Joseph Wider

Profound Cardioprotection With Chloramphenicol Succinate in the Swine Model of Myocardial Ischemia-Reperfusion Injury

Background— Emerging evidence suggests that “adaptive” induction of autophagy (the cellular process responsible for the degradation and recycling of proteins and organelles) may confer a cardioprotective phenotype and represent a novel strategy to limit ischemia-reperfusion injury. Our aim was to test this paradigm in a clinically relevant, large animal model of acute myocardial infarction. Methods and Results— Anesthetized pigs underwent 45 minutes of coronary artery occlusion and 3 hours of reperfusion. In the first component of the study, pigs received chloramphenicol succinate (CAPS) (an agent that purportedly upregulates autophagy; 20 mg/kg) or saline at 10 minutes before ischemia. Infarct size was delineated by tetrazolium staining and expressed as a % of the at-risk myocardium. In separate animals, myocardial samples were harvested at baseline and 10 minutes following CAPS treatment and assayed (by immunoblotting) for 2 proteins involved in autophagosome formation: Beclin-1 and microtubule-associated protein light chain 3-II. To investigate whether the efficacy of CAPS was maintained with “delayed” treatment, additional pigs received CAPS (20 mg/kg) at 30 minutes after occlusion. Expression of Beclin-1 and microtubule-associated protein light chain 3-II, as well as infarct size, were assessed at end-reperfusion. CAPS was cardioprotective: infarct size was 25±5 and 41±4%, respectively, in the CAPS-pretreated and CAPS-delayed treatment groups versus 56±5% in saline controls (P<0.01 and P<0.05 versus control). Moreover, administration of CAPS was associated with increased expression of both proteins. Conclusion— Our results demonstrate attenuation of ischemia-reperfusion injury with CAPS and are consistent with the concept that induction of autophagy may provide a novel strategy to confer cardioprotection.

Acute induction of autophagy as a novel strategy for cardioprotection: getting to the heart of the matter.

There is no question that necrosis and apoptosis contribute to cardiomyocyte death in the setting of myocardial ischemia-reperfusion. Indeed, considerable effort and resources have been invested in the development of novel therapies aimed at attenuating necrotic and apoptotic cell death, with the ultimate goal of applying these strategies to reduce infarct size and improve outcome in patients suffering acute myocardial infarction (MI) or ‘heart attack’. However, an issue that remains controversial is the role of autophagy in determining the fate of ischemic-reperfused cardiomyocytes: i.e., is induction of autophagy detrimental or protective? Recent data from our group obtained in the clinically relevant, in vivo swine model of acute MI provide novel evidence of a positive association between pharmacological upregulation of autophagy (achieved by administration of chloramphenicol succinate (CAPS)) and increased resistance to myocardial ischemia-reperfusion injury.

Ischemic conditioning: the challenge of protecting the diabetic heart

The successful clinical translation of novel therapeutic strategies to attenuate lethal myocardial ischemia-reperfusion injury and limit infarct size has been identified as a major unmet need, and is of particular importance in patients with type-2 diabetes. There is a wealth of preclinical evidence that ischemic conditioning (encompassing the three paradigms of preconditioning, postconditioning and remote conditioning) is profoundly cardioprotective and, via up-regulation of endogenous signaling cascades, renders the heart resistant to infarction. However, current phase II trials aimed at exploiting ischemic conditioning for the clinical treatment of myocardial ischemia-reperfusion injury have yielded mixed results, possibly reflecting the emerging concern that the efficacy of conditioning-induced cardioprotection may be compromised in the diabetic heart. Our goal in this review is to provide a summary of our present understanding of the effect of type-2 diabetes on the infarct-sparing effect of ischemic conditioning, and the challenges of limiting ischemia-reperfusion injury in the diabetic heart.

Synergistic Inhibition of Both P2Y1 and P2Y12 Adenosine Diphosphate Receptors As Novel Approach to Rapidly Attenuate Platelet-Mediated Thrombosis

