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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 11, 2005 as doi:10.1096/fj.04-3664fje. |
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,1
* Unit of Cardiac and Experimental Surgery, Careggi Hospital, Florence;
Department of Preclinical and Clinical Pharmacology, University of Florence, Italy;
Department of Anatomy, Histology and Forensic Medicine, University of Florence, Italy;
Unit of Nuclear Medicine, Careggi Hospital, Florence Italy; and
|| Prosperius Institute, Florence, Italy
1Correspondence: University of Florence, Dept. Anatomy, Histology and Forensic Medicine, Viale G. Pieraccini, 6. I-50139 Florence, Italy. E-mail: daniele.bani{at}unifi.it
SPECIFIC AIMS
Relaxin, best known for its effects on reproduction, has recently been validated as a cardiotropic hormone, being produced by the heart and acting on specific heart receptors. Our previous studies in rodents subjected to cardiac ischemia/reperfusion have shown that relaxin, given preventatively before ischemia, reduces myocardial injury and increases survival of the infarcted animals. From the clinicians’ viewpoint, these studies raise the intriguing question as to whether relaxin is also able to afford cardioprotection when ischemia has already induced its adverse effects on the myocardium, thus being of use as a drug for acute myocardial infarction. We designed the present study to test the therapeutic potential of recombinant human relaxin in a swine model of ischemia/reperfusion-induced acute myocardial infarction that reproduces the event of a patient with acute myocardial infarction subjected to primary transcutaneous coronary angioplasty (PTCA). Relaxin is therefore given as an adjunctive drug associated to PTCA with the aim at reducing reperfusion injury and enhancing myocardial salvage, thus preserving left ventricular function.
PRINCIPAL FINDINGS
1. Relaxin reduces the main serum and tissue indicators of ischemia/reperfusion-induced myocardial injury
Male swine, 30–40 kg, were anesthesized by ketamine (0.2 mL/kg b.wt.), intubated orotracheally, and ventilated mechanically with O2 and 1.5% isoflurane. Acute myocardial infarction was induced by a classical ischemia/reperfusion method. Ischemia (30 min) was obtained by transient ligation of the left anterior descending coronary artery after the 2nd diagonal branch, followed by reperfusion for 3 h. Human recombinant relaxin (Connetics Co., Palo Alto, CA, USA) was given for 20 min as a 1 mL/min continuous infusion into the right atrium, starting at reperfusion, to recreate the clinical event of a patient subjected to PTCA and adjunctive therapy in the same surgical session. We tested three increasing doses: 1.25, 2.5, and 5 µg/kg b.wt (Fig. 1
). Administration of relaxin at the higher doses (2.5 and 5 µg b.wt.) caused a marked statistically significant reduction of the main serum markers of myocardial cell damage—namely, myoglobin, CK-MB, and troponinT—which rose markedly in the control animals given the vehicle alone (Fig. 2
A–C). Compared with the vehicle-treated controls, relaxin reduced in a dose-related fashion the tissue parameters of cardiomyocyte apoptosis (caspase 3, TUNEL-positive cells). Moreover, at 2.5 and 5 µg/kg b.wt., relaxin decreased the ultrastructural signs of cardiomyocyte damage and dysfunction. In fact, in the vehicle-treated animals most cardiomyocytes showed cytoplasmic edema, hypercontraction of myofibrils, shrinkage of the plasma membrane, and mitochondrial swelling; in some cases, plasma membrane rupture and nuclear karyolysis were observed. None of these alterations can be observed upon relaxin treatment, in which cardiomyocytes had almost normal features, apart from moderate attenuation of sarcomeric Z and M lines and sporadic cytoplasmic and mitochondrial swelling.
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2. Relaxin acts by reducing oxygen free radical-mediated myocardial injury and inflammatory leukocyte recruitment
The relaxin-induced cardioprotection appears to involve a reduction of oxygen free radical-mediated myocardial injury, as indicated by the observation that relaxin caused a dose-related decrease of malondialdehyde (MDA), an indicator of membrane lipid peroxidation, and of tissue calcium overload, a marker of free radical-mediated plasma membrane dysfunction, in the myocardial tissue samples. This effect of relaxin is likely due to the marked reduction of the recruitment of inflammatory leukocytes within the reperfused myocardium, as indicated by the dose-related decrease in myocardial myeloperoxidase (MPO), a typical leukocyte enzyme.
