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Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca2+) is implicated in hypertrophic signaling pathways, the specific role of Ca2+ influx through the L-type Ca2+ channel (ICa-L) has been controversial and is the topic of this study. To determine if and how sustained increases in ICa-L induces PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the β2a subunit of Ca2+ channels (β2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a β2a-GFP.
In vivo, β2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume and cross sectional area and the expression of PCH markers in a time- and dose- dependent manner. PCH was associated with a hypercontractile phenotype including enhanced ICa-L, fractional shortening, peak Ca2+ transient, at the myocyte level, greater ejection fraction and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of β2a in cultured AFVMs increased ICa-L, cell volume, protein synthesis, NFAT and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of ICa-L, intracellular Ca2+, calcineurin, CaMK II and SERCA.
Increasing ICa-L is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca2+ pools were shown to be involved.
Pathological cardiac hypertrophy (PCH) is an independent risk factor for myocardial infarction, arrhythmia, and subsequent heart failure . It occurs in response to hemodynamic stress such as hypertension, myocardial infarction (MI) and vavular diseases . Pathological cardiovascular stress increases the contractility demands of the heart and its resident myocytes, which is achieved by activating the sympathetic nervous system . Sympathetic neurohormones activate protein kinas A (PKA) to increase Ca2+ influx, SR Ca2+ uptake, storage, and release to increase the amplitude of the systolic Ca2+ transients and contractility . Persistent activation of these signaling pathways also activates Ca2+/calmodulin dependent kinases (CaMK) which is associated with PCH .
Ca2+ regulates many hypertrophic pathways and well known examples are the Ca2+-regulated calcineurin/NFAT and CaMK/HDAC pathways . However, the proximal source of Ca2+ that induces PCH is still not well understood. Ca2+ influxes through the Cav1.2/L-type Ca2+ channels (ICa-L) [5–7], Cav3.2/α1H T-type Ca2+ channels , and transient receptor potential channels (TRPC)  have all been proposed to contribute to the pool of Ca2+ that activates hypertrophic pathways. In cardiac myocytes, ICa-L is the major Ca2+ influx and under physiological condition, ICa-L does not activate PCH. Under pathological conditions, activated neurohumoral systems increase ICa-L which is a likely source of Ca2+ to regulate hypertrophic signaling in vivo. This idea is supported by those studies that have shown a necessary role of enhanced ICa-L for the myocyte hypertrophy induced by phenylephrine (PE) , endothelin-1 (ET-1) , isoproterenol , angiotensin II , elevated extracellular KCl  and stretch . ICa-L is also able to activate key hypertrophic signaling molecules such as PKC  in cardiomyocytes. Cav1.2 channel blockers have been shown to reduce cardiac hypertrophy [6, 16] but the exact mechanism is not clear. More recently, it has been shown that reducing the expression of the Cavβ gene decreases ICa-L and blunts hypertrophy induced by transverse aortic constriction (TAC) in adult rats . We have also shown that Cavβ2a overexpression leads to cardiac hypertrophy at the age of 4 months when heart failure phenotype is present in the HE mice . Other Ca2+ influx pathways also seem to be a source of hypertrophic Ca2+, since the loss of Cav3.2/α1H  or TRPCs  blunts cardiac hypertrophy induced by TAC. Therefore, different routes of Ca2+ influx may synergically serve as the source for myocyte hypertrophy . The fact that Cav3.1/α1G overexpression in the mice is antihypertrophic rather than prohypertrophic show the complex nature of Ca2+ mediated induction of PCH.
We used transgenic mice with cardiac specific overexpression of the β2asubunit of the L-type Ca2+ channel and cultured adult feline ventricular myocytes (AFVM) and neonatal rat ventricular myocytes (NRVM) with enhanced ICa-L by overexpressing the β2a subunit to: (1), determine whether increased ICa-L was sufficient to induce myocyte hypertrophy; (2) test if enhanced ICa-L could exacerbate PCH induced by TAC; and (3) determine the signaling cascades for myocyte hypertrophy induced by enhanced ICa-L. Our results show that increasing ICa-L is sufficient to induce myocyte hypertrophy by activation of the calcineurin/NFAT and CaMK II/HDAC signaling pathways. Both cytosolic and SR/ER-nuclear envelop Ca2+ pools were shown to be involved.
