A robust meta analysis of randomized
A robust meta-analysis of 22 randomized clinical trials found that application of ischemic preconditioning strategies reduce ventricular arrhythmias . Additionally, it was demonstrated that remote ischemic perconditioning (perRIC) induces immediate cardioprotection during ongoing ischemia by attenuation of ST-segment elevation in a clinically relevant large-animal model . Accordingly, our results demonstrated that RE significantly reduced the incidence of aberrant ST-segment and the severity of arrhythmias. Clinically, ST-segment elevation MI (STEMI) is an important predictor and major emergency manifestation of ischemic heart disease , given that this aberrant ECG waveform is a typical electrical sign of acute MI . Along with these findings, we showed that the severity of electrical dysfunction correlates with the extent of MI size in NE rats and RE animals. Therefore, despite our substantial evidence that RE enhances heart tolerance against arrhythmias generation through eNOS signaling, there is an array of underlying pro-arrhythmogenic mechanisms, unique to the level of IR injury, to be deciphered [51,52]. Indeed, NO may act as a paracrine factor released from endothelial diacylglycerol kinase that affects cardiac function , as it is synthesized by both nNOS and eNOS. In the heart, approximately 80% of the total eNOS expression is found in endothelial cells with the remaining 20% found in cardiomyocytes . However, the ratio of nNOS expression between these cell types has not been demonstrated. Although a recent study has shown a key role of the endothelium in eNOS-dependent cardioprotection after long-term aerobic training , our data indicate that a single session of RE triggers acute cardioprotection through eNOS signaling by an endothelial cell-independent pathway. While endothelial-myocardial crosstalk may exist, this discrepant result might be explained by the maturation of protective effects induced by long-term exercise training, supporting the notion that cardiomyocytes produce their own NO. Furthermore, an elegant study comparing transgenic mice with systemic (using an endothelium enhancer element) versus cardiomyocyte-specific eNOS overexpression demonstrated that site-specific gene therapy provides more robust outcomes in limiting MI-related dysfunctions . However, further studies are certainly needed to validate whether RE triggers acute cardioprotection through a cardiac-dependent mechanism. In cardiomyocytes, nNOS and eNOS are spatially distributed throughout the cell creating pools of highly compartmentalized NO . Moreover, nNOS and eNOS have not only different localization in cardiomyocytes, but also opposite effects on cardiomyocyte contractility [55,56]. Our data supports the idea that eNOS plays a major role in acute cardiac dysfunction induced by IR injury, whereas nNOS does not appear to be involved. Indeed, previous studies have shown that exercise training significantly influences NO production by increasing eNOS expression [27,33,57,58] or phosphorylation at activation residues [59,60] in vascular arteries. Despite the increased eNOS activity after IR injury in NE rat hearts, which might yield augmented NO bioavailability, we demonstrated that this active form is primarily uncoupled, thereby representing an additional source of ROS, as here observed. Several studies have provided consistent evidence that eNOS uncoupling plays a pivotal role in many cardiac diseases [32,33] due to oxidative damage. In accordance, we demonstrated that RE prevented eNOS uncoupling induced by IR, thereby explaining the maintenance of cardiac ROS and carbonylated protein levels as shown in healthy hearts. Furthermore, it is well-known that during pro-oxidant conditions, such as acute MI, the excessive generation of ROS further contributes to reducing NO bioavailability. Uncoupled eNOS activity has been mainly attributed to the oxidation of BH4 to dihydrobiopterin (BH2), deficiency of NOS substrate (L-arginine), and regulatory interaction with binding partners [33,61]. Moreover, a recent study showed that part of the vascular benefits of long-term exercise training in HF rats involves the upregulation of GTP cyclohydrolase 1, a rate-limiting enzyme involved in the biosynthesis BH4 . Our final evidence supporting the notion RE mediates acute cardioprotection by limiting eNOS uncoupling is the fact that the massive production of ROS induced by IR was fully restored to control levels when NE hearts were treated with BH4. Importantly, BH4 treatment did not affect the generation of ROS in hearts preconditioned by RE, thereby indicating no physiological needs of a supplemental eNOS cofactor for proper enzymatic activity.