Ca2+ InfluxeCInduced Sarcoplasmic Reticulum Ca2+ Overload Causes Mitochondrial-Dependent Apoptosis in Ventricular Myocytes
http://www.100md.com
循环研究杂志 2005年第11期
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
the Department of Internal Medicine (S.T.P., J.D.M.), University of Arkansas for Medical Sciences, Little Rock.
Abstract
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 2a subunit-green fluorescent protein (GFP) fusion gene (Ad2a) to increase Ca2+ influx or with AdGFP as a control. Significantly fewer 2a-VMs (21.4±5.6%) than GFP-VMs (99.6±1.7%) were viable at 96 hours. A fraction of 2a-VMs (20.8±1.8%) contracted spontaneously (SC-2a-VMs), and viability was significantly correlated with the percentage of SC-2a-VMs. Higher percentages of apoptotic nuclei, DNA laddering, and cytochrome C release were detected in 2a-VMs. This apoptosis was prevented with pancaspase or caspase-3 or caspase-9 inhibitors. L-type calcium current (ICa-L) density was greater in 2a-VMs (23.4±2.8 pA/pF) than in GFP-VMs (7.6±1.6 pA/pF). SC-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 2a-VMs from apoptosis. These results show that persistent increases in Ca2+ influx through the ICa-L enhance contractility but lead to apoptosis through a mitochondrial death pathway if SR Ca2+ overload is induced.
Key Words: L-type calcium channel 2a subunit apoptosis ventricular myocyte primary culture
Introduction
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,5eC7 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 apoptosis11eC13 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 2a subunit of the L-type Ca2+ channel (Cav1.2 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 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 Ad2a-infected feline VMs (2a-VMs) had increased L-type calcium current (ICa-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
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 Cav1.22a-GFP fusion gene (Ad2a) 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 2a overexpression-induced cell death. ICa-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
Specificity of the Toxic Effect of 2a Overexpression
The effects of AdGFP and Ad2a 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 2a induced spontaneous contractions in a fraction of VMs, and the percentage of spontaneous contracting myocytes (SC-2a-VMs) increased with MOI (Figure 1B). There was a significant reduction in the viability of 2a-VMs with increased MOI. The percentage of SC-2a-VMs was significantly correlated with 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.
Transfection Efficiency of AdGFP and Ad2a 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 Ad2a, respectively. Expressed GFP was primarily distributed in the cytosol, whereas the 2a-GFP fusion protein was mainly concentrated on the surface membrane and T-tubules (Figure 1C and 1D). This 2a expression profile was slightly different from what was observed by Colecraft et al,20 who reported expressed 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 2a Induces Cell Death and Spontaneous Contraction in 2a-VMs
Overexpression of Cav1.2 2a caused a significant increase in cell death at 48, 72, and 96 hours after infection (Figure 1G). The viability of 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 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-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 2a-VMs was significantly correlated with the percentage of SC-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 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 2a-VMs. The percentage of TUNEL positive nuclei was significantly greater in VMs infected with Ad2a 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 2a-VMs (Figure 2B).
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 e蘭ol/L and ApoBlock 20 e蘭ol/L) and the caspase-3 specific inhibitor (z-DEVD-fmk 10 e蘭ol/L) increased 2a-VMs viability to >90% (Figure 2D) and decreased the percentage of SC-2a-VMs (Figure 2E). These results strongly suggest that the cell death induced by 2a overexpression is caused by caspase 3-dependent apoptosis. Notably, all 3 caspase inhibitors also significantly decreased the percentage of SC-2a-VMs, suggesting that activated caspases feed back to enhance the Ca2+ overload caused by increased Ca2+ influx.13
Overexpression of Cav1.2 2a Enhances Ca2+ Current and SR Ca2+ in Cultured VMs
2a overexpression is thought to significantly increase ICa-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 2a-VMs was characterized by measuring the density and voltage-dependent activation of ICa-L, contraction magnitude, and Ca2+ transient amplitude. ICa-L density was significantly greater (P<0.05) in 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 [V0.5, d] of Cav1.2 in 2a-VMs versus GFP-VMs: eC19.8±2.2 mV versus eC6.3±2.2mV, P<0.001; Figure 3). There was no significant difference (P=0.32) in ICa-L density between SC-2a-VMs (eC18.8±6.1 pA/pF, N=3) and quiescent 2a-VMs (eC25.3±3.4 pA/pF, N=7).
