E. A. Savino

Involvement of mitochondrial permeability transition, glutathione status, pentose phosphate pathway and oxidative damage in the protective effect of fasting on the ischaemic-reperfused rat heart.

1 Fasting, which increases the catabolism of fatty acids, gives functional protection to the ischaemic–reperfused heart. To obtain further knowledge of this cardioprotective effect, changes in mitochondrial permeability transition (MPT) were measured by the entrapment of 2‐deoxy‐[3H]‐glucose (2‐DG). We also assessed whether MPT is associated with changes in glutathione status, the activity of glucose‐6‐phosphate‐dehydrogenase (G6PDH) and tissue oxidative damage, estimated by the measurement of Thiobarbituric acid‐reactive substances (TBARS). 2 Spontaneously beating hearts of fed and 24 h fasted rats were Langendorff perfused with Krebs’–Ringer bicarbonate solution (10 mmol/L glucose) and exposed to 25 min global ischaemia, followed by 30 min reperfusion. 3 Ischaemia–reperfusion resulted in a fourfold increase in mitochondrial entrapment of 2‐DG in the fed group. This response was 29% lower in the fasted group, but there were no concomitant changes in total retention of 2‐DG in the heart. Fasting increased the activity of G6PDH by a factor of 1.4 and caused a 2.8‐fold increase in the ratio of reduced glutathione to oxidized glutathione (GSH : GSSG) at the end of the pre‐ischaemic period. Ischaemia–reperfusion did not affect G6PDH activity, but reduced the GSH : GSSG ratio in both the fed and fasted groups by 50%. Therefore, the GSH : GSSG ratio remained higher in the fasted group. Fasting also decreased cellular levels of TBARS by approximately 25%. Lipolysis of endogenous triacylglycerol was increased during the pre‐ischaemic period in the fasted group. 4 These data suggest that the enhancement of fatty acid catabolism that occurs in fasting activates mechanisms that tend to reduce oxidative damage and limit MPT.

Effects of the AMP-activated protein kinase inhibitor compound C on the postconditioned rat heart

Ischemic postconditioning (IPOC) protects the myocardium from ischemic–reperfusion injury, improving functional recovery and cell viability. This protection is concurrent with stimulation of glycogen breakdown, increased mitochondrial ATP synthesis and content, maintenance of reduced-to-oxidized glutathione ratio (GSH/GSSG), and decreased oxidative damage. The present study’s objective was to assess whether these effects are associated with increased resistance to mitochondrial permeability transition pore (MPTP) opening. The effects of the AMP-activated protein kinase (AMPK) inhibitor, compound C (CC), were measured to investigate association with AMPK. Mitochondria removed from postconditioned hearts required higher calcium levels to induce MPTP opening. Improved functional recovery, increased glycogen mobilization, maintenance of the GSH/GSSG ratio, decreased oxidative damage, and increased resistance to MPTP opening were abrogated when the hearts were postconditioned in the presence of CC, without affecting preservation of cell viability. Although AMPK appears to play a role in IPOC, it would not be the major cellular mediator.

Influence of fasting on the effects of diazoxide in the ischemic-reperfused rat heart

