Cardioprotective effects of L-glutamine in an ischemic rat heart model

概要

（研究の目的）
L-glutamine has been shown to have cardioprotective effects in animal and human models of ischemia-reperfusion injury. However, the optimal timing of glutamine administration remains unclear. This study was designed to examine the hypothesis that L-glutamine administered before and after myocardial ischemia provides better cardioprotection than when administered after ischemia only.

（方法）
Eighteen hearts of Male Wistar rats (50-300 g) were exposed to 15 minutes of ischemia using the Langendorff system. Hearts were randomly assigned to three groups. Group one received Krebs-Henseleit (KH) buffer over 20 minutes before ischemia and during 20 minutes of reperfusion (Control, n = 6), group two received KH buffer containing 2.5 mmol/L glutamine during reperfusion (Post-Gln, n = 6) and group three was given KH buffer containing 2.5 mmol/L glutamine before and after the ischemic insult (Pre+Post-Gln, n = 6). During ischemia, the heart was paced at 222 beats/min. Heart rate, coronary flow, maximum of left ventricular derivative of pressure development (dP/dt max), and left ventricular (LV) end-diastolic pressure were recorded at 5, 10, 15 and 20 minutes post-reperfusion. Rate-pressure product was calculated from LV systolic pressure and heart rate (RPP = LV systolic pressure×Heart Rate). Myocardial levels of O-linked β-N-acetylglucosamine (O-GlcNAc) and heat shock protein 70 (HSP70) were measured by Western blotting technique after 20 minutes of reperfusion.

（結果）
The LV dp/dt max in the Pre+Post-Gln group was significantly elevated as compared to the Post-Gln group after 10 minutes of reperfusion and was significantly higher than in the control group at all-time points. The LVEDP in the Pre+Post-Gln group was significantly lower than in the control group at 5, 15 and 20 minutes after reperfusion. Myocardial expression of O-GlcNAc was increased in the Pre+Post-Gln group (P <0.01 vs. control) without showing any differences in HSP70.

（考察）
The results of the present study indicate that L-glutamine, administered before and continued after myocardial ischemia improved post-ischemic cardiac performance indicating cardioprotective effects.

These benefits of L-glutamine pre-conditioning are consistent with and complement the observations made by previous studies. In an isolated working heart model, Wischmeyer et al. showed that a single-dose of L-glutamine, given intraperitoneally 18 hours before ischemia, improved cardiac output up to one hour after the insult. Bolotin et al. reported that pre-emptive administration of L-glutamine preserved cardiac output and coronary blood flow, with L-glutamine administered four hours prior to ischemia being superior to pre-treatment performed 14 hours earlier. Liu et al. demonstrated that pre-ischemic L-glutamine infusion over 30 minutes resulted in better functional recovery of the myocardium after one hour of reperfusion. Khogali et al. showed that administration of L-glutamine either before or after ischemia improved cardiac function 35 minutes following the insult. Our observations underscore the potential benefits of L-glutamine pre-conditioning, and further suggest that administration of L-glutamine shortly before and after ischemia may restore myocardial performance even sooner, i.e. after five minutes after reperfusion.

The HBP involving O-GlcNAc and HSP70 has been proposed as one mechanism underlying the beneficial effects of several pharmacological agents in the context of myocardial ischemia-reperfusion injury. The cardioprotective influence of volatile anesthetics (isoflurane), for example, has been linked to the activation of this cascade.

In the body, a small percentage (<5%) of glucose is metabolized through the HBP to synthesize the co-enzyme uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) facilitating glycosylation, via O-Linked β-N-acetylglucosamine (O-GlcNAc), of more than 80 key regulatory proteins within the cell nucleus and cytoplasma including transcription factors, kinases, and phosphatases. The expression of O-GlcNAc has been also related to the production of HSP70 which belongs to a family of important regulators of protein assembly, folding, and transport that are produced in response to stress.

(考察：続き)
As HSP70 suppresses the secretion of pro-inflammatory mediators, one could hypothesize that L-glutamine exerts its cardioprotective action through the activation of the HBP system, O-GlcNAc and the anti-inflammatory properties of HSP70. Indeed, in the present study, the expression of O-GlcNAc 20 minutes after the ischemic insult was stimulated by pre- and post-ischemic administration of L-glutamine. The HSP70 expression at the same time point, however, was not significantly affected. This discrepancy can be explained by the sampling times in our experimental protocol. Glycosylation processes, similar to phosphorylation, typically occur rapidly, within minutes. In contrast, the synthesis of proteins involved in the HBP such as HSP70 typically takes longer and may also induce long-lasting effects over hours and even days, as observed in LPS-treated cardiomyocytes and diabetic rats exposed to L-glutamine.

We acknowledge several limitations of this study. Because myocardial oxygen consumption was not measured, the impact of changes in oxygen supply and balance cannot be excluded. The expression of O-GlcNAc and HSP70 expression was simultaneously measured shortly after ischemia and reperfusion. Later assessment of HSP70 perhaps would have also shown an impact of L-glutamine on this protein.

（結論）
In this rat heart model of myocardial ischemia, glutamine pre-conditioning followed by post-ischemic administration improved cardiac function indicating cardioprotective effects, possibly mediated by O-GlcNAc.