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(Circulation Research. 2005;97:1009.)
© 2005 American Heart Association, Inc.

Cellular Biology

Ca²⁺ Influx–Induced Sarcoplasmic Reticulum Ca²⁺ Overload Causes Mitochondrial-Dependent Apoptosis in Ventricular Myocytes

Xiongwen Chen, Xiaoying Zhang, Hajime Kubo, David M. Harris, Geoffrey D. Mills, Jed Moyer, Remus Berretta, Sabine Telemaque Potts, James D. Marsh, Steven R. Houser

From the Cardiovascular Research Center (X.C., H.K., D.M.H., G.D.M., J.M., R.B., S.R.H.) and Departments of Physiology (D.M.H., G.D.M., S.R.H.) and Microbiology and Immunology (X.Z.), Temple University School of Medicine, Philadelphia, Pa; and the Department of Internal Medicine (S.T.P., J.D.M.), University of Arkansas for Medical Sciences, Little Rock.

Correspondence to Dr Steven Houser, Department of Physiology, Temple University School of Medicine, 3400 N Broad St, Philadelphia, PA 19140. E-mail steven.houser{at}temple.edu

	Abstract

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Increases in Ca²⁺ influx through the L-type Ca²⁺ channel (LTCC, Cav1.2) augment sarcoplasmic reticulum (SR) Ca²⁺ loading and the amplitude of the cytosolic Ca²⁺ transient to enhance cardiac myocyte contractility. Our hypothesis is that persistent increases in Ca²⁺ influx through the LTCC cause apoptosis if the excessive influx results in SR Ca²⁺ overload. Feline ventricular myocytes (VMs) in primary culture were infected with either an adenovirus (Ad) containing a rat Cav1.2 ß_2a subunit-green fluorescent protein (GFP) fusion gene (Adß_2a) to increase Ca²⁺ influx or with AdGFP as a control. Significantly fewer ß_2a-VMs (21.4±5.6%) than GFP-VMs (99.6±1.7%) were viable at 96 hours. A fraction of ß_2a-VMs (20.8±1.8%) contracted spontaneously (SC-ß_2a-VMs), and viability was significantly correlated with the percentage of SC-ß_2a-VMs. Higher percentages of apoptotic nuclei, DNA laddering, and cytochrome C release were detected in ß_2a-VMs. This apoptosis was prevented with pancaspase or caspase-3 or caspase-9 inhibitors. L-type calcium current (I_Ca-L) density was greater in ß_2a-VMs (23.4±2.8 pA/pF) than in GFP-VMs (7.6±1.6 pA/pF). SC-ß_2a-VMs had higher diastolic intracellular Ca²⁺ (Indo-1 ratio: 1.1±0.1 versus 0.7±0.03, P<0.05) and systolic Ca²⁺ transients (1.89±0.27 versus 0.80±0.08) than GFP-VMs. Inhibitors of Ca²⁺ influx, SR Ca²⁺ uptake and release, mitochondrial Ca²⁺ uptake, mitochondrial permeation transition pore, calpain, and Bcl-2-associated X protein protected ß_2a-VMs from apoptosis. These results show that persistent increases in Ca²⁺ influx through the I_Ca-L enhance contractility but lead to apoptosis through a mitochondrial death pathway if SR Ca²⁺ overload is induced.

Key Words: L-type calcium channel • ß_2a subunit • apoptosis • ventricular myocyte • primary culture

	Introduction

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Increased myocyte contractility is a central feature of the cardiac response to hypertension, valvular disease, and myocardial infarction.^1,2 Increased myocyte Ca²⁺ both causes enhanced contractility and contributes to the associated pathological hypertrophy.³ In this study, we explore the possibility that increased Ca²⁺ influx also promotes apoptosis.

Apoptosis is a critical component of myocyte death after myocardial infarction, persistent hemodynamic stress, and aging and in congestive heart failure.⁴ The contributions of increased intracellular Ca²⁺ ([Ca²⁺]_i), which is needed to support enhanced contractility during hemodynamic stress, to myocyte apoptosis have not been adequately explored. A critical role for excessive Ca²⁺ influx through the L-type calcium channel (LTCC or Cav1.2) and/or the Na⁺/Ca²⁺ exchanger in cardiomyocyte apoptosis induced by adrenergic agonists,^5–7 angiotensin II,⁸ and ischemia/reperfusion⁹ has been established. A modulatory role for Ca²⁺ influx through the B-type Ca²⁺ channel in cardiomyocyte apoptosis induced by ceramide has also been reported.¹⁰ In nonmyocytes, increased Ca²⁺ influx has been associated with both apoptosis^11–13 and protection from apoptosis.¹⁴ These studies document a Ca²⁺ dependence of apoptotic signaling pathways. What remains unclear is if increased Ca²⁺ influx alone is sufficient to induce myocyte apoptosis and, if so, what pathways link excessive myocyte Ca²⁺ influx to apoptosis.

