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Cardiovascular Research 2005 65(2):436-445; doi:10.1016/j.cardiores.2004.10.009
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Copyright © 2004, European Society of Cardiology

{alpha}1A- but not {alpha}1B-adrenergic receptors precondition the ischemic heart by a staurosporine-sensitive, chelerythrine-insensitive mechanism

Boyd R. Rorabaugha, Sean A. Rossa,1, Robert J. Gaivina, Robert S. Papaya, Dan F. McCunea, Paul C. Simpsonb and Dianne M. Pereza,*

aDepartment of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA
bDepartment of Cardiology, University of California, San Francisco, CA 94143, United States

* Corresponding author. Tel.: +1 216 444 2058; fax: +1 216 444 9263. Email address: perezd{at}ccf.org

Received 25 June 2004; revised 30 September 2004; accepted 7 October 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Brief periods of ischemia stimulate an endogenous mechanism in the heart that protects the myocardium from subsequent ischemic injury. {alpha}1-Adrenergic receptors (ARs) have been implicated in this process. However, the lack of sufficiently selective antagonists has made it difficult to determine which {alpha}1-AR subtype protects the heart from ischemic injury. The goal of this study was to identify the {alpha}1-AR subtype that is involved in ischemic preconditioning.

Methods: We developed transgenic mice that express constitutively active mutant (CAM) forms of the {alpha}1A-AR or the {alpha}1B-AR regulated by their endogenous promoters. Hearts isolated from transgenic and non-transgenic mice were perfused by the Langendorff method using an ischemic preconditioning perfusion protocol or a non-preconditioning perfusion protocol prior to 30-min ischemia and 40-min reperfusion. Contractile function was continuously monitored through an intraventricular balloon.

Results: The contractile function of non-transgenic hearts perfused according to the ischemic preconditioning protocol completely recovered from 30-min ischemia. However, non-transgenic hearts perfused according to the non-preconditioning protocol recovered only 60% of their contractile function. The contractile function of CAM {alpha}1A-AR hearts, but not CAM {alpha}1B-AR hearts, completely recovered from 30-min ischemia even though they were perfused according to the non-preconditioning protocol. Thus, CAM {alpha}1A-AR hearts, but not CAM {alpha}1B-AR hearts, were inherently preconditioned against ischemic injury. Staurosporine, but not chelerythrine, completely reversed the preconditioning effect of CAM {alpha}1A-ARs.

Conclusions: These data demonstrate that {alpha}1A-ARs protect the heart from ischemic injury through a staurosporine-sensitive signaling pathway that is independent of protein kinase C.

KEYWORDS Alpha 1 adrenergic receptors; Ischemic preconditioning; Transgenic mouse; Ischemia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
The high metabolic rate of the heart causes the myocardium to be sensitive to ischemic injury. However, Murry et al. [1] discovered that brief episodes of ischemia induced prior to a prolonged ischemic event protect the heart from ischemic injury through a process called ischemic preconditioning. Therefore, the development of drugs that mimic ischemic preconditioning may be useful for protecting the heart from injury during myocardial infarction, coronary artery bypass surgery, heart transplant, or other situations in which the heart becomes ischemic.

Previous studies provide evidence that norepinephrine is involved in ischemic preconditioning. Depletion of norepinephrine from sympathetic neurons abolishes ischemic preconditioning, and tyramine-induced release of norepinephrine from sympathetic neurons mimics ischemic preconditioning [2,3]. Ischemic preconditioning is also mimicked by the {alpha}1-adrenergic receptor (AR) agonist phenylephrine and blocked by the {alpha}1-AR antagonist prazosin [3], suggesting that ischemic preconditioning is mediated by {alpha}1-adrenergic receptors (ARs). Despite these data, some investigators have reported that adrenergic stimulation does not protect the heart from ischemic injury. Sebbag et al. [4] found that the {alpha}1-AR agonist methoxamine did not precondition the dog heart, and Bugge and Ytrehus [5] reported that {alpha}1-AR blockade did not abolish ischemic preconditioning in the rat heart. Thus, the role of norepinephrine in ischemic preconditioning has been a source of controversy.

