Amanda Lochner

Effect of ethanol on metabolism and function of perfused rat heart

Abstract The effect of ethanol (100 and 200 mm.) was studied on the metabolism and function of isolated, perfused rat hearts from control and ethanol-treated rats. Ethanol (200 mm.) increased incorporation of palm tate-C 14 uptake into tissue lipids, while C 4 O 2 formation was decreased, indicating a shift from exogenous to endogenous fuel. Long-term administration of ethanol resulted in a significant change in the pattern of myocardial palmitate metabolism as shown by the increased incorporation of palmintate-C 14 into tissue lipids. Ethanol depressed work performance and tension development of control rat hearts. Long-term administration of ethanol had no effect on the work performance of the perfused rat heart, but an increase in t-PH was suggestive evidence of an impairment in contractility.

Ischemic Preconditioning and the β-Adrenergic Signal Transduction Pathway

Background—Previous studies from our laboratory showed cyclic increases in tissue cAMP during a multiple-cycle preconditioning (PC) protocol, followed by attenuated cAMP accumulation during sustained ischemia. The aim of this study was to determine whether ischemia-induced activation of the β-adrenergic signaling pathway could act as a trigger in eliciting protection. Methods and Results—Isolated perfused rat hearts were preconditioned by 3×5 minutes of global ischemia, interspersed by 5 minutes of reperfusion. β-Adrenergic responsivity was assessed by measurement of tissue cAMP generation after β-adrenergic agonist administration at the end of the PC protocol. Tissue cAMP, adenylyl cyclase, and protein kinase A (PKA) activities and β-adrenergic receptor characteristics were assessed at different times. The role of cAMP generation in eliciting PC was studied by investigation of functional recovery during reperfusion after 25 minutes of global ischemia after (1) cAMP increases in the trigger period were pr...

Chronic melatonin consumption prevents obesity‐related metabolic abnormalities and protects the heart against myocardial ischemia and reperfusion injury in a prediabetic model of diet‐induced obesity

Abstract:  Obesity, a major risk factor for ischemic heart disease, is associated with increased oxidative stress and reduced antioxidant status. Melatonin, a potent free radical scavenger and antioxidant, has powerful cardioprotective effects in lean animals but its efficacy in obesity is unknown. We investigated the effects of chronic melatonin administration on the development of the metabolic syndrome as well as ischemia–reperfusion injury in a rat model of diet‐induced obesity (DIO). Male Wistar rats received a control diet, a control diet with melatonin, a high‐calorie diet, or a high‐calorie diet with melatonin (DM). Melatonin (4 mg/kg/day) was administered in the drinking water. After 16 wk, biometric and blood metabolic parameters were measured. Hearts were perfused ex vivo for the evaluation of myocardial function, infarct size (IFS) and biochemical changes [activation of PKB/Akt, ERK, p38 MAPK, AMPK, and glucose transporter (GLUT)‐4 expression). The high‐calorie diet caused increases in body weight (BW), visceral adiposity, serum insulin and triglycerides (TRIG), with no change in glucose levels. Melatonin treatment reduced the BW gain, visceral adiposity, blood TRIG, serum insulin, homeostatic model assessment index and thiobarbituric acid reactive substances in the DIO group. Melatonin reduced IFS in DIO and control groups and increased percentage recovery of functional performance of DIO hearts. During reperfusion, hearts from melatonin‐treated rats had increased activation of PKB/Akt, ERK42/44 and reduced p38 MAPK activation. Chronic melatonin treatment prevented the metabolic abnormalities induced by DIO and protected the heart against ischemia–reperfusion injury. These beneficial effects were associated with activation of the reperfusion injury salvage kinases pathway.

p38 and JNK have distinct regulatory functions on the development of apoptosis during simulated ischaemia and reperfusion in neonatal cardiomyocytes

