Exosomes are nanometer-sized vesicles released from cells into the blood, where they can transmit signals throughout the body. Shown to act on the heart, exosomes' composition and the signaling pathways they activate have not been explored. We hypothesized that endogenous plasma exosomes can communicate signals to the heart and provide protection against ischemia and reperfusion injury. This study sought to isolate and characterize exosomes from rats and healthy volunteers, evaluate their cardioprotective actions, and identify the molecular mechanisms involved. The exosome-rich fraction was isolated from the blood of adult rats and human volunteers and was analyzed by protein marker expression, transmission electron microscopy, and nanoparticle tracking analysis. This was then used in ex vivo, in vivo, and in vitro settings of ischemia-reperfusion, with the protective signaling pathways activated on cardiomyocytes identified using Western blot analyses and chemical inhibitors. Exosomes exhibited the expected size and expressed marker proteins CD63, CD81, and heat shock protein (HSP) 70. The exosome-rich fraction was powerfully cardioprotective in all tested models of cardiac ischemia-reperfusion injury. We identified a pro-survival signaling pathway activated in cardiomyocytes involving toll-like receptor (TLR) 4 and various kinases, leading to activation of the cardioprotective HSP27. Cardioprotection was prevented by a neutralizing antibody against a conserved HSP70 epitope expressed on the exosome surface and by blocking TLR4 in cardiomyocytes, identifying the HSP70/TLR4 communication axis as a critical component in exosome-mediated cardioprotection. Exosomes deliver endogenous protective signals to the myocardium by a pathway involving TLR4 and classic cardioprotective HSPs.

Many patients with ischaemic heart disease also have diabetes. As myocardial infarction is a major cause of mortality and morbidity in these patients, treatments that increase cell survival in response to ischaemia and reperfusion are needed. Exosomes—nano‐sized, lipid vesicles released from cells—can protect the hearts of non‐diabetic rats. We previously showed that exosomal HSP70 activates a cardioprotective signalling pathway in cardiomyocytes culminating in ERK1/2 and HSP27 phosphorylation. Here, we investigated whether the exosomal cardioprotective pathway remains intact in the setting of type II diabetes. Exosomes were isolated by differential centrifugation from non‐diabetic and type II diabetic patients, from non‐diabetic and Goto Kakizaki type II diabetic rats, and from normoglycaemic and hyperglycaemic endothelial cells. Exosome size and number were not significantly altered by diabetes. CD81 and HSP70 exosome markers were increased in diabetic rat exosomes. However, exosomes from diabetic rats no longer activated the ERK1/2 and HSP27 cardioprotective pathway and were no longer protective in a primary rat cardiomyocytes model of hypoxia and reoxygenation injury. Hyperglycaemic culture conditions were sufficient to impair protection by endothelial exosomes. Importantly, however, exosomes from non‐diabetic rats retained the ability to protect cardiomyocytes from diabetic rats. Exosomes from diabetic plasma have lost the ability to protect cardiomyocytes, but protection can be restored with exosomes from non‐diabetic plasma. These results support the concept that exosomes may be used to protect cardiomyocytes against ischaemia and reperfusion injury, even in the setting of type II diabetes.

Mitochondria are key organelles for ATP production in cardiomyocytes, which is regulated by processes of fission and fusion. We hypothesized that the mitochondria fusion protein dynamin-related protein 1 (Drp1) inhibition, attenuates ischemia-reperfusion (I/R) injury through modifications in mitochondrial metabolism. Rats were subjected to I/R through coronary artery ligation, and isolated cardiomyocytes were treated with an ischemia-mimicking solution. In vivo, cardiac function, myocardial infarction area, and mitochondrial morphology were determined, whereas in vitro, viability, mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption rate (OCR) were assessed. In both models, an adenovirus expressing Drp1 dominant-negative K38A (Drp1K38A) was used to induce Drp1 loss-of-function. Our results showed that I/R stimulated mitochondrial fission. Myocardial infarction size and cell death induced by I/R were significantly reduced, whereas cardiac function after I/R was improved in Drp1K38A-treated rats compared with controls. Drp1K38A-transduced cardiomyocytes showed lower OCR with no decrease in intracellular ATP levels, and on I/R, a larger decrease in OCR with a smaller reduction in intracellular ATP level was observed. However, proton leak-associated oxygen consumption was comparatively higher in Drp1K38A-treated cardiomyocytes, suggesting a protective mitochondrial uncoupling effect against I/R. Collectively, our results show that Drp1 inhibition triggers cardioprotection by reducing mitochondrial metabolism during I/R.