Three different {alpha}1-AR subtypes ({alpha}1A-AR, {alpha}1B-AR, {alpha}1D-AR) have been cloned [6–9], and two subtypes ({alpha}1A-AR and {alpha}1B-AR) are present in the myocardium [10]. However, the lack of {alpha}1-AR subtype-selective antagonists has made it difficult to identify the physiological roles of individual {alpha}1-AR subtypes in the heart and other tissues. To circumvent this problem, we developed transgenic mice that express constitutively active mutant (CAM) {alpha}1A-ARs or CAM {alpha}1B-ARs under the regulation of their endogenous {alpha}1A-AR or {alpha}1B-AR promoters, respectively. These constitutively active receptors are expressed only in tissues that endogenously express their wild-type counterparts. This transgenic mouse model provides a way to investigate the function of {alpha}1A-ARs and {alpha}1B-ARs in the heart and other tissues that endogenously express these receptors without the need for {alpha}1-AR subtype-selective ligands.

In the present study, we used the Langendorff isolated heart model to determine whether expression of CAM {alpha}1A-ARs or CAM {alpha}1B-ARs protects the heart from ischemic injury. Hearts isolated from CAM {alpha}1A-AR mice and CAM {alpha}1B-AR mice exhibited similar {alpha}1-AR densities and similar inositol 1,4,5-triphosphate (IP3) concentrations. However, only the CAM {alpha}1A-AR protected the heart from ischemic injury.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Generation of transgenic mice
The generation and genotyping of CAM {alpha}1B-AR transgenic mice has been described elsewhere [11]. Tissue-specific distribution of the CAM {alpha}1A-AR was achieved using the murine {alpha}1A-AR gene promoter [12] to drive expression of a cDNA that encodes a CAM form of the rat {alpha}1A-AR (Fig. 1). The CAM {alpha}1A-AR mutant contains an M292L mutation in transmembrane domain 6 and the A271E mutation located in the third intracellular loop [13,14]. The Case Western Reserve University Transgenic Core facility injected approximately 200 copies of each transgene into the pronuclei of one-cell B6/CBA mouse embryos, which were surgically implanted into pseudo-pregnant female mice. Founder mice were identified and subsequent generations were genotyped by southern analysis of genomic DNA. F2 mice were mated to homozygosity. This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23). In addition, the animal research committee of The Cleveland Clinic Foundation approved all animal protocols used in this study.


Figure 1
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  		Fig. 1 Vector construct used to generate CAM {alpha}1A-AR transgenic mice. The rat CAM {alpha}1A-AR, driven by the mouse {alpha}1A-AR promoter, was cloned into the Not I restriction site of the pUC 4417 plasmid. A 2.6 kb [32P]-labeled DNA probe was used to identify transgenic mice with southern blots.

 
2.2. Membrane preparation and radioligand binding
The protocols used for membrane preparation and radioligand binding have been previously described [15]. Briefly, tissues were homogenized for 30 s with a polytron in ice-cold buffer A (10 mM HEPES, pH 7.4, 250 mM sucrose, 5 mM EGTA, 0.5 mM dithiothreitol, 10 µM phenylmethylsulfonylflouoride, 64 µM benzamidine, and 720 units/l bacitracin), and then homogenized with 20 strokes of a Dounce homogenizer. The homogenate was centrifuged at 1000 x g for 5 min to remove unhomogenized debris, and the supernatant was centrifuged for 1 h at 32,000 x g. The pellet was resuspended in buffer B (20 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM EGTA, 12.5 mM MgCl2, 0.5 mM dithiothreitol, 10 µM phenylmethylsulfonylflouoride, 64 µM benzamidine, and 720 units/l bacitracin) and centrifuged again at 32,000 x g for 1 h. The centrifugation–resuspension procedure was repeated twice before the membrane was resuspended in buffer B supplemented with 10% glycerol. The protein concentration of this homogenates was measured using the Bradford assay, and the homogenate was stored at –70°C. Some tissues required a slight deviation from this general protocol. Homogenized heart, skeletal muscle, and tongue underwent 15-min incubation in 0.5 M KCl prior to the first 32,000 x g centrifugation. Liver homogenates were separated with a solution of 70% buffer A and 30% Percoll prior to the first 32,000 x g centrifugation [15].

