Martín Donato

Role of the parasympathetic nervous system in cardioprotection by remote hindlimb ischaemic preconditioning

•  What is the central question of this study? Ischaemia–reperfusion of peripheral tissues protects the heart from subsequent myocardial ischaemia–reperfusion‐induced injury and cardiac dysfunction, a phenomenon referred to as ‘remote ischaemic preconditioning’ (rIPC). This study addressed whether activation of sensory afferent nerves in the ischaemic hindlimb and vagal efferent nerves innervating the heart mediate rIPC. •  What is the main finding and its importance? Spinal cord section, bilateral vagotomy or blockade of muscarinic cholinergic receptors in vivo abolished rIPC and cardioprotection measured in vitro. Electrical stimulation of the vagus nerve induced cardioprotection, thus mimicking rIPC. The finding that sensory and parasympathetic neural mechanisms mediate rIPC confirms and extends previous results, with implications for translational studies in patients with coronary artery disease.

Adenosine and cardioprotection during reperfusion — an overview

Ischemic heart disease includes a number of entities that have been grouped in accordance with physiopathology and evolutive criteria. In recent years ‘new’ ischemic syndromes have been described. Within the ‘new’ ischemic syndromes, ventricular post-ishemic dysfunction — also known as’ stunned myocardium’ — is worth mentioning. In this route, several studies have suggested that reperfusion per se could cause cellular injury (reperfusion injury). In previous years, a protective effect on the injury caused by ischemia and reperfusion in the heart has been attributed to adenosine. These effects have been documented in different experimental in vivo and in vitro models. Thus, the administration of exogenous adenosine, or agonists of adenosine receptors prior to ischemia reduces the size of the infarction, improves the recovery of the ventricular function during reperfusion (attenuating stunning) and prolongs the time period to the ischemic contracture. However, focusing on a potential therapeutic application, it is of the utmost importance to find this protection and learn the mechanisms involved when procedures are applied during early reperfusion.

Role of matrix metalloproteinase‐2 in the cardioprotective effect of ischaemic postconditioning

The activation of matrix metalloproteinases (MMPs) contributes to myocardial injury at the onset of reperfusion; however, their role in ischaemic postconditioning is unknown. The aim of the present study was to examine the effects of ischaemic postconditioning on MMP activity in isolated rabbit hearts. The isolated rabbit hearts were subjected to 30 min of global ischaemia followed by 180 min of reperfusion (I/R group; n= 8). In the ischaemic postconditioning group (n= 8), a postconditioning protocol was performed (2 cycles of 30 s reperfusion–ischaemia). In other experiments, we added doxycycline, an MMP inhibitor, at 25 (n= 7) or 50 μmol l−1 (n= 8) during the first 2 min of reperfusion. Coronary effluent and left ventricular tissue were collected during pre‐ischaemic conditions and at different times during the reperfusion period to measure MMP–2 activity and cardiac protein nitration. We evaluated ventricular function and infarct size. In the I/R group, infarct size was 32.1 ± 5.2%; Postcon reduced infarct size to 9.5 ± 3.8% (P < 0.05) and inhibited MMP–2 activity during reperfusion. The administration of doxycycline at 50 μmol l−1 inhibited MMP–2 activity and cardiac protein nitration and reduced the infarct size to 9.7 ± 2.8% (P < 0.05). A lower dose of doxycycline (25 μmol l−1) failed to inhibit MMP–2 activity and did not modify the infarct size. Our results strongly suggest that ischaemic postconditioning may exert part of its cardioprotective effects through the inhibition of MMP–2 activity.

Rosuvastatin Given During Reperfusion Decreases Infarct Size and Inhibits Matrix Metalloproteinase-2 Activity in Normocholesterolemic and Hypercholesterolemic Rabbits

There is evidence that statin treatment before ischemia protects myocardium from ischemia/reperfusion injury. The objective is to determine whether rosuvastatin administered during reperfusion modifies infarct size and the recovery of postischemic ventricular dysfunction in normocholesterolemic and hypercholesterolemic rabbits. In addition, we also evaluated the role of matrix metalloproteinase type 2 (MMP)-2 activation. Langendorff-perfused rabbit hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion. In group 2, we added rosuvastatin after 30 minutes of ischemia and from the beginning of reperfusion. In group 3, an MMP inhibitor (doxycycline) was administered during the first 2 minutes of reperfusion. Finally, we repeated these groups but in hypercholesterolemic rabbits (groups 4, 5, and 6). The infarct size was 16.6% ± 3.9% in group 1 and 25.6% ± 2.7% in group 4. Rosuvastatin reduced infarct size to 4.5% ± 1.1% and 6.1% ± 1.5% in groups 2 and 5, respectively (P < 0.05). Rosuvastatin significantly decreased MMP-2 activity during reperfusion, and doxycycline induced an inhibition of MMP-2 activity and a reduction of infarct size in normocholesterolemic (4.9% ± 0.9%) and hypercholesterolemic animals (8.3% ± 1.6%) (P < 0.05). Rosuvastatin reduces infarct size and attenuates MMP-2 activity. These data and the correlation between MMP-2 and infarct size suggest that MMP-2 plays an important role in the mechanisms of cardioprotection afforded by rosuvastatin.

