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Experimental Physiology 91.4 pp 773-780
DOI: 10.1113/expphysiol.2006.033894
© The Physiological Society 2006
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Rapid electrical stimulation induces early activation of kinase signal transduction pathways and apoptosis in adult rat ventricular myocytes

Yukio Kuramochi1, Xinxin Guo1, Douglas B. Sawyer1 and Chee Chew Lim1

1 Molecular Stress Response Unit, Whitaker Cardiovascular Institute, Department of Medicine, Boston University Medical Center, Boston, MA, USA


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Chronic tachycardia in patients and rapid pacing in animal models induce myocardial dysfunction and initiate a cascade of compensatory adaptations that are ultimately unsustainable, leading to ventricular enlargement and failure. The molecular pathogenesis during the early stages of tachycardia-induced cardiomyopathy, however, remains unclear. We utilized our previously reported cell culture pacing system to directly assess phosphatidylinositol-3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signalling of adult rat ventricular myocytes (ARVM) in response to rapid electrical stimulation. Freshly isolated ARVMs were maintained quiescent (0 Hz), or continuously stimulated at 5 (normofrequency) and 8 Hz (rapid frequency). Pacing resulted in an increase in mitochondrial respiration, assessed by mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 48 h. Rapid pacing at 8 Hz significantly increased cell injury and death as assessed by Trypan Blue uptake, creatine phosphokinase release, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Pacing at 5 Hz induced early, but weak, activation of Akt and protein kinase 38 (p38). Rapid pacing further augmented the early activation of Akt and p38, and induced extracellular signal-related kinase (Erk) and c-jun amino terminal kinase (JNK) activation. Incubation of ARVM with PI3K inhibitor LY294002 resulted in a twofold increase of TUNEL-positive cells under all pacing conditions examined. In conclusion, rapid pacing has immediate and detrimental consequences for cardiomyocyte survival, with pro-apoptotic pathways (e.g. JNK, p38) able to overwhelm antiapoptotic signalling (PI3K/Akt, Erk). The rapid pacing methodology described in this report will be particularly useful in determination of cell signalling pathways associated with tachycardia-induced cardiomyopathy.

(Received 14 March 2006; accepted after revision 28 April 2006; first published online 4 May 2006)
Corresponding author C. C. Lim: Molecular Stress Response Unit, 650 Albany Street, X-314, Boston University Medical Center, Boston, MA 02118, USA. Email: cheelim{at}bu.edu


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Human and in vivo animal studies have shown that tachycardia induces a host of pathogenic alterations, including myocyte hypertrophy, apoptosis, fibrotic deposition and infiltration of inflammatory cells, with consequent development of heart failure (Spinale et al. 1994; Valente et al. 1998; Li et al. 2001; Nakamura et al. 2002; Hanna et al. 2004). The use of in vivo rapid pacing models has provided valuable insight into the mechanistic events of tachycardia-induced heart failure (Li et al. 2001; Nakamura et al. 2002; Qin et al. 2003; Schulz et al. 2003). In vivo models, however, are often associated with confounding variables, complicating interpretation of signalling events at the cellular level. We previously reported the use of a commercially available in vitro cell culture pacing system, optimized to continuously pace adult rat ventricular myocytes (ARVM), in the absence of cell stress at normal and low rat heart rates (5 and 2 Hz; Kuramochi et al. 2004). Using the same pacing system, this study was designed to examine the effect of supraphysiological pacing frequencies on myocyte survival and stress signalling. We compared the effect of stimulation of ARVM at three different frequencies: quiescent (0 Hz), normofrequency (5 Hz), and rapid frequency (8 Hz). We demonstrate that rapid pacing induces early c-jun amino terminal kinase (JNK) activation and enhances cytotoxicity in cardiomyocytes. Furthermore, concomitant activation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway partly attenuates but is unable to completely overcome cytotoxicity in these rapidly paced myocytes.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Myocyte preparation, electrical stimulation and treatment

