Christian Mouas

Experimental evaluation of Celsior, a new heart preservation solution.

An original heart preservation solution (Celsior) has been developed, the formulation of which has been designed to fulfil two major objectives: (1) to combine the general principles of hypothermic organ preservation with those specific for the myocardium, and (2) to offer the possibility of being used not only as a storage medium but also as a perfusion fluid during initial donor heart arrest, poststorage graft reimplantation and early reperfusion. The major principles addressed by the Celsior formulation include (1) prevention of cell swelling (by mannitol and lactobionate), (2) prevention of by the Celsior formulation include (1) prevention of cell swelling (by mannitol and lactobionate), (2) prevention of oxygen-derived free radical injury (by reduced glutathione, histidine and mannitol), and (3) prevention of contracture by enhancement of energy production (glutamate) and limitation of calcium overload (high magnesium content, slight degree of acidosis). Two experimental preparations were used: The isolated isovolumic buffer-perfused rat heart model and the heterotopic rabbit heart transplantation model. In isolated heart experiments, hearts were arrested with and stored in Celsior for 5 h at 4 degrees C and subsequently reperfused for 1 h. A similar protocol was used in the transplantation experiments except that the total ischemic time was approximately 1 1/2 h longer (corresponding to 6 h of storage followed by the 25 additional minutes of cold ischemia required for graft implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased cardiac types I and III collagen mRNAs in aldosterone-salt hypertension.

Cardiac fibrosis is one of the deleterious events accompanying hypertension that may be implicated in the progression toward heart failure. To determine the mechanisms involved in fibrosis and the role of hemodynamic versus humoral factors, we studied the expression of genes involved in hypertrophy and fibrosis in the heart of rats treated with aldosterone for 2 months with addition of 1% NaCl and 0.3% KCl in water. This treatment induced arterial hypertension, a moderate left ventricular hypertrophy, and a decrease in plasma thyroxine. Equatorial sections of hearts from treated rats showed numerous foci of proliferating nonmuscular cells and a biventricular fibrosis. Computerized videodensitometry demonstrated an increase of collagen volume fraction by 152% and 146% and of the ratio of the perivascular collagen area and vascular area by 86% and 167% in left and right ventricles, respectively. As measured by slot blot, this cardiac fibrosis was accompanied by an increase in alpha 1-I procollagen mRNA by 75% and 160% (P < .01) and in alpha 1-III mRNA by 76% and 319% (P < .01) in left and right ventricles, respectively. Atrial natriuretic peptide mRNA was induced only in the hypertrophied left ventricle. We conclude that fibrosis is occurring and involves pretranslational regulation of collagen synthesis. Whereas hypertrophy and atrial natriuretic peptide mRNA increase are restricted to the left ventricle, fibrosis is initiated in both ventricles, supporting the hypothesis that this cardiac response is independent of hemodynamic factors.

Opening of potassium channels: the common cardioprotective link between preconditioning and natural hibernation?

BACKGROUND The tolerance of hibernating mammals to cold hypoxia is related to a factor similar to agonists of delta-opioid receptors. This study was designed to assess whether activation of these receptors could reproduce the protection conferred by ischemic preconditioning and whether such cardioprotection was similarly mediated by an opening of ATP-sensitive potassium (KATP) channels. METHODS AND RESULTS Thirty-two isolated rat hearts were arrested with and stored in Celsior at 4 degrees C for 5 hours before being reperfused for 2 hours. They were divided into 4 equal groups. Group 1 hearts served as controls. In group 2, ischemic preconditioning was elicited by two 5-minute global ischemia periods interspersed with 5 minutes of reperfusion before arrest. In group 3, hearts were pharmacologically preconditioned with a 15-minute infusion of the delta-opioid receptor agonist D-Ala2-D-Leu5-enkephalin (DADLE; 200 micromol/L). In group 4, the protocol was similar to group 3 except that infusion of DADLE was preceded by infusion of the KATP blocker glibenclamide (50 micromol/L). The salutary effects of both forms of preconditioning were primarily manifest as a better preservation of diastolic function, a reduced myocardial edema, and reduced creatine kinase leakage. This protection was abolished by administration of glibenclamide before DADLE. CONCLUSIONS These data suggest that activation of delta-opioid receptors improves recovery of cold-stored hearts to a similar extent as ischemic preconditioning, most likely through an opening of KATP channels. This provides a rationale for improving the preservation of hearts for transplantation by pharmacologically duplicating the common pathway to natural hibernation and preconditioning.