Abstract. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (SI/R) were used as an in vitro model to delineate the role(s) of extracellular signal–regulated kinase (ERK), p38 and c–Jun NH2–terminal protein kinase (JNK), as well as PKB in apoptosis. Exposure of the myocytes to SI (simulated ischaemia – energy depletion induced by KCN and 2-deoxy-D–glucose) reduced cell viability, as measured by the 3–[4,5–dimethylthiazol–2–yl]–2,5–diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis as evidenced by caspase–3 activation and poly(ADP–ribose) polymerase (PARP) cleavage. However, morphological evidence of increased apoptosis, detected by staining with Hoechst 33342, was only seen in response to reperfusion. This suggests that although ischaemic conditions are sufficient to induce cellular markers of apoptosis (PARP cleavage and caspase–3 activation), reperfusion is required to complete the apoptotic pathway in these cells. Furthermore, SI resulted in a rapid, strong, biphasic activation of p38 concomitant with a weak and transient activation of the two ERK isoenzymes, p42/p44–MAPK. Reperfusion for 5 minutes resulted in a strong phosphorylation of p42/p44–MAPK, while no additional p38 activation was seen at this stage. On the other hand, p46/p54–MAPK (JNK) was phosphorylated in response to 5 minutes of reperfusion only and not during SI alone. A peak of PKB/Akt (Ser473) activity was seen within 5 minutes of exposure to SI, whereas PKB/Akt (Thr308) phosphorylation remained at the baseline level. Both PKB/Akt phosphorylation sites (Ser473 and Thr308) were phosphorylated after 5 minutes of reperfusion. Inhibition of PI–3–kinase activity, using wortmannin, decreased phosphorylation on both sites during SI. However, only SI/R-induced PKB/Akt phosphorylation on Thr308 was reduced by wortmannin. Myocytes pre–treated with SB203580, a p38–inhibitor, displayed a significant increase in cell viability [63.67 ± 1.85 to 84.33 ± 4.8% (p < 0.05)] and attenuation of the apoptotic index during SI/R [22.6 ± 2.94% to 9 ± 0.43% (p < 0.001)], while SP600125, a specific JNK inhibitor, caused a significant increase in caspase–3 activation [1.66 ± 0.03 fold to 2.56 ± 0.27 fold (p < 0.001)] and apoptotic index [22.6 ± 2.94% to 32.75 ± 6.13% (p < 0.05)]. However, PD98059, an ERK inhibitor, failed to affect apoptosis during SI/R. Inhibition of PI–3–kinase prevented the increase in mitochondrial viability usually observed during reperfusion. Interestingly, wortmannin caused a significant increase in PARP cleavage during reperfusion, but had no effect on caspase–3 activation or the apoptotic index. Our results suggest that p38 has a pro–apoptotic role while JNK phosphorylation is protective in our cell model and that these kinases act via caspase–3 to prevent or promote cell survival in response to SI/R–induced injury.

MELATONIN AND THE METABOLIC SYNDROME: A TOOL FOR EFFECTIVE THERAPY IN OBESITY-ASSOCIATED ABNORMALITIES?

The metabolic syndrome (MetS) is a cluster of metabolic abnormalities associated with increased risk for cardiovascular diseases. Apart from its powerful antioxidant properties, the pineal gland hormone melatonin has recently attracted the interest of various investigators as a multifunctional molecule. Melatonin has been shown to have beneficial effects in cardiovascular disorders including ischaemic heart disease and hypertension. However, its role in cardiovascular risk factors including obesity and other related metabolic abnormalities is not yet established, particularly in humans. New emerging data show that melatonin may play an important role in body weight regulation and energy metabolism. This review will address the role of melatonin in the MetS focusing on its effects in obesity, insulin resistance and leptin resistance. The overall findings suggest that melatonin should be exploited as a therapeutic tool to prevent or reverse the harmful effects of obesity and its related metabolic disorders.

Ischemic preconditioning: infarct size is a more reliable endpoint than functional recovery.

