeling on stress. There are several reports showing that reexpression of cardiac fetal genes is not associated with cardiac hypertrophy but rather with the pathological features. For example, SCN5A heterozygous mice show upregulation of β-MHC and -SA with no hypertrophy.41 Increased level of ANF expression is associated with tissue pathology but not necessary to the degree of cardiac hypertrophy.42 Intriguingly in a genetic mouse model with a yellow fluorescent protein–β-MHC fusion gene, reexpression of β-MHC has been found to occur predominantly in myocytes associated with regions with perivascular and interstitial fibrosis during hypertrophy.43
The Ca2+/calmodulin serine/threonine protein phosphatase calcineurin plays a central role in the development of pathological cardiac hypertrophy. Our studies show that cardiac hypertrophy responses are impaired in Cav3.2–/– mice, NFAT activity in these mice is blunted following 2-week TAB, and the T-currents reexpress in myocytes from banded WT mice, implying Cav3.2 T-channels are also involved in the activation of calcineurin–NFAT signaling and essential for pathological cardiac hypertrophy. Although a proximal relationship among calcineurin–NFAT, Ca2+ handling, and downstream effectors remains largely unclear, our coimmunoprecipitation work shows that the Cav3.2 T-channels could associate with calcineurin and thus play a role in the hypertrophic signaling. Several recent studies have demonstrated that TRPC1, TRPC3, and TRPC6 are involved in calcineurin/NFAT activation and cardiac hypertrophy in rodent models.8–11 TRPC6 and TRPC3 transgenic mice exhibit cardiomyopathy associated with increased NFAT activity and with an increased susceptibility to stimulation by pressure overload.8,11 Furthermore, the promoter region of TRPC6 contains 2 NFAT binding sites, which suggests that positive feedback is involved in the regulation of the calcineurin–NFAT–TRPC6 signaling pathway.11 We hypothesized that reexpressed Cav3.2 might induce cardiac hypertrophy by activating the calcineurin–NFAT pathway. Indeed, our analysis of mice harboring a transgenic NFAT-Luc reporter showed that the activation of NFAT is blunted in the Cav3.2–/– left ventricle following pressure overload. The notion is also upheld by the recent in vitro study showing that T-type Ca2+ blockers, kurtoxin and efonidipine, could prevent bovine serum–induced neonatal mouse cardiomyocyte hypertrophy through an inhibition of calcineurin–NFAT activation.44 Interestingly, TRPC6 transcript levels have been reported to increase 3 weeks after TAB.11 Moreover, following aortic banding in the rat, there is a 2-day delay in the association of calcineurin with calmodulin,45 which coincides with the time point at which cardiac NFAT-Luc reporter activity is upregulated in mice.39 This result suggests that calcineurin is activated as early as 2 days after stimulation. It is possible that hypertrophic stimuli lead to rapid activation of Cav3.2 channels, which then activate calcineurin–NFAT signaling at the initial stage of hypertrophic development, and that this, in turn, induces the sustained hypertrophic responses that are associated with TRPC channel upregulation.
In summary, in using a molecular genetic approach, we have demonstrated that Cav3.2 plays an important role in the development of cardiac hypertrophy induced by aortic banding and Ang II infusion and that Cav3.2 is required for activation of the calcineurin/NFAT pathway. The next steps in better understanding the development of cardiac hypertrophy include further evaluating the role of Cav3.2 inhibition in other genetic and acquired models of cardiac hypertrophy and identifying any Cav3.2-associated molecules that are involved in the regulation of cardiac hypertrophy.
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Acknowledgments |
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We thank Dr Jeffery D. Molkentin (University of Cincinnati) for kindly providing us the NFAT-Luc reporter mice. We are grateful to the Pathology Core at the Institute of Biomedical Sciences, Academia Sinica, Taiwan for technical assistance.
Sources of Funding
C.-C.C. was supported by grants from the Academia Sinica (AS95IBMS6) and the National Science Council, Taiwan (95-2320-B-001-030, 95-2320-B-001-036). C.-S.C. was a recipient of postdoctoral fellowship from Academia Sinica, Taiwan.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to the study.
Original received August 3, 2008; revision received December 17, 2008; accepted December 18, 2008.
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