Phenylephrine, vasopressin and angiotensin II as determinants of proto-oncogene and heat-shock protein gene expression in adult rat heart and aorta.

The expression of two oncogenes (conc) c-myc and c-fos, coding for nuclear proteins which play a regulatory role in growth and differentiation, and of two genes coding for two heat shock proteins (HSP) 68 (molecular weight 68,000) and 70 (molecular weight 70,000), which have a protective function during stress, have been investigated by Northern blot analysis of the total RNA, extracted from adult rat ventricle and aorta. (1) The two onc transcripts are absent from these tissues but their expression can be enhanced by a pretreatment with cycloheximide. (2) The HSP70 is, in part, constitutive, while HSP68 is not; both are thermo-inducible in an isolated coronary perfused rat heart. (3) The four messenger RNA (mRNA) are expressed in both ventricles and aorta, 1 or 2 hours after i.p. injection of 6 mg/kg phenylephrine or 12 IU/kg of vasopressin. (4) They are also induced by a continuous or discontinuous injection of angiotensin II (7.5 micrograms/kg per min) for 1-2 h, but only in the aorta. The lack of ventricular response to angiotensin II in rat ventricles has been attributed to the lack of angiotensin II receptors in this tissue. This indicates that, in addition to mechanical factors, circulating hormones which have in common the use of the phosphoinositol pathway, may activate the expression of genes coding for regulatory proteins. This may play a role in the genesis of both ventricular and aortic hypertrophy.

Preconditioning with potassium channel openers: ☆ ☆☆ ★ ★★ ♢: A new concept for enhancing cardioplegic protection?

Ischemic preconditioning defines an adaptive endogenous mechanism in which a brief episode of reversible ischemia renders the heart more resistant to a subsequent period of sustained ischemia. Because the cardioprotective effects of ischemic preconditioning might be mediated by an activation of adenosine triphosphate-sensitive potassium channels, this study was designed to assess whether these effects could be duplicated by the preischemic administration of a potassium channel opener. Fifty isolated isovolumic buffer-perfused rat hearts underwent 45 minutes of normothermic potassium arrest followed by 1 hour of reperfusion. They were divided into five equal groups that differed with regard to the preconditioning regimen: Group 1 hearts were left untreated and served a controls; in group 2, preconditioning was achieved with 5 minutes of total global ischemia followed by 5 minutes of buffer reperfusion before cardioplegic arrest; in group 3, the preconditioning stimulus consisted of a 5-minute infusion of the potassium channel opener nicorandil (10 mumol/L) followed by 5 minutes of drug-free buffer perfusion before arrest; group 4 hearts underwent a similar protocol except that the infusion of nicorandil was preceded by that of the potassium channel blocker glibenclamide (10 mumol/L); group 5 hearts were ischemically preconditioned like those of group 2 except that the no-flow preconditioning period was also preceded by a 5-minute infusion of glibenclamide (50 mumol/L). The results demonstrate that ischemic preconditioning significantly improved contractility and reduced contracture during reperfusion, as compared with results in control hearts. These protective effects were duplicated by pretreatment with nicorandil but were abolished when the drug was antagonized by a prior infusion of glibenclamide. Likewise, the glibenclamide-induced blockade of potassium channels largely blunted the beneficial effects of ischemic preconditioning. These data suggest that opening of adenosine triphosphate-sensitive potassium channels substantially contributes to preconditioning-induced cardiac protection in a surgically relevant model of global ischemia and, consequently, that the use of potassium channel openers like nicorandil could be an effective means of enhancing cardioplegic protection.