Calcium-tolerant adult rat ventricular myocytes (ARVM) were obtained from hearts of male Sprague–Dawley rats (240–260 g) as previously described (Communal et al. 1998). All procedures were approved by the Institutional Animal Care and Use Committee at Boston University School of Medicine and conducted in accordance with the NIH guidelines for animal care. Briefly, animals were anaesthetized with sodium pentobarbitone (50 mg kg–1 I.P.) and heparinized (1000 IU kg–1 I.V.), and their hearts were aseptically removed into an ice-cold modified cardioplegic solution (KB solution, in mmol l–1: KOH, 85; KCl, 30; KH2PO4, 30; MgSO4, 3; EGTA, 0.5; Hepes, 10; L-glutamic acid, 50; and taurine, 20; at pH 7.4, pH adjusted using NaOH). The hearts were retrogradely perfused on a Langendorff apparatus with Tyrode solution (in mmol –1: NaCl, 137; KCl, 5.4; CaCl2, 1.2; MgCl2, 0.5; Hepes, 10; and glucose, 10; at pH 7.4, pH adjusted using NaOH) for 5 min at 37°C. The perfusion solution was switched to a nominally Ca2+-free Tyrode solution for 6 min and then to a nominally Ca2+-free Tyrode solution containing 0.02% protease (Sigma) and 0.06% collagenase A (Boehringer Mannheim). After 10–15 min, the enzymatic solution was washed out with KB solution for an additional 5 min. After perfusion, cells from the left ventricle were released by shaking the tissue. The cells were filtered through a 150 nm mesh and allowed to settle (40 min) in KB solution. The cells were then resuspended in DMEM (Gibco), layered over 60 µg ml–1 BSA (Sigma) to separate ventricular myocytes from non-myocytes, and allowed to settle for 10–15 min. Cells were resuspended in DMEM supplemented with albumin, creatine, carnitine, and taurine (ACCT media): 2 mg ml–1 BSA, 2 mmol l–1 L-carnitine, 5 mmol l–1 creatine, 5 mmol l–1 taurine, 100 IU ml–1 penicillin and 100 mg ml–1 streptomycin. Adult rat ventricular myocytes were plated at densities of 80–150 myocytes mm–2 on four-well rectangular plates or 40 mm x 22 mm glass coverslips precoated with laminin (Becton-Dickinson). One hour after cell preparation, medium was changed to ACCT media supplemented with 100 mmol l–1 ascorbic acid. Adult rat ventricular myocytes were stimulated with carbon electrodes using a culture cell pacer system from IonOptix (Milton, MA, USA). Stimulus parameters were 6.6 V cm–1, and the duration was 2 ms, with alternating polarity. Electrical stimulation was performed at 0, 5 and 8 Hz. Incubation with the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 (Calbiochem) was for 60 min prior to electrical stimulation.

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–1 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, pH adjusted using NaOH).

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 (Kang et al. 2000). Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay using In Situ Cell Death Detection Kit (Roche). The percentages of non-viable Trypan Blue-positive cells and TUNEL-positive cells were determined by randomly counting 300 cells in each well or coverslip.

Western blotting

HSP70 and HSP90 antibodies were from Stressgen, while antiactin was obtained from Sigma. Antibodies against phospho-Akt, Akt, phospho-extracellular signal-related kinase 1/2 (Erk1/2) and phospho-p38 were from New England Biolab. Anti-Erk2, phospho-JNK, total p38 and JNK were from Santa Cruz Biotechnology. Myocytes were lysed with modified RIPA buffer (1% non-ionic detergent P4O (NP-40), 50 mM Tris-HCl, 1 mM EDTA, 0.25% deoxycholic acid (DOC), 150 mM NaCl, 1 mM phenylmethylsulphonyl fluoride, 1 µg ml–1 leupeptin, 1 µg ml–1 pepstatin, 1 µg ml–1 aprotinin and 1 mM sodium orthovandidate). Aliquots representing 30–50 µg 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, Rockford, IL, USA), quantification was performed by densitometry.

