Question

Aminooxyacetate was administered to pre-ischemic cardiac cells to determine the effect of on ischemia-reperfusion injury. Note that mitochondrial preservation is essential to reduce ischemia-reperfusion injury. What is the potential outcome of this experiment?

Answer 1

The interpretation is

Ischemic disorders, such as myocardial infarction, stroke, and peripheral vascular disease, are the most common causes of debilitating disease and death in westernized cultures. The extent of tissue injury relates directly to the extent of blood flow reduction and to the length of the ischemic period, which influence the levels to which cellular ATP and intracellular pH are reduced. By impairing ATPase-dependent ion transport, ischemia causes intracellular and mitochondrial calcium levels to increase (calcium overload). Cell volume regulatory mechanisms are also disrupted by the lack of ATP, which can induce lysis of organelle and plasma membranes. Reperfusion, although required to salvage oxygen-starved tissues, produces paradoxical tissue responses that fuel the production of reactive oxygen species (oxygen paradox), sequestration of proinflammatory immunocytes in ischemic tissues, endoplasmic reticulum stress, and development of postischemic capillary no-reflow, which amplify tissue injury. These pathologic events culminate in opening of mitochondrial permeability transition

pores as a common end-effector of ischemia/reperfusion (I/R)-induced cell lysis and death. Emerging concepts include the influence of the intestinal microbiome, fetal programming, epigenetic changes, and microparticles in the pathogenesis of I/R. The overall goal of this review is to describe these and other mechanisms that contribute to I/R injury. Because so many different deleterious events participate in I/R, it is clear that therapeutic approaches will be effective only when multiple pathologic processes are targeted. In addition, the translational significance of I/R research will be enhanced by much wider use of animal models that incorporate the complicating effects of risk factors for cardiovascular disease.

Mitochondrial dysfunction contributes to I/R

Oxygen lack during low blood flow states inhibits the flow of electrons through the mitochondrial respiratory chain. As a result, ADP phosphorylation to ATP by the F1F0 ATPase cannot occur (192, 303). Indeed, under these conditions of inhibited electron transfer, the ATP synthase actually operates in a reverse mode (ATP hydrolase), hydrolyzing the little remaining ATP in an attempt to maintain Δψm (192, 572). As consequence of these two events, ATP levels decrease very rapidly when ischemia is induced. Interestingly, selective inhibition of mitochondrial F1F0 ATPase slows the rate of ATP loss during ischemia but improves restoration of cellular ATP levels on reperfusion and limits infarct size (303). Impaired oxidative phosphorylation induced by ischemia also inhibits the breakdown and/or oxidation of fatty acids (707). As toxic fatty acids accumulate within affected cells, they fuel inflammatory arachidonic acid metabolite generation (810) and promote mitochondrial PTP opening (195). Because mitochondrial bioenergetic changes are essential early warning signs for impending ischemic conditions, monitoring patient bioenergetic health index shows potential as a new biomaker (123).

As previously mentioned, mitochondria are important sources of oxidative stress in I/R, with excessive ROS being generated by the electron transport chain and the mitochondrial outer membrane proteins p66Shc and MAOs, and several other mitochondrial proteins including mitochondrial NOX4 (172, 406) (Fig. 8). Superoxide produced under physiologic conditions via complexes I and III of the electron transport chain under physiologic conditions is neutralized by SOD. However, the increased leakage of superoxide during ischemia, especially at complex I, overwhelms cellular antioxidant defenses (172, 477, 628, 729). Restoration of oxygen delivery when blood flow is reestablished further compounds these sequelae.

Several other mitochondrial ROS sources have been described, including α-ketoglutarate dehydrogenase (αKGDH), electron-transfer flavoprotein, pyruvate dehydrogenase, glycerophosphate dehydrogenase, and ROS modulator 1 (Romo1). There is evidence that αKGDH can be a major source of oxidants when the NADH/NAD ratio is high, as occurs in I/R. Indeed, increased phosphorylation of this enzyme has been reported in female rats, which reduces ROS generation by αKGDH by mitochondria and cardiomyocytes isolated from these animals after A/R, providing a potential explanation for the lower risk for cardiovascular disease in premenopausal females (464, 628). Roles for the other mitochondrial proteins mentioned above in I/R-induced ROS generation have not yet been studied. However, given the central role of TNF in I/R injury, it is tempting to speculate that ROS modulator 1 may play an important role in postischemic ROS generation by mitochondria because this recently described protein has been shown to link TNF signaling to apoptotic cell death via mitochondrial oxidant production (419, 420).

