Fatiha Moukdar

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.

Reduction of early reperfusion injury with the mitochondria-targeting peptide Bendavia

We recently showed that Bendavia, a novel mitochondria-targeting peptide, reduced infarction and no-reflow across several experimental models. The purpose of this study was to determine the therapeutic timing and mechanism of action that underlie Bendavia’s cytoprotective property. In rabbits exposed to in vivo ischemia/reperfusion (30/180 min), Bendavia administered 20 minutes prior to reperfusion (0.05 mg/kg/h, intravenously) reduced myocardial infarct size by ∼50% when administered for either 1 or 3 hours of reperfusion. However, when Bendavia perfusion began just 10 minutes after the onset of reperfusion, the protection against infarction and no-reflow was completely lost, indicating that the mechanism of protection is occurring early in reperfusion. Experiments in isolated mouse liver mitochondria found no discernible effect of Bendavia on blocking the permeability transition pore, and studies in isolated heart mitochondria showed no effect of Bendavia on respiratory rates. As Bendavia significantly lowered reactive oxygen species (ROS) levels in isolated heart mitochondria, the ROS-scavenging capacity of Bendavia was compared to well-known ROS scavengers using in vitro (cell-free) systems that enzymatically generate ROS. Across doses ranging from 1 nmol/L to 1 mmol/L, Bendavia showed no discernible ROS-scavenging properties, clearly differentiating itself from prototypical scavengers. In conclusion, Bendavia is a promising candidate to reduce cardiac injury when present at the onset of reperfusion but not after reperfusion has already commenced. Given that both infarction and no-reflow are related to increased cellular ROS, Bendavia’s protective mechanism of action likely involves reduced ROS generation (as opposed to augmented scavenging) by endothelial and myocyte mitochondria.

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.

Reversal of Voltage-Dependent Erectile Responses in the Zucker Obese-Diabetic Rat by Rosuvastatin-Altered RhoA/Rho-kinase Signaling

INTRODUCTION The combination of independent risk factors for erectile dysfunction, obesity, hypertension, and diabetes are collectively manifested in a condition known as metabolic syndrome X (MSX). However, the regulatory mechanisms responsible for the erectile dysfunction (ED) are not fully understood. Clinical studies suggest that a pleiotropic effect of statins ability to enhance vascular relaxation might be through an impact on nitric oxide signaling or through a regulation of RhoA activation. AIM We hypothesized that regulatory aspects of short-term statin therapy involve the alteration of the RhoA/Rho-kinase signaling cascade and will reverse the ED seen in a rat model of MSX. MAIN OUTCOME MEASURES The magnitude and sensitivity of the voltage-dependent maintenance of intracavernosal blood pressure and mean arterial blood pressure. These responses were correlated with tissue protein and mRNA expression levels of RhoA and Rho kinases. METHODS Erectile function was evaluated by assessing voltage-dependent stimulation of the cavernosal nerve in 16-20 weeks old lean and obese-diabetic Zucker rats treated with 5 mg/kg/day of rosuvastatin intraperitoneally for 3 days. Cavernosal tissue RhoA and Rho-kinases expression levels were evaluated by real-time reverse transcriptase-polymerase chain reaction, Western blot. RESULTS The voltage-dependent erectile responses were suppressed by >30% in the obese-diabetic Zucker rat. The 3-day treatment with rosuvastatin partially restored the erectile response. The Rho-kinase inhibitor, H-1152, dose dependently increased the erectile responses and shifted the voltage sensitivity with statin treatment. Analysis of protein expression levels suggested elevation of RhoA and Rho kinases in obese-diabetics and statin treatment lowering Rho-kinase II. The RhoA and Rho-kinase II mRNA levels were significantly reduced in the rosuvastatin-treated obese-diabetic animals. CONCLUSIONS These results support a hypothesis that short-term statin therapy may lower RhoA/Rho-kinase expression levels and improve cavernosal blood pressure response to Rho-kinase inhibition and voltage-stimulation, and reversing an augmented vasoconstricted state associated with diabetes and/or hypertension in MSX.

Cardioprotection via preserved mitochondrial structure and function in the mPer2-mutant mouse myocardium.

We have previously shown that myocardial infarct size in nonreperfused hearts of mice with a functional deletion of the circadian rhythm gene mPer2 (mPer2-M) was reduced by 43%. We hypothesized that acute ischemia-reperfusion injury (I/R = 30 min I/2 h R) would also be reduced in these mice and that ischemic preconditioning (IPC) (3 × 5 min cycles) before I/R, which enhances protection in wild-type (WT) hearts, would provide further protection in mPer2-M hearts. We observed a 69 and 75% decrease in infarct size in mPer2-M mouse hearts compared with WT following I/R and IPC, respectively. This was coincident with 67% less neutrophil infiltration and 57% less apoptotic cardiomyocytes. IPC in mPer2-M mice before I/R had 48% less neutrophil density and 46% less apoptosis than their WT counterparts. Macrophage density was not different between WT and mPer2-M I/R, but it was 45% higher in mPer2-M IPC mouse hearts compared with WT IPC. There were no baseline differences in cardiac mitochondrial function between WT and mPer2-M mice, but, following I/R, WT exhibited a marked decrease in maximal O₂ consumption supported by complex I-mediated substrates, whereas mPer2-M did not, despite no difference in complex I content. Moreover, cardiac mitochondria from WT mice exhibited a very robust increase in ADP-stimulated O₂ consumption in response to exogenously added cytochrome c, along with a high rate of reactive oxygen species production, none of which was exhibited by cardiac mitochondria from mPer2-M following I/R. Taken together, these findings suggest that mPer2 deletion preserves mitochondrial membrane structure and functional integrity in heart following I/R injury, the consequence of which is preservation of myocardial viability. Understanding the mechanisms connecting cardiac events, mitochondrial function, and mPer2 could lead to preventative and therapeutic strategies for at risk populations.

Are Post-Operative Arrhythmias in Patients With Metabolic Syndrome a Sign of Dysfunctional Mitochondria?∗

Post-operative atrial fibrillation (POAF) is the most common cause of morbidity after coronary surgery, with an incidence that increases with patient age [(1,2)][1]. The burdens of POAF include increased hospitalization stays/costs and poorer patient prognosis. Patients experiencing POAF have an