Cardiovascular Research

Cardiovascular Research 2007 75(3):584-595; doi:10.1016/j.cardiores.2007.04.008

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Copyright © 2007, European Society of Cardiology

Activation of _1B-adrenoceptors alleviates ischemia/reperfusion injury by limitation of mitochondrial Ca²⁺ overload in cardiomyocytes

Hong Gao¹, Le Chen¹ and Huang-Tian Yang^*

Laboratory of Molecular Cardiology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) and Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai, 200025, China

^* Corresponding author. Laboratory of Molecular Cardiology, Institute of Health Sciences, SIBS, CAS and SJTUSM, 225 Chong Qing Nan Rd., #1 Bulg., Shanghai 200025, China. Tel./fax: +86 21 63852593. htyang{at}sibs.ac.cn

Received 26 November 2006; revised 3 April 2007; accepted 16 April 2007

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Supplementary data References

 
Objective Activation of ₁-adrenergic receptors (₁-ARs) mimics ischemic preconditioning (IP). However, the subtypes of ₁-ARs involved and the protective mechanisms are not entirely clear. Here we tested the hypothesis that preservation of mitochondrial integrity, in particular, Ca²⁺ homeostasis via the epsilon isoform of protein kinase C (PKC) and mitoK_ATP channels, may underlie the basis of _1B-AR-triggered cardioprotection.

Methods Indo-1 fluorescence in adult rat cardiomyocytes was used as an index of cytosolic ([Ca²⁺]_c) or mitochondrial free Ca²⁺ concentration ([Ca²⁺]_m), and cell shortening was measured simultaneously. Cells were subjected to 20 min of simulated ischemia followed by 30 min of reperfusion (I/R).

Results Activation of a₁-ARs by phenylephrine significantly decreased I/R-induced [Ca²⁺]_c and [Ca²⁺]_m overload, mitochondrial cytochrome c release and ATP reduction, and improved Ca²⁺ transients and cell shortening. These protective effects were markedly inhibited by blockade of _1B-AR (chloroethylclonidine) but not _1A-AR (5'-methylurapidil) or _1D-AR (BMY 7378). Moreover, phenylephrine-afforded protection on the [Ca²⁺]_m, [Ca²⁺]_c, and cell shortening was lost when mitoK_ATP channels were inhibited with 5-hydroxydecanoate and PKC with PKC V_1–2. However, PKC V_1–2 did not affect the mitoK_ATP channel opener diazoxide-induced protection on these parameters.

Conclusions These findings indicate that phenylephrine-induced protection on [Ca²⁺]_m homeostasis is mediated by selective activation of _1B-AR via mitoK_ATP channel opening and PKC activation. Mitochondrial function appears to be a determinant of [Ca²⁺]_c and contractile function during I/R injury.

KEYWORDS ₁-adrenergic receptors; Intracellular Ca²⁺ concentration; Mitochondria; Cell contraction; Protein kinase C

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Supplementary data References

 
Ischemic preconditioning (IP) [1] is a powerful way to trigger intrinsic adaptive responses to protect the heart against subsequent ischemia/reperfusion (I/R) injury [2–4]. The cardioprotection can also be conferred by pharmacological preconditioning, such as activation of ₁-adrenergic receptors (₁-ARs) [4]. Therefore, an understanding into the role of ₁-ARs in mediating cardioprotection could not only provide new insight into the endogenous defense mechanisms that limit I/R injury, but also help for the development of pharmacological approaches to protect the heart from ischemic injury during myocardial infarction, coronary artery bypass surgery, or other ischemic situation.

Activation of ₁-ARs with agonists can mimic IP to improve postischemic myocardial dysfunction [4,5] and reduce infarct size [5–7]. The protective effects can be abolished through depletion of endogenous catecholamines or blockage of ₁-ARs [4,6,8]. However, the precise cellular mechanisms by which ₁-ARs exert cardioprotection are not fully understood. Three ₁-AR subtypes (_1A-AR, _1B-AR, and _1D-AR) are present in the myocardium [9]. Early studies showed that _1A-AR fails to protect the heart from ischemic injury by using a selective _1A-AR agonist methoxamine or an antagonist 5-methylurapidil (5-MU), whereas a selective _1B-AR antagonist chloroethylclonidine (CEC) is able to abolish IP protection in rabbits and rats [7,10], indicating that the _1B-AR may be the mediator. However, recent studies showed that _1A- but not _1B-AR protects the heart from I/R-induced contractile dysfunction or infarction by using transgenic mice expressing constitutively active mutant (CAM) _1A-AR or _1B-AR [11,12]. Thus, the role of ₁-AR subtypes in the cardioprotection remains controversial.