Saturation binding was performed using the {alpha}1-AR-selective radioligand 2-[β-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]-tetralone ([125I]-HEAT). Membrane protein (10 µg) was incubated at room temperature for 1 h in buffer B containing various concentrations of [125I]-HEAT (total volume=500 µl). Nonspecific binding was determined using 0.1 mM phentolamine. Bound and free [125I]-HEAT were separated by trapping the membranes on a Whatman GF/C filter and washing the filter with buffer B using a cell harvester.

2.3. Measurement of inositol-1,4,5-trisphosphate (IP3)
Tissues isolated from unheparanized mice were weighed, chopped into small pieces, and incubated for 1 h at 37 °C in serum free Dulbecco's Modified Eagle Medium containing 100 mM LiCl. The tissues were centrifuged for 5 min at 1000 x g, and the pellet was resuspended in ice-cold 1 M trichloroacetic acid (5 ml/g tissue) and homogenized with a polytron. The homogenate was centrifuged for 10 min at 1000 x g (4 °C), and a solution of 1,1,2-trichloro-1,2,2-trifluoroethane/tri-n-octylamine (3/1) was used to extract the trichloroacetic acid from the supernatant. The IP3 in this extract was measured using an IP3 radioreceptor assay kit from Perkin Elmer Life Sciences (Boston, MA) according to the manufacturer's protocol.

2.4. Langendorff heart preparation and measurement of cardiac function
Age-matched mice (males and females, 3–8 months of age) were heparanized and sacrificed by cervical dislocation. Hearts were immediately excised and perfused with Krebs–Henseleit solution according to our previously described protocol [16]. Contractile function of the left ventricle was measured using an intraventricullar fluid-filled balloon connected to a pressure transducer. The balloon was inflated to achieve an end-diastolic pressure of 4–8 mm Hg. All hearts were unpaced and were allowed to stabilize for at least 30 min before being exposed to normothermic global ischemia according to the ischemic preconditioning protocol or the non-preconditioning protocol shown in Fig. 2. Staurosporine- or chelerythrine-treated hearts were perfused with 50 nM staurosporine or 10 µM chelerythrine for 40 min prior to the 30 min of ischemia. These concentrations were chosen based on a previous study that used staurosporine and chelerythrine to inhibit PKC in isolated mouse hearts [17] and on the reported affinity (IC50) of staurosporine (5 nM) and chelerythrine (0.66 µM) for protein kinase C [18,19]. Data were continually recorded with a Powerlab 4SP data acquisition system (AD Instruments).


Figure 2
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  		Fig. 2 Ischemic preconditioning and non-preconditioning perfusion protocols. Isolated hearts were perfused for 30 min before being subjected to the ischemic preconditioning (A) or non-preconditioning (B) perfusion protocols. Hearts were preconditioned against ischemic injury by four sets (4-min ischemia and 6-min reperfusion) of ischemia–reperfusion prior to a 30-min ischemic episode followed by 40 min of reperfusion. Non-preconditioned hearts were continuously perfused prior to 30-min ischemia and 40-min reperfusion. Periods of perfusion (P) are indicated by white boxes and periods of ischemia (I) are indicated by black boxes.

 
2.5. Statistical analysis
Analysis of variance and Tukey's multiple comparisons test were used to compare receptor densities, IP3 concentrations, and cardiac functional parameters of transgenic and non-transgenic mice. A probability value <0.05 was considered statistically significant. Graphpad Prism software (San Diego, Ca) was used for all data analyses.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. General characterization of transgenic animals
Three groups of mice were used in this study: non-transgenic, CAM {alpha}1A-ARs, and CAM {alpha}1B-ARs. We have previously published the initial characterization of the CAM {alpha}1B-AR mice [11,15,16]. These mice develop autonomic failure and cardiac hypertrophy after 12 months of age. However, younger mice were used in this study before these symptoms developed. The CAM {alpha}1A-AR mice were viable, had normal litter sizes, had no gross phenotypic abnormalities at birth, and have not displayed any altered autonomic phenotype.