Cardiac myocytes specific (α-MHC promoter) with inducible (tetracycline-activator (tTA)) β2a mouse lines with high (HE) and low expression (LE) levels were established [17, 20]. β2a increases the open probability and membrane trafficking of the pore-forming Cav1.2α1c subunit. Mice with both β2a and tTA transgenes (double transgenic, DTG) and off doxycycline (DOX, a derivative of tetracycline) were used as the experimental group and mice with single transgene (STG) or no transgene (wild-type, WT) were used as controls (Ctr). Since our previous study has shown that HE mice develop heart failure with associated hypertrophy at the age of 4 months (4m), we used 3-month (3m) old Cavβ2a HE mice to avoid the possibility that PCH in HE was secondary to heart failure. LE mice were used at the age of 4 months. The controls for HE and LE were 3-month old FVB and 4-month old FVB mice, respectively. Our preliminary studies showed that there was no difference in most of the measured parameters between 3m old and 4m old FVB control mice and thus those measurements in these two age groups of controls were pooled. The investigation conformed to the NIH Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee at Temple University.
To quantitate the expression and phosphorylation of the major Ca2+ handling proteins in the animal hearts or cultured myocytes, standard Western blot procedures were performed with antibodies against GAPDH, phospholamban (PLB), phosphorylated PLB at ser16 (pSer16-PLB), phosphorylated PLB at threonine17 (pThr17 PLB), Na+/Ca2+ exchange 1 (NCX1), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), Cav1.2α1c, and ryanodine receptor type 2 (RyR2). The antibodies were purchased from Millipore (PLB, α1c and NCX1), Badrilla Ltd. (pSer16PLB and pThr17 PLB), and Sigma (SERCA2a and GAPDH), respectively. Immunoblots were visualized with a chemiluminescent reagent (Lumigen PS-3, GE Healthcare UK Ltd., UK) and a Fujifilm LAS-4000 imaging system (Fujifilm Life Science USA). The target proteins were then analyzed with the Multi Gauge software (Fujifilm Life Science USA). The amount of the proteins were normalized to the internal control, GAPDH. The phosphorylation levels of PLB were evaluated by normalizing the phosphorylated PLB to the total PLB amount.
Normal adult feline VMs (AFVMs) , neonatal rat VMs (NRVMs)  and adult mouse VMs  were isolated as described previously. Myocytes were cultured and infected with adenoviruses (AdGFP or Adβ2a or AdHDAC and AdNFAT) at the desired multiplicity of infection (MOI) as described previously .
The increases in the cell volume, protein/DNA ratio and cell surface area are features of myocyte hypertrophy. The volumes of freshly isolated mouse VMs and detached AFVMs that were infected with adenoviruses for 4 days were measured with a Beckman Z2 Coulter Counter . The protein and DNA were isolated from the same sample with Trizol reagent (Invitrogen) and determined with the Biorad RC DC Protein Assay Kit II (Biorad) and a UV spectrometer, respectively. NRVMs were stained with Rodamine-philloidin (Sigma-Aldrich) for measuring surface area and for sarcomere organization examination.
Echocardiography (ECHO) was performed with a VisualSonics Vevo 770 machine. Mice were anesthetized with 2% isoflurane initially and then 1% during the ECHO procedure. Hearts were viewed in the short-axis between the two papillary muscles and analyzed in M-mode.
HW/BW ratios of LE (4m) and HE (3m) mice were measured. Standard histology techniques were used. Five slides from the middle portion of each heart were stained for FITC-labeled lectin (Sigma-Aldrich) and DAPI (Invitrogen) and then imaged. The cross sectional areas of 50 myocytes with round shape and clear nuclei were measured for each slide. To reveal collagen deposition (fibrosis), Mason’s trichrome staining was done with tissue sections and fibrotic area was quantitated with ImageJ.