As described above, I/R-induced opening of the mitochondrial PTP is a final end effector in the plethora of events in the progression to cell death during reperfusion. This pore is quiescent during ischemia because it is inhibited by acidotic conditions. However, I/R-induced mitochondrial Ca2+ overload coupled with the enormous increase in ROS production associated with the reintroduction of molecular oxygen cause the mitochondrial PTP to open (192–194, 603) (Fig. 8). Because of the open mitochondrial PTP is large, readily allowing molecules up to 1.5 kD in size to cross the channel, a massive flux of H+ions pass back into the mitochondrial matrix, thereby dissipating the Δψm, uncoupling the electron transport chain and inhibiting ATP synthesis (37, 315). At the same time, water is driven osmotically into the organelle, causing excessive swelling that can proceed to rupture.

Although the protein constituents of the mitochondrial PTP and how they interact to control its permeability have not been definitely identified, adenine nucleotide translocase, mitochondrial phosphate carrier, and cyclophilin-D are leading structural candidates (13, 35, 36, 316, 723). Despite this uncertainty, the development of cyclophilin-D inhibitors has allowed examination of the role of the mitochondrial PTP in I/R injury. Inhibition of this putative pore constituent has been shown to mitigate I/R-induced cell death (13, 149, 192, 570, 646, 690, 723). The development of CypD-deficient mice and their use in I/R studies has confirmed the aforementioned pharmacologic inhibitor studies (39, 187, 693). Mitochondrial DNA is another target in I/R (77).

Mitochondria form intercommunicating tubular networks that are linked to the cytoskeleton and are tethered to the endoplasmic reticulum via a network of membrane contact sites. Recent work indicates that mitochondrial tubular networks provide a conductive pathway dependent on proton-motive force for ultrarapid energy distribution within the cell (269). In mammalian cells, the endoplasmic reticulum membrane contact sites with mitochondria are closely apposed (gap distance of 6–15 nm between ER and mitochondrial membranes), cover about 2% to 5% of mitochondrial surface area, and function to establish tethering domains that enable exchange of signals or metabolites (e.g., Ca2+ and lipids) between these organelles (635). These dynamic organelles also undergo cycles of division (fission) and fusion, processes that can become unbalanced in pathologic states to produce alterations in mitochondrial morphology and function (137). Large networks of fused mitochondria appear with loss of fission. On the other hand, excessive fission produces small, fragmented mitochondria, a requisite step for extrinsic apoptotic cell death. Since ischemia-induced reductions in ATP levels and increased mitochondrial ROS production promote fission of these organelles, the ensuing fragmentation of mitochondria contribute to postischemic apoptotic cell death. Moreover, inhibition of mitochondrial fission reduces I/R-induced mitochondrial fragmentation and prevents opening of the mitochondrial PTP (605). Postischemic endothelial dysfunction may also involve this mechanism, since H/R induces mitochondrial fission and fragmentation in cultured endothelial cells (268). It is interesting to note that ER membrane contact sites define the location of fission on mitochondria by controlling where fission machinery assembles (635). Although not yet explored in I/R, these results suggest that therapeutic targeting of membrane contacts sites to modulate mitochondrial fission/fusion may represent a novel approach for clinical cardioprotection in I/R.

Concluding Remarks and Perspectives

Age-adjusted cardiovascular mortality has declined dramatically over the past several decades. This has been accompanied by major improvements in discharge disposition, decreases in the likelihood of readmission, and an impressive reduction in hospitalization rates for patients with or at risk for cardiovascular disease and stroke. Advances in pharmaceuticals (e.g., thrombolytic agents, antiplatelet drugs, beta blockers, and angiotensin converting enzyme inhibitors and receptor blockers), aggressive management of risk factors for cardiovascular disease, development of approaches to restore tissue perfusion (e.g., PCI and cardiopulmonary bypass), improved patient education and awareness, enhancements in quality of care (via more rapid risk stratification, timeliness of treatment, and hospital process performance analysis to ensure appropriate application of proven interventions), the discovery of sensitive blood indicators, and development of sophisticated imaging methodologies to detect subclinical disease years before symptomatic presentation all have contributed to this success. During this same period, intensive research has uncovered several major concepts regarding the mechanisms of I/R including: (i) the discovery that short bouts of I/R activate cell survival programs (ischemic conditioning) that limit lethal I/R injury (and indicating that there is a bimodal or hormetic response to I/R), (ii) reperfusion paradoxically amplifies cell injury and death, which occurs by mechanisms that are distinct from those induced by ischemia per se, (iii) uncovering of multiple death modalities that contribute to I/R-induced cell death, many of which occur by programmed sequences of events, (iv) fetal exposure to stressors incur programming events that enhance the susceptibility to cardiovascular disease and I/R syndromes later in life, and (v) numerous, complex, and highly interactive mechanisms underlie the pathogenesis of I/R. These include, but are not limited to, disruption in ion transport mechanisms that result in cellular calcium overload, overexuberant production of ROS and RNOS, inflammation, protein kinase activation, development of ER and mitochondrial dysfunction, epigenetic alterations in gene expression, formation of protein cleavage products, development of no-reflow, roles for the gut microbiome, and genomic/metabolomic/lipidomic contributions to clinical phenotypes.