3.2. Tissue-specific expression of CAM {alpha}1A-AR and CAM {alpha}1B-ARs
Saturation binding experiments with [125I]-HEAT were used to determine the {alpha}1-AR tissue distribution and magnitude of transgene overexpression in membranes prepared from skeletal muscle, tongue, heart, spleen, lung, brain, kidney, and liver. {alpha}1-AR density (Bmax) was significantly increased (1.4–4.5-fold) in the heart, brain, kidney, and liver of CAM {alpha}1A-AR and CAM {alpha}1B-AR mice compared to those of non-transgenic mice (Fig. 3A). The {alpha}1-AR density was also significantly elevated in CAM {alpha}1B-AR lung and spleen but was not elevated in lung and spleen from CAM {alpha}1A-AR mice. Specific binding was not detected in either transgenic or non-transgenic skeletal muscle or tongue, two tissues known to not express {alpha}1-ARs.


Figure 3
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  		Fig. 3 Expression and constitutive activity of CAM {alpha}1A-ARs and CAM {alpha}1B-ARs in mouse tissues. Saturation binding (A) was performed using [125I]-HEAT to determine the density (Bmax) of {alpha}1-ARs in tissues of transgenic and non-transgenic mice. *Indicates a significant difference (p<0.05) compared to non-transgenic tissues, and {dagger} indicates a significant difference compared to CAM {alpha}1B-AR tissues. Data represent the mean ± S.E.M. of six to nine experiments performed in duplicate. IP3 concentrations (B) were measured in tissues from transgenic and non-transgenic mice. IP3 was normalized to wet tissue weight. Asterisks indicate a significant difference (p<0.05) compared to non-transgenic tissues. Data represent the mean ± S.E.M. of six to seven experiments performed in duplicate.

 
3.3. Constitutive activity of CAM {alpha}1A-ARs and CAM {alpha}1B-ARs
Constitutive activity of CAM {alpha}1A-ARs and CAM {alpha}1B-ARs was determined by measuring the IP3 concentration in the heart, liver, kidney, and brain of non-transgenic and transgenic mice in the absence of exogenous agonists. IP3 concentrations were significantly elevated (p<0.05) in transgenic hearts, brains, and livers compared to those of non-transgenic mice (Fig. 3B). IP3 concentrations were 1.7–2.1-fold higher in transgenic kidneys compared to non-transgenic kidneys. However, this was not statistically significant.

3.4. Pre-ischemic parameters of cardiac function
The Langendorff isolated heart preparation was used to measure cardiac function in non-transgenic and transgenic hearts. This method has been well established in other ischemic preconditioning studies [5,18–20] and was used in the present study because it enabled us to investigate cardiac {alpha}1-ARs without interference from the autonomic nervous system. Pre-ischemic (basal) parameters of cardiac contractility (developed pressure, max+dP/dT, max–dP/dT, and contractile rate) were similar in non-transgenic, CAM {alpha}1A-AR, and CAM {alpha}1B-AR hearts despite 2–3-fold {alpha}1-AR overexpression and significantly elevated IP3 concentrations in transgenic hearts (Table 1). Coronary flow rates (non-transgenic=3.3 ± 0.5 ml/min, CAM {alpha}1A-AR=2.7 ± 0.4 ml/min, CAM {alpha}1B-AR=2.4+1.0 ml/min) and heart weights (non-transgenic=178 ± 10 mg, CAM {alpha}1A-AR=208 ± 12 mg, CAM {alpha}1B-AR=167 ± 9 mg) were also similar in transgenic and non-transgenic hearts.


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  		Table 1 Pre-ischemic cardiac parameters of hearts isolated from transgenic and non-transgenic mice

 
3.5. Post-ischemic recovery of cardiac contractile function
Non-transgenic hearts were perfused according to either the ischemic preconditioning protocol (Fig. 2A) or the non-preconditioning protocol (Fig. 2B). Following 30-min ischemia, hearts that were perfused according to the non-preconditioning protocol recovered only 60% of their pre-ischemic inotropic function (developed pressure, max+dP/dT, and max–dP/dT) and 86% of their pre-ischemic contractile rate during 40 min of post-ischemic reperfusion (Fig. 4). In addition, end diastolic pressure increased to 42 ± 10 mm Hg in non-preconditioned hearts after 5 min of reperfusion (Fig. 5) and remained elevated compared to preconditioned hearts after 40-min reperfusion, indicating that contracture had developed and the myocardium was unable to completely relax. In contrast, all inotropic parameters (developed pressure, max+dP/dT, and max–dP/dT) of non-transgenic hearts that were perfused according to the preconditioning protocol returned to pre-ischemic levels within 5 min of reperfusion. Preconditioned hearts also demonstrated a significantly smaller increase in end diastolic pressure (peaked at 17 ± 4 mm Hg following 5-min reperfusion) compared to non-preconditioned hearts (Fig. 5). These data are consistent with previous studies which have demonstrated that brief periods of ischemia protect the heart from ischemic injury.