Total mRNA was extracted from snap-frozen ventricular tissues using Trizol reagent and quantitated by a UV spectrometer. Real-time PCR was done with the SYBR Green Real Time PCR kit (Applied Biosystems, Carlsbad, CA) and an Eppendorff Mastercycler RT-PCR machine. ANF and β-myosin heavy chain (β-MHC) mRNAs were measured with GAPDH as the internal control. The primers were (5′ to 3′): ANF: forward: tgccggtagaagatgaggtc and reverse: tgcttttcaagagggcagat; β-MHC: forward: acaagctgcagctgaaggtgaa and reverse: aagagctactcctcattcaggccctt; GAPDH: forward: tgcaccaccaactgcttag and reverse: gatgcagggatgatgttc.
β2a transgenic mice were bred with transgenic mice with luciferase reporter gene under the control of NFAT promoter.
Cavβ2a LE mice at the age of 6 weeks were used for TAC  to avoid any effects of a basal phenotype. Briefly, the transverse aortic arch was visualized through a median sternotomy and 7-0 silk ligation was tied around the aorta with a 26-gauge needle between the right brachiocephalic and left common carotid arteries with subsequent removal of the needle to make the constriction. Hearts were studied 4 weeks following the operation.
A nonspecific caspase inhibitor, z-VAD-fmk (10μM, BD Biosciences) was used to inhibit myocyte apoptosis when Adβ2a at high MOIs (50 and 100) was used. Inhibitors of Cav1.2 (Nifedipine, 13μM), CaMK II (KN-93 1μM, Sigma), calcineurin (FK 506 (1μM) and Cyclosporine A (CsA, 5μM), Sigma) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (Thapsigargin, TSG, 10nM) were also used.
Data in the text are reported as mean±SEM. When appropriate, paired and unpaired T-test, ANOVA or ANOVA for repeated measures were used to detect significance with SAS 9.0 (SAS Institute Inc.). P values of ≤0.05 were considered significant. “n” is the number of cells examined and “N” is the number of cell cultures or the number of animals used.
To determine if persistent increases in ICa-L can induce PCH in vivo, we established DTG lines with low (LE, 3.1 fold) and high (HE, 7.4 fold) β2a expression . Previously we found that the expression of β2a in our DTG mice is stable at about 4m in LE and at 3m in HE mice, when animals were off DOX at weaning (3 weeks) . ICa-L, myocyte fractional shortening, and Ca2+ transients were measured in myocytes isolated from 4m LE or 3m HE mice. ICa-L density was greater in β2a-DTG VMs than in controls and was graded with β2a expression (Figure 1A & B). Fractional shortening and Ca2+ transient amplitudes (at 0.5Hz) were significantly greater in DTG-VMs, indicating enhanced Ca2+ handling (Figure 1C, D & E), due to increased Ca2+ influx and resultant increases in SR Ca2+ content (Figure 1D) that was the highest in the HE myocytes. The diastolic Ca2+ was not significantly different between control and DTG myocytes (0.5Hz). Western analysis was used to quantitate the expression of Ca2+ handing proteins: total PLB, pSer16 PLB, pThr17 PLB, SERCA2, RyR2, NCX and α1c. No difference in the expression of these proteins was observed in 3-month and 4- month control mice and thus all the control data were pooled (Figure 1F and G). No change of the expression of total PLB, pSer16 PLB, NCX, and α1c was found in β2a-DTG LE and HE mice comparing to control mice. The expression of SERCA was increased in HE hearts and pThr17 PLB was increased in both LE and HE hearts, showing that the hypercontractility in LE and HE myocytes is associated with activated CaMKII. The expression of RyR2, was significantly decreased in both LE and HE hearts (Figure 1E and F). These data indicate that there are compensatory changes in some but not all Ca2+ handling proteins in LE and HE hearts with Cavβ2a induced increases in Ca2+ influx.
