Ultrafine Particulate Matter Increases Cardiac Ischemia/Reperfusion Injury via Mitochondrial Permeability Transition Pore.

Ultrafine particulate matter (UFP) has been associated with increased cardiovascular morbidity and mortality. However, the mechanisms that drive PM-associated cardiovascular disease and dysfunction remain unclear. We examined the impact of oropharyngeal aspiration of 100 µg UFP from the Chapel Hill, NC, air shed in Sprague-Dawley rats on cardiac function, arrhythmogenesis, and cardiac ischemia/reperfusion (I/R) injury using a Langendorff working heart model. We found that exposure to UFP was capable of significantly exacerbating cardiac I/R injury without changing overall cardiac function or major changes in arrhythmogenesis. Cardiac I/R injury was attenuable with administration of cyclosporin A (CsA), suggesting a role for the mitochondrial permeability transition pore (mPTP) in UFP-associated cardiovascular toxicity. Isolated cardiac mitochondria displayed decreased Ca2+ buffering before opening of the mPTP. These findings suggest that UFP-induced expansion of cardiac I/R injury may be a result of mPTP Ca2+ sensitization resulting in increased mitochondrial permeability transition and potential initiation of mPTP-associated cell death pathways.

Pulmonary instillation of multi-walled carbon nanotubes promotes coronary vasoconstriction and exacerbates injury in isolated hearts.

The growing use of multi-walled carbon nanotubes (MWCNTs) across industry has increased human exposures. We tested the hypothesis that pulmonary instillation of MWCNTs would exacerbate cardiac ischaemia/reperfusion (I/R) injury. One day following intratracheal instillation of 1, 10 or 100 µg MWCNT in Sprague-Dawley rats, we used a Langendorff isolated heart model to examine cardiac I/R injury. In the 100 µg MWCNT group we report increased premature ventricular contractions at baseline and increased myocardial infarction. This was associated with increased endothelin-1 (ET-1) release and depression of coronary flow during early reperfusion. We also tested if isolated coronary vascular responses were affected by MWCNT instillation and found trends for enhanced coronary tone, which were dependent on ET-1, cyclooxygenase, thromboxane and Rho-kinase. We concluded that instillation of MWCNTs promoted cardiac injury and depressed coronary flow by invoking vasoconstrictive mechanisms involving ET-1, cyclooxygenase, thromboxane and Rho-kinase.

Stage of the estrous cycle does not influence myocardial ischemia-reperfusion injury in rats (Rattus norvegicus).

Even though cardiovascular disease is the leading cause of death for men and women, the vast majority of animal studies use male animals. Because female reproductive hormones have been associated with cardioprotective states, many investigators avoid using female animals because these hormones are cyclical and may introduce experimental variability. In addition, no studies have investigated the specific effects of the estrous cycle on cardiac ischemic injury. This study was conducted to determine whether the estrous cycle stage influences the susceptibility to ischemic injury in rat hearts. Estrous cycle stage was determined by using vaginal smear cytology, after which hearts underwent either in vivo (surgical) or ex vivo (isolated) ischemia-reperfusion injury. For in vivo studies, the left anterior coronary artery was ligated for 25 min of ischemia and subsequently released for 120 min of reperfusion. Infarct sizes were 42% ± 6%; 49% ± 4%; 40% ± 9%; 47% ± 9% of the zone-at-risk for rats in proestrus, estrus, metestrus, and diestrus, respectively. For ex vivo studies, isolated, perfused hearts underwent global ischemia and reperfusion for 25 and 120 min, respectively. Similar to our in vivo studies, the ex vivo rat model showed no significant differences in susceptibility to infarction or extent of cardiac arrhythmia according to estrous stage. To our knowledge, these studies provide the first direct evidence that the stage of estrous cycle does not significantly alter cardiac ischemia-reperfusion injury in rats.

Redox-dependent increases in glutathione reductase and exercise preconditioning: role of NADPH oxidase and mitochondria.