Genetic inhibition of calcineurin induces diastolic dysfunction in mice with chronic pressure overload

Calcineurin is a Ca(2+)/calmodulin-dependent protein phosphatase that induces myocardial growth in response to several physiological and pathological stimuli. Calcineurin inhibition, induced either via cyclosporine or genetically, can decrease myocardial hypertrophy secondary to pressure overload without affecting left ventricular (LV) systolic function. Since hypertrophy can also affect LV diastolic function, the goal of this study was to examine the effects of chronic pressure overload (2 wk aortic banding) in transgenic (Tg) mice overexpressing Zaki-4beta (TgZ), a specific endogenous inhibitor of calcineurin, on LV diastolic function. As expected, in the TgZ mice with calcineurin inhibitor overexpression, aortic banding reduced the degree of LV hypertrophy, as assessed by LV weight-to-body weight ratio (3.5 + or - 0.1) compared with that in non-Tg mice (4.6 + or - 0.2). LV systolic function remained compensated in both groups with pressure overload. However, the LV end-diastolic stress-to-LV end-diastolic dimension ratio, an index of diastolic stiffness and LV pressure half-time and isovolumic relaxation time, two indexes of isovolumic relaxation, increased significantly more in TgZ mice with aortic banding. Protein levels of phosphorylated phospholamban (PS16), sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, phosphorylated ryanodine receptor, and the Na(+)/Ca(2+) exchanger were also reduced significantly (P < 0.05) in the banded TgZ mice. As expected, genetic calcineurin inhibition inhibited the development of LV hypertrophy with chronic pressure overload but also induced LV diastolic dysfunction, as reflected by both impaired isovolumic relaxation and increased myocardial stiffness. Thus genetic calcineurin inhibition reveals a new mechanism regulating LV diastolic function.

Preischemic efferent vagal stimulation increases the size of myocardial infarction in rabbits. Role of the sympathetic nervous system

rabbits. Role of the sympathetic nervous system Bruno Buchholz , Martin Donato , Virginia Perez , Flavio C. Ivalde , Christian Hocht , Emiliano Buitrago , Manuel Rodriguez , Ricardo J. Gelpi a,⁎,2 a Institute of Cardiovascular Physiopathology and Department of Pathology, School of Medicine, University of Buenos Aires, Argentina b Department of Pharmacology, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina

Ischemic postconditioning: mechanisms, comorbidities, and clinical application

Abstract Since ischemic heart disease (IHD) is a major cause of mortality and heart failure, novel therapeutic strategies are expected to improve the clinical outcomes of patients with acute myocardial infarction. Brief episodes of ischemia/reperfusion performed at the onset of reperfusion can reduce infarct size; a phenomenon termed “ischemic postconditioning.” Extensive research has determined that different autacoids (e.g., adenosine, bradykinin, opioid, etc.) and cytokines, their respective receptors, kinase signaling pathways, and mitochondrial modulation are involved in ischemic conditioning. Modification of these factors by pharmacological agents mimics the cardioprotection by ischemic postconditioning. Here, the potential mechanisms of ischemic postconditioning, the presence of comorbidities, and the possible extrapolation to the clinical setting are reviewed. In the near future, large, multicentered, randomized, placebo-controlled, clinical trials will be required to determine whether pharmacological and/or ischemic postconditioning can improve the clinical outcomes of patients with IHD.

High cholesterol diet effects on ischemia–reperfusion injury of the heart

Ischemic heart disease is the leading cause of morbi-mortality in developed countries. Both ischemia-reperfusion injury and mechanisms of cardioprotection have been studied for more than 50 years. It is known that the physiopathological mechanism of myocardial ischemia involves several factors that are closely related to its development, of which hypercholesterolemia is one of the main ones. Therefore, the objective of this review was to elucidate the effects of a high-cholesterol diet on normal ventricular function and ischemia-reperfusion injury associated phenomenon such as post-ischemic ventricular dysfunction (stunned myocardium). Although there exist many studies considering several aspects of this physiopathological entity, the majority were carried out on normal animals. Thus, experiments carried out on hypercholesterolemic models are controversial, in particular those evaluating different mechanisms of cardioprotection such as ischemic preconditioning and postconditioning, and cardioprotection granted by drugs such as statins, which apart from exerting a lipid-lowering effect, exert pleiotropic effects providing cardioprotection against ischemia-reperfusion injury. These controversial results concerning the mechanisms of cardioprotection vary according to quality, composition, and time of administration of the high-cholesterol diet, as well as the species used in each experiment. Thus, to compare the results it is necessary to take all of these variables into account, since they can change the obtained results.