Ejection fraction (EF) and fractional shortening (FS) were not different at 2m between the three groups but were increased in the HE mice at 3m and in LE mice at 4m (Figure 2B). HE animals developed depressed cardiac EF and FS at 4m which continued to decline at 6m. In the LE mice, EF and FS remained greater than controls at these ages (4m and 6m) (Figure 2C & D). The thicknesses of diastolic left ventricular posterior wall (LVPWd) and anterior/interventricular septal wall (IVSd) were not significantly different between the groups at 2m but increased in the LE mice and increased (3m) and then decreased (4–6m) in the HE mice (Figure 2E and F). The left ventricular internal diameter during diastole (LVIDd) was not different between the control and LE mice although it tended to be smaller in the LE mice consistent with concentric LV hypertrophy. Diastolic LVID in HE mice continued to increase during the age of 4–6m, indicating LV dilation (Figure 2G). In agreement with the changes in the wall thickness, the LV mass estimated by ECHO increased steadily in the LE and HE mice. The LV mass/BW was the highest in the HE (HE>LE>ctr). (Figure 2H and I).
HW/BW ratios were also directly measured in excised hearts at 3m in HE and 4m in LE mice (Figure 2). At these ages, HW/BW was significantly increased in LE and HE (12.8% and 19.3%) versus control mice (Figure 3B). Ventricular weight to body weight (VW/BW) was increased by 15.7% and 40.9% in LE and HE β2a hearts (Figure 3A & C). At the tissue level, fibrosis (light blue staining in Figure 3D) was observed in the hearts of DTG mice. The cross sectional areas of VMs in LE and HE were 26.9% and 79.6% larger than in controls, respectively (Figure 3E & F). Ventricular myocyte volume was greater in LE (18.7%) and HE (42.8%) versus control mice (Figure 3G). The mRNA expression of PCH markers, ANF and β-MHC, was not significantly different between control and LE mice but were significantly increased in HE mice at 3m. At 4m, mRNA expression of both genes was increased to similar extents in HE and LE mice (Figure 3H & I). NFAT activity was significantly increased in β2a hearts (Figure 3J). These results show a strong association between β2a induced increases in contractility and cardiac hypertrophy.
At the age of 6 weeks, control and LE mice were subjected to transverse aortic constriction (TAC). TAC induced a high mortality in HE (>60%) and these animals were not studied. The initial survival rates for control and LE mice were not different and were about 80~85%. After 4 weeks of TAC, LE mice had greater HW/BW ratio and greater increases in myocyte cross sectional area than controls (Figure 4A&B). LE mice had depressed cardiac pump function evidenced by decreased ejection fraction (Figure 4C) and increased lung weight to body weight ratio, an index of pulmonary edema (Figure 4D). Similar to isoproterenol infusion as we reported previously , TAC caused much more severe fibrosis in the LE mice after TAC (Figure 4E and F).
Myocyte hypertrophy in vivo is a complex process. To more directly test the role of β2a and ICa-L in myocyte hypertrophy, we employed in vitro AFVM and NRVM culture systems to study the relationship between increased ICa-L and myocyte hypertrophy.
Increasing the multiplicity of infections (MOIs, 0, 5, 100) of Adβ2a (AdGFP as the control) graded ICa-L in AFVMs at 48 hours post infection (Table 1). The amplitudes of unloaded contractions and Ca2+ transients were also enhanced with the increased β2a but diastolic Ca2+ was not changed. The SR Ca2+ content was significantly increased and was graded with β2a expression. These results show that the Ca2+ handling phenotype of β2a mouse myocytes is reproduced in AFVMs infected with Adβ2a.