Intracellular Ca²⁺ overload is suggested to be one of the main factors involved in I/R injury [13]. Cumulated evidence show that mitochondrial ([Ca²⁺]_m) and cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) overload is correlated with mitochondrial dysfunction, contractile dysfunction, and cell death [14–17]. A limitation of [Ca²⁺]_m and [Ca²⁺]_c overload during I/R improves recovery of postischemic contraction in whole hearts [16] and in cardiomyocytes [18,19]. However, whether [Ca²⁺]_m homeostasis plays an essential role in the development of [Ca²⁺]_c overload and contractile dysfunction due to I/R and in the cardioprotection against these alterations remains unclear. In addition, activation of ₁-ARs during Ca²⁺ preconditioning protects the heart against Ca²⁺ overload injury [20], but direct evidence that activation of ₁-ARs limits I/R-induced intracellular Ca²⁺ overload is lacking.

Stimulation of ₁-ARs activates the epsilon isoform of protein kinase C (PKC) in different tissues, including myocardium [20]. Opening of mitochondrial ATP-sensitive potassium (mitoK_ATP) channels is associated to the limitation of [Ca²⁺]_m overload during I/R [21,22]. Moreover, translocation of PKC may be related to the modulation of mitoK_ATP channel opening [23]. However, the links between ₁-AR subtypes and the effects of PKC on the opening of mitoK_ATP channels as well as on the [Ca²⁺]_c and [Ca²⁺]_m homeostasis have not yet been clarified.

Therefore, the aims of this study were (i) to characterize the protective effects of ₁-AR activation against simulated I/R-induced [Ca²⁺]_m overload and alterations in [Ca²⁺]_c homeostasis and contraction in cardiomyocytes; (ii) to identify the subtype of ₁-ARs implicated in the protection; (iii) to clarify the importance of PKC and mitoK_ATP channels in ₁-AR-protected intracellular [Ca²⁺] ([Ca²⁺]_i) homeostasis and contraction; (iv) to examine whether the protection on the mitochondria, in particular [Ca²⁺]_m via opening of mitoK_ATP channels plays a key role in ₁-AR-improved [Ca²⁺]_c homeostasis and contraction during I/R.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Supplementary data References

 
The investigation conforms with the guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996), and animal procedures were also approved by the Institutional Review Board of the Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine.

2.1 Isolation of ventricular myocytes
Left ventricular myocytes were isolated from adult male Sprague–Dawley rat hearts by using a standard enzymatic method as previously described [24], and >85% of isolated rod-shape myocytes were Ca²⁺-tolerant.

2.2 Measurement of [Ca²⁺]_c, [Ca²⁺]_m, and cell shortening
For measuring [Ca²⁺]_c, cells were incubated with a Ca²⁺ indicator indo-1AM (5 µM, Molecular Probes, USA) at 25 °C for 10 min [19,24]. It was confirmed that 80% of the fluorescence located in cytosol by sequentially using 5 µM and 25 µM of digitonin to selectively release components from the cytosol and mitochondria, respectively (data supplement 1) [25]. For measuring [Ca²⁺]_m, different cells were incubated with indo-1AM (5 µM) for 30 min at 35 °C, which was designed to load indo-1 preferentially and heavily into mitochondria as previously described [26]. Then indo-1AM loaded cells were perfusing with 200 µM MnCl₂ for 20 min to quench the companion cytosolic fluorescence signal, but it did not alter cell contraction during the experiment period [25]. Ca²⁺ transients gradually decreased and became imperceptible within 20 min. Mn²⁺-resistant fluorescence originated predominantly from mitochondria (>90%, see data supplement 1). The loaded myocytes were perfused at least 30 min to wash out residual Indo-1 before initiating the study. Cells were electrical stimulated at 0.5 Hz, except during ischemia. The ratio of emitted fluorescence at 405 and 485 nm was recorded as an indicator of [Ca²⁺]_i [24]. The cells were simultaneously illuminated with red light (650–750 nm) through the bright-field path of the microscope (Olympus, Tokyo, Japan). Cell shortening, measured simultaneously with either [Ca²⁺]_c or [Ca²⁺]_m [18,19,24], was detected with an optical edge-detector, collected using a charge-coupled device camera, and analyzed by IonWizard 4.4 software in Length mode (IonOptix Co., MA, USA).

