Whitaker Cardiovascular Institute and Cardiovascular Division, Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118
Submitted 21 July 2003 ; accepted in final form 25 September 2003
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ABSTRACT |
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adult rat ventricular myocytes; apoptosis; -adrenergic receptor; electrical stimulation
NRG and its receptors, erbB2 and erbB4, are expressed in cardiac microvascular endothelium and in ventricular myocytes, respectively (53). The importance of NRG-erbB signaling pathway in the heart is underscored by targeted gene disruption studies that reveal that NRG and the erbB2/B4 receptors are essential for normal cardiac development (15, 26, 32). NRG induces erbB2 and erbB4 receptor phosphorylation in cardiac myocytes, leading to activation of phosphatidylinositol-3-kinase (PI3K) and Akt (21), a pathway with important metabolic and cytoprotective functions (2, 16, 53). NRG induces hypertrophy and suppresses baseline apoptosis in neonatal and adult rat ventricular myocytes (NRVM and ARVM) (2, 53). Accordingly, NRG expression in the postnatal heart may serve as an important growth and survival factor.
Chronic -AR stimulation is associated with adverse effects on cardiac structure and function, including myocyte loss by both apoptosis and necrosis (6, 12, 28).
-AR stimulation of ARVM in vitro induces apoptosis (8), and overexpression of
-AR leads to progressive cardiac dysfunction in association with myocyte apoptosis (5, 14). The mechanism for
-AR-stimulated apoptosis appears to involve JNK- and oxidative stress-induced activation of the mitochondrial death pathways (38). The adverse effects of chronic
-AR stimulation have been implicated in the beneficial effects of
-AR blockers in patients with contractile dysfunction (3, 37, 43).
In this study, we used a commercially available cell culture pacing system to maintain myocyte contractile activity. This system was optimized to stimulate contraction of myocytes without altering basal levels of cell stress and signaling. We demonstrate that electrical stimulation sensitizes myocytes to -AR-stimulated activation of JNK as well as cytotoxicity. Furthermore, we found that NRG protected myocytes from
-AR-induced apoptosis via a PI3K-dependent pathway in both quiescent and paced cells.
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METHODS |
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Cell treatment. The recombinant NRG, glial growth factor 2 (courtesy of M. Marchionni), was used at 10 ng/ml. L-Norepinephrine (NE; Sigma) was used at 1 µM for -AR stimulation (NE
) and at 10.0, 1.0, and 0.1 µM for
-AR stimulation (NE
) after pretreatment with either propranolol (2 µM; Sigma) or prazosin (100 nM; Sigma), respectively. Preincubation with the PI3K inhibitor LY-294002 (Calbiochem) was for 60 min before NRG treatment. Myocytes were treated with ionomycin (Sigma) at 10 µM. To evaluate the cell signaling, cells were maintained in culture room for study. For cell viability and apoptosis studies in response to
-AR stimulation, cell treatments were started 2 h after plating.
Measurement of mitochondrial respiration. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction was used as a measurement of mitochondrial respiration. Cells were incubated with 0.2 mg/ml of MTT in the culture media at 37°C for 2 h. Cells were lysed with DMSO, and absorbance at 575 nm was measured after addition of Sorensen's glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5).
Cell viability and apoptosis. Cell viability was assessed by measurement of creatine kinase (CK) release into culture media (CK-10, Sigma) and by trypan blue uptake (22). Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay using in situ cell death detection kit (Roche). The percentage of nonviable trypan blue-positive cells and TUNEL-positive cells was determined by randomly counting 300 cells in each well or coverslip.
Western blot analysis. Heat shock protein (HSP)70 and HSP90 antibodies were from Stressgen, whereas anti-actin was obtained from Sigma. Antibodies against phospho-Akt, Akt, phospho-Erk1/2, and phospho-p38 were from New England Biolab. Anti-Erk2, p38, phospho-JNK, and JNK were from Santa Cruz Biotechnology. Cytochrome c antibody was from Calbiochem.
For the detection of cytochrome c release from mitochondria, cytosol fraction is extracted with digitonon lysis buffer (17). The residual organellae are lysed with modified RIPA buffer (1% NP-40, 50 mM Tris·HCl, 1 mM EDTA, 0.25% DOC, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin, 1 mM sodium orthovandidate). Aliquots representing 5 µg of protein from digitonin-permeabilized cytosolic and noncytosolic fractions were used. To detect other proteins, 50- to 100-µg aliquots of total cell lysates were used. Sample proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane (Bio-Rad). After membrane development with ECL reagent (Pierce), quantification was performed by densitometry (Molecular Analyst, Bio-Rad).
