Effect of ischemia-reperfusion on the post-rest inotropy of isolated perfused rat heart

This paper aims to study of the effects of ischemia‐reperfusion on the post‐rest inotropy and to characterize post‐rest B1:B2 ratio as an index of intracellular Ca²⁺ overload. When the rest interval between the cardiac beats is increased, the magnitude of the post‐rest beats is increased. First beat (B1) is maximally potentiated with exponental decline of the second (B2) and subsequent beats, thereby establishing a normal B1:B2 ratio of post‐ rest inotropy of the cardiac muscle. The rest potentiation of B1 and subsequent decay in the magnitude B2 is thought to develop from the time‐dependent changes in the Ca²⁺‐uptake and release from the sarcoplasmic reticulum (SR). Ca²⁺‐kinetics of SR can be modulated by a variety of interventions which produce Ca²⁺ loading of the SR. Methods: Isolated perfused (K‐H buffer, 34°C) rat hearts were paced at 1 Hz (steady state frequency). Interbeat intervals between 1s and 10s were introduced and the recovery in the left ventricular contractile force (Pmax) of post‐rest B1 and B2 for each interval was recorded. Their relative relationship was computed and compared under control and experimental conditions. Results: High extracellular Ca²⁺ (2.50 to 7.0 mM) or low extracellular Na⁺ (50% of control), and ischemia (60 min, 34°C) ‐ reperfusion (30 min, 34°C) caused the reversal of the control relationship of the B1 to B2, with B2 being more potentiated than B1, accompanied by the appearance of after‐contractions during the rest intervals of 4s or more. The mean (± SE) control B1:B2 ratio (at 4s interval) of 1.12 ± 0.05 was significantly (P<0.001) reduced to 0.93 ± 0.07; 0.89 ± 0.01; and 0.96 ± 0.02 after high Ca²⁺ (6 mM) perfusion, low Na⁺(50%) perfusion and ischemia‐reperfusion respectively. Simultaneous perfusion with ryanodine (1 μM) abolished the after‐contractions and significantly increased the reduced ratios. The time course of changes in B1:B2 ratio after graded ischemia‐reperfusion showed a significant fall in the ratio between 30 and 60 min of ischemia. A parallel change in Pmax and a significant rise in the left ventricular end‐diastolic pressure, indicating an irreversible phase of the injury was recorded. No significant changes in B1:B2 ratio were detected during the reversible phase (<30 min) of the ischemia‐reperfusion injury. Conclusions: Ischemia‐reperfusion induces significant alterations in the relative ratio of the post‐rest contractions of the left ventricle in isolated perfused rat heart. The altered ratios were characterized to predict the irreversibility of the reperfusion injury and to index the extent of Ca²⁺‐loading of the sarcoplasmic reticulum.     

Ischemic preconditioning is not additive to preservation with hypothermia or crystalloid cardioplegia in the globally ischemic rat heart

The aim of this study was to evaluate the additive protective efficiency of ischemic preconditioning when used in combination with conventional clinically relevant cardioprotective methods of hypothermia or hypothermic cardioplegia during sustained global ischemia.Isolated rat hearts were aorta-perfused with Krebs-Henseleit buffer and were divided into six groups (n = 10 each). Group I: Ischemia at 34°C for 60 min; Group PC+I: preconditioned (PC) ischemia at 34°C, 2 episodes of 5 min ischemia and 10 min reperfusion at 34°C followed by I; Group HI: hypothermic ischemia at 10°C for 60 min; Group PC+HI: preconditioned (PC) hypothermic ischemia, 2 episodes of 5 min ischemia and 10 min reperfusion at 34°C followed by HI; Group CPL+HI: single dose of 'Plegisol' cardioplegia followed by HI; Group PC+CPL+HI: preconditioned hypothermic cardioplegia, followed by CPL+HI. At the end of 60 min ischemia, all the hearts were reperfused at 34°C for 30 min when post-ischemic recovery in left ventricular contractile function and coronary vascular dynamics was computed and compared.There was a significant depression in the post-ischemic recovery of developed pressure (P_max), positive derivative of pressure (+dp/dt), negative derivative of pressure (-dp/dt) and heterometric autoregulation (HA) of contractile force in all the groups, with no major differences between the groups. Left ventricular end-diastolic pressure (LVEDP) was significantly elevated after I at 34°C. Preconditioning (PC+I) prevented the rise in the LVEDP and this was accompanied by a significant reduction in the release of purine metabolises in the coronary effluents, particularly adenosine, during the immediate reperfusion period. Hypothermia (HI) provided essentially the same level of metabolic and mechanical preservation as offered by PC+I. Combination of hypothermia with preconditioning (PC+HI) or cardioplegia (PC+CPL+HI), did not further enhance the preservation. Post-ischemic recovery in the regional contractile function (segment shortening, %SS) followed nearly identical pattern to global (P_max) recovery. Post-ischemic recovery in coronary flow (CF) was significantly reduced and coronary vascular resistance (CVR) was significantly increased in all the groups. Myogenic autoregulation (transient and sustained) was generally enhanced indicating increased vascular reactivity. Preconditioning did not alter the time-course of these changes.Preconditioned ischemia (34°C) preserved left ventricular diastolic functions and prevented the contracture development after sustained ischemia reperfusion at 34°C. This protective effect of preconditioning was possibly mediated by the reduction in the breakdown of purine metabolises. Hypothermia alone or in combination with crystalloid cardioplegia prevented the irreversibility of the ischemic injury but produced contractile and vascular stunning which was not improved by ischemic preconditioning. The results of this study indicate that preconditioning when combined with hypothermia or hypothermic cardioplegia offered no significant additional protection.     