Figure 4
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  		Fig. 4 Expression of the CAM {alpha}1A-AR enhances post-ischemic contractile function of the ischemic mouse heart. Hearts isolated from CAM {alpha}1A-AR, CAM {alpha}1B-AR, and non-transgenic mice were perfused according to either the ischemic preconditioning protocol or the non-preconditioning protocol shown in Fig. 2. Developed pressure (A), heart rate (B), max+dP/dT (C), and max–dP/dT (D) were continuously monitored during 30-min ischemia and 40-min reperfusion. All data (except heart rate) were normalized to pre-ischemic levels of contractile function measured 1 min before the 30-min ischemic period. All parameters of contractile function (developed pressure, heart rate, max+dP/dT, and max–dP/dT) were significantly elevated (p<0.05) in preconditioned non-transgenic hearts (n=10) compared to non-preconditioned non-transgenic hearts (n=10) after 5 min of reperfusion. Post-ischemic contractile function of CAM {alpha}1A-AR hearts (n=7) was significantly greater than that of non-preconditioned non-transgenic hearts after 30-min reperfusion. Contractile function of non-preconditioned CAM {alpha}1B-AR hearts (n=5) was not significantly different from non-transgenic non-preconditioned hearts at any time point during the reperfusion. Data represent the mean ± S.E.M.

 

Figure 5
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  		Fig. 5 Expression of the CAM {alpha}1A-AR prevents the development of contracture in the ischemic mouse heart. Hearts isolated from CAM {alpha}1A-AR, CAM {alpha}1B-AR, and non-transgenic mice were perfused according to either the ischemic preconditioning protocol or the non-preconditioning protocol shown in Fig. 2. End diastolic pressure was continuously monitored during 30-min ischemia and 40-min reperfusion. End diastolic pressure was significantly greater (p<0.05) in non-preconditioned non-transgenic hearts (n=9) compared to preconditioned non-transgenic hearts (n=11) and non-preconditioned CAM {alpha}1A-AR hearts (n=8) at all time points after 10-min reperfusion. End diastolic pressure of non-preconditioned CAM {alpha}1B-AR hearts (n=5) was not significantly different from non-transgenic non-preconditioned hearts at any time point during the reperfusion. Data represent the mean ± S.E.M.

 
To determine whether expression of CAM {alpha}1A-ARs or CAM {alpha}1B-ARs protects the heart from ischemic injury, we perfused transgenic hearts according to the non-preconditioning perfusion protocol (Fig. 2B) before a 30-min ischemic episode. In CAM {alpha}1A-AR hearts, all parameters of cardiac contractility returned to pre-ischemic values within 30 min of reperfusion (Fig. 4). Developed pressure, max+dP/dT, max–dP/dT, and contractile rate were significantly greater (p<0.05) in CAM {alpha}1A-AR hearts perfused according to the non-preconditioning protocol compared to their non-transgenic counterparts after 30 min of reperfusion (Fig. 4). CAM {alpha}1A-AR hearts were also able to relax more completely during diastole (end diastolic pressure peaked at 15 ± 4 mm Hg) compared to non-transgenic hearts (Fig. 5). Despite the fact that CAM {alpha}1A-AR hearts were not subjected to the ischemic preconditioning protocol, the post-ischemic contractile function of these hearts was similar to that of non-transgenic hearts that were preconditioned by ischemia. Thus, hearts expressing CAM {alpha}1A-ARs were intrinsically preconditioned against ischemic injury. However, developed pressure, max+dP/dT, and –dP/dT recovered at a slower rate in CAM {alpha}1A hearts than in preconditioned non-transgenic hearts. This suggests that the {alpha}1A-AR does not act alone in mediating ischemic preconditioning.