Statistical analysis. Results are expressed as means ± SD of at least three different experiments. One-way ANOVA was used for multiple comparison, with Bonferroni posttest analysis. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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Chronic electrical pacing does not change MAPK activity and expression of HSP70/90 at baseline. We examined the effect of electrical stimulation on cell "stress" using mitogen- and stress-activated protein kinases (Erk, JNK, p38) and HSP70/90 expression as stress indicators after 24 h of pacing. At this time point, there was no evidence of either kinase activation or changes in HSP expression (Figs. 2 and 3).
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Electrical stimulation enhances NE-induced JNK phosphorylation without altering p38 activation. We examined short-term responses to NRG, NE
, or NE
stimulation in quiescent and paced ARVM for 24 h. NRG-stimulated Erk1/2 and Akt activation as well as
-AR-stimulated Erk1/2 phosphorylation were similar in quiescent and paced cells (Fig. 2, B and C). In contrast, pacing augmented
-AR-stimulated activation of JNK (Fig. 3A). Phosphorylation of p38 in response to NE
was not different in paced vs. quiescent cells (Fig. 3B).
Pacing augments NE cytotoxicity. The increased JNK activation by NE
in paced myocytes suggests greater sensitivity to NE
in the setting of electrical pacing. We therefore examined the morphological alterations and apoptosis of ARVM with or without NE
(10 µM NE) for 18 h. Cell shape did not change by electrical stimulation alone, but the combination of NE
and pacing increased the number of rounded cells with membrane-blebs (Fig. 4A) as well as TUNEL-positive cells in a frequency-dependent manner (Fig. 4B). We examined the effect of pacing on the cytotoxic threshold of [NE
]µM, comparing NE
-induced cell death at 18 h as well as activation of JNK and p38 at 15 min. In quiescent myocytes, cell death was only increased at the high [NE
]µM tested (10 µM), whereas in paced myocytes cell death occurred at [NE
]µM as low as 0.1 µM (Fig. 4C). Similarly, while 0.1 µM NE
did not activate JNK in quiescent ARVM, there was robust activation of JNK in ARVM paced at 5 Hz (Fig. 5A). Furthermore, we treated cells with the Ca2+ ionophore ionomycin at 10 µM to determine if JNK activation is in response to increased [Ca2+]i and observed immediate JNK phosphorylation (Fig. 5B). This result suggests the possibility that the increased JNK activation seen in paced myocytes in response to NE
is due to a rise in [Ca2+]i.
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NRG protects ARVM from NE-induced apoptosis through PI3K pathway activation. We examined the effect of NRG on
-AR-stimulated apoptosis. At [NE
]0.1µM in paced myocytes, NRG protected ARVM from apoptosis (Fig. 6, A-C). At [NE
]10µM, however, quiescent but not paced cells were rescued by NRG pretreatment (Fig. 6D). Thus NRG suppression of NE
-induced cell apoptosis is dependent on the pacing frequency and [NE
]µM.
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NRG protection from NE apoptosis was prevented by the PI3K inhibitor LY-294002 (Fig. 6, A-C). NE
-induced cytochrome c release from mitochondria and caspase-3 activation (Fig. 7), steps required for the apoptotic cascade (38), were also suppressed by NRG in a PI3K-dependent manner.
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DISCUSSION |
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Consistent with increased contractility, mitochondrial respiration as measured by MTT uptake was significantly higher in paced ARVM than quiescent cells after 48 h of electrical stimulation. This is consistent with work in NRVM showing that electrical pacing produced cellular maturation with increased mitochondrial content and activity (50, 51). Although mitochondrial content was not measured in the present study, we found that mRNA expression of muscle-specific carnitine palmytoyltranferase-I, an enzyme for long-chain fatty acid oxidation in mitochondria (7), is increased in ARVM after 48 h of pacing by real-time PCR analysis (Kuramochi Y, unpublished observation). However, higher mitochondrial uptake of MTT did not completely correlate with cell viability and apoptosis in this study. Further work is needed, perhaps with metabolic substrate alterations, to better optimize cell survival in paced myocytes.
Chronic electrical stimulation with this system at frequencies up to 5 Hz did not induce changes in HSP70/90 expression nor activation of Erk1/2, p38, and JNK, which we interpret as evidence for minimal cell stress induced by pacing. This is in contrast to reports that short-term pacing induces JNK activation in NRVM (30, 46). This discrepancy may be explained by differences in the cell pacer device, timing of experiments, and conditions employed (11), or cell phenotype studied. Certainly, electrical stimulation at sufficient currents will induce oxidation of lipid and media, with deleterious effects on cell stress. The lack of baseline activation of stress signaling with preserved responses to ligand-activated signaling, in this case NRG and NE, shows the feasibility of using this pacing system to study myocyte hypertrophy under these more physiological conditions.
