Henry M. Honda

Role of the Mitochondrial Permeability Transition in Myocardial Disease

Mitochondria play a key role in determining cell fate during exposure to stress. Their role during ischemia/reperfusion is particularly critical because of the conditions that promote both apoptosis by the mitochondrial pathway and necrosis by irreversible damage to mitochondria in association with mitochondrial permeability transition (MPT). MPT is caused by the opening of permeability transition pores in the inner mitochondrial membrane, leading to matrix swelling, outer membrane rupture, release of apoptotic signaling molecules such as cytochrome c from the intermembrane space, and irreversible injury to the mitochondria. During ischemia (the MPT priming phase), factors such as intracellular Ca2+ accumulation, long-chain fatty acid accumulation, and reactive oxygen species progressively increase mitochondrial susceptibility to MPT, increasing the likelihood that MPT will occur on reperfusion (the MPT trigger phase). Because functional cardiac recovery ultimately depends on mitochondrial recovery, cardioprotection by ischemic and pharmacological preconditioning must ultimately involve the prevention of MPT. Investigations into this area are beginning to unravel some of the mechanistic links between cardioprotective signaling and mitochondria.

Mitochondria and Ischemia/Reperfusion Injury

Abstract: Cardiac ischemia/reperfusion injury results in a variable mixture of apoptotic, necrotic, and normal tissue that depends on both the duration and severity of ischemia. Injury can be abrogated by activation of protective pathways via ischemic and pharmacologic preconditioning. Mitochondria serve as final arbiters of life and death of the cell as these organelles not only are required to generate ATP but also can trigger apoptosis or necrosis. A key mechanism of mitochondrial injury is by the mitochondrial permeability transition (MPT) that has been shown to occur at reperfusion. The article hypothesizes that ischemia/reperfusion promotes MPT in two phases: (1) MPT priming during ischemia occurs as progressive inner mitochondrial membrane leak is accompanied by depressed electron transport in the setting of fatty acid accumulation and loss of cytochrome c and antioxidants; and (2) Triggering of MPT at reperfusion is determined by the interplay of mitochondrial membrane potential (ΔΨm) with mitochondrial matrix Ca, reactive oxygen species, and pH. It has been found that strategies that promote mitochondrial recovery such as pharmacologic preconditioning by diazoxide are mediated by K+‐dependent regulation of matrix volume and ΔΨm, resulting in improved efficiency of ATP synthesis as well as prevention of cytochrome c loss. If mitochondria fail to recover, then MPT and hypercontracture can result as ΔΨm depolarization waves regeneratively cross the cell (0.1 to 0.2 μm/s).

Protection of cardiac mitochondria by diazoxide and protein kinase C: Implications for ischemic preconditioning

Mitochondrial ATP-sensitive K (mitoKATP) channels play a central role in protecting the heart from injury in ischemic preconditioning. In isolated mitochondria exposed to elevated extramitochondrial Ca, Pi, and anoxia to simulate ischemic conditions, the selective mitoKATP channel agonist diazoxide (25–50 μM) potently reduced mitochondrial injury by preventing both the mitochondrial permeability transition (MPT) and cytochrome c loss from the intermembrane space. Both effects were blocked completely by the selective mitoKATP antagonist 5-hydroxydecanoate. The protective effect against Ca-induced MPT was most evident under conditions in which the ability of electron transport to support membrane potential (Δψm) was decreased and inner membrane leakiness was increased moderately. Under these conditions, mitoKATP channel activity strongly regulated Δψm, and diazoxide prevented MPT by inhibiting the driving force for Ca uptake. Phorbol 12-myristate 13-acetate mimicked the protective effects of diazoxide, unless 5-hydroxydecanoate was present, indicating that protein kinase C activation also protects mitochondria by activating mitoKATP channels. Because Δψm recovery ultimately is required for heart functional recovery, these results may explain how mitoKATP channel activation mimics ischemic preconditioning by protecting mitochondria as they pass through a critical vulnerability window during ischemia/reperfusion.

Systematic characterization of the murine mitochondrial proteome using functionally validated cardiac mitochondria

Mitochondria play essential roles in cardiac pathophysiology and the murine model has been extensively used to investigate cardiovascular diseases. In the present study, we characterized murine cardiac mitochondria using an LC/MS/MS approach. We extracted and purified cardiac mitochondria; validated their functionality to ensure the final preparation contains necessary components to sustain their normal function; and subjected these validated organelles to LC/MS/MS‐based protein identification. A total of 940 distinct proteins were identified from murine cardiac mitochondria, among which, 480 proteins were not previously identified by major proteomic profiling studies. The 940 proteins consist of functional clusters known to support oxidative phosphorylation, metabolism, and biogenesis. In addition, there are several other clusters, including proteolysis, protein folding, and reduction/oxidation signaling, which ostensibly represent previously under‐appreciated tasks of cardiac mitochondria. Moreover, many identified proteins were found to occupy other subcellular locations, including cytoplasm, ER, and golgi, in addition to their presence in the mitochondria. These results provide a comprehensive picture of the murine cardiac mitochondrial proteome and underscore tissue‐ and species‐specification. Moreover, the use of functionally intact mitochondria insures that the proteomic observations in this organelle are relevant to its normal biology and facilitates decoding the interplay between mitochondria and other organelles.

