Myocyte contractile activity modulates norepinephrine cytotoxicity and survival effects of neuregulin-1{beta}

Yukio Kuramochi, Chee Chew Lim, Xinxin Guo, Wilson S. Colucci, Ronglih Liao, and Douglas B. Sawyer

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


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of this study is to test the hypothesis that mechanical and electrical activity in adult rat ventricular myocytes (ARVM) alters responses to proapoptotic and prosurvival ligands. The effects of electrical stimulation on myocyte survival, stress signaling, response to {beta}-adrenergic receptor ({beta}-AR)-stimulated apoptosis, and neuregulin-1{beta} (NRG) were examined. Electrical stimulation (6.6 V/cm; 0, 2, and 5 Hz; 2-ms duration; alternating polarity) of ARVM resulted in more than 70% capture. Although ARVM paced for 48 h showed higher mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (P < 0.05, 0 vs. 2 and 5 Hz), electrical stimulation had little effect on cell survival assessed by trypan blue uptake, CPK release, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Electrical stimulation for 24 h did not induce stress response (heat shock protein 70, 90) nor stress kinase (Erk, JNK, p38) activation. NRG stimulation of Erk and Akt was similar between paced and quiescent cells. Pacing sensitized myocytes to {beta}-AR-stimulated JNK phosphorylation and cell death with 0.1 µM norepinephrine (NE) in paced myocytes causing equivalent cytotoxicity to 10 µM NE in quiescent cells. NRG suppressed {beta}-AR-induced apoptosis through a phosphatidylinositol-3-kinase-dependent pathway in both paced and quiescent cells, although it is overwhelmed by high-NE concentration in paced cells. Thus myocyte contractility modulates both NE cytotoxicity as well as the cytoprotective effect of NRG. These results demonstrate the feasibility and importance of using electrically paced cardiomyocytes in primary culture when examining the signaling pathways of cell survival.

adult rat ventricular myocytes; apoptosis; {beta}-adrenergic receptor; electrical stimulation


ADULT RAT VENTRICULAR MYOCYTES (ARVM) are quiescent in primary culture. Although much is known about proapoptotic and prosurvival signaling in these quiescent cells, the effects of electrical pacing on cell survival have not been examined. The purpose of this study is to test the hypothesis that mechanical and electrical activity in ARVM alters responses to proapoptotic and prosurvival ligands. We investigated the effect of electrical pacing on {beta}-adrebergic receptor ({beta}-AR) stimulation and neuregulin-1{beta} (NRG) as proapoptotic and prosurvival stimuli, respectively.

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 {beta}-AR stimulation is associated with adverse effects on cardiac structure and function, including myocyte loss by both apoptosis and necrosis (6, 12, 28). {beta}-AR stimulation of ARVM in vitro induces apoptosis (8), and overexpression of {beta}-AR leads to progressive cardiac dysfunction in association with myocyte apoptosis (5, 14). The mechanism for {beta}-AR-stimulated apoptosis appears to involve JNK- and oxidative stress-induced activation of the mitochondrial death pathways (38). The adverse effects of chronic {beta}-AR stimulation have been implicated in the beneficial effects of {beta}-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 {beta}-AR-stimulated activation of JNK as well as cytotoxicity. Furthermore, we found that NRG protected myocytes from {beta}-AR-induced apoptosis via a PI3K-dependent pathway in both quiescent and paced cells.


    METHODS
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 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell preparation and electrical stimulation. ARVM were isolated as previously reported (8) and plated at densities of 80–150 myocytes/mm2 on four-well rectangular plates or 40 x 22-mm glass coverslips precoated with laminin (Becton-Dickinson). One hour after cell preparation, medium was changed to DMEM supplemented with albumin, creatine, carnitine, and taurine (ACCT media), with the addition of ascorbic acid (100 µM). ARVM were stimulated with carbon electrodes using a culture cell pacer system from IonOptix (Milton, MA). Stimulus parameters were 6.6 V/cm (48), and the duration was 2 ms, with alternating polarity. Electrical stimulation was performed at 0, 2, and 5 Hz. Under these conditions, we obtained ~70% capture of myocytes.

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 {alpha}-AR stimulation (NE{alpha}) and at 10.0, 1.0, and 0.1 µM for {beta}-AR stimulation (NE{beta}) 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 {beta}-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.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic electrical stimulation increases mitochondrial activity with minimal effect on cell survival. The effects of electrical pacing on myocyte survival have not previously been reported. We therefore compared survival of ARVM in culture with or without electrical stimulation for up to 48 h. Electrical stimulation was performed at 0, 2, and 5 Hz using 2-ms pulse duration and 6.6-V/cm stimuli with alternating polarity. These conditions result in more than 70% capture of myocytes. MTT uptake was not different between unpaced and paced myocytes after 24 h of pacing. By 48 h, the MTT uptake was higher in the paced myocytes (P < 0.05, 0 vs. 2 and 5 Hz). Although MTT uptake is a measure of cell viability, we saw no systematic difference in trypan blue uptake and CK release in the culture media. The MTT results must therefore reflect an increase in mitochondrial respiration per cell in the paced myocytes. As CK release and trypan blue uptake do not detect early changes in apoptosis, we measured the number of TUNEL-positive cells. We found no difference in the number of TUNEL-positive myocytes among conditions at 24 h. By 48 h, myocytes paced at 2 Hz for 48 h demonstrated a small but significant reduction in apoptosis compared with both unpaced myocytes and those stimulated at 5 Hz (Fig. 1). Despite this small difference, we conclude that there is little, if any, effect of electrical pacing on cell survival.



