Enrique A. Savino

Role of endogenous nitric oxide in classic preconditioning in rat hearts.

Ischemic preconditioning (IPC) protects the heart against subsequent sustained ischemia reperfusion (RP). Despite many triggers and signaling pathways, which seem to be involved in IPC, the IPC-mechanisms remain a controversial issue. One of them is endogenous production of nitric oxide (NO). To assess the role of NO in IPC and its relation with glycogen and glycolysis, the effects of inhibiting NO synthase with L-NAME (50 microM) were examined in IPC rat hearts perfused with medium containing 10 mM glucose. Left ventricular developed pressure-rate product (RPP) and end diastolic pressure (EDP), lactate and glycogen contents, and cell viability were measured. Global ischemia (25 min) was followed by 30 min RP. IPC consisted in one cycle of 3 min ischemia-5 min RP. IPC reduced EDP and improved RP recovery of RPP. L-NAME had no effects on the non-IPC group but abolished these effects of IPC. IPC reduced ischemic decrease of glycogen and the acceleration of glycolysis, and improved cell viability. L-NAME did not affect these effects of IPC. The results suggest that NO is ineffective on the noxious effects of ischemia-RP in non-IPC hearts and on the effects of IPC on cell viability, glycogenolysis and glycolysis whereas it is only involved in functional protection.

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.

Beneficial Effects of Oxfenicine on the Hypoxic Rat Atria

During hypoxia the isolated rat atria released lactate into the bathing medium and underwent a rise of resting tension and a decline of the peak developed tension and pacemaker frequency. The atria from 24 h fasted rats, which oxidize faster their endogenous triacylglycerol pool, showed greater functional disturbances during hypoxia and a smaller recovery after reoxygenation than those from fed rats. Oxfenicine, which is a selective inhibitor of carnitine palmitoyltransferase I, attenuated the rise of resting tension and improved the post-hypoxic recovery of peak tension in the atria from fasted rats. The decline of the pacemaker frequency as well as the lactate output were not altered by the inhibitor. Present data show that oxfenicine ameliorated some of the hypoxic functional disturbances. Inasmuch lactate output did not change and these effects manifested only in the atria predisposed to the utilization of endogenous lipids, it may be inferred that oxfenicine preserved the atrial functions through the inhibition of fatty acid oxidation.

Effects of hypoxic preconditioning on the hypoxicreoxygenated atria from fed and fasted rats

Hypoxic preconditioning (PC) was studied using rat atria set up isometrically in 10 mM dextrose medium and paced at 1 Hz, applying three different protocols wherein fed and 24-h fasted rats were used in protocols 1 and 2 and only the fed in protocol 3. In protocol 1, PC was achieved applying a 5 min hypoxia followed by 10 min of reoxygenation before the onset of a 60 min hypoxia and 60 min reoxygenation. In protocol 2 the 5 min and a posterior 45 min hypoxia were applied in the absence of dextrose whereas in the 10 min and 60 min reoxygenation periods dextrose was present. In protocol 3, two cycles of 5 min dextrose-free hypoxic periods were applied before the sustained hypoxia (dextrose-free) and reoxygenation periods (10 min and final 45 min, both in the presence of dextrose). In the control groups of all protocols, the equilibration periods were prolonged to compensate the duration of PC. In the control groups of protocols 1 and 2, the sustained hypoxia evoked greater disturbances of contractility and a smaller post-hypoxic recovery in the fasted than in the fed rat atria. In protocol 1, PC markedly reduced the rise in resting tension and improved the post-hypoxic recovery in the fasted rat atria whereas in the fed rat atria protective effects were small and brief. In protocol 2, PC evoked a small reduction of contracture only in the atria from fasted rats and in protocol 3, PC exacerbated the hypoxic disturbances. These data suggest that PC effects depend both on the severity of the PC stress and the sustained hypoxia; and that PC does not require coronary flow.ResumenSe estudia el precondicionamiento hipóxico (PC) en registros isométricos de aurículas de rata montadas isométricamente en medio con glucosa 10 mM y estimuladas a 1 Hz. Se aplican tres protocolos usando ratas alimentadas o en ayunas de 24-h en los 1 y 2, y sólo alimentadas en el 3. En el protocolo 1, el PC consiste en 5 min de hipoxia seguido de 10 min de reoxigenación antes de aplicar 60 min de hipoxia y reoxigenación de 60 min. En el protocolo 2, se aplica 5 min de hipoxia en medio sin sustrato y la hipoxia ulterior de 45 min también en medio sin sustrato, que se repone al reoxigenar durante los períodos de 10 y 60 min. En el protocolo 3, se aplican dos ciclos de 5 min de hipoxia sin sustrato seguidos de 10 min de reoxigenación con sustrato antes de la hipoxia de 45 min sin sustrato y 60 min de reoxigenación con sustrato. En los tres protocolos, la duración del período de equilibrio de los grupos controles se prolonga para compensar la duración del PC. La hipoxia prolongada en los grupos controles de los protocolos 1 y 2 provoca un mayor deterioro de la contractilidad y menor recuperación al reoxigenar en las aurículas de ratas en ayunas. En el protocolo 1, el PC reduce marcadamente la contractura y mejora la recuperación al reoxigenar en las ratas en ayunas mientras que en las alimentadas, todos los efectos beneficiosos son pequeños y efímeros. En el protocolo 2, el PC sólo produce una leve reducción de la contractura en los animales en ayunas y en el protocolo 3, el PC resulta francamente nocivo. Los resultados sugieren que los efectos del PC dependen de la severidad del estímulo precondicionante y de la hipoxia prolongada y que el PC no requiere la circulación coronaria.

