Predicting functional recovery from ischemia in the rat myocardium

Abstract

Depletion of high-energy phosphates, accumulation of inorganic phosphate and intracellular acidosis have each been proposed as important events in the transition from reversible to irreversible ischemic injury. To assess whether each variable is predictive of functional recovery on reperfusion, these were measured in the isolated isovolumic rat heart using 31P NMR. Perfused hearts were subjected to either 10, 12 or 40 min of normothermic ischemia followed by 40 min of reperfusion. Hearts were then freeze-clamped for further analysis of phosphate metabolites by NMR and ion chromatography. High-energy phosphates, Pi, phosphomonoesters and pH were measured by 31P NMR spectroscopy at 2 minute intervals. Heart rate and developed pressure were monitored simultaneously. All hearts undergoing 10 min of ischemia and 40% of hearts subjected to 12 min of ischemia demonstrated good functional recovery. The remainder of hearts ischemic for 12 min went into contracture on reperfusion with little return of function. Hearts subject to 40 min of ischemia went into ischemic contracture and showed no recovery on reperfusion. Intracellular pH, [ATP], and [Pi] measured prior to reperfusion did not predict the extent of recovery. However, phosphomonoesters were detected prior to reperfusion in all hearts that did not recover well, but were not observed in hearts that showed good mechanical recovery. Analysis of tissue extracts by 31P NMR and ion chromatography indicated that the most prominent components of the phosphomonoesters were glucose 6-phosphate, alpha-glycerol phosphate and AMP. In conclusion, of the various phosphorus metabolites that can be measured by 31P NMR, only one group, the phosphomonoesters, was predictive of functional recovery.

Adenylate kinase AK1 knockout heart: energetics and functional performance under ischemia-reperfusion

“…After reperfusion, hearts were labeled with 18 O, which was introduced for 30 s with the K-H buffer supplemented with 30% of 18 O-labeled H2O (Isotec) as described previously (30). Hearts were freeze-clamped, pulverized under liquid N 2, and extracted in a solution containing 0.6 M HClO4 and 1 mM EDTA (30,31).…”

“…Protein content was determined with a DC Protein Assay kit (Bio-Rad). Extracts were neutralized with 2 M KHCO 3 and used to determine 18 O incorporation. Rapid 18 O labeling of Pi, ␥-ATP, and creatine phosphate (CrP) was determined with 18 Oassisted 31 P NMR spectroscopy (30), whereas slow 18 O labeling of ␤-ADP and ␤-ATP was determined by mass spectrometry (14,43).…”

“…18 O labeling of ␤-ADP and ␤-ATP was quantified by mass spectrometry (13,14). Cellular ATP and ADP were purified by high-performance liquid chromatography (Hewlett-Packard 1100 series HPLC system) with a Mono Q HR 5/5 ion-exchange column (Pharmacia Biotech).…”

“…Samples that contained phosphoryls of ␤-ADP and ␤-ATP, as glycerol 3-phosphate, were converted to respective trimethylsilyl derivatives with Tri-Sil/BSA (Pierce) as the derivatization agent. The 18 O enrichment of phosphoryls in glycerol 3-phosphates was determined with a Hewlett-Packard 5973 gas chromatograph-mass spectrometer operated in the select ion-monitoring mode. Mass ions (m/z) of 357, 359, 361, and 363 that corresponded to phosphoryl species of 16 O, 18 O1, 18 O2, and 18 O3 were monitored in trimethylsilyl derivatives of ␤-ADP and ␤-ATP (13,17).…”

Myocardial ischemia-reperfusion and cardioprotection: a study with microdialysis in a porcine model

“…These findings may indicate that lactate is accumulated intracellularly. Another metabolic fate of glucose during myocardial ischemia may be to the accumulation of glucose-6-phosphate and glycerol-3-phosphate (Jeffrey 1992). The G-3-P may further be hydrolyzed and recovered as glycerol (de Groot 1994).…”