In contrast to CAM {alpha}1A-AR hearts, reperfused CAM {alpha}1B-AR hearts exposed to the non-preconditioning protocol (Fig. 2B) recovered only 55% of their pre-ischemic contractile function (developed pressure, max+dP/dT, and max–dP/dT) (Fig. 4). Recovery of these inotropic parameters was not significantly different from non-transgenic hearts also perfused according to the non-preconditioning protocol. In addition, expression of CAM {alpha}1B-ARs failed to protect the heart from contracture, as the end diastolic pressure of reperfused hearts was not significantly different from that of non-transgenic hearts that were perfused according to the non-preconditioning protocol (Fig. 5). Thus, unlike expression of CAM {alpha}1A-ARs, expression of CAM {alpha}1B-AR did not protect the heart from ischemic injury.

Two types of ischemic preconditioning have been reported. Early preconditioning protects the heart from ischemic injury that occurs immediately following the preconditioning stimulus and lasts for 1 h. A second type of preconditioning, called delayed preconditioning, has also been reported. Delayed preconditioning protects the heart from ischemic injury that occurs between 24 and 72 h following the preconditioning stimulus [21]. To determine the time frame of CAM {alpha}1A-AR-mediated preconditioning, we perfused CAM {alpha}1A-AR hearts with 1 µM prazosin for 40 min prior to 30-min ischemia and 40-min reperfusion (Fig. 6). This acute prazosin treatment had no effect on post-ischemic recovery of contractile function. To determine whether subchronic prazosin treatment could inhibit CAM {alpha}1A-AR-mediated preconditioning, we injected mice with prazosin (2.4 µmol/kg) once daily for 3 days. Hearts were then isolated and perfused according to the non-preconditioning protocol on the fourth day. This prazosin treatment protocol has been used by other investigators for subchronic blockade of {alpha}1-ARs in rats [18]. In contrast to acute prazosin treatment, subchronic treatment significantly decreased the preconditioning effect of CAM {alpha}1A-AR expression. These data suggest that CAM {alpha}1A-AR-mediated preconditioning requires more than 40 min but less than 72 h to develop. This is consistent with delayed preconditioning.


Figure 6
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  		Fig. 6 Subchronic, but not acute prazosin treatment, abolishes the preconditioning effect of the CAM {alpha}1A-AR in the ischemic mouse heart. CAM {alpha}1A-AR hearts were perfused according to the non-preconditioning protocol. Prazosin treatment was performed by perfusing the heart with 1 µM prazosin for 40 min prior to 30-min ischemia (acute) or by injecting the mouse with 2.4 µmol prazosin/kg body weight once daily for 3 days (subchronic) prior to harvesting and perfusing the heart. All data were normalized to pre-ischemic levels of contractile function measured 1 min before the 30-min ischemic period. Developed pressure (A), max+dP/dT (B), and max–dP/dT (C) were significantly decreased after 30-min reperfusion in the subchronic prazosin-treated CAM {alpha}1A-AR hearts (n=8) compared to the nontreated CAM {alpha}1A-AR hearts (n=7). Acute prazosin treatment (n=3) had no significant effect on post-ischemic recovery of contractile function.

 
The signal transduction pathways involved in ischemic preconditioning have not been well defined. However, protein kinase C is thought to be an important player in this process [22,23]. To investigate the signaling pathways involved in the preconditioning of CAM {alpha}1A-AR hearts, we perfused CAM {alpha}1A-AR hearts with the PKC-selective inhibitor chelerythrine (10 µM) or the broad spectrum kinase inhibitor, staurosporine (50 nM) for 40 min prior to a 30-min ischemic episode. Hearts were then reperfused for 40 min in the absence of these kinase inhibitors. Staurosporine or chelerythrine had no effect on pre-ischemic contractile function. However, staurosporine completely abolished the cardioprotective effects of the CAM {alpha}1A-AR. Developed pressure, max+dP/dT, and max–dP/dT were significantly depressed in staurosporine-pretreated CAM {alpha}1A-AR hearts compared to CAM {alpha}1A-AR hearts that were not pretreated with this kinase inhibitor (Fig. 7). The CAM {alpha}1A-AR was also unable to prevent the development of contracture of ischemic hearts in the presence of staurosporine (data not shown). These data suggest that the cardioprotective effect of CAM {alpha}1A-AR expression is mediated by a staurosporine-sensitive, chelerythrine-insensitive mechanism.