2.3 Experimental protocols
After equilibration, the myocytes were randomly assigned to one of the following groups (Fig. 1).

Group 1: normal and vehicle controls. The myocytes (n=10) were perfused with modified K–H solution containing (mM) 129 NaCl, 4 KCl, 20 NaHCO₃, 0.9 NaH₂PO₄, 0.5 MgSO₄, 10 glucose, and 1.8 CaCl₂, gassed with 95% O₂/5% CO₂ (pH 7.4) at 35 ° during the entire experimental period. In vehicle control (n=8), DMSO (0.01%, 15 min/60 min washout), as used in the diazoxide protocol, did not affect [Ca²⁺]_c, [Ca²⁺]_m or cell shortening (data not shown).

Group 2: control I/R. To measure [Ca²⁺]_c, [Ca²⁺]_m and cell shortening under I/R, a cellular model of simulated I/R was used as previously described [18,19,27]. Briefly, myocytes were perfused with ischemic solution, containing (mM) 123.0 NaCl, 8.0 KCl, 6.0 NaHCO₃, 0.9 NaH₂PO₄, 0.5 MgSO₄, 20.0 Na-lactate, and 1.8 CaCl₂, gassed with 95% N₂/5% CO₂ (pH 6.8), for 20-min followed by 30-min reperfusion with modified K–H solution. Left ventricular myocytes from the same heart were harvested for protein extraction and ATP content analysis.

Group 3: effects of ₁-ARs and the subtypes. Cells were perfused with propranolol (1 µM), a β-AR antagonist, for 5-min before and during the treatment of phenylephrine (a ₁-AR agonist), to avoid any interference induced by β-adrenergic stimulation during phenylephrine treatment.

Group 3A: phenylephrine+I/R. Phenylephrine (10 µM) for 5-min/10-min washout prior to I/R.

Group 3B: 5-MU+phenylephrine+I/R. 5-MU (1 µM), a _1A-AR selective antagonist, 15-min prior to and during phenylephrine treatment followed by a 10-min washout before I/R.

Group 3C: CEC+phenylephrine+I/R. CEC (10 µM), an irreversible _1B-AR antagonist, for 15-min/15-min washout prior to phenylephrine and a 10-min washout after phenylephrine treatment.

Group 3D: BMY 7378+phenylephrine+I/R. BMY 7378 (1 µM), a _1D-AR selective antagonist, 15-min prior to and during phenylephrine followed by a 10-min washout before I/R.

Group 3E: antagonist alone+I/R. To investigate whether antagonists would affect Ca²⁺ transients or cell shortening, β-AR antagonist propranolol and _1A-, _1B- or _1D-AR selective antagonist 5-MU, CEC, or BMY 7378 was perfused with the same concentration and treatment time as used in the corresponding treatment group (n=10–12/group). No significant differences were detected in the parameters examined between the non-drug control and antagonist alone treated groups (Fig. 1 in data supplement).

Group 4: roles of mitoK_ATP channel opening in the protection.

Group 4A: diazoxide (Dia)+I/R. Diazoxide (100 µM), a selective mitoK_ATP channel opener, for 15-min prior to I/R.

Group 4B: 5-hydroxydecanoate (5-HD)+phenylephrine+I/R. 5-HD (100 µM), a selective mitoK_ATP channel blocker, for 15-min prior to and during phenylephrine treatment, followed by a 10-min washout before I/R.

Group 4C: 5-HD+Dia+I/R. 5-HD (100 µM) for 15-min prior to and during diazoxide treatment before I/R.

Group 4D: 5-HD+I/R. 5-HD (100 µM) for 30-min before I/R. This treatment did not affect Ca²⁺ transients and cell shortening relative to the drug-free control (n=11, Fig. 1 in data supplement).

Group 5: roles of PKC in the protection.

Group 5A: PKC V1–2+I/R. PKC V_1–2 (10 µM), a specific PKC inhibitor, for 20-min/10-min washout prior to I/R.

Group 5B: PKC V_1–2+phenylephrine+I/R. Same as group 5A, but phenylephrine was added during the last 5-min treatment of PKC V_1–2, followed by a 10-min washout before I/R.