Despite this enhanced mechanistic understanding of I/R injury from preclinical studies, there has been very little success in transforming these discoveries into new adjuvant therapies with proven efficacy in relevant patient populations (89, 109, 399, 427, 474, 565, 597, 614, 645, 809). Indeed, no new treatment to reduce infarct size has emerged since the advent of thrombolysis and angioplasty. This disappointing translation of mechanistic findings relates in part to the limitations imposed by conducting evaluative trials in patients with advanced disease that may be beyond salvage, which also imposes a short time window for improving outcomes. Perhaps more importantly, much of the preclinical work conducted to date has been accomplished in young, healthy animals, whereas patients usually present with coexisting risk factors. Another factor, which is only now emerging as a consideration, is contributions from the host’s microbiome. Because the microbiome is influenced by a host of factors that are rarely controlled for, microbiota composition differences may contribute in a major way to discrepant findings in the literature. A fourth explanation relates to therapeutic focus on a single contributory mechanism in the setting of multi-factorial pathological processes that sum to produce tissue injury and death. Indeed, the large number of contributory factors in the pathogenesis of I/R injury argues against the concept of single drug intervention that has characterized the approaches adopted in basic research, by the pharmaceutical industry, and in clinical trials. On the other hand, studies investigating the mechanisms underlying the protective actions of ischemic preconditioning have shown that this intervention targets multiple pathologic processes in I/R, raising the hope that pharmacologic agents that mimic its powerful cardioprotective effects might prove more effective. In addition, since conditioning activates cell signaling programs to upregulate the expression of several survival proteins, gene therapy approaches based on these discoveries have also shown promise. However, while success of initial small scale trials indicated that such conditioning-based approaches might improve clinical outcomes (16, 126, 823, 834), larger trials have failed to confirm efficacy (399, 614, 645, 796, 881). This led to proposals to better standardize protocols for therapeutic intervention by conditioning approaches, more extensive and rigorous experimental validation of new targets in preclinical work before patient testing is considered, and to design clinical trials with an eye toward decreasing patient heterogeneity with regard to cardiovascular disease phenotype (399, 881). Until only the last decade or so, I/R studies in general and work focused on conditioning mechanisms was conducted largely in young, healthy animals. This experimental focus, when coupled with the growing body of evidence indicating that the cardioprotective effects of conditioning strategies are subverted by the presence of comorbid risk factors, indicates that the design of preclinical studies should include models that better mirror patient phenotype in those suffering adverse cardiovascular events. Another point of this discussion is to recognize that ischemic disorders have complex, multifactorial etiologies that are pathophysiologically heterogeneous but highly interactive. As such, prevailing paradigms have to be constantly evaluated, updated, and adjusted by new evidence, with care taken not to become entrenched or biased by current dogma.

The application of stem cell therapy holds great promise because it targets repair and regenerative processes in postischemic tissues, thereby avoiding the issues described above that have contributed to the failure of treatments directed at mechanisms directly causing injury in I/R. Indeed, injection of adult stem cells into hearts and brains damaged by I/R was shown to improve function and facilitate beneficial remodeling (78, 607, 786, 906). While it was presumed that these effects were due to differentiation of the engrafted stem cells into cardiac myocytes and vascular cells, other studies failed to demonstrate such plasticity (42, 577). In addition, adult stem cells show poor survivability after injection into the harsh milieu present in postischemic tissues (270). Moreover, sufficient numbers of cardiac myocytes cannot be generated from the relatively small numbers of stem cells that are injected (346). Based on this information, it was recently suggested that adult stem cells improve function and promote reparative remodelling via their release of paracrine mediators such as growth factors and chemokines (346). The beneficial actions of such paracrine factors appear to be related to release of cytoprotective molecules, immunomodulatory effects, promotion of cardiomyocyte proliferation, alterations in ECM remodeling that limit fibrosis, stimulation of angiogenesis, activation of tissue resident stem cells to differentiate into cardiac myocytes, and may involve release of exosomes and microvesicles (346). Because of the complex multifactorial processes that participate in postischemic healing, it is clear that these paracrine mechanisms are highly dynamic and require exquisite temporal and spatial organization to effect repair and regeneration. Understanding these multifaceted, dynamically phased, and highly pleiotropic mechanisms will be a major focus of future research to improve stem cell-mediated tissue repair and regeneration after I/R.