Excessive [Ca²⁺] within mitochondria can induce apoptosis by opening the mitochondrial permeability transition pore (mPTP).¹⁵ This occurs at higher mitochondrial Ca²⁺ levels than those that match myocyte energy supply and demand in normal cardiomyocytes.¹⁶ Excessive cytoplasmic [Ca²⁺] has been linked to apoptosis via activation of Ca²⁺/calmodulin-dependent calcineurin, which leads to dephosphorylation of Bcl-2 antagonist of cell death protein (BAD), which in turn promotes translocation of Bcl-2-associated X protein (BAX) to mitochondria and oligomerization of BAX and/or BAD at the mitochondrial outer membrane (OMM), where they induce cytochrome C release. This apoptotic signaling cascade is involved in the apoptosis caused by excessive adrenergic activation of cardiac myocytes.⁶ Disturbances of Ca²⁺ regulation by the sarcoplasmic reticulum (SR) also can play an important role in apoptosis.¹⁵ SR (or endoplasmic reticulum, ER) Ca²⁺ overload and depletion have been shown to activate calpains, which induce apoptosis through BAD, BH3 (Bcl-2 homology domain 3)-interacting domain death agonist protein (Bid), and caspase 12.⁴ SR (or ER) Ca²⁺ depletion also causes the release of DNases (eg, DNase I) from SR (or ER) to induce apoptosis.¹⁷ Increased intracellular Ca²⁺ may activate a calmodulin-dependent kinase II (CaMK II) to induce apoptosis by a yet to be determined mechanism.⁷ These studies suggest a strong link between abnormal myocyte Ca²⁺ handling, mitochondrial dysfunction, and apoptosis.

The hypothesis of this study is that persistent increases in Ca²⁺ influx through the LTCC cause mitochondrial-dependent apoptosis by inducing SR Ca²⁺ overload in cardiomyocytes. Adenoviral gene transfer of the ß_2a subunit of the L-type Ca²⁺ channel (Cav1.2 ß_2a) was used to increase Ca²⁺ influx into cultured adult feline ventricular myocytes (VMs), which, unlike adult rat and mouse myocytes,¹⁸ have low diastolic [Ca²⁺] and low SR Ca²⁺ content in culture.¹⁹ Cav1.2 ß_2a is an accessory subunit that chaperones the pore-forming Cav1.2 _1c to the surface membrane, increases _1c open probability, and shifts _1c voltage-dependent activation to more negative membrane potentials.²⁰ The present results show that Adß_2a-infected feline VMs (ß_2a-VMs) had increased L-type calcium current (I_Ca-L) density, had increased contractility, developed spontaneous contractions associated with SR Ca²⁺ overload,²¹ and had an increased rate of apoptosis. This apoptosis appears to involve SR-mediated mitochondrial Ca²⁺ overload.

	Materials and Methods

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Cultured adult feline VMs obtained from the left ventricle (up to 4 days) were infected with adenovirus (Ad) containing a green fluorescent protein (GPP) gene (AdGFP) as a control or a Cav1.2ß_2a-GFP fusion gene (Adß_2a) at a multiplicity of infection (MOI) of 100 to study the effect of persistent increase in Ca²⁺ influx on VM survival. In situ DNA nick end labeling (TUNEL) assay, 4',6-diamidino-2-phenylindole-2 HCl (DAPI) staining, DNA laddering, and caspase inhibitors were used to determine the role of apoptosis in ß_2a overexpression-induced cell death. I_Ca-L,²² contractions, and intracellular Ca²⁺ were¹⁹ measured as described previously. Drugs modulating Ca²⁺ handling and apoptotic pathways and Western blot were used to study the mechanism of Ca²⁺-induced apoptosis. Detailed description of Materials and Methods can be found in Data Supplement online at http://circres.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Specificity of the Toxic Effect of ß_2a Overexpression
The effects of AdGFP and Adß_2a MOIs of 1, 10, 50, 100, 500, and 1000 on myocyte viability and spontaneous contractions were tested (Figure 1A). GFP-VM viability at 96 hours after infection was not significantly different from uninfected VMs, showing no toxicity up to an MOI of 500. Only 0.04% of GFP-VMs contracted spontaneously, and this was not different from uninfected controls (Figure 1A). Overexpression of ß_2a induced spontaneous contractions in a fraction of VMs, and the percentage of spontaneous contracting myocytes (SC-ß_2a-VMs) increased with MOI (Figure 1B). There was a significant reduction in the viability of ß_2a-VMs with increased MOI. The percentage of SC-ß_2a-VMs was significantly correlated with ß_2a-VM viability (R²=0.80, P<0.01). An MOI of 100 was used in all subsequent experiments because this appears to be below the level associated with nonspecific toxic effects of adenoviral infection.