Group 5C: PKC V_1–2+diazoxide+I/R. PKC V_1–2 (10 µM), 15-min alone/15-min combination treatment with diazoxide prior to I/R.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Experimental protocols in rat left ventricular myocytes. Phe, phenylephrine (1-AR agonist); 5-MU, 5-methylurapidil (1A-AR antagonist); CEC, chloroethylclonidine (1B-AR antagonist); BMY 7378 (1D-AR antagonist); Dia, diazoxide (mitoKATP channel opener); 5-HD, 5-hydroxydecanoate (mitoKATP channel inhibitor); PKC V1-2 (PKC inhibitor).

 
Phenylephrine, propranolol, 5-MU, CEC, BMY 7378, diazoxide, 5-HD were from Sigma and PKC V_1–2 was from Research Biochemical Int. Diazoxide was dissolved in DMSO. All others were dissolved in deionized distilled water.

2.4 Western blot analysis
2.4.1 Sample preparation
For examining translocation of PKC, cytosolic and particulate fraction was obtained from isolated myocytes [28]. For analysis of cytochrome c, cells were fractionated into mitochondrial and cytoplasmic compartments [29]. The detailed method was in data supplement 3. Protein content was determined by Bradford method.

2.4.2 Western blotting for PKC and cytochrome c
To examine translocation of PKC, the cytosol (95 µg) and particulate (70 µg) fractions were electrophoresed on 8% polyacrylamide gels. For cytochrome c detection, mitochondrial (35 µg) and cytosolic fractions (20 µg) were electrophoresed on 15% polyacrylamide gels. The proteins were electroblotted onto polyvinylidenedifluoride membranes and probed with the anti-PKC antibody (1:5,000, Sigma, USA) or anti-cytochrome c antibody (1:2,000, Santa Cruz, USA), and the binding of the primary antibodies was detected by peroxidase-conjugated second anti-mouse antibody (1:2,000, Sigma, USA). The immunoreactions were visualized using an enhanced-chemiluminescent detection kit (Amersham Pharmacia Biotech, UK) and the bands were quantified with densitometry.

2.5 ATP content
As previously described [30], harvested cells were resuspended in cold homogenization buffer containing 50 mM potassium fluoride, 10 mM EDTA, and 30% glycerol, pH 7.0. The cell extract was used to measure ATP content with an ATP bioluminescence assay kit (Boehringer Mannheim, USA) according to the manufacturer's protocol. The values were expressed as nmoles/mg of protein.

2.6 Statistical analysis
Data are presented as mean±S.E.M. Statistical significance was determined using ANOVA or repeated ANOVA for multiple comparisons or repeated measurements. P<0.05 was regarded as statistically significant.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Supplementary data References

 
3.1 Characteristics of [Ca²⁺]_m, [Ca²⁺]_c, and cell shortening
The resting [Ca²⁺]_c and [Ca²⁺]_m significantly increased during I/R in myocytes. The rise of [Ca²⁺]_m by ischemia was earlier than that of the [Ca²⁺]_c (resting [Ca²⁺]_c vs. [Ca²⁺]_m at 1 and 5 min of ischemia: 103.0±0.7% vs. 106.7±0.9% and 112.7±1.7% vs. 123.8±1.9% of preischemic values, P<0.05 and P<0.01), although both of them increased to a similar level at 30 min of reperfusion (R30, Fig. 2A, B). The amplitude of Ca²⁺ transients and cell shortening was suppressed by 46.9±6.4% and 52.6±5.7% at R30 (Fig. 3A, D), accompanied with a significant decrease of the maximum upstroke velocity (V_max) and prolonged decay rate () of Ca²⁺ transients (Fig. 3B, C) and cell shortening (Fig. 3E, F). The data reveal that I/R-induced [Ca²⁺]_c and [Ca²⁺]_m overload is associated with the contractile dysfunction.

View larger version (47K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Cytosolic ([Ca2+]c) and mitochondrial free Ca2+ concentration ([Ca2+]m) during preischemia and I/R. Number of myocytes in each group is indicated in parentheses. Pre, preischemia. **P<0.01 vs. control I/R group; ##P<0.01, ###P<0.001 vs. corresponding phenylephrine or diazoxide alone group.

 

View larger version (52K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effects of phenylephrine on dynamics of cytosolic Ca2+ transients and cell shortening in the presence or absence of 5-MU, CEC or BMY 7378. Analysis of the amplitude (A, B), maximum upstroke velocity (Vmax, C, D) and decay rate (, E, F) of Ca2+ transients (left) and cell shortening (right). R30, 30 min of reperfusion. Number of myocytes in each group is indicated in parentheses. **P<0.01 vs. corresponding control; ##P<0.01 vs. phenylephrine values.