Abstract.The search for the mechanism of preconditioning-induced cardioprotection has been hampered by controversial results obtained by workers using different animal species, experimental models, protocols and endpoints. The aim of this study was to evaluate the role of the perfusion model (retrograde vs working), the infarct size and severity of ischaemia (regional vs global) as well as the endpoint (functional recovery vs infarct size) in preconditioning. The isolated perfused rat heart was preconditioned by 3 × 5 min global ischaemia, followed by different periods of regional or global ischaemia and reperfusion. Ischaemic preconditioning of working hearts resulted in increased functional recovery after 25–35 min global ischaemia, while retrogradely perfused hearts showed no significant improvement (except after 30 min global ischaemia). In addition, the percentage reduction in functional performance during reperfusion observed in the latter group was significantly less than in working hearts. Hearts were also subjected to regional ischaemia, perfused in either retrograde or working mode and infarct size determined. Regionally ischaemic working as well as retrogradely perfused hearts when preconditioned showed a significant increase in functional recovery after 35 min ischaemia only. In contrast to global ischaemia, the percentage recovery in mechanical performance of regionally ischaemic hearts was not affected by the mode of perfusion. Preconditioning of working hearts caused a significant reduction in infarct size after both 30 and 35 min ischaemia. However, preconditioned retrogradely perfused hearts showed a significant decline in infarct size after 35 min regional ischaemia only. In conclusion, the effect of the perfusion mode on functional recovery is dependent on the size and severity of ischaemia. It also affects the ischaemic time at which infarct size reduction by prior preconditioning occurs in the retrogradely perfused heart.

A role for the RISK pathway and KATP channels in pre‐ and post‐conditioning induced by levosimendan in the isolated guinea pig heart

Background and purpose: Myocardial reperfusion injury prevents optimal salvage of the ischaemic myocardium, and adjunct therapy that would significantly reduce reperfusion injury is still lacking. We investigated whether (1) the heart could be pre‐ and/or post‐conditioned using levosimendan (levosimendan pre‐conditioning (LPC) and levosimendan post‐conditioning (LPostC)) and (2) the prosurvival kinases and/or the sarcolemmal or mitochondrial KATP channels are involved.

A potential role for angiotensin II in obesity induced cardiac hypertrophy and ischaemic/reperfusion injury.

BackgroundThe mechanisms for obesity induced myocardial remodelling and subsequent mechanical dysfunction are poorly understood. There is good evidence that angiotensin II and TNFα have strong growth promoting properties and are elevated with obesity. In addition, these two peptides may interact to exacerbate myocardial ischaemic/reperfusion injury.HypothesisObesity increases systemic and myocardial renin–angiotensin system (RAS) activity and TNFα levels and contributes to obesity induced cardiac remodelling and ischaemic/reperfusion injury.MethodsMale Wistar rats were placed on a standard rat chow diet or cafeteria diet for 16 weeks. Two additional groups of rats received the respective diets and losartan (30 mg/ kg/d) in their drinking water. Hearts were perfused on the isolated working rat heart perfusion system and mechanical function was documented before and after 15 min normothermic total global ischaemia. Blood and myocardial samples were collected for angiotensin II, TNFα and NADPH oxidase activity determinations.ResultsThe rats on the cafeteria diet became obese compared to rats on the standard rat chow (438 ± 5.9 g vs 393 ± 7.3 g for control, p < 0.05). Obesity was associated with elevated serum angiotensin II (0.050 ± 0.015 pmol/ml vs. 0.035 ± 0.003 pmol/ml, p < 0.05) and TNFα levels (42.8 ± 5.93 pg/ml vs. 13.18 ± 2.50 pg/ml, p < 0.05), and increased heart to body weight ratios (3.1 ± 0.04 mg/g vs. 2.8 ± 0.03 mg/g, p < 0.05). Losartan had no effect on body weight but decreased basal myocardial angiotensin II and TNFΑ levels as well as heart to body weight ratio in the obese and lean controls (2.5 ± 0.05 mg/g and 2.6 ± 0.04 mg/g relative to their controls, p < 0.05). Hearts from obese rats had lower reperfusion aortic outputs (AO) than their concurrent controls (18.42 ± 1.17 ml/min vs. 27.8 ± 0.83 ml/min, p < 0.05). Losartan improved aortic output recoveries in obese rats (23.0 ± 1.71 ml/min, p < 0.05).ConclusionsObesity increased serum angiotensin II and TNFα levels, blood pressure, and heart weight to body weight ratios. These changes were associated with decreased basal and post–ischaemic myocardial mechanical function. Chronic AT1 receptor antagonism prevented the adverse changes in heart weight, mechanical function and susceptibility to ischaemic/reperfusion injury. Although current data do not exclude additional mechanisms for obesity induced cardiac remodelling, they suggest that angiotensin II may contribute to obesity induced cardiac remodelling and ischaemic/reperfusion injury.

