No abstract

Background-The signaling pathways that control ischemia/reperfusion-induced cardiomyocyte apoptosis in heart have not been fully defined. In this study, we investigated whether Akt signaling has a role in the antiapoptotic pathways of preconditioning against hypoxia/reoxygenation (H/R). Methods and Results-Primary cultures of adult rat ventricular myocytes (ARVMs) were subjected to preconditioning (PC) by exposing the cells to 10 minutes of hypoxia followed by 30 minutes of reoxygenation. Non-PC and PC myocytes were subjected to 90 minutes of hypoxia followed by 120 minutes of reoxygenation. Hypoxic-PC protected the myocytes from subsequent H/R injury, as evidenced by decreased apoptosis and LDH release and increased cell viability. H/R-induced cytochrome c release and activation of caspase-3 and -9 were blocked by PC. This protective effect was inhibited by treating the cells with LY294002 (50 mol/L), a PI3 kinase inhibitor, for 10 minutes before and during PC. PC also induced phosphorylation of Akt and BAD. Protein levels of Bcl-2 in mitochondria were maintained in PC. ARVMs were infected with either a control adenovirus (Adeno lac-Z), an adenovirus expressing dominantnegative Akt, or an adenovirus expressing constitutively active Akt. Ectopic overexpression of constitutively active Akt protected ARVMs from apoptosis induced by hypoxia/reoxygenation compared with Adeno lac-Z. In contrast, dominant negative Akt overexpression abolished the antiapoptotic effect of PC. Conclusions-Our data demonstrated that in adult cardiomyocytes, the antiapoptotic effect of PC against H/R requires Akt signaling leading to phosphorylation of BAD, inhibition of cytochrome c release, and prevention of caspase activation.

No abstract

The signal cascade that triggers and mediates ischemic preconditioning (IPC) remains unclear. The present study investigated the role of the Src family of tyrosine kinases in IPC. Isolated and buffer-perfused rat hearts underwent IPC with three cycles of 5-min ischemia and 5-min reperfusion, followed by 30-min ischemia and 120-min reperfusion. The Src tyrosine kinase family-selective inhibitor PP1 was administered between 45 and 30 min before ischemia (early PP1 treatment) or for 15 min before IPC [early PP1-preconditioning (PC) treatment]. PP1 was also administered for 5 min before the sustained ischemia (late PP1 treatment) or after IPC (late PP1-PC treatment). Src kinase was activated after 30 min of ischemia in both the membrane and cytosolic fractions. Src kinase was also activated by IPC but was attenuated after the sustained ischemia. Early and late PP1 treatment inhibited Src activation after the sustained ischemia and reduced infarct size. Early PP1-PC inhibited Src activation after IPC but not after the sustained ischemia and blocked cardioprotection afforded by IPC. Late PP1-PC treatment abrogated IPC-induced activation of Src and protein kinase C (PKC)-epsilon in the membrane but not in the cytosolic fraction. This treatment modality abrogated Src activation after the sustained ischemia and failed to block cardioprotection afforded by IPC. These results suggest that Src kinase activation mediates ischemic injury but triggers IPC in the position either upstream of or parallel to membrane-associated PKC-epsilon.

adenosine is an important mediator of ischemic preconditioning (IPC), its relative contribution to IPC remains unknown. Because adenosine is formed through the hydrolysis of ATP, the present study investigated the role of ATP and adenosine in IPC. Isolated and buffer-perfused rat hearts underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. The rate-pressure product (RPP) 30 min after reperfusion was taken as an endpoint of functional protection. Interstitial fluid (ISF) adenine nucleotides and adenosine were measured by cardiac microdialysis techniques. Inhibition of IPC-induced recovery of RPP was partial by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (SPT; 100 M) or by the structurally distinct P2Y purinoceptor antagonists suramin (300 M) or reactive blue (RB; 10 M) but was additive when SPT was given with suramin or RB. The P2X antagonist pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulfonic acid tetrasodium (50 M) had no effect on functional protection. The improved functional recovery was not significantly affected by an ecto-5Ј-nucleotidase inhibitor, ␣,␤-methylene adenosine diphosphate (AMP-CP; 100 M), alone but was inhibited by AMP-CP plus SPT, suramin, or RB. ISF ATP and adenosine increased temporarily by 10-fold during IPC. AMP-CP augmented the increase in ISF ATP associated with the decrease in ISF adenosine. There was a reciprocal correlation between the ISF concentration of ATP and adenosine in preconditioned hearts. In addition, there was a significant correlation between ISF adenosine and ATP and the inhibitory potency of SPT and suramin or RB against functional protection conferred by IPC. These results suggest that extracellular ATP and adenosine play a complementary role in IPC through P2Y purinoceptors and adenosine receptors, respectively. cardiac microdialysis ADENOSINE is known to play an important role in mediating ischemic preconditioning (IPC) in many species, including the rabbit, pig, dog, and human (33). However, the argument against the role of adenosine as the principal mediator for IPC in the rat heart has been provided by several investigators (26, 32). In addition, although ecto-5Ј-nucleotidase plays a major role in extracellular adenosine formation during IPC (16-18), the inhibitory effect of an ecto-5Ј-nucleotidase inhibitor, ␣,␤-methylene adenosine diphosphate (AMP-CP), on myocardial protection conferred by IPC is controversial (31). Thus there is circumstantial evidence suggesting that adenosine is not the sole mediator of IPC.Extracellular ATP is a local regulator of physiological functions in the cardiovascular system. ATP is released to the interstitial space from endothelial cells by mechanical and chemical stimuli such as bradykinin, acetylcholine, and serotonin (1, 42) and from sympathetic and parasympathetic perivascular nerves (3) and cardiomyocytes (9) in response to ischemia or hypoxia. ATP was indeed found in the coronary effluent of the isolated and perfused heart during hypoxia and postischemic reper...