HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury.

RATIONALE: Recent evidence indicates that histone deacetylase enzymes (HDACs) contribute to ischemia reperfusion (I/R) injury, and pan-HDAC inhibitors have been shown to be cardioprotective when administered either before an ischemic insult or during reperfusion. We have shown previously that selective inhibition of class I HDACs provides superior cardioprotection when compared to pan-HDAC inhibition in a pretreatment model, but selective class I HDAC inhibition has not been tested during reperfusion, and specific targets of class I HDACs in I/R injury have not been identified. OBJECTIVE: We hypothesized that selective inhibition of class I HDACs with the drug MS-275 (entinostat) during reperfusion would improve recovery from I/R injury in the first hour of reperfusion. METHODS AND RESULTS: Hearts from male Sprague-Dawley rats were subjected to ex vivo I/R injury±MS-275 class I HDAC inhibition during reperfusion alone. MS-275 significantly attenuated I/R injury, as indicated by improved LV function and tissue viability at the end of reperfusion. Unexpectedly, we observed that HDAC1 is present in the mitochondria of cardiac myocytes, but not fibroblasts or endothelial cells. We then designed mitochondria-restricted and mitochondria-excluded HDAC inhibitors, and tested both in our ex vivo I/R model. The selective inhibition of mitochondrial HDAC1 attenuated I/R injury to the same extent as MS-275, whereas the mitochondrial-excluded inhibitor did not. Further assays demonstrated that these effects are attributable to a decrease in SDHA activity and subsequent metabolic ROS production in reperfusion. CONCLUSIONS: We demonstrate for the first time that HDAC1 is present within the mitochondria of cardiac myocytes, and mitochondrial HDAC1 contributes significantly to I/R injury within the first hour of reperfusion.

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

Histone Deacetylases Exert Class-Specific Roles in Conditioning the Brain and Heart Against Acute Ischemic Injury.

Ischemia-reperfusion (IR) injury comprises a significant portion of morbidity and mortality from heart and brain diseases worldwide. This enduring clinical problem has inspired myriad reports in the scientific literature of experimental interventions seeking to elucidate the pathology of IR injury. Elective cardiac surgery presents perhaps the most viable scenario for protecting the heart and brain from IR injury due to the opportunity to condition the organs prior to insult. The physiological parameters for the preconditioning of vital organs prior to insult through mechanical and pharmacological maneuvers have been heavily examined. These investigations have revealed new insights into how preconditioning alters cellular responses to IR injury. However, the promise of preconditioning remains unfulfilled at the clinical level, and research seeking to implicate cell signals essential to this protection continues. Recent discoveries in molecular biology have revealed that gene expression can be controlled through posttranslational modifications, without altering the chemical structure of the genetic code. In this scenario, gene expression is repressed by enzymes that cause chromatin compaction through catalytic removal of acetyl moieties from lysine residues on histones. These enzymes, called histone deacetylases (HDACs), can be inhibited pharmacologically, leading to the de-repression of protective genes. The discovery that HDACs can also alter the function of non-histone proteins through posttranslational deacetylation has expanded the potential impact of HDAC inhibitors for the treatment of human disease. HDAC inhibitors have been applied in a very small number of experimental models of IR. However, the scientific literature contains an increasing number of reports demonstrating that HDACs converge on preconditioning signals in the cell. This review will describe the influence of HDACs on major preconditioning signaling pathways in the heart and brain.

Selective inhibition of class I but not class IIb histone deacetylases exerts cardiac protection from ischemia reperfusion.