3. Relaxin increases myocardial viability and reduces heart contractile dysfunction
Functional evaluation of myocardial salvage was carried out by cardiac single-photon emission computed tomography (SPECT) using 201Thallium (201Tl+), a competitor for K+, to label viable cardiac cells. The radionuclide solution (201TlCl, 3 mCi/111 MBq) was injected as a bolus in the left atrium 10 min before the end of reperfusion. The SPECT protocol used allowed us to evaluate the volume of the myocardium rendered ischemic and to identify and measure the irreversibly damaged myocardial tissue area.
Relaxin treatment resulted in an overall protection of the ischemic/reperfused myocardium, as shown by the striking reduction of irreversibly injured myocardial tissue that did not pick up the viability tracer 201Tl+ (Fig. 3
). The defect of 201Tl+ captation by the ventricular wall was 52.3% ± 2.6 in the vehicle-treated swine and dropped to 32.1% ± 6.2 in the swine given 5 µg/kg b.wt. relaxin (Student’s t test: P>0.05). Functionally, the relaxin-induced myocardial salvage improved the contractile performance of the heart, as indicated by the long-lasting, stable increase in cardiac index observed upon 5 µg/kg b.w. relaxin. No significant changes in the trend of heart rate and systemic blood pressure were observed between the relaxin-treated and control animals.
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DISCUSSION
The present study shows that recombinant human relaxin, given as a drug to counteract reperfusion-induced adverse effects in a swine model of acute myocardial infarction, affords a clear-cut protection to the heart as compared with the vehicle-treated controls. In fact, relaxin, administered at reperfusion, causes a reduction of the release of myoglobin, CK-MB and troponin T into the blood, the classical myocardium-specific proteins currently used to monitor heart injury in infarcted patients, as well as a decrease in the assayed tissue markers of cardiomyocyte apoptosis (caspase 3, TUNEL assay) and in the histopathological signs of cardiomyocyte injury and contractile dysfunction (myofibril hypercontraction). Relaxin reduces the extension of the irreversibly injured myocardial tissue that did not pick up the viability tracer 201Tl+. In the meantime, relaxin is able to sustain the contractile performance of the heart by preventing the decrease in cardiac output that usually occurs in the vehicle-treated swine. The relaxin-induced cardioprotection appears to involve a reduction of oxygen free radical-mediated damage, as suggested by the observed decrease of tissue MDA and calcium overload, likely due to the marked reduction of the recruitment of inflammatory leukocytes (MPO) within the reperfused myocardium. The mechanism of action of relaxin likely relies on an early protection at the level of coronary vessels. In fact, at reperfusion, relaxin could counteract the endothelium-mediated initiation of microcirculatory failure and inflammation, thereby decreasing no-reflow phenomenon and inflammatory myocardial damage. However, the possibility that relaxin could induce cardiomyocytes to become resistant to reperfusion-induced injury should not be ruled out. Clear-cut evidence for specific relaxin receptor expression has been given for atrial but not ventricular cardiomyocytes. Recently, however, a glucocorticoid-like, plasma membrane receptor-independent action of relaxin has been described: this could account for a direct protective effect of relaxin on ventricular cardiomyocytes.
Results of this study highlight the therapeutic potential of human relaxin, given as an adjunctive drug to catheter-based reperfusion, to reduce reperfusion injury and enhance heart salvage in acute myocardial infarction, and provides background to future clinical trials. They provide an useful indication of the relaxin dose that could be used in clinical studies: 5 µg/kg b.wt. afforded the best myocardial protection in our swine model.
Based on the current findings, relaxin appears more promising than other drugs that have been used to reduce ischemia-reperfusion injury, such as antioxidants. These drugs raised enthusiastic expectations when applied to cellular systems but gave rise to conflicting results and little or no concrete benefit when used to relieve heart damage and dysfunction by myocardial infarction, either in animal models or in clinical practice. At variance with antioxidants, relaxin could exert a much broader spectrum of protective actions, including the reduction of inflammatory leukocyte and platelet response, inhibition of the release of proinflammatory and arrhythmogenic mediators by mast cells, and the dilatation of coronary blood vessels potentially able to potentiate the direct and collateral circulation to the infarction area and thereby remove harmful substances generated locally by inflammation, reperfusion biochemistry, and cell demise. Relaxin could show advantages over adenosine, which is currently under clinical investigation for acute myocardial infarction. In fact, adenosine A1 receptor agonists have been found to cause the heart to become resistant to ischemia, but receptor activation should precede ischemia to afford protection. Hence, A1 receptor agonists should be maximally effective when administered preventatively before ischemia. The present study shows that relaxin can exert cardioprotection when given at reperfusion, thus offering a good chance as an adjunctive drug for PTCA.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3664fje;
This article has been cited by other articles:
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