We then tested if VMs infected with β2a were hypertrophied. Myocyte volume and protein synthesis (protein/DNA ratio) were compared in β2a- versus in GFP-AFVMs. GFP overexpression did not have a significant effect on AFVM volume but β2a increased AFVM volume in an Adβ2a MOI-dependent fashion (Figure 5A). The increases in myocyte volume induced by β2a overexpression were 10% to 20% (Figure 5B). Adβ2a infection of AFVM at an MOI of 5 caused protein/DNA ratio to increase by 33.8±7.0% (Figure 5C) compared to AdGFP. β2a infection of NRVMs (1123.5±30.1μm2) increased NRVM surface area (52.4%) more than AdGFP (737.0±23.9μm2) and induced more organized sarcomeres (Figure 5D). These data suggest that increases in Ca2+ influx through Cav1.2 induce cardiac myocyte hypertrophy.
We tested whether the pathways involving calcineurin (CaN)/NFAT3 and the CaMK II/HDAC5 were activated. AFVMs were co-infected with adenoviruses containing an NFATc4 (NFAT3)-GFP fusion gene (MOI=100) or an HDAC5-GFP (MOI=100) fusion gene and Adβ2a (MOI=5) or AdGFP (MOI=5). The GFP fluorescence from AdGFP or Adβ2a was weak at 48 hours post infection and did not interfere with the strong NFAT-GFP and HDAC-GFP fluorescence. Co-infection with AdNFAT-GFP and AdGFP resulted in strong fluorescence that was evenly distributed in the cytoplasm of AFVMs (Figure 6A) and some AFVMs with slightly green nuclei like in some of GFP-AFVMs, possibly due to baseline CaN activity. In AFVMs co-infected with both Adβ2a and AdNFAT3, the majority VMs had bright green nuclei (Figure 6B & C). These results show that the CaN/NFAT3 pathway is activated after increased Ca2+ influx.
HDAC, a repressor of hypertrophic signaling, is found in the nucleus under basal conditions and translocates into the cytosol when it is phosphorylated by CaMK II or other kinases. The % of AFVMs in which HDAC5 was translocated to the cytoplasm (% of nuclei without HDAC5) was significantly higher in β2a-AFVMs than in GFP-AFVMs (Figure 6D, E & F). Increased CaMK II activity may be responsible for the HDAC5 translocation from the nucleus in β2a infected myocytes, as indicated by the increased PLB phosphorylation at Thr17. (Figure 6G).
Treatments of β2a -AFVMs with a Cav1.2 blocker (nifedipine, 10μM), an intracellular Ca2+ buffer (BAPTA-AM, 1μM), CaN inhibitors (CsA, 5μM and FK 506, 1μM), and a CaMK II inhibitor (KN93, 1μM), all prevented β2a-induced increases in myocyte volume (Figure 7A), protein/DNA ratio (Figure 7B) and β2a-induced NFAT translocation (Figure 7C). Similarly, inhibition of CaMK II with KN93 abolished the HDAC5 translocation induced by β2a (Figure 7D). These results suggest that the myocyte hypertrophy observed in β2a-myocytes is mediated by increases in Ca2+ influx and subsequent activation of CaN/NFAT and CaMK II/HDAC signaling pathways.
Phenylephrine (PE), a hypertrophic agonist, increased myocyte volume, NFAT and HDAC translocation in AFVMs (Figure 7) infected with both Adβ2a and AdGFP. However, phenylephrine did not further increase these hypertrophic parameters in β2a-AFVMs.
SR Ca2+ may be involved in myocyte hypertrophy by providing local release of Ca2+ from the perinuclear envelope into the nucleus to induce HDAC translocation  and/or by releasing Ca2+ into the cytoplasm . Inhibiting SERCA with thapsigargin (TSG) significantly increased diastolic Ca2+ and reduced SR Ca2+ content in both GFP and β2a-VMs (Table 1). TSG also abolished Ca2+ transients in cultured myocytes. It also blocked β2a-induced myocyte hypertrophy (Figure 7A and B) and the translocation of HDAC from the nucleus to the cytoplasm (Figure 7D). However, TSG did not block NFAT translocation in β2a-AFVMs (Figure 7C) and in GFP-AFVMs TSG promoted NFAT nuclear translocation without inducing hypertrophy. These data suggest that SR Ca2+ is necessary for β2a-mediated HDAC translocation and hypertrophy but not necessary for NFAT translocation.