Endothelial cell dynamics under pulsating flows: significance of high versus low shear stress slew rates (d(tau)/dt).

AbstractShear stress modulates endothelial cell (EC) remodeling via realignment and elongation. We provide the first evidence that the upstroke slopes of pulsatile flow, defined as shear stress slew rates (positive ∂τ/∂τ affect significantly the rates at which ECs remodel. We designed a novel flow system to isolate various shear stress slew rates by precisely controlling the frequency, amplitude, and time-averaged shear stress τave of pulsatile flow. Bovine aortic endothelial cell (BAEC) monolayers were exposed to three conditions: (1) pulsatile flow (1 Hz) at high slew rate (293 dyn/cm2 s), (2) pulsatile flow (1 Hz) at low slew rate (71 dyn/cm2 s), and (3) steady laminar flow at ∂τ/∂t=0. All of the three conditions were operated at τave=50{dyn/cm}2. BAEC elongation and alignment were measured over 17 h. We were able to demonstrate the effects of shear stress slew rates ∂τ/∂t on EC remodeling at a fixed spatial shear stress gradient (∂τ/∂x). We found that pulsatile flow significantly increased the rates at which EC elongated and realigned, compared to steady flow at ∂τ/∂t=0. Furthermore, EC remodeling was faster in response to high than to low slew rates at a given tau τave

Induction of Monocyte Binding to Endothelial Cells by MM-LDL Role of Lipoxygenase Metabolites

Treatment of human aortic endothelial cells (EC) with minimally oxidized LDL (or minimally modified LDL, MM-LDL) produces a specific pattern of endothelial cell activation distinct from that produced by LPS, tumor necrosis factor-alpha, and interleukin-1, but similar to other agents that elevate cAMP. The current studies focus on the signal transduction pathways by which MM-LDL activates EC to bind monocytes. We now demonstrate that, in addition to an elevation of cAMP, lipoxygenase products are necessary for the MM-LDL response. Treatment of EC with inhibitors of the lipoxygenase pathway, 5,8,11, 14-eicosatetraynoic acid (ETYA) or cinnamyl-3, 4-dihydroxy-alpha-cyanocinnamate (CDC), blocked monocyte binding in MM-LDL-treated EC (MM-LDL=118+/-13%; MM-LDL+ETYA=33+/-4%; MM-LDL+CDC=23+/-4% increase in monocyte binding) without reducing cAMP levels. To further investigate the role of the lipoxygenase pathway, cellular phospholipids were labeled with arachidonic acid. Treatment of cells for 4 hours with 50 to 100 microg/mL MM-LDL, but not native LDL, caused a 60% increase in arachidonate release into the medium and increased the intracellular formation of 12(S)-HETE (approximately 100% increase). There was little 15(S)-HETE present, and no increase in its levels was observed. We demonstrated that 12(S)-HETE reversed the inhibitory effect of CDC. We also observed a 70% increase in the formation of 11,12-epoxyeicosatrienoic acid (11, 12-EET) in cells treated with MM-LDL. To determine the mechanism of arachidonate release induced by MM-LDL, we examined the effects of MM-LDL on intracellular calcium levels. Treatment of EC with both native LDL and MM-LDL caused a rapid release of intracellular calcium from internal stores. However, several pieces of evidence suggest that calcium release alone does not explain the increased arachidonate release in MM-LDL-treated cells. The present studies suggest that products of 12-lipoxygenase play an important role in MM-LDL action on the induction of monocyte binding to EC.

Altered Proteome Biology of Cardiac Mitochondria Under Stress Conditions

Myocardial ischemia-reperfusion induces mitochondrial dysfunction and, depending upon the degree of injury, may lead to cardiac cell death. However, our ability to understand mitochondrial dysfunction has been hindered by an absence of molecular markers defining the various degrees of injury. To address this paucity of knowledge, we sought to characterize the impact of ischemic damage on mitochondrial proteome biology. We hypothesized that ischemic injury induces differential alterations in various mitochondrial subcompartments, that these proteomic changes are specific to the severity of injury, and that they are important to subsequent cellular adaptations to myocardial ischemic injury. Accordingly, an in vitro model of cardiac mitochondria injury in mice was established to examine two stress conditions: reversible injury (induced by mild calcium overload) and irreversible injury (induced by hypotonic stimuli). Both forms of injury had a drastic impact on the proteome biology of cardiac mitochondria. Altered mitochondrial function was concomitant with significant protein loss/shedding from the injured organelles. In the setting of mild calcium overload, mitochondria retained functionality despite the release of numerous proteins, and the majority of mitochondria remained intact. In contrast, hypotonic stimuli caused severe damage to mitochondrial structure and function, induced increased oxidative modification of mitochondrial proteins, and brought about detrimental changes to the subproteomes of the inner mitochondrial membrane and matrix. Using an established in vivo murine model of regional myocardial ischemic injury, we validated key observations made by the in vitro model. This preclinical investigation provides function and suborganelle location information on a repertoire of cardiac mitochondrial proteins sensitive to ischemia reperfusion stress and highlights protein clusters potentially involved in mitochondrial dysfunction in the setting of ischemic injury.

