End Reperfusion Research Articles

Malonate protection against acute infarction in isolated mouse hearts is lost in the presence of insulin

Abstract Background & Objectives Malonate, an inhibitor of mitochondrial succinate dehydrogenase (SDH), is a promising novel protectant against acute ischaemia-reperfusion injury (IRI) through reduction of ischaemic succinate accumulation and ROS production during early reperfusion. Malonate is still protective when given only at reperfusion, making it an ideal agent for treating AMI patients. However, numerous studies demonstrated that the metabolic milieu could affect the potency of cardioprotective treatments. Therefore, in this study, we explored whether and how the severity of cardiac I/R injury and cardioprotection by malonate is affected by the metabolic milieu. We hypothesized that protection by malonate is affected by metabolic milie. Methods Isolated Langendorff-perfused hearts (n=8 to 19 per group) of C57Bl/6N mice were perfused with perfusate to which substrates and hormones were added in a step-wise manner: 1) glucose (G), 2) glucose + glutamine (GG), 3) glucose + glutamine + palmitate (GGP), or 4) glucose + glutamine + palmitate + insulin (GGPI), and subjected to 30 min ischaemia (I) and 90 min reperfusion (R). Heart were treated with malonate (5mM) or vehicle (saline) during the first 5 min of reperfusion only. Cardiac function of the left ventricle was monitored by a balloon in the left ventricle. Coronary effluent was collected at 7 time points of reperfusion to detect the activity of lactate dehydrogenase (LDH) as an indicator of cell death. At the end reperfusion, hearts were used for infarct size determination through TTC staining. Data are presented as mean ± SD. Results Going from glucose-only perfusate to a perfusate containing all examined substrates and hormones, the severity of cardiac I/R injury increased: infarct size (%) went from 31.9 ± 10.9% to 37.7 ± 10.6% to 52.3 ± 14.2%, and finally to 68 ± 9.9 %. Similar trends for increased IRI were observed for LDH, End-Diastolic Pressure (EDP) and recovery of the Rate-Pressure Product (RPP). When compared with vehicle control, malonate significantly decreased IS% (control vs malonate: 31.9 ± 10.9% vs. 23.3 ± 4.8%, 37.7 ± 10.6% vs. 20.9 ± 8.5%, 68 ± 9.9% vs. 40.2 ± 9.2 %), and increased the RRP recovery (64.1 ± 16.6 %vs. 83.3 ± 17.9 %, 55.6 ± 11% vs. 72.3 ± 4%, 39.3 ± 15.6% vs. 55.9 ± 8.3%) in the G, GG and GGP group. The EDP was significantly decreased by malonate in the G and GG group (32.8 ± 14.3 mmHg vs. 20.9 ± 9.8 mmHg, 35.1 ± 8.7 mmHg vs. 22.2 ± 8.6 mmHg ). LDH was only significantly decreased by malonate in the GG group (35.2 ± 12.9 vs. 17.1 ± 7.7 U / GWW). Most importantly, adding insulin to the perfusate nullified protection by malonate for all four outcomes. Conclusions The severity of cardiac I/R injury is lowest with glucose-only perfusate, and increased with increased complexity of the metabolic milieu. Although malonate is a strong protectant against cardiac IRI for most metabolic milieus, in the isolated heart, protection is lost in the presence of insulin.