 
3.2 Effects of ₁-ARs and the subtypes on [Ca²⁺]_m, [Ca²⁺]_c, and cell shortening
We examined whether activation of ₁-ARs would prevent I/R-induced alterations in [Ca²⁺]_m, [Ca²⁺]_c, and cell contraction by using an ₁-AR agonist phenylephrine. During preischemia, no significant difference in resting [Ca²⁺]_c and [Ca²⁺]_m was detected between the control and phenylephrine-treated groups, whereas I/R-induced [Ca²⁺]_c and [Ca²⁺]_m overload was markedly suppressed by 65.7±5.6% and 74.3±3.8% at R30 in phenylephrine-treated cells (Fig. 2A, B). Cell shortening was increased slightly by phenylephrine and it returned to baseline levels by the end of a 10-min washout (Fig. 3A, B and Fig. 2 in data supplement). However, I/R-inhibited amplitude, upstroke V_max and decay rate of Ca²⁺ transients and cell shortening were markedly attenuated in phenylephrine-treated cells at R30 (Fig. 3). Selective _1A-, _1B-, and _1D-AR antagonists 5-MU, CEC, and BMY 7378 did not affect these parameters during the preischemic phase. However, phenylephrine-triggered protection was inhibited by CEC but not by 5-MU and BMY 7378 (Figs. 2A, B and 3), indicating that _1B-AR plays a key role in the protective effect triggered by activation of ₁-ARs.

3.3 Roles of mitoK_ATP channels in phenylephrine-mediated protection on [Ca²⁺]_m, [Ca²⁺]_c, and cell shortening
We then examined whether mitoK_ATP channels is involved in the ₁-AR-prevented [Ca²⁺]_m overload and whether limitation of [Ca²⁺]_m overload during I/R is correlated to the ₁-AR-mediated protection on the [Ca²⁺]_c and contraction. Diazoxide, a selective opener of mitoK_ATP channels, significantly attenuated I/R-induced resting [Ca²⁺]_c and [Ca²⁺]_m overload by 88.3±7.3% and 63.7±4.9% (Fig. 2C, D) and improved dynamics of Ca²⁺ transients and cell shortening at R30 (Fig. 4). 5-HD, a selective blocker of mitoK_ATP channels, totally blocked phenylephrine- and diazoxide-induced protection against I/R-induced alteration in resting [Ca²⁺]_c and [Ca²⁺]_m (Fig. 2C, D) as well as Ca²⁺ transients and cell shortening (Fig. 4). Therefore, activation of ₁-ARs prevents the development of I/R-induced [Ca²⁺]_m overload via opening of mitoK_ATP channels, which may subsequently contribute to the protection in the [Ca²⁺]_c and cell contraction.

View larger version (54K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Involvement of mitoKATP channels in phenylephrine-mediated regulation of cytosolic Ca2+ transients and cell shortening. Analysis of the amplitude (A, B), upstroke Vmax (C, D), and decay rate (E, F) of Ca2+ transients (left) and cell shortening (right). R30, 30 min of reperfusion. Number of myocytes in each group is indicated in parentheses. **P<0.01 vs. corresponding control; ##P<0.01, ###P<0.001 vs. the group indicated.

 
3.4 Roles of PKC in phenylephrine-mediated protections on [Ca²⁺]_m, [Ca²⁺]_c, and cell shortening
To identify the role of PKC in ₁-AR-mediated protection, we examined the link between the translocation of PKC and the ₁-AR subtype implicated during I/R. Particulate/cytosol ratio of PKC during preischemia was 0.44±0.17, and it was increased to 0.91±0.25 at R30 (P<0.05). The particulate fraction of PKC was markedly increased by phenylephrine during preischemia and I/R, but it was increased more during I/R. Selective _1A-, _1B- or _1D-AR antagonist 5-MU, CEC or BMY 7378 had a similar inhibitory effect in phenylephrine-increased particulate/cytosol ratio of PKC at preischemia, whereas only CEC significantly inhibited the translocation of PKC in phenylephrine-treated I/R cells (Fig. 5).

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Translocation of PKC from the cytosol to the particulate fraction during preischemia and I/R. Upper panel, representative immunoblots of PKC. Low panel, summarized data. n=4 each. *P<0.05, **P<0.01 vs. corresponding preischemic values; ##P<0.01, ###P<0.001 vs. corresponding phenylephrine values.