This investigation aimed to asses whether the mitochondrial ATP-sensitive potassium channel opener diazoxide could reproduce the protection conferred by ischemic preconditioning and to ascertain whether its effects are associated with changes in glycogen breakdown and glycolytic activity. Hearts of fed and 24-h fasted rats were perfused with 10 mM glucose containing medium and exposed to 25 min no-flow ischemia plus 30 min reperfusion. Diazoxide (10 μM) perfusion was begun 10 min before ischemia and continued throughout the experiment. Fasting accelerated reperfusion recovery of contraction, reduced the post-ischemic contracture and decreased lactate accumulation during ischemia but had no effects on glycogen levels and cellular viability. Diazoxide, did not affect glycogen catabolism but improved reperfusion recovery of contraction. Furthermore, diazoxide reduced ischemic lactate accumulation and contracture amplitude only in the fed group whereas it improved cell viability in the fed and fasted groups. These data indicate that: 1) reduced lactate production which may attenuate myocyte acidification might explain, at least in part, the beneficial effects of diazoxide on mechanical function, although data obtained with the fasted rat hearts indicate that other mechanisms must be involved as well; 2) the reduction of lactate production occurring in the fed group, does not seem to be related to glycogenolysis; and 3) since diazoxide improved cell viability in the fasted rat group where it did not reduce glycolytic activity, other mechanisms may be responsible for this cytoprotective effect.ResumenSe estudia en este trabajo si el diazóxido, que activa los canales mitocondriales de potasio sensibles al ATP, reproduce los efectos del precondicionamiento isquémico y si tales efectos se asocian con cambios en la glucogenolisis y la actividad glicolítica. Corazones perfundidos de ratas alimentadas y tras 24 h de ayuno se exponían a 25 min de isquemia con reperfusión de 30 min. La perfusión con diazóxido (10 μM) comenzaba 10 min antes de la isquemia y continuaba durante todo el experimento. El ayuno acelera la recuperación de la contracción al reperfundir, reduce la contractura post-isquémica y disminuye la acumulación isquémica de lactato, pero no tiene efectos sobre los niveles de glucógeno y la viabilidad celular. El diazóxido no afecta a la glucogenolisis, pero mejora la recuperación post-isquémica de la contracción. Además, el diazóxido reduce la acumulación isquémica de lactato y la amplitud de la contractura sólo en el grupo alimentado, pero mejora la viabilidad celular en ambos grupos. Los resultados indican que: 1) la reducción de la producción de lactato, que atenuaría la acidificación del miocito, explicaría, al menos en parte, los efectos beneficiosos del diazóxido sobre la función mecánica, aunque los resultados de las ratas en ayunas indican la implicación de otros mecanismos; 2) la reducción de la producción de lactato obtenida en el grupo alimentado no parece estar relacionada con la glucogenolisis; y 3) como el diazóxido mejora la viabilidad celular en los corazones de ratas en ayunas en los cuales no reduce la glicólisis, otros mecanismos serían responsables del efecto citoprotector

The effects of 4-pentenoic and pentanoic acids on the isolated rat atria

The peak developed tension and the pacemaker frequency of the isolated atria from fed and fasted rats, declined progressively during the incubation in a glucose-free medium containing 2-deoxyglucose. The atria from fed rats exhibited a faster decline than those from fasted rats, which was associated to a slower triacylglycerol lipolysis. 4-Pentenoic acid inhibited the lipolysis of both groups of atria but did not alter the atrial contractile performance. However, it enhanced the decline of the pacemaker frequency in the atria from fasted rats whereas, in contrast, it alleviated the decline in the fed atria. n-Pentanoic acid ameliorated the impairment of the contractile and pacemaker activities in both groups of atria, without affecting the lipolysis. It was concluded that, since the inhibition of the intramyocardial lipolysis did not correlate with changes of the atrial functions, 4-pentenoic acid was not appropriate to assess about the contribution of endogenous triacylglycerol to the maintenance of the atrial contractile and pacemaker activities.

Effects of 3-methyladenine on isolated left atria subjected to simulated ischaemia-reperfusion.

Although autophagy is a prominent feature of myocardial ischaemia and reperfusion, its functional significance is unclear and controversial. In order to gain a deeper insight into the role of autophagy in myocardial ischaemia‐reperfusion, we explored the effects of the pharmacological inhibitor of autophagy 3‐methyladenine (3‐MA). Isolated rat atria subjected to simulated 75‐min ischaemia/75‐min reperfusion (Is‐Rs) in the presence or absence of 3‐MA were used. The LC3‐II/LC3‐I ratio, an indicator of autophagosome formation, did not increase after ischaemia either in the presence or absence of 3‐MA, but there was significant enhancement during reperfusion, which was prevented by the presence of 3‐MA. The autophagy inhibitor also increased p62 protein, one of the specific substrates degraded through the autophagy‐lysosomal pathway. Electron micrographs showed double membrane autophagosome‐like structures during reperfusion, which were absent in atria subjected to Is‐Rs in the presence of 3‐MA. These findings suggest that this agent inhibited the autophagic flux under the present experimental conditions. Inhibition of autophagy during Is‐Rs was accompanied by a high incidence of tachyarrhythmias during reperfusion, and a decrease in the maximal inotropic response to β‐adrenergic and to calcium stimulation at the end of Is‐Rs. Deterioration of mitochondrial morphology and function, without affecting cell viability, was observed in atria subjected to Is‐Rs in the presence of 3‐MA. The present results suggest an association between the inhibition of autophagy and functional alterations of the cells that have undergone sublethal stress, and have been able to recover in this experimental model of ischaemia–reperfusion.