Myocardial triggers involved in remote ischemic preconditioning activation

New Findings What is the central question of this study? Ischemia/reperfusion of peripheral tissues protects the heart from subsequent myocardial ischemia/reperfusion injury, a phenomenon referred to as remote ischemic preconditioning (rIPC). This study evaluated the possible myocardial triggers of rIPC. What is the main finding and its importance? rIPC reduces infarct size through a vagal pathway and a mechanism involving Akt and eNOS phosphorylation, opening of mitochondrial K+ATP channels and increasing of mitochondrial H2O2 production. All these phenomena occur prior to the myocardial ischemia, therefore they could act as “triggers” of rIPC. Aims: It has been proposed that remote ischemic preconditioning (rIPC) activates a parasympathetic neural pathway. However, the myocardial rIPC intracellular mechanism remains unclear. Here we characterized some of the intracellular signals participating as rIPC triggers. Methods and Results: Isolated rat hearts were subjected to 30 minutes of global ischemia and 120 minutes of reperfusion (Non-rIPC). In a second group, before the isolation of the heart, a rIPC protocol (3 cycles of hindlimb ischemia/reperfusion) was performed. The infarct size was measured with tetrazolium staining. Akt and eNOS expression/phosphorylation, and mitochondrial H2O2 production were evaluated at the end of rIPC protocol, before myocardial ischemia/reperfusion. rIPC significantly decreased the infarct size and induced Akt and eNOS phosphorylation. The protective effect on infarct size was abolished by cervical vagal section (CVS), L-NAME (NO synthesis inhibitor) and 5-HD (mK+ATP channels blocker). Mitochondrial production of H2O2 was increased by rIPC while it was abolished by CVS, L-NAME and 5-HD. Conclusions: rIPC activates a parasympathetic vagal pathway and a mechanism involving the Akt and eNOS phosphorylation, the opening of mK+ATP, and the release of H2O2 by the mitochondria. These entire phenomena occur prior to the myocardial ischemia, which could act as triggers of rIPC. This article is protected by copyright. All rights reservedWhat is the central question of this study? Ischaemia–reperfusion of peripheral tissues protects the heart from subsequent myocardial ischaemia–reperfusion injury, a phenomenon referred to as remote ischaemic preconditioning (rIPC). This study evaluated the possible myocardial triggers of rIPC. What is the main finding and its importance? Remote ischaemic preconditioning reduces infarct size through a vagal pathway and a mechanism involving phosphorylation of Akt and endothelial nitric oxide synthase, opening of mitochondrial ATP‐dependent K+ channels and an increase in mitochondrial H2O2 production. All these phenomena occur before the myocardial ischaemia; hence, they could act as ‘triggers’ of rIPC.

Myocardial triggers involved in activation of remote ischaemic preconditioning.

New Findings What is the central question of this study? Ischemia/reperfusion of peripheral tissues protects the heart from subsequent myocardial ischemia/reperfusion injury, a phenomenon referred to as remote ischemic preconditioning (rIPC). This study evaluated the possible myocardial triggers of rIPC. What is the main finding and its importance? rIPC reduces infarct size through a vagal pathway and a mechanism involving Akt and eNOS phosphorylation, opening of mitochondrial K+ATP channels and increasing of mitochondrial H2O2 production. All these phenomena occur prior to the myocardial ischemia, therefore they could act as “triggers” of rIPC. Aims: It has been proposed that remote ischemic preconditioning (rIPC) activates a parasympathetic neural pathway. However, the myocardial rIPC intracellular mechanism remains unclear. Here we characterized some of the intracellular signals participating as rIPC triggers. Methods and Results: Isolated rat hearts were subjected to 30 minutes of global ischemia and 120 minutes of reperfusion (Non-rIPC). In a second group, before the isolation of the heart, a rIPC protocol (3 cycles of hindlimb ischemia/reperfusion) was performed. The infarct size was measured with tetrazolium staining. Akt and eNOS expression/phosphorylation, and mitochondrial H2O2 production were evaluated at the end of rIPC protocol, before myocardial ischemia/reperfusion. rIPC significantly decreased the infarct size and induced Akt and eNOS phosphorylation. The protective effect on infarct size was abolished by cervical vagal section (CVS), L-NAME (NO synthesis inhibitor) and 5-HD (mK+ATP channels blocker). Mitochondrial production of H2O2 was increased by rIPC while it was abolished by CVS, L-NAME and 5-HD. Conclusions: rIPC activates a parasympathetic vagal pathway and a mechanism involving the Akt and eNOS phosphorylation, the opening of mK+ATP, and the release of H2O2 by the mitochondria. These entire phenomena occur prior to the myocardial ischemia, which could act as triggers of rIPC. This article is protected by copyright. All rights reservedWhat is the central question of this study? Ischaemia–reperfusion of peripheral tissues protects the heart from subsequent myocardial ischaemia–reperfusion injury, a phenomenon referred to as remote ischaemic preconditioning (rIPC). This study evaluated the possible myocardial triggers of rIPC. What is the main finding and its importance? Remote ischaemic preconditioning reduces infarct size through a vagal pathway and a mechanism involving phosphorylation of Akt and endothelial nitric oxide synthase, opening of mitochondrial ATP‐dependent K+ channels and an increase in mitochondrial H2O2 production. All these phenomena occur before the myocardial ischaemia; hence, they could act as ‘triggers’ of rIPC.