View larger version (48K): [in this window] [in a new window]   Figure 1. Effects of ß2a overexpression on VM viability and spontaneous contraction. A, AdGFP did not cause cell death during a 4-day period in primary culture at an MOI up to 500. Almost no spontaneously contracting myocytes were observed. B, Adß2a caused cell death and a significant percentage of VMs exhibited spontaneous contractions. VM viability decreased with MOIs. C and D, Representative cells infected with AdGFP or Adß2a at the MOI of 100 at 48 hours after infection. The expressed GFP is evenly distributed in the cytosol. The expressed ß2a-GFP fusion protein primarily localizes to surface membranes and T-tubules. E and F. Representative fields of VMs infected with AdGFP and Adß2a 96 hours after infection (200x). G, Overexpressing Cav1.2 ß2a (MOI of 100) caused a progressive decrease in the viability of VMs at 48, 72, and 96 hours after infection compared with overexpressing GFP. H and I, Overexpressing Cav1.2 ß2a significantly and progressively increased percentage of SC-ß2a-VMs at 24, 48, 72, and 96 hours after infection. The VM viability of ß2a-VMs was significantly correlated with the percentage of SC-ß2a-VMs (J). *P<0.05; **P<0.01.

Transfection Efficiency of AdGFP and Adß_2a and Distribution of Overexpressed Proteins
The percentage of GFP-expressing rod-shaped VMs among total rod-shaped VMs was measured at 48 hours after infection. With an MOI of 100, the transfection efficiencies were 97.1±6.0% and 96.0±1.3% for AdGFP and Adß_2a, respectively. Expressed GFP was primarily distributed in the cytosol, whereas the ß_2a-GFP fusion protein was mainly concentrated on the surface membrane and T-tubules (Figure 1C and 1D). This ß_2a expression profile was slightly different from what was observed by Colecraft et al,²⁰ who reported expressed ß_2a on the surface membrane but not on T-tubules. The possible reasons for this difference could be the significantly lower MOI (100 versus 10 000) used in our study and/or the fact that we examined cellular localization at 48 versus 18 hours.

Overexpression of Cav1.2 ß_2a Induces Cell Death and Spontaneous Contraction in ß_2a-VMs
Overexpression of Cav1.2 ß_2a caused a significant increase in cell death at 48, 72, and 96 hours after infection (Figure 1G). The viability of ß_2a-VMs (21.4±5.6%, n=27) was significantly lower (P<0.05) than that of the GFP-VMs (99.6±1.7%, n=27) at 96 hours (Figure 1). At 24 hours after infection, there was no significant decrease in viability of ß_2a-VMs, likely due to the fact that more than 24 hours was needed for the transfected gene to be expressed at the abundance sufficient to induce cell death. The percentage of SC-ß_2a-VMs was increased at 24 hours after infection and increased further with time (20.8±1.8% at 96 hours after infection). The viability of ß_2a-VMs was significantly correlated with the percentage of SC-ß_2a-VMs (R²=0.66, P<0.0001; Figure 1J). Spontaneous contractions in VMs occur when the SR accumulates Ca²⁺ above the threshold level (SR Ca²⁺ overload) at which spontaneous SR Ca²⁺ release occurs.²¹

The Role of Apoptosis in Cell Death Induced by ß_2a Overexpression
DAPI staining, TUNEL assay, and DNA laddering techniques were used to determine the contribution of apoptosis to cell death in GFP-VMs and ß_2a-VMs. The percentage of TUNEL positive nuclei was significantly greater in VMs infected with Adß_2a versus AdGFP at 48, 72, and 96 hours after infection (Figure 2). DNA laddering was used to confirm apoptosis. A weak pattern of DNA laddering was observed in GFP-VMs but a much stronger DNA laddering pattern was found in ß_2a-VMs (Figure 2B).

View larger version (28K): [in this window] [in a new window]   Figure 2. Apoptosis induced by ß2a-overexpression. A, Apoptosis was detected with DAPI staining and TUNEL assay. B, DNA laddering revealed with DNA gel electrophoresis. Lane 1: 100bp ladder marker. VMs infected with AdGFP or Adß2a 72 hours after infection. C, Apoptotic rate determined by the increase in the percentage of TUNEL positive nuclei versus the value at time 0. D, ß2a-VM viability was significantly increased in the absence (CTR) or presence of 2 general caspase inhibitors (ApoBlock and z-VAD-fmk) and 1 caspase-3 inhibitor (z-DEVD-fmk). E, Effect of caspase inhibitors on the percentage of spontaneously contracting ß2a-VMs. All asterisks represent significance between the CTR group and the treated groups at the same time point. *P<0.05; **P<0.01.