Signal transduction mechanisms involved in cardiac preconditioning: Role of Ras-GTPase, Ca2 +/calmodulin-dependent protein kinase II and epidermal growth factor receptor

It is well established that brief episodes of ischemia/reperfusion (I/R) [preconditioning (PC)] protect the myocardium from the damage induced by subsequent more prolonged I/R. However, the signaling pathways activated during PC or I/R are not well characterized. In this study, the role of Ras-GTPase, tyrosine kinases (TKs), epidermal growth factor receptor (EGFR) and Ca^{2 +}/calmodulin-dependent protein kinase II (CaMK II) in mediating PC in a perfused rat heart model was investigated. A 40-min episode of global ischemia in perfused rat hearts produced significantly impaired cardiac function, measured as left ventricular developed pressure (P_max) and left ventricular end-diastolic pressure (LVEDP), and impaired coronary hemodynamics, measured as coronary flow (CF) and coronary vascular resistance (CVR). PC significantly enhanced cardiac recovery after I/R. Combination of PC and FPT III (Ras-GTPase inhibitor FPT III; 232 ng/min for 6 days) treatment did not produce any additive benefits as compared to PC alone. In contrast, PC-induced improvements in cardiac function after I/R were significantly attenuated by pretreatment with genistein (1mg/kg/day for 6 days), a broad-spectrum inhibitor of TKs, or AG1478 (1mg/kg/day for 6 days), a specific inhibitor of EGFR tyrosine kinase or KN-93 (578 ng/min for 6 days), a CaMK II inhibitor, before PC. These observations suggest that PC and FPT III pretreatment may produce cardioprotection via similar mechanisms. Present results also indicate that activation of TKs and specifically activation of EGFR-mediated TKs and CaMK II-mediated regulation of calcium homeostasis are part of the PC mechanisms that improve recovery after I/R. (Mol Cell Biochem 268: 175–183, 2005)     

Tissue acetylsalicylic acid esterase activity in rats with acute and chronic liver damage from carbon-tetrachloride and ethanol.

The hydrolysis of acetylsalicylic acid (ASA) in vivo by serum and various tissues of rats with experimentally produced acute and chronic toxic liver damage, was estimated by spectrophotometric measurement of salicylic acid (SA) appearance. It was inferred from the data obtained that the liver tissue helped to maintain normal ASA esterase activity in the blood which would otherwise be affected by liver damage.     

The protective effects of 17beta-estradiol against ischemia–reperfusion injury and its effect on pacing postconditioning protection to the heart

The role of pacing postconditioning (PPC) in the heart protection against ischemia–reperfusion injury is not completely understood. The aim of this study was to investigated if 17-β-estradiol (estrogen, E2), endogenous atrial natriuretic peptide (ANP), endogenous brain natriuretic peptide (BNP), and tumor necrosis factor-alpha (TNF-α) are involved in PPC-mediated protection. Langendorff perfused female Wistar rat hearts were used for this study. Hearts challenged with regional ischemia for 30 min subjected to no further treatment served as a control. The PPC protocol was 3 cycles of 30 s pacing alternated between the right atrium and left ventricle (LV). Protection was assessed by recovery of LV contractility and coronary vascular–hemodynamics. Ischemia induced a significant (P?<?0.05) deterioration in the heart function compared with baseline data. PPC alone or in combination with short-term E2 treatment (E2 infusion at the beginning of reperfusion) significantly (P?<?0.05) improved the heart functions. Short-term E2 treatment post-ischemically afforded protection similar to that of PPC. However, long-term E2 substitution for 6 weeks completely attenuated the protective effects of PPC. Although no changes were noted in endogenous ANP levels, PPC significantly increased BNP expression level and decreased TNF-α in the cardiomyocyte lysate and coronary effluent compared to ischemia and controls. Our data suggested a protective role for short-term E2 treatment similar to that of PPC mediated by a pathway recruiting BNP and downregulating TNF-α. Our study further suggested a bad influence for long-term E2 substitution on the heart as it completely abrogated the protective effects of PPC.     