Coronary flow as a determinant of c-myc and c-fos proto-oncogene expression in an isolated adult rat heart

Chronic cardiac overload induces quantitative and qualitative changes of the phenotype which finally adapt the myocardium to its new functional requirements (Swynghedauw, 1986). It has been proposed that both stretch and enhanced isometric tension could trigger these modifications (Peterson and Lesch, 1972), but until now no real messenger has been found. In a search for signals which may account for these changes, we decided to investigate the expression of two proto-oncogenes, c-fos and c-myc, coding for nuclear proteins (review in Adamson, 1987) because several of their properties are consistent with their possessing a role in the transduction of extracellular growth signals to the cell interior. We report here that, in adult rat heart, expression of the c-fos and c-myc proto-oncogene was both sequentially and transitorily increased when, in a beating heart but not in an arrested heart, the coronary flow (and/or pressure) was augmented. This was studied in an isolated, coronary perfused heart preparation, a model in which the initial conditions of a cardiac overload may be mimicked in such a way that protein synthesis is stimulated by increasing the coronary perfusion pressure (Kira et al., 1984).

Is potassium channel opening an effective form of preconditioning before cardioplegia

BACKGROUND Opening of adenosine triphosphate-sensitive potassium channels might be one of the mechanisms by which preconditioning preserves the myocardium against ischemic damage. The present study was therefore designed to compare the protective efficacy of ischemic preconditioning with that of pharmacologic preconditioning involving the use of a potassium channel opener in a surgically relevant model of cold cardioplegic arrest. METHODS Thirty isolated isovolumic rat hearts were subjected to 2 hours of potassium arrest at an average myocardial temperature of 23 degrees C, followed by 1 hour of reperfusion. Three groups (n = 10 per group) were studied: (1) control (no prearrest intervention); (2) ischemic preconditioning, achieved with 5 minutes of noflow ischemia followed by 5 minutes of reperfusion before arrest; and (3) pharmacologic preconditioning, achieved with a 5-minute infusion of the potassium channel opener nicorandil (10 mumol/L) followed by 5 minutes of drug-free perfusion before arrest. Standard functional indices were measured at multiple times during reperfusion, at the end of which pressure-volume curves were constructed and compared with those obtained at baseline. RESULTS Both ischemically and pharmacologically preconditioned hearts recovered systolic and diastolic function to a significantly greater extent than the controls. There was no difference in the recovery patterns between the forms of preconditioning. However, analysis of the postischemic pressure-volume curves demonstrated that nicorandil-preconditioned hearts incurred the smallest losses of compliance throughout the ischemia-reperfusion sequence. CONCLUSIONS The protective effects of a standard ischemic preconditioning challenge on functional recovery after an episode of moderately hypothermic cardioplegic arrest can be duplicated by pharmacologic opening of adenosine triphosphate-sensitive potassium channels. This finding may be of clinical relevance because of the availability of potassium channel openers, such as nicorandil, for human use.

Increased injury of hypertrophied myocardium with ischemic arrest: Preservation with hypothermia and cardioplegia

Many patients undergoing cardiac surgery have some degree of myocardial hypertrophy. To assess the response of hypertrophied myocardium to simulated cardiac surgery, left ventricular hypertrophy was induced in rats by aortic banding, and ventricular function was measured by means of the isolated, isovolumic heart perfusion technique. The hypertrophied hearts had a greater susceptibility to ischemic injury than nonhypertrophied control hearts, as manifested by a greater degree of diastolic contracture during the recovery period after 30 minutes of ischemic arrest at 37 degrees C. Hypothermia without cardioplegia during a 2-hour arrest did not completely preserve diastolic function in the hypertrophied hearts, but cardioplegia combined with hypothermia completely protected the hypertrophied hearts against 2 hours of ischemia. The results suggest a need for both hypothermic and cardioplegic preservation techniques in patients with myocardial hypertrophy who have cardiac surgical procedures requiring a significant period of myocardial ischemia.