Excessive Ca²⁺ influx through Cav1.2 has been shown to induce both necrosis and apoptosis depending on the energetic status of the cell.¹³ Activation of caspases is a critical step in apoptosis. The general caspase inhibitors (z-VAD-fmk 10 µmol/L and ApoBlock 20 µmol/L) and the caspase-3 specific inhibitor (z-DEVD-fmk 10 µmol/L) increased ß_2a-VMs viability to >90% (Figure 2D) and decreased the percentage of SC-ß_2a-VMs (Figure 2E). These results strongly suggest that the cell death induced by ß_2a overexpression is caused by caspase 3-dependent apoptosis. Notably, all 3 caspase inhibitors also significantly decreased the percentage of SC-ß_2a-VMs, suggesting that activated caspases feed back to enhance the Ca²⁺ overload caused by increased Ca²⁺ influx.¹³

Overexpression of Cav1.2 ß_2a Enhances Ca²⁺ Current and SR Ca²⁺ in Cultured VMs
ß_2a overexpression is thought to significantly increase I_Ca-L by increasing the open probability of Cav1.2 and trafficking of the pore-forming Cav1.2 _1C subunit to the sarcolemma.²⁰ Ca²⁺ handling in ß_2a-VMs was characterized by measuring the density and voltage-dependent activation of I_Ca-L, contraction magnitude, and Ca²⁺ transient amplitude. I_Ca-L density was significantly greater (P<0.05) in ß_2a-VMs (23.4±2.8 pA/pF, n=10) than in GFP-VMs (7.6±1.6 pA/pF, n=11), and the voltage-dependence of activation was shifted to negative potentials (half-maximum activation voltage [V_{0.5, d}] of Cav1.2 in ß_2a-VMs versus GFP-VMs: –19.8±2.2 mV versus –6.3±2.2mV, P<0.001; Figure 3). There was no significant difference (P=0.32) in I_Ca-L density between SC-ß_2a-VMs (–18.8±6.1 pA/pF, N=3) and quiescent ß_2a-VMs (–25.3±3.4 pA/pF, N=7).

View larger version (27K): [in this window] [in a new window]   Figure 3. Effect of ß2a overexpression on ICa-L in cultured VMs. A, Representative ICa-L current recorded. B and C, ICa-L density was significantly greater in ß2a-VMs than in GFP-VMs, and the voltage-dependency of activation was significantly shifted to the negative voltages. *P<0.05; **P<0.01.

Field-stimulated myocyte fractional shortening (Figure 4) was significantly (P<0.01) greater in ß_2a-VMs (9.8±1.2%, n=28, N=4) than in GFP-VMs (3.2±0.6%, n=24, N=4). Fractional shortening in both spontaneously contracting (12.8±2.0%, n=10, N=4, P<0.001) and quiescent ß_2a-VMs (8.2±1.4%, n=18, N=4, P<0.05) was significantly greater than in GFP-VMs (3.2±0.6%, n=24, N=4), although the difference between quiescent and spontaneously contracting ß_2a-VMs was not significant (P=0.09) (Figure 4A and 4B). Peak systolic Ca²⁺ was significantly greater in ß_2a-VMs versus GFP-VMs (1.72±0.22, n=24, N=4 versus 0.80±0.08, n=28, N=4, P<0.01) (Figure 4C). Both quiescent (1.48±0.15, n=18, N=4) and spontaneously contracting ß_2a-VMs (1.89±0.25, n=10, N=4) had higher peak Ca²⁺ than in GFP-VMs. There was no significant difference in peak [Ca²⁺]_i between SC-ß_2a-VMs and quiescent ß_2a-VMs. Diastolic Ca²⁺ in SC-ß_2a-VMs (1.07±0.12, n=10, N=4) was significantly greater (Figure 4C) than in quiescent ß_2a-VMs (0.73±0.04, n=18, N=4) and GFP-VMs (0.73±0.03, n=24, N=4).

View larger version (28K): [in this window] [in a new window]   Figure 4. Effect of ß2a overexpression on contractility and Ca2+ handling. A, Representative contractions and [Ca2+]i transient from GFP-VMs, SC-ß2a-VMs, and Q-ß2a-VMs. B, Fractional shortening is significantly greater in ß2a-VMs than in GFP-VMs. C, When diastolic Ca2+ concentration of all ß2a-VMs were compared with that of GFP-VMs, there was no significant difference between the groups (P=0.06). Diastolic Ca2+ was significantly higher in SC-ß2a-VMs than in GFP-VMs and quiescent ß2a-VMs (Q-ß2a-VMs), however. Peak systolic Ca2+ was significantly higher in all ß2a-VMs than in GFP-VMs. There was no significant difference in peak Ca2+ between SC-ß2a-VMs and quiescent ß2a-VMs. *P<0.05; **P<0.01.

These results show that infecting adult feline myocytes with Cav1.2 ß_2a increases I_Ca-L and shifts its voltage-dependence of activation to negative potentials, which should increase Ca²⁺ influx through Cav1.2 in resting ß_2a-VMs, thereby causing high SR Ca²⁺ loads. The increased cellular Ca²⁺ then appears to induce apoptosis through the activation of caspases (Figure 2).