Role of Endothelial Nitric Oxide Synthase in the Regulation of SREBP Activation by Oxidized Phospholipids

Oxidized-1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), found in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to synthesize chemotactic factors, such as interleukin (IL)-8. Previously, we demonstrated that the sustained induction of IL-8 transcription by Ox-PAPC was mediated through the activation of sterol regulatory element–binding protein (SREBP). We now present evidence for the role of endothelial nitric oxide synthase (eNOS) in the activation of SREBP by Ox-PAPC. Ox-PAPC treatment of EC induced a dose- and time-dependent activation of eNOS, as measured by phosphorylation of serine 1177, dephosphorylation of threonine 495, and the conversion of l-arginine to l-citrulline. Activation of eNOS by Ox-PAPC was regulated through a phosphatidylinositol-3-kinase/Akt-mediated mechanism. These studies also demonstrated that pretreatment of EC with NOS inhibitor, N&ohgr;-nitro-l-arginine-methyl ester (L-NAME), significantly inhibited Ox-PAPC–induced IL-8 synthesis. Because SREBP activation had been previously shown to regulate IL-8 transcription by Ox-PAPC, we examined the effects of L-NAME on Ox-PAPC–induced SREBP activation. Our data demonstrated that Ox-PAPC–induced SREBP activation and expression of SREBP target genes were significantly reduced by pretreatment with L-NAME. Interestingly, treatment of EC with NO donor, S-nitroso-N-acetylpenicillamine, did not activate SREBP, suggesting that NO alone was not sufficient for SREBP activation. Rather, our findings indicated that superoxide (O2·−), in combination with NO, regulated SREBP activation by Ox-PAPC. We found that Ox-PAPC treatment generated O2·− through an eNOS-mediated mechanism and that mercaptoethylguanidine, a peroxynitrite scavenger, reduced SREBP activation by Ox-PAPC. Taken together, these findings propose a novel role for eNOS in the activation of SREBP and SREBP-mediated inflammatory processes.

A role for NADPH oxidase 4 in the activation of vascular endothelial cells by oxidized phospholipids

Previous studies from our group have demonstrated that oxidized 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) activates over 1000 genes in human aortic endothelial cells (HAECs). Prominent among these are genes regulating inflammation, cholesterol homeostasis, antioxidant enzymes, and the unfolded protein response. Previous studies from our lab and others suggested that transcriptional regulation by Ox-PAPC may be controlled, at least in part, by reactive oxygen species. We now present evidence that Ox-PAPC activation of NADPH oxidase 4 (NOX4) is responsible for the regulation of two of these important groups of genes: those controlling inflammation and those involved in sterol regulation. Our data demonstrate that Ox-PAPC increases reactive oxygen species formation in HAECs as seen by DCF fluorescence. NOX4 is the major molecule responsible for this increase because downregulation of NOX4 and its components (p22(phox) and rac1) blocked the Ox-PAPC effect. Our data show that Ox-PAPC did not change NOX4 transcription levels but did induce recruitment of rac1 to the membrane for NOX4 activation. We present evidence that vascular endothelial growth factor receptor 2 (VEGFR2) activation is responsible for rac1 recruitment to the membrane. Finally, we demonstrate that knockdown of NOX4 and its components rac1 and p22(phox) decreases Ox-PAPC induction of inflammatory and sterol regulatory genes, but does not affect Ox-PAPC transcriptional regulation of other genes for antioxidants and the unfolded protein response. In summary, we have identified a VEGFR2/NOX4 regulatory pathway by which Ox-PAPC controls important endothelial functions.

A complex flow pattern of low shear stress and flow reversal promotes monocyte binding to endothelial cells.

Predilection sites for atherosclerosis within the vasculature are characterized by low shear stress and flow reversal. In this study, endothelial cells were exposed to a complex flow pattern that was characterized by particle velocity determination. Bovine aortic endothelial cells exposed to low shear stress and flow reversal demonstrated higher levels of monocyte binding compared to endothelial cells exposed to one-directional flow. In addition, endothelial cells exposed to low shear stress and flow reversal responded to inflammatory stimuli with substantial increases in monocyte binding, similar to that seen in cells exposed to one-directional flow. These findings suggest a mechanism by which areas of low shear stress and flow reversal are predisposed to the development of atherosclerotic lesions.