 
The roles of PKC in the [Ca²⁺]_m, [Ca²⁺]_c, and cell shortening were analyzed by using PKC selective inhibitor PKC V_1–2. Neither PKC V_1–2 alone nor PKC V_1–2 plus phenylephrine or diazoxide affected [Ca²⁺]_m, [Ca²⁺]_c and cell shortening during preischemia (Fig. 2E, F). However, PKC V_1–2 exacerbated I/R-induced resting [Ca²⁺]_c and [Ca²⁺]_m overload, and completely abrogated phenylephrine-mediated protection against I/R-induced [Ca²⁺]_c and [Ca²⁺]_m overload (Fig. 2E, F) as well as depression of the Ca²⁺ transients (Fig. 6A–C) and cell contraction (Fig. 6D–F). In contrast, PKC V_1–2 did not affect diazoxide-mediated protection on these parameters (Figs. 2E, F and 6). These findings establish that activation of PKC is implicated in ₁-AR-mediated protection against I/R-induced alterations in [Ca²⁺]_m, [Ca²⁺]_c and cell contraction. Moreover, the mitoKATP channel-mediated cardioprotection may be subsequent to the activation of PKC.

View larger version (53K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effects of PKC in phenylephrine-mediated regulation of cytosolic Ca2+ transients and cell shortening. Analysis of the amplitude (A, B), upstroke Vmax (C, D) and decay rate (E, F) of Ca2+ transients (left) and cell shortening (right). R30, 30 min of reperfusion. Number of myocytes in each group is indicated in parentheses. **P<0.01 vs. corresponding control values; ##P<0.01 vs. corresponding phenylephrine values.

 
3.5 Contribution of ₁-AR subtypes to phenylephrine-mediated protection on mitochondrial integrity
To gain further insight into the interrelationships among activation of _1B-AR subtype, PKC, mitoKATP channels, and mitochondrial integrity during I/R, we investigated effects of ₁-AR subtype antagonists and PKC or mitoK_ATP channel inhibitors on mitochondrial cytochrome c release, a marker of mitochondrial damage [31], and ATP content. The cytochrome c in cytosol was lower in preischemic cardiomyocytes, but it substantially increased during I/R, accompanied with a decrease of cytochrome c in mitochondrial fraction (Fig. 7A). However, this release was prevented in the cells treated with either diazoxide or phenylephrine. The protection was abrogated by the mitoK_ATP channel blocker 5-HD (Fig. 7A). In addition, phenylephrine-mediated protection was also blocked by either PKC V_1–2 (PKC inhibitor) or CEC (_1B-AR antagonist), but not by _1A- or _1D-AR antagonist 5-MU or BMY 7378 (Fig. 7A).

View larger version (54K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Mitochondria cytochrome c release and cell ATP contents. A, Representative immunoblots of cytochrome c (Cyto-c) in cytosolic and mitochondrial extracts at preischemia and 30 min of reperfusion. Similar results were obtained in at least four independent experiments. B, Analysis of different treatments on cell ATP contents at preischemia, 20 min of ischemia and 30 min of reperfusion. Number of independent experiments is indicated in parentheses. *P<0.05, **P<0.01 vs. corresponding control values; #P<0.05, ##P<0.01 vs. phenylephrine or diazoxide alone values.

 
During preischemia, ATP contents of cardiomyocytes were similar among groups with or without pharmacological interventions. However, ATP contents significantly decreased at 20-min ischemia and 30-min reperfusion, and the decreases were markedly attenuated by diazoxide or phenylephrine. The protection was blocked by 5-HD. In addition, PKC V_1–2 or CEC, but not 5-MU or BMY 7378, completely abrogated phenylephrine-induced protection on the ATP content (Fig. 7B). Altogether, these data provide further support that activation of _1B-AR can protect mitochondrial integrity during I/R via activation of PKC and opening of mitoK_ATP channels.