Field-stimulated myocyte fractional shortening (Figure 4) was significantly (P<0.01) greater in 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 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 2a-VMs was not significant (P=0.09) (Figure 4A and 4B). Peak systolic Ca2+ was significantly greater in 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 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-2a-VMs and quiescent 2a-VMs. Diastolic Ca2+ in SC-2a-VMs (1.07±0.12, n=10, N=4) was significantly greater (Figure 4C) than in quiescent 2a-VMs (0.73±0.04, n=18, N=4) and GFP-VMs (0.73±0.03, n=24, N=4).
These results show that infecting adult feline myocytes with Cav1.2 2a increases ICa-L and shifts its voltage-dependence of activation to negative potentials, which should increase Ca2+ influx through Cav1.2 in resting 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 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 2a-VMs (Figure 5). Nifedipine (13 e蘭ol/L), a Cav1.2 blocker, prevented apoptosis and BAPTA-AM (1 e蘭ol/L), a cell permeable Ca2+ buffer, largely protected 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 e蘭ol/L and caffeine 1 mmol/L) partially rescued 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 2a overexpression. It is worth noting that higher concentrations of some SR-modifying drugs induced rather than blocked apoptosis in GFP-VMs and 2a-VMs. For example, 10 e蘭ol/L BAPTA-AM, 1 e蘭ol/L thapsigargin, and 10 mmol/L caffeine induced apoptosis in GFP-VMs within 2 days and in 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
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 2a-VMs. No pS16-PLB was detected in either group, but the phosphorylation at T17 was significantly greater in 2a-VMs (Figure 5 C and 5D). The increased phosphorylation of PLB at T17 should increase SERCA activity24 and promote SR Ca2+ overload in 2a-VMs.
CaMK II is known to be involved in myocyte apoptosis7 induced by -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 e蘭ol/L; KN 93, 1 e蘭ol/L; and AIP, 20 e蘭ol/L) were used to test the role of CaMK II in apoptosis induced by 2a overexpression (Figure 5A). KN 62 (VM viability: 90.1±3.0%) and KN 93 (VM viability: 99.5±3.8%) almost fully protected 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 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 2a-Induced Apoptosis in Cultured VMs
Experiments with z-DEVD-fmk suggest that activation of caspase-3 is a critical step in 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 2a-overexpression (Figure 6A). In addition, significant amounts of cytochrome C were found in the cytosolic fraction of 2a-VMs but not GFP-VMs (Figure 6C), documenting a central role of mitochondria in Ca2+-mediated apoptosis.
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 2a-VM viability, suggesting that both the mPTP and the BAX/BAK complex are involved in 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 e蘭ol/L dantrolene (inhibiting both ryanodine and IP3 receptors) prevented spontaneous contractions (SR Ca2+ release) and significantly reduced apoptosis in 2a-VMs (Figure 7A and 7B). These data show that Ca2+ release from an overloaded SR is necessary and sufficient to induce apoptosis in 2a-VMs.
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 e蘭ol/L), did not alter 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 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 e?calpain (micro-calpain, activated by Ca2+ in micromolar range) was found to be cleaved in 2a-VMs at 48 hours after infection (Figure 6D).
Discussion
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 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 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 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 Ad2a MOI used and the use of cultured rat VMs, which have SR Ca2+ overload when unpaced.21
The Role of SR Ca2+ Overload in 2a-VM Apoptosis
The present experiments show that persistent increases in Ca2+ influx through Cav1.2 in 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 2a-induced apoptosis is supported by the fact that reduced SR Ca2+ loading (nifedipine, 1 e蘭ol/L ryanodine, and 1 mmol/L caffeine) or blocking SR Ca2+ release (10 nmol/L ryanodine and 1 e蘭ol/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 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 2a-VMs from apoptosis. Direct activation of cytosolic Ca2+-dependent DNase I also does not appear to play a role in 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 2a-VMs.
The Role of the Mitochondria in 2a-VM Apoptosis
Many aspects of the current study, including cytochrome C release into the cytosol, support a central role of mitochondria in 2a overexpression-induced myocyte apoptosis. In addition, 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 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 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 2a-VMs (eC78.0±6.6 mV; n=9, among which 3 were spontaneously contracting) versus GFP-VMs (eC74.6±7.5 mV; n=9). These RPs are similar to those measured in freshly isolated feline VMs (eC74.9±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 activity5,6 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 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 e?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.).