Figure 7
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  		Fig. 7 Staurosporine abolishes the preconditioning effect of CAM {alpha}1A-AR in the ischemic mouse heart. CAM {alpha}1A-AR hearts were perfused according to the non-preconditioning protocol and staurosporine (50 nM) or chelerythrine (5 µM) was perfused through the heart for 40 min immediately prior to the 30-min period of ischemia. Hearts were not perfused with staurosporine or chelerythrine during the 40-min post-ischemic reperfusion period. Developed pressure (A), heart rate (B), max+dP/dT (C), and max–dP/dT (D) were continuously monitored during 30-min ischemia and 40-min reperfusion. All data (except heart rate) were normalized to pre-ischemic levels of contractile function measured 1 min before the 30-min ischemic period. Developed pressure, max+dP/dT, and max–dP/dT of staurosporine-treated CAM {alpha}1A-AR hearts were significantly decreased (p<0.05) in staurosporine-pretreated CAM {alpha}1A-AR hearts (n=4) compared to nontreated CAM {alpha}1A-AR hearts (n=7) at all time points after 20-min reperfusion. Chelerythrine (n=4) had no significant effect on these parameters of contractile function. Data represent the mean ± S.E.M.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Ischemic preconditioning stimulates an endogenous mechanism in the heart that protects the myocardium from ischemic injury [1]. Although {alpha}1-ARs are thought to be involved in ischemic preconditioning [2,3], it has been unclear which {alpha}1-AR subtype mediates this process. {alpha}1A-ARs and {alpha}1B-ARs are present in the mouse heart [10]. In this study, we used hearts expressing constitutively active {alpha}1A-ARs or constitutively active {alpha}1B-ARs to investigate the effects of {alpha}1-AR subtypes on ischemic preconditioning. We found that the contractile function of CAM {alpha}1A-AR hearts completely recovered from 30-min ischemia even without undergoing the ischemic preconditioning perfusion protocol. Thus, hearts expressing the CAM {alpha}1A-AR were inherently preconditioned against ischemic injury. In contrast, expression of CAM {alpha}1B-ARs provided no protection against ischemic injury.

Previous attempts to identify the {alpha}1-AR subtype that mediates ischemic preconditioning have produced mixed results. Consistent with our data, Gao et al. [24] found that expression of CAM {alpha}1B-ARs in the heart-targeted transgenic mouse does not protect the heart from ischemic injury. However, others have reported that ischemic preconditioning of rat and rabbit hearts is mediated by {alpha}1B-ARs [25–27]. It is possible that the {alpha}1-AR subtype that mediates ischemic preconditioning is different in the mouse heart than in rabbit or rat hearts. However, the conclusion that {alpha}1B-ARs precondition rabbit and rat hearts was based on the observation that ischemic preconditioning of these hearts was blocked by the alkylating agent, chloroethylclonidine (CEC), but not by the {alpha}1A-AR-selective antagonist, 5-methylurapidil (5-MU). A more recent study has shown that CEC inactivates all three {alpha}1-AR subtypes and is not an {alpha}1B-AR specific agent [28]. Therefore, it is likely that differences in the conclusions of these studies results from the fact that the currently available {alpha}1-AR antagonists demonstrate minimal selectivity for individual {alpha}1-AR subtypes.

The observation that CAM {alpha}1A-ARs, but not CAM {alpha}1B-ARs, protect the heart from ischemic injury is consistent with previous studies that have demonstrated that {alpha}1A-ARs and {alpha}1B-ARs activate different signaling pathways in cardiac myocytes [29–31]. The signaling pathways involved in ischemic preconditioning are not well understood despite almost 20 years of intensive investigation. However, PKC has been reported to be an important player in the preconditioning process [22,23]. In the present study, we demonstrated that CAM {alpha}1A-ARs protect the heart from ischemic injury through a process that is not PKC-dependent but is dependent upon another staurosporine-sensitive signaling pathway. Staurosporine inhibits several protein kinases (protein kinase A, protein kinase C, protein kinase G, cyclin-dependent kinase 1, cyclin-dependent kinase 2, extracellular regulated kinase I, phosphorylase kinase, S6 kinase, myosin light chain kinase, calmodulin-dependent kinase II, Src, and Fgr) with similar affinities (IC503–20 nM) [32–34]. Our observation that the broad spectrum kinase inhibitor staurosporine, but not the PKC-selective inhibitor chelerythrine, abolished the cardioprotective effect of the CAM {alpha}1A-AR suggests that {alpha}1A-AR-mediated preconditioning of the ischemic heart is mediated by a staurosporine-sensitive signaling pathway other than PKC. Further work is needed to identify specific {alpha}1A-AR signaling pathways that lead to ischemic preconditioning.