Abstract 284: Loss of Micu3 Confers Cardioprotection Against Cardiac Injury

Background: Mitochondrial calcium flux and signaling is integral to cardiac function and contraction. However, during pathologic conditions such as ischemic/reperfusion injury, mitochondrial calcium overload can induce the opening of the mitochondrial permeability transitioning pore (PTP), resulting in the collapse of mitochondrial membrane potential, ATP depletion, and generation of reactive oxygen species, all together leading to cell death. Hence, modulation of mitochondrial calcium and inhibition of the PTP is a promising target for cardioprotection and prevention of cardiomyocyte death. The mitochondrial calcium uniporter (MCU) complex mediates rapid mitochondrial calcium uptake. MICU3 is a regulator of the MCU complex and has been shown to be a highly potent stimulator of MCU-dependent calcium uptake in neuronal cells. We found that MICU3 is expressed in hearts and we therefore investigated the role of MICU3 in the heart. We examined the role of MICU3 in the development of hypertrophy and in a separate study we examined the response to ischemic-reperfusion (I/R) injury. Given its role in regulating mitochondrial calcium uptake, we hypothesized that loss of MICU3 confers protection against cardiac injury. Methods: Mice with global deletion of Micu3 (Micu3 -/- ) were created utilizing CRISPR-Cas9 technology. Adult knockout and littermate wild type mice were treated with Isoproterenol (15mg/kg/day) for two weeks to induce hypertrophy. Echocardiograms were performed at baseline and after treatment to assess changes in left ventricular size and function. I/R injury was studied using Langendorff ex vivo perfused heart system, exposing knockout and wild type hearts to 20 minutes of ischemia and 90 minutes of reperfusion. Hemodynamic data and infarct size were collected and compared. Student t-test and 2-way ANOVA were used for statistical analysis. Result: Micu3 -/- mice had normal cardiac function at baseline. There was no sex difference in cardiac function. Micu3 -/- mice continued to show normal function after 2 weeks of treatment with Isoproterenol, whereas wild type mice exhibited depressed function (median FS 35% vs. 24% p = 0.0001, EF 64% vs. 50% p = 0.0001). Wild type mice developed LV dilation from baseline (median 4.15mm vs. 4.57mm, p = 0.0014), whereas LV dimension remained stable in Micu3 -/- mice (median 4.12mm vs. 4.18mm, p= 0.9892). Micu3 - /- mice were also protected from I/R injury. Compared to wild types, Micu3 -/- hearts demonstrated less contractile dysfunction at end reperfusion (median rate pressure product 62% vs. 41%, p = 0.002), and significantly smaller infarct size (median 33% vs. 53%, p = 0.0001). Conclusion: Loss of MICU3 confers cardioprotection against ischemic reperfusion injury and Isoproterenol induced cardiac dysfunction.

Mitochondrial uncoupling does not decrease reactive oxygen species production after ischemia-reperfusion.

Cardiac ischemia-reperfusion (IR) leads to myocardial dysfunction by increasing production of reactive oxygen species (ROS). Mitochondrial H(+) leak decreases ROS formation; it has been postulated that increasing H(+) leak may be a mechanism of decreasing ROS production after IR. Ischemic preconditioning (IPC) decreases ROS formation after IR, but the mechanism is unknown. We hypothesize that pharmacologically increasing mitochondrial H(+) leak would decrease ROS production after IR. We further hypothesize that IPC would be associated with an increase in the rate of H(+) leak. Isolated male Sprague-Dawley rat hearts were subjected to either control or IPC. Mitochondria were isolated at end equilibration, end ischemia, and end reperfusion. Mitochondrial membrane potential (mΔΨ) was measured using a tetraphenylphosphonium electrode. Mitochondrial uncoupling was achieved by adding increasing concentrations of FCCP. Mitochondrial ROS production was measured by fluorometry using Amplex-Red. Pyridine dinucleotide levels were measured using HPLC. Before IR, increasing H(+) leak decreased mitochondrial ROS production. After IR, ROS production was not affected by increasing H(+) leak. H(+) leak increased at end ischemia in control mitochondria. IPC mitochondria showed no change in the rate of H(+) leak throughout IR. NADPH levels decreased after IR in both IPC and control mitochondria while NADH increased. Pharmacologically, increasing H(+) leak is not a method of decreasing ROS production after IR. Replenishing the NADPH pool may be a means of scavenging the excess ROS thereby attenuating oxidative damage after IR.