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Supplementary data References

 
In this study, we demonstrate that (i) activation of ₁-ARs with phenylephrine maintains cytosolic and mitochondrial Ca²⁺ homeostasis, improves contraction, and preserves ATP content under simulated I/R; (ii) the cardioprotection is inhibited by selective _1B-AR but not by _1A- or _1D-AR antagonists; (iii) the mitoK_ATP channel blocker 5-HD and PKC inhibitor PKC V_1–2 abolish ₁-AR-mediated cardioprotection on the [Ca²⁺]_m and ATP content, but PKC V_1–2 does not affect mitoK_ATP channel opener diazoxide-afforded protection on the mitochondria; (iv) blockage of ₁-AR-mediated protection in [Ca²⁺]_m homeostasis is accompanied with a loss of ₁-AR-mediated protection in the [Ca²⁺]_c homeostasis and cell contraction. These findings provide novel insights into the cellular mechanisms underlying the regulation of Ca²⁺ homeostasis, in particular [Ca²⁺]_m by activation of _1B-AR against I/R injury.

4.1 Activation of the ₁-ARs maintains [Ca²⁺]_c and [Ca²⁺]_m homeostasis during I/R
The observation that development of [Ca²⁺]_c and [Ca²⁺]_m overload during simulated I/R accompanied with the impaired cell contraction is consistent with our previous findings [18,19] and other reports in I/R [13] and anoxia/reoxygenation cells [32]. Several lines of evidence suggest that activation of ₁-ARs mimics IP in rats and other species [4,8,33] through the mechanisms related to nitric oxide (NO) [8], PKC isoforms [7,20] and mitoK_ATP channels [33]. However, the role of ₁-ARs on intracellular Ca²⁺ homeostasis during I/R, in particular on the [Ca²⁺]_m is unknown. The present study is the first one to demonstrate the important role of ₁-ARs in attenuating I/R-induced [Ca²⁺]_m and [Ca²⁺]_c overload through activation of PKC and mitoK_ATP channels in cardiomyocytes.

4.2 Activation of the _1B-AR protects cardiomyocytes against I/R injury
In the rat heart, _1A-AR, _1B-AR, and _1D-AR subtypes are present in an approximate proportion of 25%:45%:30% [9]. By using selective ₁-AR subtype antagonists, we provide new evidence here that activation of the _1B-AR can maintain [Ca²⁺]_i homeostasis and mitochondrial integrity and function during I/R. These findings are in agreement with the observations that _1B-but not _1A-AR is implicated in ₁-AR agonist-induced cardioprotection against postischemic contractile dysfunction and infarction [5,7,8]. However, recent studies using transgenic mice that express CAM _1A-AR or CAM _1B-AR showed that _1A-AR but not _1B-AR preconditions the ischemic heart [11,12]. It is possible that the a₁-AR subtype that mediates cardioprotection is different between the mouse and the rabbit or rat hearts. However, it is not supported by the observation that the _1B-AR also triggers the cardioprotection in the mouse heart [8]. It may be also caused by different signaling pathways associated with the cardioprotection between the transgenic and the wild-type animals. The proportions of active _1A-AR or _1B-AR are altered in these transgenic mice, which may change Ca²⁺ homeostasis during I/R as these subtypes have opposing effects on Ca²⁺ homeostasis [34]. Moreover, chronic augment of _1B-AR activity may affect β-AR signaling [35], thereby alter the regulatory mechanisms involved in pathophysiological responses such as I/R injury.

4.3 ₁-AR-prevented mitochondrial Ca²⁺ overload due to I/R is mediated by opening of mitoK_ATP channels and activation of PKC
Opening of mitoK_ATP channels appears to play an important role in maintaining [Ca²⁺]_m homeostasis during I/R [21,22,36]. This is further supported by our observation that the protective effects of ₁-ARs and diazoxide on [Ca²⁺]_m can be abolished by blockage of mitoK_ATP channels with 5-HD. It was shown that opening of mitoK_ATP channels not only impedes mitochondrial Ca²⁺ uptake by dissipates the inner mitochondrial membrane potential, but also activates cyclosporine sensitive mitochondrial Ca²⁺ release, thereby blunting [Ca²⁺]_m accumulation in both isolated mitochondria and intact myocytes [22,36]. 5-HD also appears to affect mitochondrial energy metabolism independent of its action on mitoK_ATP channels [37]. Therefore, mechanisms other than mitoKATP channel opening in ₁-AR-maintained [Ca²⁺]_m homeostasis cannot be excluded.