Increases in myocyte [Ca2+] induce PCH [8, 13, 25], but the source of the hypertrophic [Ca2+] are still not clearly defined. The conundrum is that myocyte cytosolic [Ca2+] fluctuates over a wide range during each normal heart beat, and can be increased with physiological stimuli such as exercise and pregnancy without inducing PCH . In the present study we tested the idea that persistent increases in ICa-L, the primary Ca2+ influx pathway in the heart, are sufficient to cause PCH. Our experiments showed that persistent increases in Ca2+ influx caused enhanced contractions and Ca2+ transients and these changes were associated with myocyte hypertrophy, both in-vitro and in-vivo, indicating a direct effect of increased Ca2+ influx on myocyte hypertrophy. We also showed that both NFAT and HDAC nuclear translocation were necessary for LTCC-dependent hypertrophy. NFAT translocation is more dependent on the change of cytosolic Ca2+ while HDAC translocation is more related to the increase of SR-nuclear envelope Ca2+.
Previously, we have shown that Cavβ2a DTG mice develop cardiac hypertrophy associated cardiac arrhythmia  and premature death , fibrosis, blunted β-adrenergic responses and diastolic dysfunction when stressed . Here we show that in the DTG hearts the expression of markers of pathological hypertrophy, ANF and β-MHC, are increased. These features of Cavβ2a DTG hearts indicate that the hearts of Cavβ2a DTG mice are more likely to have pathological hypertrophy. However, whether Cavβ2a LE and HE hearts undergo decompensation is depending on the extent of SR and cytosolic Ca2+ overload because LE mouse hearts remain hypercontractile up to one year while HE mice develop heart failure after the age of 4 months. Furthermore, we are not clear whether the pathological hypertrophy was secondary to myocyte apoptosis or necrosis induced by mitochondrial Ca2+-overload.
Persistent changes in the amplitude and duration of the systolic [Ca2+] transient that are specific to pathological stress could activate induce pathological hypertrophy signaling. There is reasonable evidence for this hypothesis . Our results show that persistently increasing Ca2+ influx through Cav1.2 increases the Ca2+ transient amplitude and diastolic Ca2+ at high contracting rates and activate the signaling cascades to induce pathological hypertrophy. Consistent with our study, it has been shown that LTCC blockers eliminate NRVM hypertrophy induced by many neurohormones [9–13] and stretch . In vivo, there is also evidence that reducing the increase in Ca2+ influx through Cav1.2 by knockdown of the β2 subunit  or with Ca2+ channel blockers (benidipine)  after pressure overload reduces the ensuing hypertrophy. Whether increased Ca2+ influx through Cav1.2 induces cardiac hypertrophy by enhancing contractile Ca2+ transients or through a local domain (e.g., calveolae) is not yet clear.
Some studies suggest that the source of Ca2+ for activation of hypertrophic signaling is distinct from the Ca2+ that activates myofilaments . These sources of hypertrophic Ca2+ include Ca2+ entry through T-type Ca2+ channels (TTCC) , TRP channels [9, 27] or capacitive Ca2+ entry . Many years ago we postulated that the re-expression of TTCC in diseased hearts might play a role in hypertrophic signaling . A recent report addressing this hypothesis suggests that in pressure overload, hypertrophic Ca2+ enters myocytes exclusively through α1H TTCC . These studies suggest that hypertrophic Ca2+ entry is compartmentalized to activate PCH signaling independent of changes in bulk cytoplasmic [Ca2+]. However, not all enhanced Ca2+ influx is able to induce PCH. We have shown that enhanced Ca2+ influx through the Cav3.1 (α1G) TTCC does not induce PCH and actually antagonizes cardiac hypertrophy induced by TAC .