Statistical analysis

Results are expressed as means ± S.D. of at least three different experiments. One-way ANOVA was used for multiple comparison. A value of P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Rapid electrical stimulation adversely affects cell viability

We examined the effects of rapid pacing on ARVM survival for up to 48 h (Fig. 1). Electrical stimulation at 5 and 8 Hz resulted in more than 60% capture of myocytes. For both stimulation frequencies, the percentage of myocytes captured remained the same (60%) at 24 h, while it decreased to ~50% at 48 h. MTT uptake was not different between unpaced and paced myocytes after 24 h of pacing. By 48 h, however, there was an increase in MTT uptake compared to unpaced cells (P < 0.05, 5 Hz > 8 Hz > 0 Hz, among 3 frequencies; Fig. 1A). We found no significant difference in percentage TUNEL-positive cells, Trypan Blue uptake, and CK release between quiescent myocytes and those paced at 5 Hz; however, rapid pacing at 8 Hz significantly promoted myocyte injury and death (Fig. 1B–D).


Figure 1
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  		Figure 1.  Effect of electrical stimulation on MTT uptake and cardiomyocyte injury and survival
Myocytes were maintained quiescent (0 Hz) or electrically paced at normofrequency (5 Hz) or rapid frequency (8 Hz). After 24 and 48 h, myocytes were assayed for MTT uptake (A; n = 3 for paced conditions), TUNEL staining (B; n = 3), Trypan Blue uptake (C; n = 3) and creatine kinase (CK) release in culture media (D; n = 5). All results were normalized to quiescent conditions. *P < 0.05 versus 0 and 8 Hz; {dagger}P < 0.05 versus 0 and 5 Hz; and {ddagger}P < 0.05 versus 0 Hz.

 
Rapid electrical stimulation increases stress- and mitogen-activated protein kinases

To explore potential mechanisms for the observed decrease in cell viability in the presence of rapid pacing, we examined the effect of electrical stimulation on stress- and mitogen-activated protein kinases (Akt, Erk, JNK and p38) and on heat shock protein 70/90 (HSP70/90) expression as stress indicators. Stimulation at 5 Hz increased Akt and p38 phosphorylation (P < 0.05 versus 0 Hz), at 10 and 30 min after pacing, respectively (Fig. 2A). In contrast, 8 Hz pacing further augmented the phosphorylation of Akt and p38, as well as activating Erk and JNK (P < 0.05 versus 0 Hz for all kinases studied). Similar to 5 Hz pacing, the increase in Akt and p38 phosphorylation occurred at 10 and 30 min, respectively, whereas transient Erk activation peaked at 10 min (Fig. 2B). JNK phosphorylation, in contrast, was activated at 10 min and remained elevated up to 60 min (Fig. 2B). At 8 Hz, the levels of Akt and JNK phosphorylation were statistically higher than those at 5 Hz (Fig. 2C and D). Total expression of each kinase studied did not differ significantly between this and our previous report (Kuramochi et al. 2004). Interestingly, there was no evidence of changes in HSP70/90 expression, even at rapid pacing frequency (Fig. 2A and B).


Figure 2
Figure 2
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  		Figure 2.  Effect of rapid electrical stimulation on cardiomyocyte kinase signalling
Myocytes were electrically paced at 5 (A) and 8 Hz (B) for up to 60 min, and stress signalling was examined by Western blot at the time points indicated. Blots are representative of three to four different experiments. The peak activations of each stress kinase (10 min pacing for pAkt and pErk; 30 min pacing for pJNK and pp38) were arranged side by side for comparison (C) and analysed by densitometry (D). *P < 0.05 versus 0 Hz; and {dagger}P < 0.05 versus 5 Hz, n = 3–4.

 
PI3K pathway is important for cell survival in both quiescent and paced myocytes

To elucidate the role of the PI3K pathway for paced and unpaced myocytes, ARVM were incubated in the presence and absence of the PI3K inhibitor LY294002 for 24 h. Myocyte apoptosis was examined by TUNEL staining. As shown in Fig. 3, the percentage of TUNEL-positive cells was highest in 8 Hz paced ARVM (P < 0.05 versus 0 and 5 Hz). The treatment with LY294002 resulted in a twofold increase of apoptosis in both quiescent and electrically stimulated myocytes (*P < 0.05 versus 0 and 5 Hz without LY294002, {dagger}P < 0.05 versus the others, n = 4; Fig. 3). Thus, while activation of the PI3K/Akt pathway was cytoprotective at all pacing frequencies examined, it was not able to overcome the deleterious effects of rapid electrical stimulation.