While inhibition of class I/IIb histone deacetylases (HDACs) protects the mammalian heart from ischemia reperfusion (IR) injury, class selective effects remain unexamined. We hypothesized that selective inhibition of class I HDACs would preserve left ventricular contractile function following IR in isolated hearts. Male Sprague Dawley rats (n=6 per group) were injected with vehicle (dimethylsulfoxide, 0.63mg/kg), the class I/IIb HDAC inhibitor trichostatin A (1mg/kg), the class I HDAC inhibitor entinostat (MS-275, 10mg/kg), or the HDAC6 (class IIb) inhibitor tubastatin A (10mg/kg). After 24h, hearts were isolated and perfused in Langendorff mode for 30min (Sham) or subjected to 30min global ischemia and 120min global reperfusion (IR). A saline filled balloon attached to a pressure transducer was placed in the LV to monitor contractile function. After perfusion, LV tissue was collected for measurements of antioxidant protein levels and infarct area. At the conclusion of IR, MS-275 pretreatment was associated with significant preservation of developed pressure, rate of pressure generation, rate of pressure relaxation and rate pressure product, as compared to vehicle treated hearts. There was significant reduction of infarct area with MS-275 pretreatment. Contractile function was not significantly restored in hearts treated with trichostatin A or tubastatin A. Mitochondrial superoxide dismutase (SOD2) and catalase protein and mRNA in hearts from animals pretreated with MS-275 were increased following IR, as compared to Sham. This was associated with a dramatic enrichment of nuclear FOXO3a transcription factor, which mediates the expression of SOD2 and catalase. Tubastatin A treatment was associated with significantly decreased catalase levels after IR. Class I HDAC inhibition elicits protection of contractile function following IR, which is associated with increased expression of endogenous antioxidant enzymes. Class I/IIb HDAC inhibition with trichostatin A or selective inhibition of HDAC6 with tubastatin A was not protective. This study highlights the need for the development of new strategies that target specific HDAC isoforms in cardiac ischemia reperfusion.

Sivelestat attenuates myocardial reperfusion injury during brief low flow postischemic infusion.

The neutrophil elastase inhibitor sivelestat (ONO-5046) possesses unknown mechanisms of cardioprotection when infused following global ischemia, even in the absence of neutrophils. Since myocardial ischemia-reperfusion injury is strongly associated with endothelial dysfunction and reactive oxygen species (ROS) generation during reperfusion, we have tested the hypothesis that infusion of sivelestat during postischemic low flow would preserve endothelial and contractile function and reduce infarct size through an ROS-mediated mechanism. Isolated male rat hearts, subjected to global ischemia of 25 minutes, were reperfused with low flow with or without sivelestat followed by a full flow reperfusion. Hearts treated with sivelestat showed a significant improvement of LV contractile function and a reduction in infarct size. Infusion of L-NAME (nonspecific blocker of endothelial nitric oxide synthase (eNOS)) along with sivelestat during reperfusion reversed the preservation of contractile function and infarct size. In vitro EPR spin trapping experiments showed that sivelestat treatment decreased superoxide adduct formation in bovine aortic endothelial cells (BAECs) subjected to hypoxia-reoxygenation. Similarly, dihydroethidine (DHE) staining showed decreased superoxide production in LV sections from sivelestat-treated hearts. Taken together, these results indicate that sivelestat infusion during postischemic low flow reduces infarct size and preserves vasoreactivity in association with decreased ROS formation and the preservation of nitric oxide.

Hyperoxemic reperfusion after prolonged cardiac arrest in a rat cardiopulmonary bypass resuscitation model.

BACKGROUND: The effect of hyperoxygenation at reperfusion, particularly in the setting of cardiac arrest, remains unclear. This issue was studied in a prolonged cardiac arrest model consisting of 25 min cardiac arrest in a rat resuscitated with cardiopulmonary bypass (CPB). The objective of this study was to determine the effect of hyperoxygenation following prolonged cardiac arrest resuscitation on mitochondrial and cardiac function. METHODS: Male Sprague-Dawley rats (400-450 g) were anesthetized with ketamine and xylazine and instrumented for closed chest cardiopulmonary bypass (CPB). Following a 25-min KCl-induced cardiac arrest, the animals were resuscitated by CPB with 100% oxygen. Three minutes after successful return of spontaneous circulation (ROSC), the animals received either normoxemic reperfusion (CPB with 40-50% oxygen) or hyperoxemic reperfusion (CPB with 100% oxygen) for 1 h. Post-resuscitation hemodynamics, cardiac function, mitochondrial function and immunostaining of 3-nitrotyrosine were compared between the two different treatment groups. RESULTS: At 1 h after ROSC, the hyperoxemic reperfusion group had a significant higher mean arterial pressure, less metabolic acidosis and better diastolic function than the normoxemic reperfusion group. Cardiac mitochondria from the hyperoxemic reperfusion group had a higher respiratory control ratio (RCR) and cardiac tissue showed less nitroxidative stress compared to the normoxemic reperfusion group. CONCLUSIONS: One hour of hyperoxemic reperfusion after 25 min of cardiac arrest in an in vivo CPB model resulted in significant short-term improvement in myocardial and mitochondrial function compared with 1h of normoxemic reperfusion. This myocardial response may differ from previously reported post-arrest hyperoxia mediated effects following shorter arrest times.