The Effects of 4-Pentenoic and Pentanoic Acids on the Hypoxic Rat Atria

When exposed to hypoxia, the isolated rat atria released lactate into the bathing medium and underwent a rise in resting tension and a decline of the contractions frequency. In some of them, it also occurred a complete cessation of the pacemaker activity. Atria from 24-h fasted rats, when compared to those from fed ones, exhibited a lower lactate output, a higher rise in resting tension, a faster decay of the contraction frequency and an increased occurrence of atrial arrest. In both the fed and fasted rats atria, some triacylglycerol lipolysis remained throughout the hypoxic incubation. Addition of 2 mM 4-pentenoic acid abolished the lipolytic activity and reduced lactate output in both groups of atria. In the fed rats atria it also accelerated the decrease of the pacemaker frequency. Pentanoic acid reduced lactate output in both groups of atria and in those from fed rats it did not alter lipolysis but increased the rise in resting tension, the decline of the pacemaker frequency and the occurrence of atrial arrest. Present data indicate that although 4-pentenoic acid inhibits fatty acid oxidation and endogenous lipolysis, it was not able to reduce the noxious effects of hypoxia. Since the effects of 4-pentenoic acid were rather similar to those of fasting and pentanoic acid, they might be ascribed to the accumulation of its own oxidative metabolites which could be detrimental for the hypoxic atria.

Effects of wortmannin on cardioprotection exerted by ischemic preconditioning in rat hearts subjected to ischemia-reperfusion

Ischemic preconditioning (IPC) is one of the most powerful interventions to reduce ischemia-reperfusion injury. The aim of the present study was to investigate the involvement of the phosphatidylinositol-3-kinases (PI3Ks) family in cardioprotection exerted by IPC and the relationship between preservation of mitochondrial morphology and ATP synthesis capacity. In this regard, macroautophagy (autophagy) is considered a dynamic process involved in the replacement of aged or defective organelles under physiological conditions. IPC consisted of four 5-min cycles of ischemia-reperfusion followed by sustained ischemia. Wortmannin (W), a PI3K family inhibitor, was added to the perfusion medium to study the involvement of autophagy in the beneficial effects of IPC. In the present study, LC3-II/I expression was significantly increased in the IPC group when compared with the control group. The hearts subjected to IPC showed greater degradation of p62 than control groups, establishing the existence of an autophagic flow. Electron microscopy showed that IPC preserves the structural integrity of mitochondria after ischemia and at the end of reperfusion. Moreover, hearts subjected to IPC exhibited increased mitochondrial ATP synthesis. The beneficial effects of IPC were abolished by W in all trials of this study, abolishing the differences between the IPC and control groups. These results suggest that IPC could partly reduce injury by ischemia-reperfusion (I/R) by decreasing mitochondrial damage and promoting autophagy. Since W is a nonspecific inhibitor of the PI3Ks family, further research is required to confirm participation of PI3K in the response to IPC.

Involvement of energetic metabolism in the effects of ischemic postconditioning on the ischemic-reperfused heart of fed and fasted rats

The effects of ischemic-postconditioning (IPOC) on functional recovery and cell viability of ischemic-reperfused hearts from fed and fasted rats were studied in relation to triacylglycerol and glycogen mobilization, ATP content, glucose-6-phosphate dehydrogenase activity and reduced/oxidized glutathione (GSH/GSSG). Oxidative damage was estimated by measuring thiobarbituric acid reactive substances (TBARS). IPOC improved contractile recovery and cell viability in the fed but attenuated them in the fasted hearts. In both groups ischemia lowered glycogen. IPOC further reduced it. Triacylglycerol remained unchanged during ischemia-reperfusion in both groups, but triacylglycerol mobilization was activated by IPOC in the fasted group. ATP was increased by IPOC in the fed hearts, but lowered in the fasted ones, which appeared to be associated with the rates of ATP synthesis in isolated mitochondria. In the fed hearts IPOC raised glucose-6-phosphate dehydrogenase activity and GSH/GSSG, and lowered TBARS. These results suggest that IPOC effects are associated with changes in the ATP supply, mobilization of energy sources and glutathione antioxidant ratio.

Effects of 5-hydroxydecanoate and ischemic preconditioning on the ischemic-reperfused heart of fed and fasted rats.