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Fig. 1. Chronic electrical stimulation of adult rat ventricular myocytes (ARVM) increases 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake with minimal change in myocyte viability. A: results of MTT assay are normalized to quiescent conditions (n = 8). *P < 0.05 vs. 0 Hz. B: percentage of apoptosis assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (n = 5). *P < 0.05 vs. 0 and 5 Hz at 48 h. C: percentage of cell death of unfixed cells assessed by trypan blue uptake (n = 6). D: CPK assay of culture media (n = 6). P = not significant.

 

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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Fig. 2. Electrical stimulation has minimal effect on cell stress and does not alter neuregulin-1{beta} (NRG)-activated Erk1/2 and Akt signaling. Myocytes are left quiescent or electrically paced for 24 h. Basal expression of heat shock protein (HSP)70 and HSP90 (n = 5; A) and levels of phosphorylated Erk1/2 and Akt are evaluated by Western blot analysis after 15 min of NRG treatment (n = 6; B) or {alpha}-adrenergic receptor (AR) stimulation [1 µM norepinephrine (NE) with a pretreatment of propranorol; see METHODS] (n = 4; C).

 


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Fig. 3. Electrical stimulation modulates NE{beta}-induced JNK activation. Myocytes are left quiescent or electrically paced for 24 h. Levels of phosphorylated JNK (n = 6; A) and p38 (n = 4; B) are examined after 15 min of stimulation with NE{beta} at 10 µM. *P < 0.05 vs. 0 Hz with {beta}-AR stimulation.

 

Electrical stimulation enhances NE{beta}-induced JNK phosphorylation without altering p38 activation. We examined short-term responses to NRG, NE{alpha}, or NE{beta} stimulation in quiescent and paced ARVM for 24 h. NRG-stimulated Erk1/2 and Akt activation as well as {alpha}-AR-stimulated Erk1/2 phosphorylation were similar in quiescent and paced cells (Fig. 2, B and C). In contrast, pacing augmented {beta}-AR-stimulated activation of JNK (Fig. 3A). Phosphorylation of p38 in response to NE{beta} was not different in paced vs. quiescent cells (Fig. 3B).

Pacing augments NE{beta} cytotoxicity. The increased JNK activation by NE{beta} in paced myocytes suggests greater sensitivity to NE{beta} in the setting of electrical pacing. We therefore examined the morphological alterations and apoptosis of ARVM with or without NE{beta} (10 µM NE) for 18 h. Cell shape did not change by electrical stimulation alone, but the combination of NE{beta} 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{beta}]µM, comparing NE{beta}-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{beta}]µM tested (10 µM), whereas in paced myocytes cell death occurred at [NE{beta}]µM as low as 0.1 µM (Fig. 4C). Similarly, while 0.1 µM NE{beta} 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{beta} is due to a rise in [Ca2+]i.



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Fig. 4. Pacing sensitizes ARVM to NE{beta}-induced apoptosis. Myocytes are plated and paced at 0, 2, and 5 Hz. One hour after starting electrical stimulation, {beta}-AR stimulation (with 10 µM NE) of myocytes is performed for 18 h. A: microscopic images with or without {beta}-AR stimulation show increased numbers of rounded cells under paced conditions (original magnification: x200). B: percentage of TUNEL-positive myocytes with or without {beta}-AR stimulation (n = 4; *P < 0.05 vs. 0 and 5 Hz with NE{beta}, {dagger}P < 0.05 vs. 0 and 2 Hz with NE{beta}). C: myocytes were treated with NE{beta} at 0.1, 1.0, and 10.0 µM for 18 h. Then, cell viability was accessed by trypan blue uptake (n = 4; *P < 0.05 between 0 and 5 Hz, {dagger}P < 0.05 among 3 frequencies).

 


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Fig. 5. Pacing sensitizes ARVM to NE{beta}-induced JNK activation. Myocytes are left quiescent or electrically paced for 24 h. Phosphorylated-JNK, phosphorylated-p38, and total JNK are examined by Western blot analysis with [NE{beta}]0.1µM for 15 min (A) and after incubation with ionomycin (10 µM) for indicated periods of times (B). Blots are representative of different 3 experiments.