SR Ca²⁺ Overload Mediates ß_2a Overexpression-Induced Apoptosis in Cultured VMs
Drugs with clearly defined effects on Ca²⁺ handling were used to explore the relationship between increased Ca²⁺ and apoptosis in ß_2a-VMs (Figure 5). Nifedipine (13 µmol/L), a Cav1.2 blocker, prevented apoptosis and BAPTA-AM (1 µmol/L), a cell permeable Ca²⁺ buffer, largely protected ß_2a-VMs from apoptosis. Drugs that prevent SR Ca²⁺ overload by inhibiting SERCA (thapsigargin 10 nmol/L) or by inducing SR Ca²⁺ leak (ryanodine 1 µmol/L and caffeine 1 mmol/L) partially rescued ß_2a-VMs from apoptosis and reduced their rate of spontaneous contraction. These results strongly implicate SR Ca²⁺ overload and spontaneous Ca²⁺ release in the apoptosis induced by ß_2a overexpression. It is worth noting that higher concentrations of some SR-modifying drugs induced rather than blocked apoptosis in GFP-VMs and ß_2a-VMs. For example, 10 µmol/L BAPTA-AM, 1 µmol/L thapsigargin, and 10 mmol/L caffeine induced apoptosis in GFP-VMs within 2 days and in ß_2a-VMs at later times. These results suggest that antiapoptotic effects are produced when Ca²⁺ overload is prevented, but proapoptotic effects are induced if SR Ca²⁺ stores are depleted, potentially via an SR stress response.²³

View larger version (25K): [in this window] [in a new window]   Figure 5. Effect of drugs modulating Ca2+ handling on VM viability and the percentage of SC-ß2a-VMs induced by ß2a overexpression. A, VM viability at 96 hours after infection. All drugs tested had full or partial protection on ß2a-VMs. B, All drugs significantly decreased the percentage of SC-ß2a-VMs at 96 hours after infection. C and D, The phosphorylation of threonine17 was significantly greater in ß2a-VMs than in GFP-VMs. CTR indicates control; NIF, nifedipine, TG, thapsigargin; CAFF, caffeine; RYA, ryanodine; DANT, dantrolene. Numbers above the bars in A and B indicate times of cell cultures. *P<0.05; **P<0.01; ***P<0.001.

Increased cytosolic Ca²⁺ can activate Ca²⁺/camodulin-dependent kinase (CaMK), which in turn phosphorylates specific target proteins including phospholamban (PLB) at threonine-17.²⁴ Western blot analysis with antibodies that detect total PLB, PLB with phosphorylated serine¹⁶ (pS¹⁶-PLB), and PLB with phosphorylated threonine¹⁷ (pT¹⁷-PLB) showed that the total PLB expression was not different between GFP-VMs and ß_2a-VMs. No pS¹⁶-PLB was detected in either group, but the phosphorylation at T¹⁷ was significantly greater in ß_2a-VMs (Figure 5 C and 5D). The increased phosphorylation of PLB at T¹⁷ should increase SERCA activity²⁴ and promote SR Ca²⁺ overload in ß_2a-VMs.

CaMK II is known to be involved in myocyte apoptosis⁷ induced by ß-adrenergic stimulation, possibly via enhanced SERCA activity resulting from increased phosphorylation of PLB at T¹⁷ and subsequent SR Ca²⁺ overload. Three CaMK II (the major form of CaMK in cardiac myocytes) inhibitors (KN 62, 5 µmol/L; KN 93, 1 µmol/L; and AIP, 20 µmol/L) were used to test the role of CaMK II in apoptosis induced by ß_2a overexpression (Figure 5A). KN 62 (VM viability: 90.1±3.0%) and KN 93 (VM viability: 99.5±3.8%) almost fully protected ß_2a-VMs from apoptosis and reduced spontaneous contractions (Figure 5B). KN62 and KN93, however, are known to have nonspecific effects, including block of K⁺ channels.²⁵ Therefore, a more specific peptide inhibitor of CaMK II (AIP) was also tested and found to be protective (Figure 5A and 5B), but somewhat less so than KN62 and KN93. These results suggest that CaMK II is involved in ß_2a-induced apoptosis, at least in part by modulating Ca²⁺ influx and SR Ca²⁺ loading.²⁶ Direct modulation of other apoptotic pathways by CaMK II could not be excluded.

The Role of Mitochondria in ß_2a-Induced Apoptosis in Cultured VMs
Experiments with z-DEVD-fmk suggest that activation of caspase-3 is a critical step in ß_2a-induced apoptosis. Caspase-3 activation is thought to be the final common pathway in apoptosis. The connection between SR Ca²⁺ overload and apoptosis appears to involve an intrinsic, mitochondrial-dependent pathway, of which cytochrome C release and subsequent caspase-9 activation are characteristics.¹³ We found that the caspase-9 inhibitor z-LEHD-fmk was able to fully prevent the apoptosis induced by ß_2a-overexpression (Figure 6A). In addition, significant amounts of cytochrome C were found in the cytosolic fraction of ß_2a-VMs but not GFP-VMs (Figure 6C), documenting a central role of mitochondria in Ca²⁺-mediated apoptosis.