Measurement of hydrogen peroxide and oxidant stress in a recirculating whole blood-perfused rat heart model.

AIM OF STUDY: Isolated hearts used in the study of ischemia-reperfusion induced myocardial reactive oxygen species (ROS) have typically been perfused with crystalloid buffer. Limitations of crystalloid buffer which may exaggerate the production of ROS, include a requirement for higher oxygen tension and the absence of the intrinsic erythrocyte antioxidant defenses. Using a novel recirculating blood-perfused rat heart model, we measured H(2)O(2) concentration in the blood (as an indicator of ROS formation) and tissue glutathione concentration (an overall measure of oxidant stress) following ischemia and reperfusion. METHODS: Autologous blood was obtained and the heart isolated from pentobarbital-anesthetized male Sprague-Dawley rats and placed on a recirculating perfusion circuit with an in-line peristaltic pump and oxygenator. Blood temperature was maintained at 37°C. Hearts underwent normal perfusion for 120min (Sham Group, n=7) or 35min of normal perfusion, 25min of global ischemia, followed by 60min of reperfusion with baseline coronary blood flow levels (IR group, n=6). Oxygen delivery was compared with a group of buffer-perfused hearts perfused at 85mmHg. RESULTS: LV function in the sham group remained stable for 2h under normal physiologic oxygen conditions. The oxygen tension and coronary flow were significantly decreased but the myocardial oxygen delivery was significantly increased with blood perfusion compared with buffer perfusion. In the blood IR group, a significant increase in H(2)O(2) was seen early in reperfusion and a reduction in tissue GSH was noted at the end of reperfusion. CONCLUSION: This model offers significant physiologic advantages in the study of ischemia and reperfusion, particularly in terms of oxygen delivery, compared with the more commonly used acellular buffer-perfused isolated heart systems.

Post-cardiac arrest hyperoxia and mitochondrial function.

INTRODUCTION: Rapid post-ischemic re-oxygenation is necessary to minimize ischemic injury, but itself can induce further reperfusion injury through the induction of reactive oxygen species. Utilization of oxygen within the cell primarily occurs in the mitochondria. The objective of this study was to determine heart mitochondrial function after 1 h of controlled arterial oxygenation following cardiac arrest and restoration of spontaneous circulation (ROSC). We hypothesized that arterial hyper-oxygenation following ROSC would result in greater impairment of heart mitochondrial function. METHODS: KCl cardiac arrest was induced in anesthetized rats. Following 6.5 min of cardiac arrest, animals were resuscitated with standard thumper CPR, ventilation and epinephrine. Following ROSC, all animals were ventilated for 60 min with either 100% O(2) or 40% O(2) titrated to achieve normoxia utilizing pulse oximetry. At the end of 1 h, heart mitochondria were isolated and mitochondrial respiratory function was measured. RESULTS: Post-ROSC arterial PaO2 was 280 ± 40 in the 100% O2 group and 105 ± 10 in the 40% O2 group. One hour after ROSC, heart mitochondrial state 3 respirations and respiration control ratio (state 3/4 respiration) were significantly reduced from baseline in animals ventilated with 100% O(2), but not with 40% O(2). CONCLUSION: Post-ROSC arterial hyperoxia after a short cardiac arrest exacerbates impaired mitochondrial function. The overall clinical significance of these findings is unclear and requires additional work to better understand the role of post-arrest hyperoxia on cardiac and mitochondrial function.

Preservation of mitochondrial function with cardiopulmonary resuscitation in prolonged cardiac arrest in rats.

