INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

ISCHEMIC PRECONDITIONING (IP) by brief episodes of ischemia-reperfusion protects the myocardium from the damage induced by a subsequent more prolonged ischemia. Several triggers and mediators of IP have been identified, whereas the final end effector is still unknown (15). Protein kinase C (PKC) is an established mediator of IP in mice and rabbits, whereas other PKC isoforms may be more important in other species (15). PKC is involved in signaling complexes with at least 36 proteins (1, 9, 18), among them connexin43 (Cx43) (10), an integral protein of myocardial gap junctions. Activated PKC colocalizes with Cx43 and contributes to phosphorylation of Cx43 in rats (3) and humans (2), which might then modulate gap junction transmission characteristics and intercellular communication. Indeed, in isolated mouse hearts, uncoupling of gap junctions using heptanol abolished infarct size reduction by IP (8). The data on the effect of gap junction uncoupling on infarct size per se are controversial. Pretreatment with the gap junction uncoupler heptanol had no effect on infarct size in isolated rabbit hearts (6), but heptanol given during early ischemia decreased infarct size in isolated rabbit hearts (16). Also, in pigs in vivo heptanol given during early reperfusion decreased infarct size (4). Data on the importance of Cx43 for IP in vivo, however, are lacking. We therefore studied whether or not Cx43 is involved in the cardioprotection by IP using heterozygous Cx43-deficient mice.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The experimental protocols were approved by the bioethics committee of the district of Düsseldorf, Germany. Mice were handled according to the guidelines of the American Physiological Society.

We used the in situ mouse heart model developed by Guo et al. (5). Briefly, male and female C57BL/6J wild-type and heterozygous Cx43-deficient mice (B6.129-Gja1^tm1Kdr, JAX mice; Bar Harbor, ME) (12) (weight: 30.5 ± 4.5 g, age: 14-20 wk) were anesthetized with pentobarbital sodium (80 mg/kg ip) and atropine sulfate (0.04 mg/kg ip). Electrocardiogram and rectal temperature were monitored continuously. Temperature was held close to 37°C. After intubation (polyethlene-60 tubing) and with ventilation (oxygen-supplemented room air, 105 breaths/min ZOOVENT Jetsys, Universal Lung Ventilators; Netherfield, UK), a midline thoracotomy and pericardiotomy were performed. With the use of a 9.0-nylon suture, a nontraumatic balloon occluder was attached to the left anterior descending coronary artery 1 mm distal to the tip of the left auricle. Control mice (n = 8/8) underwent 30 min occlusion and 120 min reperfusion. IP was induced by 5 min occlusion and 10 min reperfusion before the sustained occlusion (1×IP, n = 8/8). Five additional Cx43-deficient mice were subjected to three cycles of 5 min occlusion and 10 min reperfusion (3×IP). At the end of the protocol, mice were heparinized, and the hearts were rapidly excised and immersed into ice-cold saline (~4°C). The aorta was cannulated and perfused with Krebs-Henseleit solution, 1% 2,3,5-triphenyltetrazolium chloride, and 10% KCl. Hearts were cut into four to five transverse slices, and infarct size was measured by planimetry and expressed as a percentage of the left ventricle (8).

For Western immunoblotting of Cx43, a primary rabbit anti-Cx43 (Zymed; San Francisco, CA) and secondary anti-rabbit IgG horseradish peroxidase-conjugated antibody (Cell Signaling; Beverly, MA) were used. Analysis of the amount of Cx43 was performed in right ventricular tissue (14.4 ± 5.3 mg) by enhanced chemiluminescence and quantitative two-dimensional densitometry (wild-type/Cx43^+/, n = 4/3 mice).

Data are means ± SD. Data analysis was performed with Sigma Stat software (Jandel Scientific; San Rafael, CA) by one-way ANOVA. A P value <0.05 was taken to indicate statistical significance.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Heart rate and temperature were not different among the five groups throughout the protocol (Table 1). The heterozygous Cx43-deficient mice had 55.5 ± 9.3% of wild-type right ventricular Cx43 expression (Fig. 1). Infarct size was reduced by IP in wild-type mice but not in heterozygous Cx43-deficient mice, not even when subjected to three IP cycles (Fig. 2).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Typical autoradiograph of right ventricular connexin43 (Cx43) in a wild-type (WT) and a heterozygous Cx43-deficient mouse (Cx43+/) (A) and densitometry data of Cx43 protein in wild-type and Cx43-deficient right ventricular tissue (B; means ± SD, *P < 0.05 vs. WT). AU, arbitrary units.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Individual infarct size data and corresponding means ± SD, *P < 0.05 vs. other groups. Open circles, males; closed circles, females. CON, 30-min occlusion, 120-min reperfusion, 1×IP/3×IP, one/three cycle(s) of 5-min occlusion, 10-min reperfusion before the sustained occlusion; LV, left ventricle.

Heterozygous Cx43-deficient mice have the same phenotype as wild-type mice (7). In the present study, heart rate was comparable between groups, and Cx43-deficient mice also had no electrocardiogram abnormalities. In the present study, 30-min occlusion and 120-min reperfusion resulted in similar infarct sizes in both genotypes. Thus Cx43 deficiency per se does not exaggerate tissue damage under sustained ischemia. In contrast, the infarct-sparing effect of IP was completely abolished in heterozygous Cx43-deficient mice. Apparently 50% of Cx43 is not sufficient to sustain the mechanism(s) of IP.

Our findings are somewhat contrary to prior findings in rat brain cells exposed to ischemia, which close their gap junctions to isolate themselves from surrounding cells (11). If this were true for cardiomyocytes, Cx43 deficiency in our study should have been cardioprotective. There is, however, good evidence that gap junctions are open at some time point during myocardial ischemia. In isolated rat cardiomyocytes and hearts, open gap junctions permit the equilibration of cytosolic concentrations of Na⁺ and Ca²⁺, thus contributing to propagation of rigor contracture (13, 14). In isolated mouse hearts, uncoupling of gap junctions using heptanol abolished IP protection, suggesting that an as-yet-unknown "survival" factor may be transmitted via gap junctions during IP (8). Also in the present study in vivo, to the extent that gap junctions remained open, the lack of IP in heterozygous Cx43-deficient mice points to the importance of intercellular communication (17) in the mechanism(s) of IP.

A number of recent investigations revealed that activated PKC forms signaling complexes with other proteins, which can be categorized as structural proteins, signaling proteins (Cx43 among them), and stress-activated proteins (10). The composition of such complexes is dynamically altered and causally involved in cardioprotective signaling (10, 19). The recruitment of Cx43 to the PKC signaling complex is associated with posttranslational modification of Cx43 during cardioprotection (10). Alternatively, disruption of the PKC-Cx43 signaling complex could act to prevent phosphorylation of some other unidentified targets associated with the complex. Obviously, a 50% reduction of functioning PKC-Cx43 signaling complexes is sufficient to completely abolish the infarct-sparing effect by IP. We propose Cx43 might be involved in cellular volume regulation, which is considered as one potential end effector of IP (15).