The other important finding here is that inhibition of PKC exacerbates I/R-induced [Ca²⁺]_m overload and totally abolishes ₁-AR-mediated protection on [Ca²⁺]_m, mitochondrial cytochrome c release and ATP content, but not the protection afford by diazoxide. Together with the earlier observations regarding PKC-induced mitoK_ATP channel opening [38,39], these findings indicate that activation of PKC by IP or ₁-ARs may mediate cardioprotection against I/R injury via maintenance of mitochondrial Ca²⁺ homeostasis and integrity through mitoK_ATP channel opening. Although it is unclear how PKC activation induces mitoK_ATP channel opening, PKC may functionally associate with mitoK_ATP channels via phosphorylation of mitoK_ATP channels or via an intermediate protein that activates mitoK_ATP channels [23].

4.4 Mitochondrial integrity is a determinant of cytosolic Ca²⁺ homeostasis and cell contraction during I/R
Accumulating evidence suggest that [Ca²⁺]_m overload is critical in the pathogenesis of ischemic or hypoxic injury [15,16,40,41]. However, it is unclear whether mitochondrial Ca²⁺ homeostasis plays a fundamental role in controlling the [Ca²⁺]_c and recovery of contraction after ischemic injury. Here, we found that (i) ischemia-induced [Ca²⁺]_m overload occurs prior to the occurrence of the [Ca²⁺]_c overload, which is consistent with the observation that [Ca²⁺]_m rises in far greater proportion than [Ca²⁺]_c [42]; (ii) limitation of I/R-induced [Ca²⁺]_m overload via mitoK_ATP channel opening can prevent [Ca²⁺]_c overload and improve contraction; (iii) abrogation of ₁-AR-conferred protection against I/R-induced [Ca²⁺]_m overload by inhibiting mitoK_ATP channel opening is associated with a loss of the protection in [Ca²⁺]_c and cell contraction. Therefore, limitation of [Ca²⁺]_m accumulation appears to play a key role in the cardioprotection, whereas the attenuation of [Ca²⁺]_c overload is more likely a secondary effect in the cardioprotection. In agreement with this hypothesis, attenuation of [Ca²⁺]_m but not [Ca²⁺]_c overload by inhibition of mitochondrial uniporter of Ca²⁺transport is responsible for the cardioprotection against I/R-induced contractile dysfunction and infarction [16,41]. Moreover, the extent of [Ca²⁺]_m rise during ischemia determines the outcome of reoxygenation [15]. Furthermore, suppression of mitochondrial Ca²⁺ uptake by NO donor possibly reduces cytosolic Ca²⁺ overload during ischemia [40]. [Ca²⁺]_m overload in I/R myocytes can lead to mitochondrial dysfunction [14,17]. For example, [Ca²⁺]_m overload triggers mitochondrial permeability transition and then causes cytochrome c release [17]. The latter appears to be partially responsible for the ischemic respiratory inhibition, which may contribute to the contractile dysfunction at reperfusion [43]. Reduced [Ca²⁺]_m overload by mitoK_ATP channel opening via reducing mitochondrial Ca²⁺ uptake might be expected to increase [Ca²⁺]_c rather than to decrease [Ca²⁺]_c overload as we observed. This paradox might be interpreted with the following possibilities. Maintained mitochondrial Ca²⁺ homeostasis is suggested to improve ATP synthesis and maintain constant volume of the mitochondrial matrix [44]. These effects would preserve mitochondrial function of providing adequate ATP supply to cytosolic ATPase, which is essential to support the action of ATP-dependent ion pumps/exchangers and receptors, such as Na⁺/K⁺ and Ca²⁺ pumps [45], Na⁺/Ca²⁺ exchangers and ryanodine receptors, to maintain cytosolic Ca²⁺ homeostasis [19,46]. Thus limitation of [Ca²⁺]_m overload during I/R may represent a potential therapeutic target.

In conclusion, our findings demonstrate that activation of _1B-AR protects cardiomyocytes against simulated I/R-induced alteration in cytosolic and mitochondrial Ca²⁺ homeostasis and cell contraction via activation of PKC and mitoK_ATP channels. The attenuation of mitochondrial Ca²⁺ overload possibly underlies the basis of ₁-AR-triggered cardioprotection. Further studies are required to confirm these findings in the I/R heart.

	Supplementary data

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Supplementary data References

 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2007.04.008.

Time for primary review 17 days

	Acknowledgements

 
This study was supported partially by Grants from Major State Basic Research Development Program of P.R. China (G2000056905), NSFC (30393133), and Knowledge Innovation Program of the CAS (KSCX1-YW-R-75).

	Notes

 
¹ These authors contributed equally to this work.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Supplementary data References

 

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