Figure 3
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  		Figure 3.  Inhibition of PI3K/Akt pathway in rapid electrically stimulated cardiomyocytes
Quiescent or electrically paced myocytes were incubated in the presence or absence of the PI3K inhibitor LY294002 for 24 h. Myocytes were subsequently fixed, and apoptosis determined by TUNEL assay. *P < 0.05 versus 0 and 5 Hz without LY294002; and {dagger}P < 0.05 versus all other conditions (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Supraphysiological heart rates are associated with progressive ventricular remodelling, and experimental literature demonstrates that rapid electrical pacing can induce a dilated cardiomyopathy in animals (Spinale et al. 1994; Valente et al. 1998; Hanna et al. 2004). To date, the vast majority of studies on tachycardia have utilized in vivo rapid atrial pacing, where typically 2–3 weeks of pacing induces heart failure with specific consequences for the cardiomyocytes, including alterations in signal transduction pathways (Schulz et al. 2003; Hanna et al. 2004; Ananthakrishnan et al. 2005), shifts in energy substrate utilization (Pierpont et al. 1993) and apoptosis (Spinale et al. 1994; Liu et al. 1995; Hanna et al. 2004). In the in vivo setting, however, the effect of rapid pacing on cardiac myocytes is confounded by extrinsic factors, such as haemodynamics, neurohormonal activation, ischaemia, and systemic and non-myocyte constituents (Pierpont et al. 1993; Osorio et al. 2002). Moreover, at the level of the single cell, the literature is scant on studies that directly assess the effect of rapid electrical stimulation on cardiomyocytes, and these reports have used either cultured atrial myocytes (HL-1 cells) or neonatal myocytes (Yonemochi et al. 2000; Yang et al. 2005). To our knowledge, our report is the first to address the immediate short-term consequences (minutes to days) of rapid pacing in adult ventricular myocytes.

The in vivo rat heart rate is approximately 300 beats min–1, while ARVM, once isolated, are mostly quiescent. This non-physiological condition acutely lowers oxygen consumption (Rose et al. 1991) and leads to changes in metabolic enzyme expression, free fatty acid uptake and contractile properties (Ellingsen et al. 1993; Ivester et al. 1995; Kato et al. 1995; Xia et al. 1997; Luiken et al. 2001). We previously reported the stimulus parameters of a commercially available cell pacer system that allowed electrical field stimulation of ARVM without molecular evidence of cell stress and worsening myocyte survival at low (2 Hz) and normal (5 Hz) frequencies (Kuramochi et al. 2004). Using the same system, this study was designed to elucidate the effects of rapid electrical stimulation (8 Hz) on myocyte survival and MAPK signalling in quiescent myocytes and in cells paced at physiological frequency.

Similar to our previous report (Kuramochi et al. 2004), mitochondrial respiration as assessed by MTT uptake was significantly higher in paced ARVM groups at 48 h when compared to quiescent cells. These results are consistent with in vitro work showing that short-term pacing produces cellular maturation with increased mitochondrial content and activity (Xia et al. 1996, 1997). Pacing at physiological frequency (5 Hz) did not induce cell death, as we previously reported (Kuramochi et al. 2004); however, we found that rapid pacing at 8 Hz increased cell cytotoxicity and apoptosis, as early as 24 h from the initiation of rapid pacing. These results imply that rapid pacing-induced cardiomyocyte death is an early event and not just a late phenomenon as described in chronic models of in vivo pacing-induced heart failure (Liu et al. 1995; Schulz et al. 2003; Ananthakrishnan et al. 2005). It is possible that energy substrate availability was limited in our model and, as a consequence, mitochondrial respiration was unable to keep up with the increased cardiomyocyte workload conditions at 8 Hz. In our previous report, however, higher mitochondrial uptake of MTT did not necessarily correlate with cell apoptosis (Kuramochi et al. 2004), and further studies are needed to fully elucidate the interaction between cardiomyocyte viability and energy production–consumption mismatch in the setting of rapid electrical stimulation.