Short- and long-term effects of melatonin on myocardial post-ischemic recovery

Abstract:  Melatonin, the chief secretory product of the pineal gland, has been shown to protect the heart against ischemia–reperfusion injury. This was attributed to its free radical scavenging and broad‐spectrum antioxidant properties. The possibility that melatonin may act via its receptor and intracellular signaling, has not yet been addressed in this regard. In all previous studies, only the acute effects of melatonin on the heart, were evaluated. The aims of the present study were to: (i) compare the acute and long‐term effects of melatonin on infarct size and functional recovery of the ischemic heart, and (ii) evaluate the role of the melatonin receptor in cardioprotection. For evaluation of the short‐term effects of melatonin on contractile recovery and infarct size, the isolated perfused working rat heart was subjected to 20 min global ischemia or 35 min regional ischemia respectively, and melatonin (25–50 μm) administered either before and during reperfusion, or before ischemia or during reperfusion after ischemia. The melatonin receptor was manipulated using luzindole and N‐acetyltryptamine. The long‐term effects of melatonin were evaluated 24 hr after melatonin administration (2.5 or 5.0 mg/kg, i.p.) or after oral administration for 7 days (20 or 40 μg/mL). Infarct size and mechanical recovery during reperfusion of the working heart were used as endpoints. Melatonin (50 μm), when administered either before and during reperfusion after ischemia or during reperfusion only, significantly improved cardiac output and work performance and reduced infarct size compared with untreated controls. Luzindole (5 μm), a melatonin receptor antagonist, abolished these cardioprotective effects. Long‐term administration of melatonin (i.p. or orally for 7 days) caused a significant reduction in infarct size of hearts subjected to 35 min regional ischemia. The cardioprotection persisted for 2–4 days after discontinuation of treatment. In summary, the results obtained suggest that melatonin induces short‐ as well as long‐term protection and that the melatonin receptor is also involved in its cardioprotective actions.

Normothermic ischemic cardiac arrest of the isolated working rat heart. Effects of time and reperfusion on myocardial ultrastructure, mitochondrial oxidative function, and mechanical recovery.

The ischemic state of the myocardium of the isolated working rat heart after induction of normothermic ischemic cardiac arrest was assessed by the interrelationship among changes in myocardial ultrastructure, mitochondrial oxidative phosphorylation, and tissue high energy phosphate contents. At all time intervals (10-40 minutes) studied, the ultrastructural changes were more severe in the subendocardium than in the subepicardium. After 25-40 minutes of normothermic ischemic cardiac arrest, the mitochondrial oxygen uptake (state 3) became increasingly depressed, particularly in mitochondria isolated from the subendocardium. Mitochondrial oxidative function, as measured in vitro, did not correlate well with mitochondrial ultrastructural damage. In addition, the effects of coronary reperfusion on the ability of the ischemic heart to recover in terms of ultrastructure, mechanical, and metabolic function were evaluated. Hearts subjected to 10-40 minutes of normothermic ischemic cardiac arrest showed almost complete ultrastructural recovery of the subepicardium upon reperfusion; regression of ultrastructural changes occurred to a lesser extent in the subendocardium. Reperfusion for 30 minutes did not alleviate the depression in mitochondrial oxidative function, while tissue ATP levels did not return to control, preischemic levels. After 20 minutes of normothermic ischemic cardiac arrest, the mechanical performance of the working heart during reperfusion was significantly depressed, compared with pre-ischemic control values. Normal ultrastructure of the subendocardium always accompanied mechanical recovery, while improvement of mitochondrial oxidative function was not essential.