Calcium influx through Cav1.2 is a proximal signal for pathological cardiomyocyte hypertrophy

Abstract

Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca(2+)) is implicated in hypertrophic signaling pathways, the specific role of Ca(2+) influx through the L-type Ca(2+) channel (I(Ca-L)) has been controversial and is the topic of this study. To determine if and how sustained increases in I(Ca-L) induce PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the β2a subunit of Ca(2+) channels (β2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a β2a-GFP were used. 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 I(Ca-L), fractional shortening, peak Ca(2+) 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 I(Ca-L), cell volume, protein synthesis, NFAT, and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of I(Ca-L), intracellular Ca(2+), calcineurin, CaMKII, and SERCA. In conclusion, increasing I(Ca-L) is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca(2+) pools were shown to be involved.

Figure 1. Enhanced Ca²⁺ handling in ventricular myocytes isolated from β2a LE and HE mice

A, examples of I_Ca-L. B, averaged I–V relationships of I_Ca-L in VMs from control (Ctr), LE and HE mice. The maximal I_Ca-L in Ctr, LE and HE myocytes were −12.77±0.84, −23.30±2.16, and −33.09±4.01pA/pF, respectively. C, Example of Ca²⁺ transients recorded with indo-1 in VMs from Ctr, LE and HE mice (0.5Hz). D, There was no difference in diastolic Ca²⁺ in control (0.63±0.02), LE (0.65±0.03) and HE (0.62±0.03) VMs but increased amplitudes of systolic Ca²⁺ in DTG VMs (ctr: 0.72±0.02; LE: 0.81±0.05; HE: 0.96±0.04). E, fractional shortening was enhanced β2a (Ctr: 5.0±0.6%; LE: 8.2±1.1%; HE: 9.6±0.9%). F, Western blots of total PLB, pSer16 PLB, pThr17 PLB, NCX, SERCA, α1c, RyR and GAPDH (internal control) in control, LE and HE heaerts. G, Averaged normalized protein expression levels. In B: @: p<0.01 vs. Ctr; #: p<0.05 vs. LE; $: p<0.05 vs. Ctr; &: p<0.01 vs. Ctr. In D & E: ^*: p<0.05; **: p<0.01. “n” is the number of cells from at least 3 animals.

Figure 1. Enhanced Ca²⁺ handling in ventricular myocytes isolated from β2a LE and HE mice

A, examples of I_Ca-L. B, averaged I–V relationships of I_Ca-L in VMs from control (Ctr), LE and HE mice. The maximal I_Ca-L in Ctr, LE and HE myocytes were −12.77±0.84, −23.30±2.16, and −33.09±4.01pA/pF, respectively. C, Example of Ca²⁺ transients recorded with indo-1 in VMs from Ctr, LE and HE mice (0.5Hz). D, There was no difference in diastolic Ca²⁺ in control (0.63±0.02), LE (0.65±0.03) and HE (0.62±0.03) VMs but increased amplitudes of systolic Ca²⁺ in DTG VMs (ctr: 0.72±0.02; LE: 0.81±0.05; HE: 0.96±0.04). E, fractional shortening was enhanced β2a (Ctr: 5.0±0.6%; LE: 8.2±1.1%; HE: 9.6±0.9%). F, Western blots of total PLB, pSer16 PLB, pThr17 PLB, NCX, SERCA, α1c, RyR and GAPDH (internal control) in control, LE and HE heaerts. G, Averaged normalized protein expression levels. In B: @: p<0.01 vs. Ctr; #: p<0.05 vs. LE; $: p<0.05 vs. Ctr; &: p<0.01 vs. Ctr. In D & E: ^*: p<0.05; **: p<0.01. “n” is the number of cells from at least 3 animals.