View larger version (27K): [in this window] [in a new window]   Figure 6. Effect of drugs modulating apoptotic pathways on VM viability and the percentage of SC-ß2a-VMs induced by ß2a overexpression. A, VM viability at 96 hours after infection. All drugs except FK 506 decreased apoptosis. B, Some, but not all drugs decreased the percentage of SC-ß2a-VMs at 96 hours after infection. C, Substantial cytochrome c (Cyt c) was found in the cytosol from ß2a-VMs but not from GFP-VMs. D, Significant amounts of cleaved µ-calpain were found in ß2a-VMs but not in GFP VMs. Ctr indicates control; RR, ruthenium red; calpain inhit III, calpain inhibitor III. Numbers above the bars in A and B indicate cell culture times. *P<0.05.

Mitochondrial cytochrome C is released into the cytoplasm through the BAX/BAK complex and/or direct rupture of the outer membrane of the mitochondria after the opening of the mPTP.¹³ All of these routes could be induced by mitochondrial Ca²⁺ overload. Inhibiting the mPTP with NIM 811 (VM viability: 57.2±2.5%) and the BAX/BAK complex with BIP-V5 (VM viability: 77.2±3.8%) increased ß_2a-VM viability, suggesting that both the mPTP and the BAX/BAK complex are involved in ß_2a-overexpression induced myocyte apoptosis (Figure 6A).

Opening of the mPTP can be induced by mitochondrial Ca²⁺ overload.¹⁵ Inhibiting mitochondrial Ca²⁺ uptake via the mitochondrial uniporter with Ru 360 and ruthenium red reduced apoptosis (Figure 6A). The mitochondrial uniporter has a low affinity for Ca²⁺ (in the mmol/L range). Therefore, only mitochondria located close to the SR Ca²⁺ release channels may be exposed to Ca²⁺ concentrations sufficient to induce apoptosis.¹³ We found that inhibiting Ca²⁺ release from the SR with 10 nmol/L ryanodine (inhibiting ryanodine receptor) or 1 µmol/L dantrolene (inhibiting both ryanodine and IP₃ receptors) prevented spontaneous contractions (SR Ca²⁺ release) and significantly reduced apoptosis in ß_2a-VMs (Figure 7A and 7B). These data show that Ca²⁺ release from an overloaded SR is necessary and sufficient to induce apoptosis in ß_2a-VMs.

View larger version (27K): [in this window] [in a new window]   Figure 7. Illustration summarizing the mechanism responsible for apoptosis induced by increasing Ca2+ influx. RyR indicates the ryanodine receptor; CaM, calmodulin; OMM, outer membrane of the mitochondrion; Cyt c, cytochrome C; CyD, cyclophilin-D; ANT, the adenine nucleotide translocator; VDAC, the voltage-dependent anion channel; and tBid, truncated Bid.

BAX and/or BAK can translocate to mitochondria and oligomerize to form pores, allowing cytochrome C release. These processes are stimulated by dephosphoryaltion of BAD by calcineurin,⁶ cleavage of Bid¹³ and BAX⁴ by calpain, and cleavage of Bid by caspase-8.¹³ Inhibition of calcineurin (FK 506, 1 µmol/L), did not alter ß_2a-induced apoptosis (Figure 6A), suggesting that calcineurin does not play a role in Ca²⁺-induced apoptosis.²⁷ Both calpain inhibitor III and the caspase-8 inhibitor z-IETD-fmk, however, significantly reduced ß_2a-induced apoptosis (Figure 6A). To further test which calpain was involved, Western blot analysis was used to detect cleaved calpain fragments, which are the active forms. No cleaved m-calpain (milli-calpain, activated by Ca²⁺ in millimolar range) fragments were detected (data not shown), but 6.1±1.8% of total µ-calpain (micro-calpain, activated by Ca²⁺ in micromolar range) was found to be cleaved in ß_2a-VMs at 48 hours after infection (Figure 6D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we show that cultured adult feline VMs have a very low rate of apoptosis and do not contract spontaneously because of low SR Ca²⁺ stores.¹⁸ Increasing Ca²⁺ influx by overexpressing Cav1.2 ß_2a caused SR Ca²⁺ overload and induced apoptosis. Our data suggest that the very high local [Ca²⁺], which results from spontaneous Ca²⁺ release from an overloaded SR, induces mitochondrial Ca²⁺ overload, cytochrome c release, and activation of caspase-3 to cause apoptosis. We speculate that an increased rate of apoptosis induced by persistent SR Ca²⁺ overload could be a significant cause of myocyte death in cardiovascular diseases that involve persistent increases in myocyte contractility, such as hypertension, and after myocardial infarction.