Type 2 diabetes mellitus (T2DM) is a highly prevalent disease worldwide. Cardiovascular disorders generated as a consequence of T2DM are a major cause of death related to this disease. Diabetic cardiomyopathy (DCM) is characterized by the morphological, functional and metabolic changes in the heart produced as a complication of T2DM. This cardiac disorder is characterized by constant high blood glucose and lipids levels which eventually generate oxidative stress, defective calcium handling, altered mitochondrial function, inflammation and fibrosis. In this context, insulin is of paramount importance for cardiac contractility, growth and metabolism and therefore, an impaired insulin signaling plays a critical role in the DCM development. However, the exact pathophysiological mechanisms leading to DCM are still a matter of study. Despite the numerous questions raised in the study of DCM, there have also been important findings, such as the role of micro-RNAs (miRNAs), which can not only have the potential of being important biomarkers, but also therapeutic targets. Furthermore, exosomes also arise as an interesting variable to consider, since they represent an important inter-cellular communication mechanism and therefore, they may explain many aspects of the pathophysiology of DCM and their study may lead to the development of therapeutic agents capable of improving insulin signaling. In addition, adenosine and adenosine receptors (ARs) may also play an important role in DCM. Moreover, the possible cross-talk between insulin and ARs may provide new strategies to reverse its defective signaling in the diabetic heart. This review focuses on DCM, the role of insulin in this pathology and the discussion of new molecular insights which may help to understand its underlying mechanisms and generate possible new therapeutic strategies.

The alpha2-adrenergic receptor agonist Dexmedetomidine (Dex) is a sedative medication used by anesthesiologists. Dex protects the heart against ischemia-reperfusion (IR) and can also act as a preconditioning mimetic. The mechanisms involved in Dex-dependent cardiac preconditioning, and whether this action occurs directly or indirectly on cardiomyocytes, still remain unclear. The endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway and endothelial cells are known to play key roles in cardioprotection against IR injury. Therefore, the aims of this work were to evaluate whether the eNOS/NO pathway mediates the pharmacological cardiac effect of Dex, and whether endothelial cells are required in this cardioprotective action. Isolated adult rat hearts were treated with Dex (10nM) for 25min and the dimerization of eNOS and production of NO were measured. Hearts were then subjected to global IR (30/120min) and the role of the eNOS/NO pathway was evaluated. Dex promoted the activation of eNOS and production of NO. Dex reduced the infarct size and improved the left ventricle function recovery, but this effect was reversed when Dex was co-administered with inhibitors of the eNOS/NO/PKG pathway. In addition, Dex was unable to reduce cell death in isolated adult rat cardiomyocytes subjected to simulated IR. Cardiomyocyte death was attenuated by co-culturing them with endothelial cells pre-treated with Dex. In summary, our results show that Dex triggers cardiac protection by activating the eNOS/NO signaling pathway. This pharmacological effect of Dex requires its interaction with the endothelium.

Ischemic preconditioning (IPC) limits myocardial infarct size through the activation of the PI3K–Akt signal cascade; however, little is known about the roles of individual PI3K isoforms in cardioprotection. We aimed, therefore, to elucidate the role of the PI3Kα isoform in cardioprotection Pharmacological PI3Kα inhibition was assessed in isolated-perfused mouse hearts subjected to ischemia/reperfusion injury (IRI), either during the IPC procedure or at reperfusion. PI3Kα inhibition abrogated the IPC-induced protective effect at reperfusion, but not when given only during the IPC protocol. These results were confirmed in an in vivo model. Moreover, pharmacological PI3Kα activation by insulin at reperfusion was sufficient to confer cardioprotection against IRI. In addition, PI3Kα was shown to be expressed and activated in mouse cardiomyocytes, mouse cardiac endothelial cells, as well as in mouse and human heart tissue. Furthermore, PI3Kα was shown to mediate its effect though the inhibition of mitochondrial permeability transition pore opening. In conclusion, PI3Kα activity is required during the early reperfusion phase to reduce myocardial infarct size. This suggests that strategies specifically enhancing the α isoform of PI3K at reperfusion promote tissue salvage and as such, and could provide a direct target for clinical treatment of IRI.