Figure 2. Cardiac hypertrophy in DTG mice

A, Examples of M-mode echocardiography images of Ctr (4-month old), LE (4-month old) and HE (3-month old) mice. B, Heart rates were not different between groups when cardiac function was evaluated by echocardiography. C & D, Cardiac EF and FS in Ctr, β2a LE and HE mice at 2m–6m. E & F, Diastolic IVS and LVPW thickness were increased in both LE and HE mice at 3m but decreased in HE mice at 4m and 6m. IVSd and LVPWd in LE mice remained greater at 4m and 6m. G, Diastolic LVID was increased in HE mice at 4m and 6m. H, LV masses in LE and HE mice kept increasing from 3m to 6m. I, No significant differences in BW were detected between groups. J, LV Mass to body weight ratios were increased in LE and HE mice after 3m. # P<0.05 between LE and Ctr; $ P<0.05 between HE and Ctr; & P<0.01 HE vs. LE; @ P<0.001 Ctr vs. LE; % P<0.001 Ctr vs. HE; ! P<0.001 LE vs. HE; * P<0.001 LE vs. HE.

Figure 2. Cardiac hypertrophy in DTG mice

A, Examples of M-mode echocardiography images of Ctr (4-month old), LE (4-month old) and HE (3-month old) mice. B, Heart rates were not different between groups when cardiac function was evaluated by echocardiography. C & D, Cardiac EF and FS in Ctr, β2a LE and HE mice at 2m–6m. E & F, Diastolic IVS and LVPW thickness were increased in both LE and HE mice at 3m but decreased in HE mice at 4m and 6m. IVSd and LVPWd in LE mice remained greater at 4m and 6m. G, Diastolic LVID was increased in HE mice at 4m and 6m. H, LV masses in LE and HE mice kept increasing from 3m to 6m. I, No significant differences in BW were detected between groups. J, LV Mass to body weight ratios were increased in LE and HE mice after 3m. # P<0.05 between LE and Ctr; $ P<0.05 between HE and Ctr; & P<0.01 HE vs. LE; @ P<0.001 Ctr vs. LE; % P<0.001 Ctr vs. HE; ! P<0.001 LE vs. HE; * P<0.001 LE vs. HE.

Figure 3. HE and LE mice develops PCH

A, Micrographs of Ctr (4 month), LE (4m) and HE (3m) hearts. The smallest division on the scale bar is 1mm. B, HW/BW ratios were significantly greater in LE (6.67±0.22mg/g) and HE (7.06±0.35mg/g) mice than in control (5.92±0.13mg/g) mice. C, VW/BW ratios were significantly greater in LE (5.63±0.11mg/g) and HE (6.86±0.90mg/g) mice than in control (4.86±0.11mg/g). D, Trichrome-staining of cardiac tissues from Ctr, LE and HE mice showed significant fibrosis (light blue staining) in LE and HE mice. E, Example images of lectin-stained cardiac tissue showing cross-sections of myocytes. Green, lectin-stained membrane; Blue: DAPI-stained nuclei. F, There were greater cross sectional areas of VMs in HE (334.1±16.5 μm²) mouse hearts than in control (186.1±9.7 μm²) and LE (236.0±5.3μm²) mouse hearts. G, VM volume was significantly larger in HE (31,660±1,723fL) than in LE (26,309±503fL) and control mice (22,165±585fL). H & I, ANF- and β-MHC- mRNA expression evaluated by real-time PCR. J, Relative NFAT activity in DTG myocytes is significantly higher than in control myocytes. One-way ANOVA was done for B, C, F and G, two-way ANOVA for H and I and student t-test for J. The number in the bars indicates the number of hearts examined.

Figure 3. HE and LE mice develops PCH

A, Micrographs of Ctr (4 month), LE (4m) and HE (3m) hearts. The smallest division on the scale bar is 1mm. B, HW/BW ratios were significantly greater in LE (6.67±0.22mg/g) and HE (7.06±0.35mg/g) mice than in control (5.92±0.13mg/g) mice. C, VW/BW ratios were significantly greater in LE (5.63±0.11mg/g) and HE (6.86±0.90mg/g) mice than in control (4.86±0.11mg/g). D, Trichrome-staining of cardiac tissues from Ctr, LE and HE mice showed significant fibrosis (light blue staining) in LE and HE mice. E, Example images of lectin-stained cardiac tissue showing cross-sections of myocytes. Green, lectin-stained membrane; Blue: DAPI-stained nuclei. F, There were greater cross sectional areas of VMs in HE (334.1±16.5 μm²) mouse hearts than in control (186.1±9.7 μm²) and LE (236.0±5.3μm²) mouse hearts. G, VM volume was significantly larger in HE (31,660±1,723fL) than in LE (26,309±503fL) and control mice (22,165±585fL). H & I, ANF- and β-MHC- mRNA expression evaluated by real-time PCR. J, Relative NFAT activity in DTG myocytes is significantly higher than in control myocytes. One-way ANOVA was done for B, C, F and G, two-way ANOVA for H and I and student t-test for J. The number in the bars indicates the number of hearts examined.