During cardiac arrest (CA), myocardial perfusion is solely dependent on cardiopulmonary resuscitation (CPR) although closed-chest compressions only provide about 10-20% of normal myocardial perfusion. The study was conducted in a whole animal CPR model to determine whether CPR-generated oxygen delivery preserves or worsens mitochondrial function. Male Sprague-Dawley rats (400-450 g) were randomly divided into four groups: (1) BL (instrumentation only, no cardiac arrest), (2) CA(15) (15 min cardiac arrest without CPR), (3) CA(25) (25 min cardiac arrest without CPR) and (4) CPR (15 min cardiac arrest, followed by 10 min CPR). The differences between groups were evaluated by measuring mitochondrial respiration, electron transport chain (ETC) complex activities and mitochondrial ultrastructure by transmission electron microscopy (TEM). The CA(25) group had the greatest impairment of mitochondrial respiration and ETC complex activities (I-III). In contrast, the CPR group was not different from the CA(15) group regarding all measures of mitochondrial function. Complex I was more susceptible to ischemic injury than the other complexes and was the major determinant of mitochondrial dysfunction. Observations of mitochondrial ultrastructure by TEM were compatible with the biochemical results. The findings suggest that, despite low blood flow and oxygen delivery, CPR is able to preserve heart mitochondrial function and viability during ongoing global ischemia. Preservation of complex I activity and mitochondrial function during cardiac arrest may be an important mechanism underlying the beneficial effects of CPR which have been shown in clinical studies.

Oxygen requirement during cardiopulmonary resuscitation (CPR) to effect return of spontaneous circulation.

BACKGROUND: Recent scientific evidence has demonstrated the importance of good quality chest compressions without interruption to improve cardiac arrest resuscitation rates, and suggested that a de-emphasis on minute ventilation is needed. However, independent of ventilation, the role of oxygen and the optimal oxygen concentration during CPR is not known. Previous studies have shown that ventilation with high oxygen concentration after CPR is associated with worse neurologic outcome. We tested the hypothesis that initial ventilation during CPR without oxygen improves resuscitation success. METHODS: Sprague-Dawley rats were anesthetized with ketamine/xylazine (IP), intubated and ventilated with room air. A KCl bolus (0.04 mg/g) was given (IV) to induce asystolic cardiac arrest and ventilation was stopped. At 6 min, CPR was started with an automated chest compressor at a rate of 200-240/min and epinephrine (0.01 mg/kg) was given 1 min later. During CPR, the ventilation rate was 50% of baseline with one of three oxygen concentrations: (1) 0% O2 (100% N2), (2) 21% O2, or (3) 100% O2. The prescribed oxygen concentration was continued for 2 min after return of spontaneous circulation (ROSC) and then all animals were switched to 100% oxygen for 1h prior to extubation. Blood gases were measured at baseline, 2 min and 1h after ROSC. Group comparisons were done using Fisher's exact test and ANOVA. RESULTS: ROSC was achieved in 1/10 (0% O2), 9/11 (21% O2) and 10/12 (100% O2, p<0.001). ROSC times after starting CPR were 80s in the 0% O2, 115+/-87 s in the 21% O2 group and 95+/-33 s in the 100% O2 group (mean+/-SD, p=0.5). Aortic end-diastolic pressure before ROSC was not different among groups. 100% oxygen ventilation in the first 2 min resulted in higher PaO2 at ROSC 2 min (109+/-44 mm Hg vs. 33+/-8 mm Hg, p<0.001). Survival to 72 h was 0/1 (0% O2), 7/9 (21% O2) and 8/10 (100% O2) with a low neurologic deficit score in both O2 groups (NDS range 5-25). CONCLUSIONS: In a mild cardiac arrest model with generally good neurologic recovery, initial CPR ventilation with no O2 did not allow for ROSC. In contrast, CPR coupled with room air or higher oxygen levels result in a high rate of ROSC with good neurologic recovery. During CPR, the level of oxygenation must be considered, which if too low may preclude initial ROSC.

Cardiovascular response to epinephrine varies with increasing duration of cardiac arrest.

OBJECTIVE: Epinephrine (adrenaline) is widely used as a primary adjuvant for improving perfusion pressure and resuscitation rates during cardiopulmonary resuscitation (CPR). Epinephrine is also associated with significant myocardial dysfunction in the post-resuscitation period. We tested the hypothesis that the cardiac effects of epinephrine vary according to the duration of cardiac arrest. METHODS AND MATERIALS: Cardiac arrest (CA) was induced in Sprague-Dawley rats with an IV bolus of KCl (40 microg/g). Three series of experiments were performed with CPR begun after 2, 4, or 6 min of cardiac arrest. Epinephrine (0.01 mg/kg) IV or placebo was given immediately in the 2 and 4 min CA groups. In the 6 min group, CPR was started after 6 min CA and epinephrine was given at 15 min if no return of spontaneous circulation (ROSC) occurred. Time to ROSC was recorded in all groups. Cardiac function was determined with trans-thoracic echocardiography at baseline, 5, 30 and 60 min after ROSC. RESULTS: After 2 min CA, 8/8 (100%) placebo animals and 8/8 (100%) epinephrine animals attained ROSC. Cardiac index was significantly increased during the first 60 min in the epinephrine group compared with the placebo group (p<0.01). After 4 min of cardiac arrest, 14/29 (48%) placebo animals and 14/16 (88%) epinephrine animals attained ROSC (p<0.01). Cardiac index after ROSC returned to baseline in both groups, although tended to be lower in the epinephrine group. After 6 min CA, 10/31 (32%) animals attained ROSC without epinephrine and 17/21 (81%) animals with epinephrine (p<0.01). Post-ROSC depression of cardiac index was greatest in the epinephrine group (p<0.05). CONCLUSIONS: As the duration of cardiac arrest increases, a paradoxical myocardial epinephrine response develops, in which epinephrine becomes increasingly more important to attain ROSC, but is increasingly associated with post-ROSC myocardial depression.

