Alicia Varela

Influence of Fasting on the Effects of Ischemic Preconditioning in the Ischemic-Reperfused Rat Heart

The effects of fasting and ischemic preconditioning (IP) on heart function of Langendorff-perfused rat hearts exposed to 25min global ischemia plus 30min reperfusion (RP), were correlated with lactate release and tissue-levels of long-chain acyl carnitine (LCCa) and CoA (LCCoA). IP was achieved by a 3min ischemia plus a 5min reperfusion cycle. Creatine kinase leakage was measured to assess the extent of cardiac injury. Fasting reduced the ischemic-induced contracture, improved RP recovery of mechanical function, reduced lactate release and increased the end-ischemia LCCoA and LCCa levels. Both in the fed and the fasted rat hearts IP delayed the pacemaker depression, reduced the amplitude of ischemic contracture and improved the RP recovery of contraction. However, IP reduced creatine kinase and lactate release only in the fed rat hearts. IP had no effects on tissue LCCa and LCCoA in both groups. These data suggest that: 1) beneficial effects of fasting may be ascribed, at least in part, to a reduced lactate production which may attenuate ischemic myocyte acidification and to the accumulation of fatty acyl esters which would favour citric acid cycle replenishment during RP. 2) beneficial effects of IP could be in part explained by the reduction of lactate production in the fed group although data obtained with the fasted rat heart indicate that another mechanisms must also be involved in the effects of IP. 3) accumulation of LCCoA and LCCa is not involved in the noxious effects of ischemia as well as in the protection effected by IP.

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.

Protection of ischaemic-reperfused rat heart by dimethylamiloride is associated with inhibition of mitochondrial permeability transition.

1 The aim of the present study was to assess whether protection afforded by the Na+/H+ exchanger blocker dimethylamiloride (DMA) is associated with inhibition of mitochondrial permeability transition (MPT). The effects of DMA were compared with those of cyclosporine (Cs) A, an inhibitor of MPT. 2 Rat hearts were Langendorff perfused with Krebs’–bicarbonate medium containing 10 mmol/L glucose and were subjected to 25 min no‐flow global ischaemia and 30 min reperfusion in the presence or absence of 10 µmol/L DMA or 0.2 µmol/L CsA. Cell viability was measured using tetrazolium stain. The MPT was determined by loading hearts with 2‐deoxy‐[3H]‐glucose (2DG), which enters mitochondria only during MPT. Total heart 2DG content as an estimation of the extent of tissue damage was also measured. To assess whether DMA has any direct effect on glycolysis, a cell‐free heart extract containing all the glycolytic enzymes was used. 3 Dimethylamiloride improved functional recovery (rate–pressure product) from 24 ± 7 to 68 ± 11% (P < 0.01) at reperfusion end, attenuated the increase in left ventricular end‐diastolic pressure (from 29 ± 7 to 6 ± 3% 10 min after reperfusion onset; P < 0.01), improved cell viability (from 21.2 ± 6.6 to 69.6 ± 7.1% at reperfusion end; P < 0.05) and lessened lactate accumulation at the end of ischaemia (119 ± 15 vs 163 ± 14 µmol/g dry weight; P < 0.05). Dimethylamiloride limited MPT : 2DG mitochondrial entrapment, being 33.1 ± 14.2 and 96.3 ± 14.0 at reperfusion end in the treated and control hearts, respectively (P < 0.05), and concomitantly raised total 2DG content (51.3 ± 4.4 vs 86.8 ± 1.7 × 103 d.p.m./g wet weight in control and treated groups, respectively; P < 0.05). Cyclosporine A improved functional recovery and attenuated the amplitude of ventricular diastolic pressure in ischaemic–reperfused hearts. It also reduced mitochondrial entrapment (67.3 ± 7.7%; P < 0.05 vs control) and increased total cell 2DG content (162.3 ± 1.3 × 103 d.p.m./g wet weight; P < 0.01 vs control) at the end of reperfusion. Dimethylamiloride did not affect glucose consumption and lactate production in the cell‐free heart extract. 4 In conclusion, DMA protects against the noxious effects of ischaemia–reperfusion and inhibits MPT, coinciding with present and previous findings concerning the effects of CsA. Dimethylamiloride also diminished lactate accumulation, although it did not exhibit any direct effect on glycolysis. These data suggest that blockade of Na+/H+ exchange by DMA attenuates the extent of MPT in ischaemic–reperfused rat heart.

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.