Effects of fasting and hypoxic preconditioning on the hypoxic-reoxygenated ventricular strips of the rat heart.

The investigation aimed to assess the effects of hypoxic preconditioning in right ventricle strips of fed and 24-h fasted rats, which display a fast fatty acid catabolism, and to ascertain whether these effects are associated with changes in the tissue levels of long-chain acylCoA and acyl carnitine and glycolytic activity. Strips were mounted isometrically in Krebs-bicarbonate solution with 10 mM dextrose and paced at 1 Hz. Strips were exposed to 30 min hypoxia and 60 min reoxygenation with or without a previous preconditioning cycle of 5 min hypoxia followed by a 10 min reoxygenation. During hypoxia the fasted rat strips underwent a greater contracture with respect to the fed group. Preconditioning reduced the contracture strength and accelerated the post-hypoxic recovery only in the fasted rat strips. Hypoxia evoked an increase in the acylCoA and acyl carnitine tissue-contents of the strips which reached higher levels in the fasted than in the fed rat groups. Preconditioning had no effects on the content of these metabolites. During hypoxia lactate output was lower in the fasted than in the fed rat strips and preconditioning abolished this decrease. These data suggest that the protective effects of hypoxic preconditioning occur in the heart tissue predisposed to the oxidation of fatty acid and can not be ascribed to changes in the accumulation of acylCoA and acyl carnitine but could be due, at least in part, to an activation of glycolysis.ResumenEn este trabajo, se trata de comparar los efectos del precondicionamiento hipóxico en bandas de ventrículo derecho entre ratas alimentadas y sometidas a 24-h de ayuno (catabolismo de ácidos grasos acelerado) y determinar si se correlacionan con los niveles tisulares de acilCoA y acil carnitina de cadena larga y la actividad glucolítica. Las bandas se montan isométricamente en Krebs-bicarbonato con glucosa 10 mM, se estimulan a 1 Hz y se someten a 30 min de hipoxia y 60 min de reoxigenación. El precondicionamiento consiste en un ciclo de 5 min de hipoxia y 10 min de reoxigenación antes de la hipoxia prolongada. La hipoxia provoca contractura, que es mayor en el grupo de ratas no alimentadas, y el precondicionamiento reduce la contractura y acelera la recuperación de la fuerza al reoxigenar sólo en el citado grupo. La hipoxia aumenta los contenidos de acilCoA y acil carnitina tanto en las bandas de ratas alimentadas como en las sometidas a ayuno, pero en éstas se alcanzan concentraciones mayores. El precondicionamiento no altera los niveles de estos metabolitos. El ayuno da lugar a menor liberación de lactato durante la hipoxia y el precondicionamiento anula este descenso. Se concluye que los efectos protectores del precondicionamiento hipóxico ocurren en el tejido ventricular predispuesto a la oxidación de ácidos grasos y no se deben a modificaciones en la acumulación de acilCoA o acil carnitina pero podrían deberse, al menos en parte, a la activación de la glucólisis.