Inhibition of peroxynitrite precursors, NO and O2, at the onset of reperfusion improves myocardial recovery.

AIM OF STUDY: Previous reports note an increase in both reactive oxygen species (ROS) and nitric oxide (*NO) at the onset of myocardial reperfusion. We tested the hypothesis that inhibition of *NO or ROS production at the time of reperfusion improves recovery of post-ischemic myocardial function. METHODS AND MATERIALS: Isolated rat hearts were perfused with temperature controlled (37.4 degrees C) modified Krebs Henseleit buffer solution at 85 mm Hg. Following 20 min of global ischemia, hearts were reperfused for the first 10 min with: (1) standard buffer (control), (2) buffer with a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), (3) buffer with superoxide dismutase (SOD) or (4) buffer with N-morpholinosydnonimine hydrochloride (SIN-1), a peroxynitrite generator. Tissue O(2) and *NO were continuously measured with thin electrochemical probes embedded in the wall of the LV. ROS was measured with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (40 mM). LV contractile function was continuously monitored. RESULTS: Recovery of LV contractile function was significantly improved in hearts initially reperfused with L-NAME and SOD and significantly depressed in hearts reperfused with SIN-1 compared with control (p<0.01, n=5-8 per group). DMPO-adduct during reperfusion (measure of ROS) was significantly decreased with SOD (p<0.001 versus L-NAME and Control, n=4 per group) and unchanged with L-NAME and SIN-1 compared with Control. With L-NAME, tissue *NO and PO(2) were significantly decreased, independent of coronary flow, during reperfusion compared with control and SIN-1. CONCLUSIONS: Inhibition of O(2)*(-) or *NO at the time of reperfusion improves early reperfusion LV function and alters tissue oxygen tension. In contrast to pre-ischemic treatments, intervention to reduce peroxynitrite generation at the onset of reperfusion can effectively improve post-ischemic myocardial recovery.

O2 delivery and redox state are determinants of compartment-specific reactive O2 species in myocardial reperfusion.

Reperfusion of the ischemic myocardium leads to a burst of reactive O(2) species (ROS), which is a primary determinant of postischemic myocardial dysfunction. We tested the hypothesis that early O(2) delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer-perfused rat hearts were loaded with the fluorophores dihydrofluorescein or Amplex red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular wall. Hearts were made globally ischemic for 20 min and then reperfused with either 95% or 20% O(2)-saturated perfusate. The same protocol was repeated in hearts loaded with dihydrofluorescein and perfused with either 20 or 5 mM glucose-buffered solution to determine relative changes in NADH and FAD. Myocardial O(2) delivery during the first 5 min of reperfusion was 84.7 +/- 4.2 ml O(2)/min with 20% O(2)-saturated buffer and 354.4 +/- 22.8 ml O(2)/min with 95% O(2) (n = 8/group, P < 0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% O(2) compared with 20% O(2). However, the resorufin signal (extracellular ROS) was significantly increased with 20% O(2) compared with 95% O(2) during reperfusion. Perfusion of hearts with 20 mM glucose reduced the (.)NADH during ischemia (P < 0.001) and the (.)ROS at reperfusion (P < 0.001) compared with 5.5 mM-perfused glucose hearts. In conclusion, initial O(2) delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high O(2) delivery promotes intracellular ROS and low O(2) delivery promotes extracellular ROS. The redox state that develops during ischemia appears to be an important precursor for reperfusion ROS production.