Objective— Unlike currently approved adenosine diphosphate receptor antagonists, the new diadenosine tetraphosphate derivative GLS-409 targets not only P2Y12 but also the second human platelet adenosine diphosphate receptor P2Y1 and may, therefore, be a promising antiplatelet drug candidate. The current study is the first to investigate the in vivo antithrombotic effects of GLS-409. Approach and Results— We studied (1) the in vivo effects of GLS-409 on agonist-stimulated platelet aggregation in anesthetized rats, (2) the antithrombotic activity of GLS-409 and the associated effect on the bleeding time in a canine model of platelet-mediated coronary artery thrombosis, and (3) the inhibition of agonist-stimulated platelet aggregation by GLS-409 versus selective P2Y1 and P2Y12 inhibition in vitro in samples from healthy human subjects before and 2 hours after aspirin intake. In vivo treatment with GLS-409 significantly inhibited adenosine diphosphate- and collagen-stimulated platelet aggregation in rats. Further, GLS-409 attenuated cyclic flow variation, that is, platelet-mediated thrombosis, in vivo in our canine model of unstable angina. The improvement in coronary patency was accompanied by a nonsignificant 30% increase in bleeding time. Of note, GLS-409 exerted its effects without affecting rat and canine hemodynamics. Finally, in vitro treatment with GLS-409 showed effects similar to that of cangrelor and the combination of cangrelor with the selective P2Y1 inhibitor MRS 2179 on agonist-stimulated platelet aggregation in human platelet-rich plasma and whole blood before and 2 hours after aspirin intake. Conclusions— Synergistic inhibition of both P2Y1 and P2Y12 adenosine diphosphate receptors by GLS-409 immediately attenuates platelet-mediated thrombosis and effectively blocks agonist-stimulated platelet aggregation irrespective of concomitant aspirin therapy.

Mitochondrial dynamics following global cerebral ischemia

Global brain ischemia/reperfusion induces neuronal damage in vulnerable brain regions, leading to mitochondrial dysfunction and subsequent neuronal death. Induction of neuronal death is mediated by release of cytochrome c (cyt c) from the mitochondria though a well-characterized increase in outer mitochondrial membrane permeability. However, for cyt c to be released it is first necessary for cyt c to be liberated from the cristae junctions which are gated by Opa1 oligomers. Opa1 has two known functions: maintenance of the cristae junction and mitochondrial fusion. These roles suggest that Opa1 could play a central role in both controlling cyt c release and mitochondrial fusion/fission processes during ischemia/reperfusion. To investigate this concept, we first utilized in vitro real-time imaging to visualize dynamic changes in mitochondria. Oxygen-glucose deprivation (OGD) of neurons grown in culture induced a dual-phase mitochondrial fragmentation profile: (i) fragmentation during OGD with no apoptosis activation, followed by fusion of mitochondrial networks after reoxygenation and a (ii) subsequent extensive fragmentation and apoptosis activation that preceded cell death. We next evaluated changes in mitochondrial dynamic state during reperfusion in a rat model of global brain ischemia. Evaluation of mitochondrial morphology with confocal and electron microscopy revealed a similar induction of fragmentation following global brain ischemia. Mitochondrial fragmentation aligned temporally with specific apoptotic events, including cyt c release, caspase 3/7 activation, and interestingly, release of the fusion protein Opa1. Moreover, we uncovered evidence of loss of Opa1 complexes during the progression of reperfusion, and electron microscopy micrographs revealed a loss of cristae architecture following global brain ischemia. These data provide novel evidence implicating a temporal connection between Opa1 alterations and dysfunctional mitochondrial dynamics following global brain ischemia.

Reduction of Infarct Size by the Therapeutic Protein TAT-Ndi1 In Vivo

Lethal myocardial ischemia–reperfusion (I/R) injury has been attributed in part to mitochondrial respiratory dysfunction (including damage to complex I) and the resultant excessive production of reactive oxygen species. Recent evidence has shown that reduced nicotinamide adenine dinucleotide–quinone internal oxidoreductase (Ndi1; the single-subunit protein that in yeast serves the analogous function as complex I), transduced by addition of the TAT-conjugated protein to culture media and perfusion buffer, can preserve mitochondrial function and attenuate I/R injury in neonatal rat cardiomyocytes and Langendorff-perfused rat hearts. However, this novel metabolic strategy to salvage ischemic-reperfused myocardium has not been tested in vivo. In this study, TAT-conjugated Ndi1 and placebo-control protein were synthesized using a cell-free system. Mitochondrial uptake and functionality of TAT-Ndi1 were demonstrated in mitochondrial preparations from rat hearts after intraperitoneal administration of the protein. Rats were randomized to receive either TAT-Ndi1 or placebo protein, and 2 hours later all animals underwent 45-minute coronary artery occlusion followed by 2 hours of reperfusion. Infarct size was delineated by tetrazolium staining and normalized to the volume of at-risk myocardium, with all analysis conducted in a blinded manner. Risk region was comparable in the 2 cohorts. Preischemic administration of TAT-Ndi1 was profoundly cardioprotective. These results demonstrate that it is possible to target therapeutic proteins to the mitochondrial matrix and that yeast Ndi1 can substitute for complex I to ameliorate I/R injury in the heart. Moreover, these data suggest that cell-permeable delivery of mitochondrial proteins may provide a novel molecular strategy to treat mitochondrial dysfunction in patients.

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia (1). Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion (2). The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) (2), as first presented in its current form in Sanderson, et al. (2008) (3), which produces reproducible injury to selectively vulnerable brain regions (3-6). The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time. In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Publisher Correction: Inhibitory modulation of cytochrome c oxidase activity with specific near-infrared light wavelengths attenuates brain ischemia/reperfusion injury

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