Figure 4. Increases in I_Ca-L promoted hypertrophic response to transverse aortic constriction (TAC)

LE mice developed more severe hypertrophy (A) and had increased myocyte cross sectional area (B) after TAC. $: p<0.05 vs. LE sham; *: p<0.05 vs. control with TAC. C & D, The fractional shortening of the heart was reduced and the lung weight to body weight was increased in the LE mice but not in control mice after TAC, indicating the development of heart failure in LE mice. E & F, raw images and averaged percentage of blue fibrotic area of Mason’s Trichrome staining of LV tissues of control and LE mice with (TAC) or without TAC (sham) procedure.

Figure 4. Increases in I_Ca-L promoted hypertrophic response to transverse aortic constriction (TAC)

LE mice developed more severe hypertrophy (A) and had increased myocyte cross sectional area (B) after TAC. $: p<0.05 vs. LE sham; *: p<0.05 vs. control with TAC. C & D, The fractional shortening of the heart was reduced and the lung weight to body weight was increased in the LE mice but not in control mice after TAC, indicating the development of heart failure in LE mice. E & F, raw images and averaged percentage of blue fibrotic area of Mason’s Trichrome staining of LV tissues of control and LE mice with (TAC) or without TAC (sham) procedure.

Figure 5. β2a overexpression induces hypertrophy of AFVMs and NRVMs

A & B, The effects of different MOIs (0, 5, 10, 50 and 100) of Adβ2a or AdGFP on myocyte volume. β2a expression significantly increased the volume of AFVMs after 4 days of culture. C, Normalized protein/DNA ratio in cells infected with Adβ2a or AdGFP at the MOI of 5. D, The effects of Adβ2a (MOI 5) or AdGFP (MOI 5) on the surface area of NRVMs. β2a overexpression increased NRVM surface area and sarcomere organization. *: p<0.05 vs. AdGFP at the same MOI; **: p<0.01 vs. AdGFP at the MOI of 5.

Figure 6. β2a overexpression induced translocation of NFAT3 and HDAC5 in cultured AFVMs

A & B, Examples of NFAT localization in GFP-VMs (A) or β2a-VMs (B). C, Averaged NFAT translocation rates in GFP-VMs and β2a-VMs. D & E, Examples of HDAC5 localization in AFVMs infected with AdGFP (D) or Adβ2a (E). F, Averaged HDAC5 translocation rates in GFP-VMs and β2a-VMs. G, Adβ2a significantly increased Thr17 phosphorylation on PLB. The bars in A, B, D and E are 15μm.

Figure 7. Effects of drugs on myocyte volume, protein/DNA ratio, NFAT and HDAC translocation in GFP- and β2a-AFVMs

Effects of a Cav1.2 blocker (nifedipine, 13μM), an intracellular Ca²⁺ chelator (BAPTA, 1μM), calcineurin (CaN) inhibitors (cyclosporine A, 5μM & FK506, 1μM), a SERCA inhibitor (thapsigargin (TSG), 10nM), a CaMK II inhibitor (KN93, 1μM) and a hypertrophy inducer (phenylephrine (PE), 10μM) on myocyte volume (A) and protein synthesis (B). The increases in myocyte volume (A) and protein/DNA ratio (B) were prevented by all drugs except PE. Nifedipine, BAPTA-AM, CsA and FK506 blocked NFAT translocation induced by β2a overexpression (C). KN93 and TSG prevented the increase of HDAC5 translocation induced by β2a overexpression but phenylephrine did not further increase HDAC5 translocation in β2a-VMs (D). ^#: p<0.05 vs. AdGFP without drug treatment; *: p<0.05 vs. β2a-VMs without drug treatment.