In chronic pacing-induced heart failure models, an increase in myocardial activation of Akt, JNK, Erk or p38 have all been reported (Schulz et al. 2003; Spragg et al. 2003; Hanna et al. 2004; Ananthakrishnan et al. 2005), and Schulz et al. (2003) were able to show that the increase in JNK and p38 MAPK activation correlated with a rise in myocyte apoptosis. By contrast, in a pacing-induced canine model of congestive heart failure (CHF), Hanna et al. (2004) found an early and sustained increase in apoptosis (as early as 24 h, in agreement with our own study), but MAPK signalling was not observed until the very late stages (5 weeks) of heart failure. We found that short-term electrical stimulation at normofrequency (5 Hz) induced in minutes a temporal and weak activation of Akt and p38 without altering cell survival. Rapid pacing, in contrast, rapidly augmented all stress- and mitogen-activated kinase phosphorylation examined in our study. Taken together, these observations suggest that tachycardia may induce a biphasic response in kinase signalling, with an early transient response as observed in our study, followed by a late increase in kinase signalling coincident with late remodelling and progression of heart failure.

One possible mechanism for the increase in myocyte apoptosis in rapidly paced ARVM is abnormal Ca2+ homeostasis. An increase in peak calcium current and diastolic calcium levels occurs in electrically stimulated myocytes (Berger et al. 1994; Holt et al. 1997), and prolonged elevation in [Ca2+]i can induce cell death (Marks, 1997; Stamm et al. 2002; Zhu et al. 2003). Furthermore, mitochondrial Ca2+ and oxidative stress have been shown to increase with pacing frequency (Miyata et al. 1991; Pattwell et al. 2004; Ananthakrishnan et al. 2005). Stimulation of ARVM with the calcium ionophore ionomycin activates JNK (Kuramochi et al. 2004) and, in a pacing induced heart failure model, antioxidants can inhibit both cardiomyocyte apoptosis and JNK phosphorylation (Qin et al. 2003). It is now widely accepted that, in cardiomyocytes, reactive oxygen species promote JNK signalling which directly activates the cytochrome c-mediated death pathway (Aoki et al. 2002; Kwon et al. 2003; Qin et al. 2003). Thus, rapid electrical stimulation of ARVM may cause a collective increase in [Ca2+]i and oxidative stress with robust stimulation of the JNK pathway and consequent activation of the mitochondrial death pathway. Akt signalling is generally considered to be antiapoptotic and, while Akt activation was unable to prevent apoptosis in rapidly paced cardiomyocytes, Akt was still protective as treatment with PI3K inhibitor LY294002 significantly impaired myocyte survival in both paced and unpaced conditions. We speculate that the beneficial action of Akt is related to shifts in energy utilization from fatty acid to glucose (Pierpont et al. 1993; Rathmell et al. 2003; Ananthakrishnan et al. 2005).

In conclusion, rapid electrical stimulation in cardiomyocytes induces early activation of stress- and mitogen-activated kinases, with detrimental consequences for survival because the pro-apoptotic effects of JNK and p38 outweigh the antiapoptotic actions of Akt and Erk. It is important to emphasize that these events occurred during the early stages of rapid cardiomyocyte pacing, and thus are not influenced by chronic myocyte remodelling observed during the latter stages of pacing-induced heart failure models. Our present observations underscore the importance of myocyte contraction rate on cell viability and survival. Moreover, this system provides a more physiological, actively contracting cell-based assay to screen for compounds that might have a beneficial effect in the setting of heart failure.


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 Introduction
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 Discussion
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   Acknowledgements
 
This study was funded by NIH grants HL03878, HL68144, a grant from the Juvenile Diabetes Research Foundation to D.B.S., and 1K01AG024056 and AHA Scientist Development Grant 0430087 N to C.C.L.




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