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Torella D, Rota M, Nurzynska D, Musso E, Monsen A, Shiraishi I, Zias E, Walsh K, Rosenzweig A, Sussman MA, Urbanek K, Nadal-Ginard B, Kajstura J, Anversa P, Leri A. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ Res. 2004; 94: 514eC524.(Xiongwen Chen, Xiaoying Z)
the Department of Internal Medicine (S.T.P., J.D.M.), University of Arkansas for Medical Sciences, Little Rock.
Abstract
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 2a subunit-green fluorescent protein (GFP) fusion gene (Ad2a) to increase Ca2+ influx or with AdGFP as a control. Significantly fewer 2a-VMs (21.4±5.6%) than GFP-VMs (99.6±1.7%) were viable at 96 hours. A fraction of 2a-VMs (20.8±1.8%) contracted spontaneously (SC-2a-VMs), and viability was significantly correlated with the percentage of SC-2a-VMs. Higher percentages of apoptotic nuclei, DNA laddering, and cytochrome C release were detected in 2a-VMs. This apoptosis was prevented with pancaspase or caspase-3 or caspase-9 inhibitors. L-type calcium current (ICa-L) density was greater in 2a-VMs (23.4±2.8 pA/pF) than in GFP-VMs (7.6±1.6 pA/pF). SC-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 2a-VMs from apoptosis. These results show that persistent increases in Ca2+ influx through the ICa-L enhance contractility but lead to apoptosis through a mitochondrial death pathway if SR Ca2+ overload is induced.
Key Words: L-type calcium channel 2a subunit apoptosis ventricular myocyte primary culture
Introduction
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,5eC7 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 apoptosis11eC13 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 2a subunit of the L-type Ca2+ channel (Cav1.2 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 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 Ad2a-infected feline VMs (2a-VMs) had increased L-type calcium current (ICa-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
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 Cav1.22a-GFP fusion gene (Ad2a) 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 2a overexpression-induced cell death. ICa-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
Specificity of the Toxic Effect of 2a Overexpression
The effects of AdGFP and Ad2a 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 2a induced spontaneous contractions in a fraction of VMs, and the percentage of spontaneous contracting myocytes (SC-2a-VMs) increased with MOI (Figure 1B). There was a significant reduction in the viability of 2a-VMs with increased MOI. The percentage of SC-2a-VMs was significantly correlated with 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.
Transfection Efficiency of AdGFP and Ad2a 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 Ad2a, respectively. Expressed GFP was primarily distributed in the cytosol, whereas the 2a-GFP fusion protein was mainly concentrated on the surface membrane and T-tubules (Figure 1C and 1D). This 2a expression profile was slightly different from what was observed by Colecraft et al,20 who reported expressed 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 2a Induces Cell Death and Spontaneous Contraction in 2a-VMs
Overexpression of Cav1.2 2a caused a significant increase in cell death at 48, 72, and 96 hours after infection (Figure 1G). The viability of 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 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-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 2a-VMs was significantly correlated with the percentage of SC-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 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 2a-VMs. The percentage of TUNEL positive nuclei was significantly greater in VMs infected with Ad2a 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 2a-VMs (Figure 2B).
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 e蘭ol/L and ApoBlock 20 e蘭ol/L) and the caspase-3 specific inhibitor (z-DEVD-fmk 10 e蘭ol/L) increased 2a-VMs viability to >90% (Figure 2D) and decreased the percentage of SC-2a-VMs (Figure 2E). These results strongly suggest that the cell death induced by 2a overexpression is caused by caspase 3-dependent apoptosis. Notably, all 3 caspase inhibitors also significantly decreased the percentage of SC-2a-VMs, suggesting that activated caspases feed back to enhance the Ca2+ overload caused by increased Ca2+ influx.13
Overexpression of Cav1.2 2a Enhances Ca2+ Current and SR Ca2+ in Cultured VMs
2a overexpression is thought to significantly increase ICa-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 2a-VMs was characterized by measuring the density and voltage-dependent activation of ICa-L, contraction magnitude, and Ca2+ transient amplitude. ICa-L density was significantly greater (P<0.05) in 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 [V0.5, d] of Cav1.2 in 2a-VMs versus GFP-VMs: eC19.8±2.2 mV versus eC6.3±2.2mV, P<0.001; Figure 3). There was no significant difference (P=0.32) in ICa-L density between SC-2a-VMs (eC18.8±6.1 pA/pF, N=3) and quiescent 2a-VMs (eC25.3±3.4 pA/pF, N=7).