The sensitivity of myocytes to the cytotoxic effects of NE was markedly altered by electrical pacing, with
100-fold decrease in the [NE
]µM necessary to induce cell death at physiological frequencies. Our findings support the notion that NE
may contribute to adverse myocardial remodeling through the induction of apoptosis in vivo. Interestingly, the [NE
]µM that induced apoptosis in paced cells was much closer to that reported in animal models of cardiac failure than that in quiescent cells (34, 36, 42), suggesting the pacing system in more closely approximating in vivo conditions. Although the exact mechanism for increased NE
sensitivity in paced ARVM remains to be elucidated, we suspect it is related, at least in part, to an increase in [Ca2+]i that occurs in electrically stimulated myocytes (4, 19). In quiescent ARVM, NE
-induced apoptosis can be inhibited with an L-type Ca2+ channel blocker (8). It is well known that prolonged [Ca2+]i elevations can induce cell death (29, 41, 45, 54). Electrical stimulation alone increases free Ca2+ content only during systole (4, 19), whereas the combination of
-AR stimulation and pacing acts synergistically to increase both systolic and diastolic [Ca2+]i through several mechanisms (54). Prolonged increases in [Ca2+]i act through one or more pathways including potentiating calmodulin-dependent protein kinase II (54), increases in sarcoplasmic reticulum Ca2+ (44), and frequency-dependent Thr17 phosphorylation of phospholamban (18). Moreover, mitochondrial Ca2+ also increases with pacing frequency in the presence of NE
(33). These mechanisms may raise endoplasmic reticulum and mitochondrial Ca2+ load and perhaps lead to apoptosis by influencing mitochondrial permeability transition pore (9, 10, 49).
We found that JNK phosphorylation in response to NE was markedly augmented in paced ARVM. The increased JNK activation paralleled the rise in cell death and is consistent with our recent finding that JNK activation is required for
-AR-induced apoptosis in ARVM (38). The calcium ionophore ionomycin also induced JNK activation, suggesting a link between increases in [Ca2+]i and JNK activation. In contrast to JNK, p38 phosphorylation after
-AR stimulation was like an "on/off" switch, independent of pacing, and by extension [Ca2+]i and metabolism. These data imply that p38 activation is more tightly coupled to the
-AR activation than JNK and hence is not influenced by environmental conditions. Further studies are necessary to fully elucidate the interaction between Ca2+ homeostasis, reactive oxygen species (ROS), and activated JNK/p38, which appear to collectively regulate the life or death of a myocyte in the presence of
-AR stimulation (25, 47, 52).
In the intact heart, of course, there are many other factors that mediate myocyte fate, including NRG. Acting through the erbB2 and erbB4 receptor tyrosine kinases, NRG activates both PI3K/Akt and Erk1/2 pathways, both of which have been implicated in modulation of cell survival (16, 53). NRG is expressed in cardiac microvascular endothelial cells (53). The finding that NRG prevents -AR-stimulated apoptosis in ARVM is consistent with the idea that NRG is among the endogenous cardiac growth factors that act to preserve cardiac structure and function in the presence of stress. The protective effect of NRG in ARVM occurs through a PI3K-dependent pathway, although it is overwhelmed by high [NE
]µM in paced myocytes. Furthermore, pretreatment of NRG suppressed the cytochrome c release from mitochondria and caspase-3 processing.
-AR-stimulated apoptosis in ARVM can be inhibited by antioxidants (35, 36), suggesting one potential mechanism for NRG protection is the modulation of oxidative stress (16), either through suppressing generation of ROS or increasing the activity of endogenous antioxidant enzymes. Thus NRG cytoprotection may be similar to the actions of insulin and insulin-like growth factor-I (IGF-I), which activate the PI3K/Akt pathway (1, 31) and inhibit ROS-stimulated apoptosis in other experimental systems.
Although the NRG/erbB signaling system shares many features with insulin, IGF-I, and other growth factors, there is an absolute requirement for NRG and its receptors for the maintenance of normal cardiac structure and function. In mice lacking erbB2 or erbB4 in the myocyte, cardiac failure develops in the absence of overt stress (15, 26, 32). These mice appear to have increased levels of myocyte loss from apoptosis. Interestingly, in a pressure overload model of myocardial failure, downregulation of erbB2 and erbB4 receptors was observed (39). Our current observations support the thesis that downregulation of NRG receptors, and therefore NRG signaling, might be mechanistic in the progression of cardiac failure in these models. Moreover, strategies to augment NRG/erbB signaling in the heart may be beneficial in preventing the progression of heart failure.
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ACKNOWLEDGMENTS |
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GRANTS
This study is funded by Grants HL-20612 to W. S. Colucci and HL-03878, HL-68144, and a grant from the Juvenile Diabetes Research Foundation (to D. B. Sawyer).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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