AIMS: We have previously shown that exercise leads to sustainable cardioprotection through a mechanism involving improved glutathione replenishment. This study was conducted to determine if redox-dependent modifications in glutathione reductase (GR) were involved in exercise cardioprotection. Furthermore, we sought to determine if reactive oxygen species generated by NADPH oxidase and/or mitochondria during exercise were triggering events for GR modulations. METHODS AND RESULTS: Rats were exercised for 10 consecutive days, after which isolated hearts were exposed to ischaemia/reperfusion (25 min/120 min). Exercise protected against infarction and arrhythmia, and preserved coronary flow. The GR inhibitor BCNU abolished the beneficial effects. GR activity was increased following exercise in a redox-dependent manner, with no change in GR protein levels. Because fluorescent labelling of GR protein thiols showed lower amounts of reduced thiols after exercise, we sought to determine the source of intracellular reactive oxygen species that may be activating GR. Subsets of animals were exercised immediately after treatment with either NADPH-oxidase inhibitors apocynin or Vas2870, or with mitoTEMPO or Bendavia, which reduce mitochondrial reactive oxygen species levels. The cardioprotective effects of exercise were abolished if animals exercised in the presence of NADPH oxidase inhibitors, in clear contrast to the mitochondrial reagents. These changes correlated with thiol-dependent modifications of GR. CONCLUSION: Adaptive cardioprotective signalling is triggered by reactive oxygen species from NADPH oxidase, and leads to improved glutathione replenishment through redox-dependent modifications in GR.

Reduction of ischemia/reperfusion injury with bendavia, a mitochondria-targeting cytoprotective Peptide.

BACKGROUND: Manifestations of reperfusion injury include myocyte death leading to infarction, contractile dysfunction, and vascular injury characterized by the "no-reflow" phenomenon. Mitochondria-produced reactive oxygen species are believed to be centrally involved in each of these aspects of reperfusion injury, although currently no therapies reduce reperfusion injury by targeting mitochondria specifically. METHODS AND RESULTS: We investigated the cardioprotective effects of a mitochondria-targeted peptide, Bendavia (Stealth Peptides), across a spectrum of experimental cardiac ischemia/reperfusion models. Postischemic administration of Bendavia reduced infarct size in an in vivo sheep model by 15% (P=0.02) and in an ex vivo guinea pig model by 38% to 42% (P<0.05). In an in vivo rabbit model, the extent of coronary no-reflow was assessed with Thioflavin S staining and was significantly smaller in the Bendavia group for any given ischemic risk area than in the control group (P=0.0085). Myocardial uptake of Bendavia was ≈25% per minute, and uptake remained consistent throughout reperfusion. Postischemic recovery of cardiac hemodynamics was not influenced by Bendavia in any of the models studied. Isolated myocytes exposed to hypoxia/reoxygenation showed improved survival when treated with Bendavia. This protection appeared to be mediated by lowered reactive oxygen species-mediated cell death during reoxygenation, associated with sustainment of mitochondrial membrane potential in Bendavia-treated myocytes. CONCLUSIONS: Postischemic administration of Bendavia protected against reperfusion injury in several distinct models of injury. These data suggest that Bendavia is a mitochondria-targeted therapy that reduces reperfusion injury by maintaining mitochondrial energetics and suppressing cellular reactive oxygen species levels. (J Am Heart Assoc. 2012;1:e001644 doi: 10.1161/JAHA.112.001644.).

Mitochondrial permeability transition in the diabetic heart: contributions of thiol redox state and mitochondrial calcium to augmented reperfusion injury.