Inhibition of the pacemaker current: a bradycardic therapy for off-pump coronary operations

BACKGROUND The accurate performance of coronary anastomoses on the beating heart requires some form of myocardial immobilization that can be achieved pharmacologically. Different classes of drugs can be used to induce bradycardia, but the most effective in this setting of off-pump operation has not yet been determined. METHODS Fifty-six isolated buffer-perfused rabbit hearts were divided into seven equal groups. Control hearts were continuously perfused throughout the experimental time course. A second group of hearts underwent 60 minutes of potassium arrest (at 37 degrees C) followed by 1 hour of reperfusion. The following pharmacologic approaches were tested in the remaining five groups: short-acting beta-blockade (esmolol, 6 x 10(-3) mol/L and 3 x 10(-4) mol/L), opening of adenosine triphosphate-dependent potassium channels (nicorandil, 10(-3) mol/L and 10(-5) mol/L), and inhibition of the pacemaker current, which largely accounts for the diastolic depolarization of sinoatrial node cells (S 16257-2, 3 x 10(-6) mol/L). Each drug was infused at a constant rate for 60 minutes, after which hearts were perfused for 1 additional hour with drug-free buffer. Heart rate and isovolumic measurements of function and coronary flow were serially taken during and after drug infusion. RESULTS The worst recovery of systolic and, moreover, diastolic function was yielded by potassium arrest. Neither esmolol nor nicorandil was able to induce a significant bradycardia. However, nicorandil did not impair function which, conversely, was markedly depressed after esmolol therapy. Significant bradycardia (p < 0.0001 versus corresponding baseline values and versus all other groups) was only achieved with pacemaker current inhibition, which was otherwise associated with an excellent preservation of contractility, diastolic function, and coronary flow. CONCLUSIONS Inhibition of the pacemaker current seems to be an effective approach for inducing intraoperative bradycardia without compromising left ventricular function or flow.

Failure of Preconditioning to Improve Postcardioplegia Stunning of Minimally Infarcted Hearts

BACKGROUND Ischemic preconditioning is an effective means of reducing myocardial infarct size, but its ability to attenuate stunning after an episode of surgically relevant global ischemia remains elusive. Likewise, the role played by adenosine in this setting has not been established conclusively. This study was designed to address these two issues. METHODS Thirty isolated, crystalloid-perfused rabbit hearts were subjected to 60 minutes of normothermic potassium arrest and 60 minutes of reperfusion. They were divided into three equal groups. The first group had no prearrest intervention and served as a control. In the second group, ischemic preconditioning was achieved with 5 minutes of zero-flow ischemia followed by 5 minutes of buffer reperfusion before arrest. In the third group, the hearts were first infused for 5 minutes with the nucleoside transport inhibitor draflazine (10(-6) mol/L), the efficacy of which was demonstrated by reversal of the normally high inosine to adenosine ratio in the coronary effluent. These hearts subsequently were given 2 additional minutes of ischemic (zero-flow) preconditioning followed by 5 minutes of reperfusion before arrest. During reperfusion, function was measured serially under isovolumic conditions. Myocardial necrosis was estimated from the release of creatine kinase after the initial 5 minutes of reflow, and the planimetrically determined extent of infarction was determined by triphenyltetrazolium chloride staining. RESULTS Baseline hemodynamic data were comparable among the three groups. Neither ischemic preconditioning alone nor ischemic preconditioning with draflazine-induced enhancement of endogenous adenosine levels improved postischemic recovery of function over that seen in control, untreated hearts. These results correlated with a minimal amount of infarction in the control group (on average, <10% of the left ventricle), which was not reduced further by either preconditioning regimen. CONCLUSIONS These data support the idea that, in the absence of substantial necrosis, ischemic preconditioning does not ameliorate postischemic stunning, which leads to the question of its usefulness in clinical cardiac operations. Although, in this model, protection was not potentiated by increasing endogenous concentrations of adenosine, it remains a worthwhile goal to identify the final effectors of the signaling pathway accounting for the otherwise demonstrated cardioprotective effects of preconditioning because of the potential for these mediators to act as effective antiischemic agents.