Field-stimulated myocyte fractional shortening (Figure 4) was significantly (P<0.01) greater in 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 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 2a-VMs was not significant (P=0.09) (Figure 4A and 4B). Peak systolic Ca2+ was significantly greater in 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 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-2a-VMs and quiescent 2a-VMs. Diastolic Ca2+ in SC-2a-VMs (1.07±0.12, n=10, N=4) was significantly greater (Figure 4C) than in quiescent 2a-VMs (0.73±0.04, n=18, N=4) and GFP-VMs (0.73±0.03, n=24, N=4).
These results show that infecting adult feline myocytes with Cav1.2 2a increases ICa-L and shifts its voltage-dependence of activation to negative potentials, which should increase Ca2+ influx through Cav1.2 in resting 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 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 2a-VMs (Figure 5). Nifedipine (13 e蘭ol/L), a Cav1.2 blocker, prevented apoptosis and BAPTA-AM (1 e蘭ol/L), a cell permeable Ca2+ buffer, largely protected 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 e蘭ol/L and caffeine 1 mmol/L) partially rescued 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 2a overexpression. It is worth noting that higher concentrations of some SR-modifying drugs induced rather than blocked apoptosis in GFP-VMs and 2a-VMs. For example, 10 e蘭ol/L BAPTA-AM, 1 e蘭ol/L thapsigargin, and 10 mmol/L caffeine induced apoptosis in GFP-VMs within 2 days and in 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
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 2a-VMs. No pS16-PLB was detected in either group, but the phosphorylation at T17 was significantly greater in 2a-VMs (Figure 5 C and 5D). The increased phosphorylation of PLB at T17 should increase SERCA activity24 and promote SR Ca2+ overload in 2a-VMs.
CaMK II is known to be involved in myocyte apoptosis7 induced by -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 e蘭ol/L; KN 93, 1 e蘭ol/L; and AIP, 20 e蘭ol/L) were used to test the role of CaMK II in apoptosis induced by 2a overexpression (Figure 5A). KN 62 (VM viability: 90.1±3.0%) and KN 93 (VM viability: 99.5±3.8%) almost fully protected 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 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 2a-Induced Apoptosis in Cultured VMs
Experiments with z-DEVD-fmk suggest that activation of caspase-3 is a critical step in 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 2a-overexpression (Figure 6A). In addition, significant amounts of cytochrome C were found in the cytosolic fraction of 2a-VMs but not GFP-VMs (Figure 6C), documenting a central role of mitochondria in Ca2+-mediated apoptosis.
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 2a-VM viability, suggesting that both the mPTP and the BAX/BAK complex are involved in 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 e蘭ol/L dantrolene (inhibiting both ryanodine and IP3 receptors) prevented spontaneous contractions (SR Ca2+ release) and significantly reduced apoptosis in 2a-VMs (Figure 7A and 7B). These data show that Ca2+ release from an overloaded SR is necessary and sufficient to induce apoptosis in 2a-VMs.
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 e蘭ol/L), did not alter 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 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 e?calpain (micro-calpain, activated by Ca2+ in micromolar range) was found to be cleaved in 2a-VMs at 48 hours after infection (Figure 6D).
Discussion
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 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 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 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 Ad2a MOI used and the use of cultured rat VMs, which have SR Ca2+ overload when unpaced.21
The Role of SR Ca2+ Overload in 2a-VM Apoptosis
The present experiments show that persistent increases in Ca2+ influx through Cav1.2 in 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 2a-induced apoptosis is supported by the fact that reduced SR Ca2+ loading (nifedipine, 1 e蘭ol/L ryanodine, and 1 mmol/L caffeine) or blocking SR Ca2+ release (10 nmol/L ryanodine and 1 e蘭ol/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 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 2a-VMs from apoptosis. Direct activation of cytosolic Ca2+-dependent DNase I also does not appear to play a role in 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 2a-VMs.
The Role of the Mitochondria in 2a-VM Apoptosis
Many aspects of the current study, including cytochrome C release into the cytosol, support a central role of mitochondria in 2a overexpression-induced myocyte apoptosis. In addition, 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 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 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 2a-VMs (eC78.0±6.6 mV; n=9, among which 3 were spontaneously contracting) versus GFP-VMs (eC74.6±7.5 mV; n=9). These RPs are similar to those measured in freshly isolated feline VMs (eC74.9±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 activity5,6 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 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 e?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.).
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