This investigation aimed to assess whether the mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) could abolish the protection conferred by fasting and ischemic preconditioning (IPC) and to ascertain whether these effects are associated with glycogen breakdown and glycolytic activity. Langendorff perfused hearts of fed and 24-h fasted rats were exposed to 25 min ischemia plus 30 min reperfusion. IPC was achieved by a 3 min ischemia plus a 5 min reperfusion cycle. 5-HD (100 μM) perfusion begun 5 min before IPC or 13 min before sustained ischemia in the non preconditioned groups. Fasting improved the reperfusion recovery of contraction, decreased the contracture and the lactate production, increased glycogenolysis and did not affect the percentage of viable tissue. 5-HD abolished the effects of fasting on the contractile recovery but did not affect the contracture. 5-HD decreased the lactate production in the fed group, increased the preischemic glycogen content in both nutritional groups and did not affect the ischemic glycogen fall. IPC improved the contractile function but prevented the contracture only in the fed group, reduced lactate accumulation and glycogenolysis and evoked an increase of the viable tissue. 5-HD abolished the effects of IPC on the contractile recovery and did not affect its effect on the contracture, lactate production, glycogenolysis and viable tissue. These data suggest that the mitocondrial ATP-sensitive potassium channel is involved in the effects of fasting and IPC on the contractile function but the other cardioprotective and metabolic effects appear evoked through other mechanisms. Also suggest that besides the inhibition of the mitochondrial potassium channel, other mechanisms mediate the effects of 5-HD.ResumenSe investiga si el 5-hidroxidecanoato (5-HD), bloqueante de los canales mitocondriales de potasio sensibles al ATP, impide la protección conferida por el ayuno y el precondicionamiento isquémico (PCI) y si estos efectos se asocian con la glucogenolisis y la actividad glicolítica. Corazones perfundidos de ratas alimentadas o en ayunas de 24 h, se expusieron a 25 min de isquemia seguidos de 30 min de reperfusión. El PCI consistió en un ciclo de 3 min de isquemia seguido de 5 min de reperfusión. La perfusión con 5-HD (100 μM) comenzó 5 min antes del PCI o 13 min antes de la isquemia sostenida en los grupos no precondicionados. El ayuno mejora la recuperación contráctil al reperfundir, disminuye la contractura y la producción de lactato, aumenta la glucogenolisis y no afecta al porcentaje de tejido viable. El 5-HD anula los efectos del ayuno sobre la recuperación de la contracción pero no afecta a la contractura. El 5-HD reduce la producción de lactato en el grupo alimentado, aumenta el contenido preisquémico de glucógeno en ambos grupos y no altera la caída del glucógeno. El PCI mejora la función contráctil pero reduce la contractura sólo en el grupo alimentado, reduce la acumulación de lactato y la glucogenolisis, y aumenta el tejido viable. Los resultados sugieren que el canal mitocondrial de potasio sensible al ATP está implicado en los efectos del ayuno y el PCI sobre la función contráctil pero los otros efectos cardioprotectores y metabólicos parecen mediados por otros mecanismos. También sugieren que además de la inhibición del canal mitocondrial de potasio, otros mecanismos median en los efectos del 5-HD.

Influence of fasting on the effects of dimethylamiloride and oxfenicine on ischaemic–reperfused rat hearts

Abstract To assess whether glycolysis, Na+–H+ exchange and oxidation of fatty acid derived from endogenous lipolysis are involved in the beneficial effects of 24-h fasting on the ischaemic – reperfused heart, it was studied the effects of inhibiting Na+ – H+ exchange using 10 μM dimethylamiloride and fatty acid oxidation using 2 mM oxfenicine, on the functional activity, lactate production and cell viability measured with tetrazolium stain. Since fasting accelerates heart fatty acid oxidation, data were compared to those from fed rats; using Langendorff perfused (glucose 10 mM) hearts of 250–350 g Wistar rats exposed to 25 min ischaemia – 30 min reperfusion. Fasting reduced the ischaemic rise of end diastolic pressure (contracture), improved recovery of contraction and lowered lactate production in comparison with the fed whereas cellular viability was similar in both groups. Dimethylamiloride improved the recovery of contraction (fed control 24 ± 9%, fed treated 68 ± 11%, P < 0.05 at the end of reperfusion), attenuated the contracture (fed control 40 ± 9%, fed treated 24 ± 11%, P < 0.05 at the beginning of reperfusion) and reduced lactate production in the fed group and increased cellular viability in both groups (fed control 21 ± 6%, fed treated 69 ± 7%, P < 0.05, and fasted control 18 ± 7%, fasted treated 53 ± 8%, P < 0.05). Oxfenicine reduced the recovery of contraction (fasted control 88 ± 6%, fasted treated 60 ± 11%, P < 0.05) and increased lactate production of fasted group and attenuated the contracture in the fed. These data suggest that beneficial effects of fasting owe, at least in part, to a lowered glycolysis probably secondary to the increased fatty acid oxidation and to the accumulation of energy supplying acyl esters. Dimethylamiloride slowing of glycolysis might explain functional improvement, whereas it seems unrelated to the protection on cell viability.