There is some evidence that the [Ca2+] released from the nuclear envelope, which is contiguous with the SR, through IP3 receptors activates the CaMK/HDAC pathway [24, 31] to activate hypertrophic signaling. Activation of IP3 receptors may be independent of the cytoplasmic [Ca2+] transient and may require activation of membrane receptors by prohypertrophic neurohormones such as angiotensin and endothelin . Our study shows that the β2a-VMs have increased SR Ca2+ content and suggests that this could be involved in induction of pathological hypertrophy. Future studies will need to resolve the role of microdomain signaling and Ca2+ loading of the SR-nuclear envelope in β2a-induced hypertrophy.
Hypertrophic pathways including CaN/NFAT, CaMKII/HDAC, PKC, and MAPK pathways are regulated by Ca2+ . We found that both the CaN/NFAT and CaMKII/HDAC pathways are activated when Ca2+ influx is increased and that inhibiting these two pathways prevented Ca2+-mediated myocyte hypertrophy in vitro. We also explored the potential involvement of these pathways in vivo by using NFAT activity reporter mice and measuring CaMK II activity. All studies suggest a critical role for CaN/NFAT and CaMKII/HDAC pathways signaling cascades in PCH induced by β2a.
Our data suggest that the SR could be a primary sensor of cell stress and changes in SR [Ca2+] are necessary for activation of both cytoplasmic and nuclear prohypertrophic signals. Persistent increases in Ca2+ influx will increase SR Ca2+ loading and release to provide the local cytoplasmic [Ca2+] required for activation of hypertrophic signaling. Since the nuclear envelope is contiguous with the SR, increasing SR Ca2+ may result in an increase in nuclear Ca2+ transients and thereafter activation of nuclear hypertrophic signaling. We used thapsigargin to inhibit SERCA and abolish SR Ca2+ uptake and subsequent SR Ca2+ release. This treatment blocked β2a-induced hypertrophy and HDAC translocation, but initiated NFAT nuclear translocation. These results suggest that elevated cytoplasmic [Ca2+] by thapsigargin activates CaN/NFAT signaling without activating nuclear CaMKII/HDAC signaling. However, we could not determine whether the blunted myocyte hypertrophy by TSG is due to the abolishment of Ca2+ transients or compartmented SR/nuclear envelope Ca2+ release. It is likely that the elimination of nuclear envelope Ca2+ release reduced CaMK II activation in the nucleus. These results also show that at least under our conditions, NFAT and HDAC translocation can be independently controlled and both molecules must be translocated for induction of PCH. Our results support a role of HDAC as a repressor of PCH and suggest it must be exported from the nucleus for NFAT to initiate hypertrophic responses. Our study does leave open the possibility that Cav1.2 directly activates calcineurin closely associated with Cav1.2 to induce NFAT translocation  and TSG activates other signaling pathways such as ER stress signaling that may blunt myocyte hypertrophy.
The role of Ca2+ influx through Cav1.2 in the induction of PCH in human diseases such as chronic hypertension or after myocardial infarction is unclear. In both of these diseases, systolic wall stress and cardiac myocyte contractility are augmented by enhancing Ca2+ influx and subsequent Ca2+ handling. The surviving myocardium after myocardial infarction is hypercontractile , in large part due to the activated sympathetic system . In addition, β2a expression has been reported to be increased in failing human hearts . These studies suggest that increases in Ca2+ entry may be a proximal cause of the PCH in humans. This is difficult to prove because LTCC antagonists usually affect the vasculature and systolic blood pressure before cardiac effects are observed . However, there are many clinical scenarios in which Ca2+ channel antagonists are used and regression of cardiac hypertrophy is induced .
Our studies suggest that excessive Ca2+ influx through Cav1.2 is a proximal source of Ca2+ for pathological hypertrophy.
Calcium (Ca) is a necessary signaling molecule for activating hypertrophic responses. However the source(s) of the proximal Ca is not clear yet. This study aims to explore whether persistently enhanced Ca influx through the L-type Ca channel (Cav1.2), a situation happening when the heart is stressed, is sufficient to induce pathological cardiac hypertrophy and what is the underlying mechanism. The highlights in this study include:
This work was supported by grants from the NIH (HL089312 to SRH and HL088243 to XC); HHMI (to JDM) and American Heart Association (AHA0730347N to XC).
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