Transgenic and knockout mouse models have also been used to investigate the role of {alpha}1-AR subtypes in regulating cardiac contractility under non-ischemic conditions. Our observation that modest (2–3-fold) overexpression of CAM {alpha}1A-AR or CAM {alpha}1B-ARs had no significant effect on pre-ischemic levels of contractility in the present study is consistent with previous reports that have shown that the {alpha}1-AR system plays a secondary role to β-ARs in regulating cardiac contractility under normal conditions. However, a recent study using {alpha}1A–{alpha}1B-AR double knockout mice demonstrated that {alpha}1-ARs are required for normal myocardial contractility [35]. In addition, other investigators have reported that vast overexpression (170-fold) of cardiac {alpha}1A-ARs enhances cardiac contractility while overexpression (43-fold) of {alpha}1B-ARs decreases cardiac contractility [36,37]. Interestingly, these data coincide with the fact that modest expression of CAM {alpha}1A-ARs, but not CAM {alpha}1B-ARs, enhanced the recovery of cardiac contractility during post-ischemic reperfusion in the present study.

The tissue distribution of CAM {alpha}1A-ARs and CAM {alpha}1B-ARs was consistent with previous studies of {alpha}1-AR subtype tissue distribution. {alpha}1-AR densities were elevated in heart, brain, kidney, and liver of both CAM {alpha}1A-AR and CAM {alpha}1B-AR mice and in spleen and lung of CAM {alpha}1B-AR mice (Fig. 3A). Rokosh and Simpson [38] reported that {alpha}1A-ARs account for a 30–58% of the {alpha}1-AR population in brain, heart, and kidney. Binding studies in {alpha}1B-AR knockout mice have demonstrated that {alpha}1B-ARs make up a large population of the {alpha}1-AR population in the liver (98%), heart (74%), and brain (32–42%) [39]. An unusual finding in our study was the high expression of CAM {alpha}1A-AR expression in the liver, a tissue that expresses predominately {alpha}1B-ARs [10,39]. However, in transgenic mice expressing a green fluorescent protein-tagged {alpha}1A-AR (under the same promoter described in this study), we have found high expression of the {alpha}1A-AR in blood vessels and in lymphocytes circulating through the bile ducts (manuscript in preparation). Thus, {alpha}1A-ARs are present in nonhepatocytes within the liver.

In summary, this is the first study to demonstrate that {alpha}1A-ARs, but not {alpha}1B-ARs, protect the mouse heart from ischemic injury. One limitation of our data is that the cardioprotective effect of CAM {alpha}1A-AR expression was measured over a relatively short time period following ischemia. Thus, it is unclear whether this effect would be of significant benefit in the long-term. Additional work is needed to determine the long-term consequences of CAM {alpha}1A-AR-mediated preconditioning and to identify the downstream signaling pathways involved in this process. This may lead to the development of drugs that mimic ischemic preconditioning and are useful for protecting the heart from ischemic injury. In addition, we believe that our transgenic mouse model will provide a valuable tool for elucidating the physiological roles of {alpha}1A-ARs and {alpha}1B-ARs in the heart and other tissues.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was funded by RO1Heart Lung 61438 (D.M.P.), a T32 Heart Lung 07914 training grant in vascular biology (B.R.R.), a National Research Service Award (D.F.M), and two local American Heart Association Fellowships (B.R.R. and S.A.R.).


   Notes
 
1 Current address: GlaxoSmithKline, Research Triangle Park, NC, United States. Back

Time for primary review 24 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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