Mitochondria from diabetic hearts are sensitized to mitochondrial permeability transition pore (PTP) opening, which may be responsible for the increased propensity for cardiac injury in diabetic hearts. The purpose of this study was to determine if redox-dependent PTP opening contributes to augmented injury in diabetic hearts, and if compounds targeted at mitochondrial PTP, ROS, and calcium influx protected diabetic hearts from injury. Hearts from control or streptozotocin-induced diabetic rats were excised for either whole-heart or isolated mitochondria experiments. Myocardial glutathione content was oxidized in diabetic hearts when compared to control, and this translated to increased oxidation of the adenine nucleotide translocase in diabetic hearts. Diabetic mitochondria displayed significantly greater sensitivity to PTP opening than non-diabetic counterparts, which was reversed with the thiol-reducing agent dithiothreitol. The thiol-oxidant diamide increased calcium sensitivity in control, but not diabetic mitochondria. Diabetic animals treated with the mitochondria-targeted ROS suppressing peptide MTP-131 also showed improved resistance to PTP opening. In separate experiments hearts underwent ex vivo ischemia/reperfusion (IR). Diabetic hearts were more susceptible to IR injury, with infarct sizes of 60 ± 4% of the area-at-risk (vs. 46 ± 2% in non-diabetics; P<0.05). Administration of the PTP blocker NIM811 (5 µM), MTP-131 (1 nM) or the mitochondrial calcium uniporter blocker minocycline (1 µM) at the onset of reperfusion reduced infarct sizes in both control and diabetic hearts. These findings suggest that augmented susceptibility to injury in the diabetic heart is mediated by redox-dependent shifts in PTP opening, and that three novel mitochondria-targeted agents administered at reperfusion may be suitable adjuvant reperfusion therapies to attenuate injury in diabetic patients.

Short-term exercise preserves myocardial glutathione and decreases arrhythmias after thiol oxidation and ischemia in isolated rat hearts.

The purpose of this study was to determine if exercise (Ex) protects hearts from arrhythmias induced by glutathione oxidation or ischemia-reperfusion (I/R). Female Sprague-Dawley rats were divided into two experimental groups: sedentary controls (Sed) or short-term Ex (10 days of treadmill running). Twenty-four hours after the last session, hearts were excised and exposed to either perfusion with the thiol oxidant diamide (200 µM) or global I/R. Ex significantly delayed the time to the onset of ventricular arrhythmia after irreversible diamide perfusion. During a shorter diamide perfusion protocol with washout, Ex significantly decreased the incidence of arrhythmia, as evidenced by a delayed time to the first observed arrhythmia, lower arrhythmia scores, and lower incidence of ventricular fibrillation. Ex hearts exposed to I/R (30-min ischemia/30-min reperfusion) also showed lower arrhythmia scores and incidence of ventricular fibrillation compared with Sed counterparts. Our finding that Ex protected intact hearts from thiol oxidation was corroborated in isolated ventricular myocytes. In myocytes from Ex animals, both the increase in H(2)O(2) fluorescence and incidence of cell death were delayed after diamide. Although there were no baseline differences in reduced-to-oxidized glutathione ratios (GSH/GSSG) between the Sed and Ex groups, GSH/GSSG was better preserved in Ex groups after diamide perfusion and I/R. Myocardial glutathione reductase activity was significantly enhanced after Ex, and this was preserved in the Ex group after diamide perfusion. Our results show that Ex protects the heart from arrhythmias after two different oxidative stressors and support the hypothesis that sustaining the GSH/GSSG pool stabilizes cardiac electrical function during conditions of oxidative stress.

High doses of ketamine-xylazine anesthesia reduce cardiac ischemia-reperfusion injury in guinea pigs.

Choosing an appropriate anesthetic protocol that will have minimal effect on experimental design can be difficult. Guinea pigs have highly variable responses to a variety of injectable anesthetics, including ketamine-xylazine (KX). Because of this variability, supplemental doses often are required to obtain an adequate plane of anesthesia. Our group studies the isolated guinea pig heart, and we must anesthetize guinea pigs prior to harvesting this organ. In this study, we sought to determine whether a higher dose of KX protected isolated guinea pig hearts against myocardial ischemia-reperfusion injury. Male Hartley guinea pigs (Crl:HA; 275 to 300 g; n = 14) were anesthetized with either of 2 doses of KX (K: 85 mg/kg, X: 15 mg/kg; or K: 200 mg/kg, X: 60 mg/kg). After thoracotomy, hearts underwent 20 min of ischemia followed by 2 h of reperfusion. The high dose of KX significantly reduced myocardial infarct size as compared with the low dose (36% ± 3% and 51% ± 6%, respectively). Furthermore, the high dose of KX improved hemodynamic function over that associated with the low dose as measured by increases in both left ventricular developed pressure (49 ± 4 and 30 ± 8 mm Hg, respectively) and maximal rate of left ventricular relaxation (-876 ± 70 and -576 ± 120 mm Hg/s, respectively). However, the high dose of KX did not alter the maximal rate of left ventricular contraction or coronary flow. These results suggest that supplementation of KX to ensure an adequate anesthetic plane may introduce unwanted variability in ischemia-reperfusion studies.