Increased Ca²⁺ Influx, SR Ca²⁺ Loading, and Myocyte Contractility
Increased Ca²⁺ current is the normal physiological mechanism to increase SR Ca²⁺ stores and the Ca²⁺ transient (increased contractility).^2,28 In the present study, we used primary cultures of adult feline VMs to test the idea that persistent increases in Ca²⁺ influx, while increasing contractility, can induce apoptosis if the Ca²⁺ influx is sufficient to cause SR Ca²⁺ overload. Importantly, myocytes from large mammals (cats, dogs, and humans) have a low SR Ca²⁺ load in the absence of pacing because they maintain low intracellular Na⁺ ([Na⁺]_i) which causes forward mode Na⁺/Ca²⁺ exchange activity to maintain low cytosolic and SR Ca²⁺ stores.¹⁸ VMs from small mammals have higher [Na]_i, resulting in increased Ca²⁺ influx (or reduced Ca²⁺ efflux) via the Na⁺/Ca²⁺ exchanger, higher cytosolic [Ca²⁺], and increased SR Ca²⁺ load, which results in spontaneous SR Ca²⁺ release, even when myocytes are not paced.^18,21 Therefore, myocytes from small mammals are poorly suited for experiments testing the effects of increased Ca²⁺ on myocyte growth or death because they are Ca²⁺ overloaded under control conditions.²¹

To specifically increase Ca²⁺ influx through the LTCC (the major Ca²⁺ channel in cardiac myocytes) into nonstimulated feline VMs, we used a noncardiac Cav1.2 ß_2a subunit because of its unique ability to shift LTCC activation to negative voltages and increase P₀. We chose this approach because traditional pharmacological agents that can increase Ca²⁺ influx through the LTCC, such as LTCC agonists Bay K 8644 and FPL-64176, are less specific, with poorly defined effects on ryanodine receptor function.²⁹

The Role of Persistent Increase in Ca²⁺ Influx in VM Apoptosis
Increased Ca²⁺ influx has been implicated in the apoptosis of cardiomyocytes induced by ischemia/reperfusion,⁹ catecholamines,^5,7 and angiotensin II⁸ and other cells.¹³ Muth et al³⁰ have observed a higher myocyte apoptotic rate in the intact heart of a line of transgenic mice overexpressing the Cav1.2 _1c subunit, suggesting a role for persistent increases in Ca²⁺ influx in apoptosis in vivo. To our knowledge, our study is the first detailed study to show that increasing Ca²⁺ influx through the LTCC by itself is sufficient to induce cardiomyocyte apoptosis and to demonstrate a clear link between dysregulated increases in Ca²⁺ influx and apoptosis.

Previously, Colecraft et al²⁰ noted >90% myocyte death induced by overexpressing Cav1.2 ß_2a in cultured rat VMs 48 hours after infection, but no mechanistic studies were performed. The mechanism of myocyte death in that study is likely to be different than in the current series of experiments because of the high Adß_2a MOI used and the use of cultured rat VMs, which have SR Ca²⁺ overload when unpaced.²¹

The Role of SR Ca²⁺ Overload in ß_2a-VM Apoptosis
The present experiments show that persistent increases in Ca²⁺ influx through Cav1.2 in ß_2a-VMs enhance contractility but can cause Ca²⁺ overload of the SR, which was significantly correlated with the induction of apoptosis (Figure 1F). Ca²⁺-mediated activation of CaMK II²⁶ with subsequent phosphorylation of the PLB to increase SERCA activity appears to be involved in SR Ca²⁺ overload (Figure 5). An essential role of spontaneous SR Ca²⁺ release in ß_2a-induced apoptosis is supported by the fact that reduced SR Ca²⁺ loading (nifedipine, 1 µmol/L ryanodine, and 1 mmol/L caffeine) or blocking SR Ca²⁺ release (10 nmol/L ryanodine and 1 µmol/L dantrolene) significantly reduced apoptosis. In addition, when Ca²⁺ influx and SR Ca²⁺ release were increased by pacing (continuously for 24 hours) myocytes in culture, apoptosis was induced in ß_2a-VMs (>90% VM death) but not in GFP-VMs (data not shown). These results are in good agreement with those studies showing that the SR/ER serves as an integral component of inducible apoptosis.²³ In this regard, overexpression of SERCA in nonmyocytes has been shown to be proapoptotic,³¹ whereas cells devoid of the SR/ER Ca²⁺ release channels (IP3 receptors) are resistant to apoptotic stimuli.³² In addition, proapoptotic bcl-2 proteins (BAX and BAK) induce apoptosis in part by increasing SR/ER Ca²⁺ content, whereas antiapoptotic bcl-2 proteins offer protection by decreasing SR/ER Ca²⁺ content.¹³ Collectively, these data support a crucial role for SR Ca²⁺ overload in apoptosis. Our results show for the first time that processes that augment SR Ca²⁺ initiate apoptosis if they cause SR Ca²⁺ overload.