Inhibition of Ras-GTPase,but not tyrosine kinases or Ca2+/calmodulin-dependent protein kinase II,improves recovery of cardiac function in the globally ischemic heart

The signaling pathways involved in ischemic heart disease are not well characterized. In this study, the roles of Ras-GTPase, tyrosine kinases (TKs) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in global ischemia and reperfusion (I/R) in a perfused rat heart model were investigated and compared to beneficial effects produced by preconditioning (PC). A 40 min episode of global ischemia followed by a 30 min reperfusion in perfused rat hearts produced significantly impaired cardiac function, measured as left ventricular developed pressure (Pmax) and left ventricular end-diastolic pressure (LVEDP), and impaired coronary hemodynamics, measured as coronary flow (CF) and coronary vascular resistance (CVR). Hearts from male Wistar rats pre-treated with the tyrosine kinase inhibitor, genistein (1 mg/kg/day for 6 days), or the CaMKII inhibitor, KN-93 (578 ng/min for 6 days), produced detrimental effects on recovery of cardiac function and coronary hemodynamics. In contrast, pre-treatment with Ras-GTPase inhibitor FPT III (232 ng/min for 6 days) significantly enhanced cardiac recovery in terms of left ventricular contractility and coronary vascular hemodynamics. Treatment with FPT III also significantly reduced expression of the sodium-hydrogen exchanger-1 (NHE-1) which was elevated during I/R as detected by Western blotting. These data suggest that TKs and CaMKII are involved in signaling pathways leading to recovery from cardiac ischemia, whereas activation of Ras-GTPase signaling pathways are critical in the development of cardiac dysfunction due to I/R.     

Chronic treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase, attenuates ischemia/reperfusion-induced cardiac dysfunction in normotensive and hypertensive diabetic animals

Diabetes is associated with increased incidence of cardiovascular disease. Mechanisms that contribute to development of diabetic cardiopathy are not well understood. Phosphatidylinositol 3-kinase (PI3K) is a family of protein kinases that play an important role in regulation of cardiac function. It has been shown that inhibition of certain PI3K enzymes may produce cardiovascular protection. The aim of the present study was to determine whether chronic treatment with LY294002, an inhibitor of PI3K, can attenuate diabetes-induced cardiac dysfunction in isolated hearts obtained from normotensive and hypertensive rats. Recovery of cardiac function after 40 min of global ischemia and 30 min of reperfusion, measured as left ventricular developed pressure, left ventricular end-diastolic pressure, coronary flow and coronary vascular resistance, was worse in hearts obtained from diabetic and/or hypertensive animals compared to their respective controls. Treatment with LY294002 (1.2 mg/kg/day) for 4 weeks significantly prevented diabetes-induced cardiac dysfunction in both normotensive and hypertensive rats. Treatment with LY294002 did not significantly alter blood pressure or blood glucose levels. These results suggest that inhibition of PI3K signaling pathways can prevent ischemia/reperfusion-induced cardiac dysfunction in normotensive and hypertensive rats without correcting hyperglycemia or high blood pressure.     

Lipid accumulation in the rat liver: a histological and biochemical study.

The sequential pattern of lipid accumulation and associated biochemical changes were studied in two commonly used experimental models of nutritional fatty liver in rats. Female rats were maintained for 8 weeks on high fat, low protein diets containing adequate methionine and choline, and drinking water ad libitum (Diet 1), or deficient in methionine and choline and containing 20% ethanol as a substitute for drinking water (Diet 2). Histologically, there was a progressive increase in liver lipids, mainly in the periportal areas. Occasional foci of liver cell necrosis with lipogranuloma formation occurred in areas of severe fatty change. These changes appeared earlier and were more marked in rats maintained on Diet 2. Electron micrographs revealed large lipid droplets in the liver cells, which sometimes contained myelin figures. The mitochondria were enlarged, distorted and appeared as amorphous structures with disorientated cristae in rats on Diet 1, whereas they had a condensed conformation in rats maintained on Diet 2. Rough endoplasmic reticulum was fragmented and degranulated particularly in rats on Diet 1, and smooth endoplasmic reticulum showed hyperplasia and vesiculation in rats on Diet 2. There was a progressive increase in the total liver lipids and triglycerides in both the groups of rats. This fatty change was accompanied by a significant increase in hepatic 3-hydroxybutyrate, acetoacetate, malate, 2-oxoglutarate, citrate, lactate, ammonia, glutamate, alanine and aspartate, and a significant decrease in oxaloacetate, urea and glucose concentrations. The mass action ratios for alanine aminotransferase, aspartate amino transferase, and glutamate dehydrogenase, generally moved in a parallel direction. Hepatic ATP content was considerably reduced accompanied by a decrease in [ATP]/[ADP] ratios and a significant increased in [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] ratios. There was a corresponding decrease in the [NAD+]/[NADH] ratios both in the cytoplasmic and mitochondrial compartments. These biochemical changes were particularly severe in rats maintained on Diet 1 and Diet 2 for 8 weeks. There was a very good relationship between impaired mitochondrial and endoplasmic reticulum functions, redox and phosphorylation states, and the relevance of their changes to the fate of fatty liver cells.