Duodenal-jejunal bypass surgery does not increase skeletal muscle insulin signal transduction or glucose disposal in Goto-Kakizaki type 2 diabetic rats.

BACKGROUND: Duodenal-jejunal bypass (DJB) has been shown to reverse type 2 diabetes (T2DM) in Goto-Kakizaki (GK) rats, a rodent model of non-obese T2DM. Skeletal muscle insulin resistance is a hallmark decrement in T2DM. The aim of the current work was to investigate the effects of DJB on skeletal muscle insulin signal transduction and glucose disposal. It was hypothesized that DJB would increase skeletal muscle insulin signal transduction and glucose disposal in GK rats. METHODS: DJB was performed in GK rats. Sham operations were performed in GK and nondiabetic Wistar-Kyoto (WKY) rats. At 2 weeks post-DJB, oral glucose tolerance (OGTT) was measured. At 3 weeks post-DJB, insulin-induced signal transduction and glucose disposal were measured in skeletal muscle. RESULTS: In GK rats and compared to sham operation, DJB did not (1) improve fasting glucose or insulin, (2) improve OGTT, or (3) increase skeletal muscle insulin signal transduction or glucose disposal. Interestingly, skeletal muscle glucose disposal was similar between WKY-Sham, GK-Sham, and GK-DJB. CONCLUSIONS: Bypassing of the proximal small intestine does not increase skeletal muscle glucose disposal. The lack of skeletal muscle insulin resistance in GK rats questions whether this animal model is adequate to investigate the etiology and treatments for T2DM. Additionally, bypassing of the foregut may lead to different findings in other animal models of T2DM as well as in T2DM patients.

Cardiac arrhythmias induced by glutathione oxidation can be inhibited by preventing mitochondrial depolarization.

We have previously proposed that the heterogeneous collapse of mitochondrial inner membrane potential (DeltaPsi(m)) during ischemia and reperfusion contributes to arrhythmogenesis through the formation of metabolic sinks in the myocardium, wherein clusters of myocytes with uncoupled mitochondria and high K(ATP) current levels alter electrical propagation to promote reentry. Single myocyte studies have also shown that cell-wide DeltaPsi(m) depolarization, through a reactive oxygen species (ROS)-induced ROS release mechanism, can be triggered by global depletion of the antioxidant pool with diamide, a glutathione oxidant. Here we examine whether diamide causes mitochondrial depolarization and promotes arrhythmias in normoxic isolated perfused guinea pig hearts. We also investigate whether stabilization of DeltaPsi(m) with a ligand of the mitochondrial benzodiazepine receptor (4'-chlorodiazepam; 4-ClDzp) prevents the formation of metabolic sinks and, consequently, precludes arrhythmias. Oxidation of the GSH pool was initiated by treatment with 200 microM diamide for 35 min, followed by washout. This treatment increased GSSG and decreased both total GSH and the GSH/GSSG ratio. All hearts receiving diamide transitioned from sinus rhythm into ventricular tachycardia and/or ventricular fibrillation during the diamide exposure: arrhythmia scores were 5.5+/-0.5; n=6 hearts. These arrhythmias and impaired LV function were significantly inhibited by co-administration of 4-ClDzp (64 microM): arrhythmia scores with diamide+4-ClDzp were 0.4+/-0.2 (n=5; P<0.05 vs. diamide alone). Imaging DeltaPsi(m) in intact hearts revealed the heterogeneous collapse of DeltaPsi(m) beginning 20 min into diamide, paralleling the timeframe for the onset of arrhythmias. Loss of DeltaPsi(m) was prevented by 4-ClDzp treatment, as was the increase in myocardial GSSG. These findings show that oxidative stress induced by oxidation of GSH with diamide can cause electromechanical dysfunction under normoxic conditions. Analogous to ischemia-reperfusion injury, the dysfunction depends on the mitochondrial energy state. Targeting the mitochondrial benzodiazepine receptor can prevent electrical and mechanical dysfunction in both models of oxidative stress.