 

NRG protects ARVM from NE{beta}-induced apoptosis through PI3K pathway activation. We examined the effect of NRG on {beta}-AR-stimulated apoptosis. At [NE{beta}]0.1µM in paced myocytes, NRG protected ARVM from apoptosis (Fig. 6, A-C). At [NE{beta}]10µM, however, quiescent but not paced cells were rescued by NRG pretreatment (Fig. 6D). Thus NRG suppression of NE{beta}-induced cell apoptosis is dependent on the pacing frequency and [NE{beta}]µM.



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Fig. 6. NRG protects ARVM from NE{beta}-induced apoptosis. A: cells paced at 5 Hz are incubated with indicated treatment for 18 h, and apoptosis is examined by TUNEL staining. NRG treatments are started 30 min before NE{beta} stimulation. Phase-contrast views of TUNEL staining (top, arrowhead means TUNEL-positive cell), light microscopic view (middle), and DAPI (bottom) staining are shown (original magnification: x200, scale bar = 100 µm). B: TUNEL staining at high magnification (x400, scale bar = 100 µm). Arrow means the TUNEL-positive cell in which chromatin condensation is obvious on DAPI staining. Top: TUNEL staining; bottom: DAPI staining. C: quantification of TUNEL-positive myocytes. In 5-Hz paced cells, [NE{beta}]0.1µM causes almost the same percentage of apoptosis as quiescent cells treated with [NE{beta}]10µM. NRG protects cells from apoptosis under both conditions. The protective effect of NRG is completely blocked by phosphatidylinositol-3-kinase inhibitor LY-294002 (LY; n = 5). *P < 0.05 vs. control (no treatment), NE{beta}, LY + NE{beta}, and LY + NRG + NE{beta} at the same frequency; {dagger}P < 0.05 vs. NE{beta}, LY + NE{beta}, and LY + NRG + NE{beta} at the same frequency; {ddagger}P < 0.05 vs. control, NE{beta}, LY + NE{beta}, and LY + NRG + NE{beta} at the same frequency. D: pretreatment with NRG for 30 min suppresses NE{beta} (10 µM)-induced apoptosis in quiescent cells but not in paced cells (n = 4). *P < 0.05 vs. control, NRG, and NRG + NE{beta} at the same frequency; {dagger}P < 0.05 vs. control and NRG.

 

NRG protection from NE{beta} apoptosis was prevented by the PI3K inhibitor LY-294002 (Fig. 6, A-C). NE{beta}-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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Fig. 7. NRG suppresses cytochrome c release from mitochondria and caspase-3 processing induced by {beta}-AR stimulation. Cells were pretreated with or without LY-294002 and/or NRG after pacing overnight, followed by {beta}-AR stimulation. Then, cells were lysed 3 h after treatment. A and B: cytosol and noncytosol fractions were analyzed by Western blot analysis. Data are representative of 4 different experiments. C: total cell lysates were blotted with anti-caspase-3 that recognizes both procaspase-3 and its processed, activated fragment.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ARVM contract about five times per second in vivo, but upon isolation and placement in cell culture they beat rarely. These nonphysiological conditions acutely lower oxygen consumption (40) and chronically lead to changes in metabolic enzyme expression (51), decline in contractile properties (13, 20, 23), and change in fatty acid uptake (27). Electrical field stimulation has been used to maintain contractile function and calcium transients in ARVM and adult feline cardiomyocytes (4, 19, 23). However, the effects of electrical field stimulation have not been considered systematically in studies of myocyte survival. Some of these pacing systems require higher voltage or longer duration, and in earlier work there appeared to be deleterious effects on cell stress (24) and survival (Sawyer DB, unpublished observation). In the present study, using a commercially available system, we were able to find stimulus parameters that allowed field stimulation in the absence of molecular evidence of cell stress, without worsening myocyte survival. In fact, under conditions of low-frequency stimulation (2 Hz), myocyte survival improved, albeit only slightly. Intuitively, it makes sense that myocyte survival would improve in vitro under more physiological conditions. We anticipate that refinement in the stimulation protocols and/or culture conditions will lead to more gains in myocyte stability and survival in culture.

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{alpha}, 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{beta} was markedly altered by electrical pacing, with ~100-fold decrease in the [NE{beta}]µM necessary to induce cell death at physiological frequencies. Our findings support the notion that NE{beta} may contribute to adverse myocardial remodeling through the induction of apoptosis in vivo. Interestingly, the [NE{beta}]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{beta} 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{beta}-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 {beta}-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{beta} (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{beta} 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 {beta}-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 {beta}-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 {beta}-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 {beta}-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 {beta}-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{beta}]µM in paced myocytes. Furthermore, pretreatment of NRG suppressed the cytochrome c release from mitochondria and caspase-3 processing. {beta}-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.


    ACKNOWLEDGMENTS
 
We thank M. Marchionni for recombinant NRG.

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).


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. B. Sawyer, Myocardial Biology Unit, Boston Medical Center, 650 Albany St., Boston, MA 02118 (E-mail: Douglas.Sawyer{at}bmc.org).

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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