Increases in cytosolic Ca²⁺ could cause apoptosis, independent of an increase in SR Ca²⁺ loading, by activating calcineurin, which dephosphorylates BAD,⁶ or by activating cytosolic Ca²⁺ dependent DNase I, which directly cleaves DNA.¹⁷ The calcineurin inhibitor (FK 506) did not protect ß_2a-VMs from apoptosis. Direct activation of cytosolic Ca²⁺-dependent DNase I also does not appear to play a role in ß_2a-induced apoptosis because inhibition of caspases with ApoBlock, z-VAD-fmk (general caspase inhibitors), and z-DEVD-fmk (a caspase-3 inhibitor) were able to fully prevent apoptosis in ß_2a-VMs.

The Role of the Mitochondria in ß_2a-VM Apoptosis
Many aspects of the current study, including cytochrome C release into the cytosol, support a central role of mitochondria in ß_2a overexpression-induced myocyte apoptosis. In addition, ß_2a overexpression-induced apoptosis was significantly reduced by inhibition of the characteristic caspase (caspase-9), the initiator of mitochondria-mediated apoptosis, by inhibiting the mPTP and BAX/BAK complex and by inhibiting Ca²⁺ uptake into the mitochondria. We speculate that the very high local [Ca²⁺]_i produced by spontaneous Ca²⁺ release from a Ca²⁺-overloaded SR causes mitochondrial Ca²⁺ overload via a mitochondrial Ca²⁺ uniporter. A connection between SR Ca²⁺ release and mitochondrial Ca²⁺ uptake in apoptosis has been shown by others.²³

The Death Receptor Pathway in ß_2a Overexpression-Induced Apoptosis
Caspase-8 activation is thought to be an indication of activation of the extrinsic (death receptor-dependent) apoptotic pathway.^4,13 Our results show that ß_2a-induced apoptosis can be reduced by caspase-8 inhibitors, consistent with interaction of components of intrinsic and extrinsic apoptotic signaling pathways.

Limitations
The present experiments were performed using primary cultures of unpaced adult feline VMs, which are known to change their characteristics (in particular, they tend to depolarize) with time in culture.³³ We did not find a significant difference in resting potential (RP) after 3 days in culture in ß_2a-VMs (–78.0±6.6 mV; n=9, among which 3 were spontaneously contracting) versus GFP-VMs (–74.6±7.5 mV; n=9). These RPs are similar to those measured in freshly isolated feline VMs (–74.9±0.8 mV, n=54). Therefore, the RP of feline VMs during the first 3 days does not appear to demonstrate significant depolarization, consistent with results by others suggesting that feline VMs are more resistant to culture-induced remodeling.³⁴

Significance of This Study
The present experiments show that increase in Ca²⁺ influx through Cav1.2 induces apoptosis when it is of sufficient magnitude to cause SR Ca²⁺ overload. These mechanisms may be involved in cardiomyocyte apoptosis induced by hypertension (during the hypercontractile, compensated hypertrophic stage), ischemia/reperfusion,⁹ and excessive adrenergic activity^5,6 and in heart failure. Whether or not apoptosis induced by excess Ca²⁺ influx is an important component of cardiac decompensation resulting from persistent cardiovascular diseases cannot be determined from these experiments. An alternative idea is that this form of apoptosis promotes myocyte turnover (from cardiac stem cells)³⁵ and is beneficial rather than detrimental. Assuming that Ca²⁺ influx-induced apoptosis leads to excess cell death and cardiac decompensation, however, therapies that limit rather than enhance SR Ca²⁺ loading might be beneficial. Another consideration is that although almost all myocytes were exposed to the same death stimuli, not all cells died. Therefore, the adult myocyte preparation used in our study might be useful in identifying mechanisms that allow myocytes to withstand apoptosis inducing signals.

Conclusion
Overexpressing a Cav1.2 ß_2a subunit in adult feline cardiomyocytes increases Ca²⁺ current, which activates CaMK II and increases Ca²⁺ uptake into the SR, increasing contractility but eventually causing SR Ca²⁺ overload. The high SR Ca²⁺ load from spontaneous SR Ca²⁺ release induces sufficient mitochondrial Ca²⁺ uptake to cause mitochondria Ca²⁺ overload. Altered cellular Ca²⁺ regulation also activates µ-calpain, which may cleave Bid into tBid (truncated Bid). tBid promotes BAX/BAK translocation and oligmerization. Mitochondrial Ca²⁺ overload and BAX/BAK complex formation on the mitochondrial outer membrane lead to cytochrome C release and caspase-9 activation. Active caspase-9 also activates caspase-8, leading to apoptotic cell death (Figure 7). We conclude that an excessive increase in Ca²⁺ influx and SR Ca²⁺ loading, as is seen in response to chronic hemodynamic stress, enhances contractility but can induce myocyte death. Therapies that prevent excessive increases in inotropy may provide benefit by reducing myocyte death.

	Acknowledgments

 
This research was supported by grants from the National Institutes of Health (HL33920 and HL66415 to S.R.H.).

	Footnotes

 
Original received June 7, 2005; revision received September 1, 2005; accepted September 28, 2005.

	References

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