Abstract 19807: Increased Triacylglycerol Synthesis in the Heart Improves Functional Recovery After Ischemia

Accumulation of cardiac lipids has been associated with cardiac dysfunction in diabetes and obesity while increased muscle lipid storage in athletes is associated with improved function. We have previously shown that cardiac specific overexpression of diacylglycerol acyltransferase 1 (MHC-DGAT1), the rate limiting enzyme for triacylglycerol (TAG) synthesis, enhances TAG synthesis, reduces ceramide formation, and rescues lipotoxic cardiomyopathy. Using MHC-DGAT1 mice, we tested whether increased TAG synthesis and storage affected cardiac responses to ischemia-reperfusion. A doubling of cardiac TAG content in MHC-DGAT1 (P<0.05; n=4) was not associated with in-vivo cardiac dysfunction or heart size. With dynamic 13C NMR spectroscopy we found a 70% increase of incorporation of 13C labeled fatty acids (FA) into the cardiac TAG pool in MHC-DGAT1 hearts. In isolated Langendorff perfused hearts, rate pressure product (RPP), was similar in MHC-DGAT1 and control (CON) hearts at baseline (BL). When subjected to 25min of no-flow ischemia (ISC), end diastolic pressure rose equivalently in both groups. However, recovery of RPP during reperfusion (REP) was significantly improved in MHC-DGAT1 compared to CON (51±6% vs 28±6%; n=5-6). At the end of REP, 13C NMR spectroscopy of heart extracts suggested decreased exogenous FA oxidation with no change in glucose oxidation in MHC-DGAT1 hearts. Cardiac TAGs, measured in freeze-clamped hearts, remained unchanged in CON during BL, at end ISC, and at end REP (n=3 per time point). Interestingly, despite ~2-fold increase in TAG in MHC-DGAT1 at BL, TAG content depleted to levels similar to CON at end ISC and REP, inferring accelerated TAG lipolysis in MHC-DGAT1 during the ischemic period. Non-esterified fatty acid (NEFA) concentration was not different between the groups at any time point. In conclusion, these results suggest that ischemia-induced activation of lipolysis in MHC-DGAT1 hearts improves functional recovery, in part, by increasing the oxidation of endogenous lipids during reperfusion. These data also suggest a potential secondary role of endogenous lipids to act as signaling molecules to modulate cardiac function.

Abstract 107: The Innate Immune NALP3 Receptor Mediates Cardiac Ischemic Preconditioning in the Isolated Heart

Objective: Recent studies indicate that the innate immune system is not only activated by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As activation triggers (cellular K+ extrusion, ROS production) for the innate immune NALP3 inflammasome protein complex overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NALP3 inflammasome is involved in IPC and acute I/R injury of the heart. Methods & Results: Baseline performance and I/R injury was investigated in isolated, Langendorff-perfused hearts from wild-type, ASC-/- and NALP3-/- mice. Baseline cardiac performance was unaffected by genetic deletion of NALP3 inflammasome components. Deletion of the ASC and NALP3 component exacerbated IR injury on mechanical parameters of the heart. However, deficiency in ASC or NALP3 was without effect on cell death (LDH release during reperfusion). In a separate series we subjected these same hearts to IPC, consisting of 3 x 5 min I/R preceding the 35 min I. IPC protected WT and ASC-/- hearts against IR injury (improved function and less cell death). However, no IPC effects on function or cell death were observed in NALP3-/- hearts. NALP3-/- hearts had significantly decreased cardiac IL-6 levels with a trend towards lower Il-1β levels at end reperfusion, suggesting abrogating of IPC through diminished IL-6 and/or Il-1β signalling. However, subsequent experiments showed that inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) was without effect on IPC in WT hearts. In contrast, neutralising IL-6 using an antibody against IL-6 in WT hearts abrogated IPC in NALP3-/- hearts. Conclusions: The data suggest that the innate immune NALP3 protein, in an NALP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6 dependent mechanism

Deletion of the Innate Immune NLRP3 Receptor Abolishes Cardiac Ischemic Preconditioning and Is Associated with Decreased Il-6/STAT3 Signaling

ObjectiveRecent studies indicate that the innate immune system is not only triggered by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As triggers for the innate immune NLRP3 inflammasome protein complex appear to overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NLRP3 inflammasome is involved in IPC and acute I/R injury of the heart.Principal FindingsBaseline cardiac performance and acute I/R injury were investigated in isolated, Langendorff-perfused hearts from wild-type (WT), ASC−/− and NLRP3−/− mice. Deletion of NLRP3 inflammasome components ASC−/− or NLRP3−/− did not affect baseline performance. The deletions exacerbated I/R-induced mechanical dysfunction, but were without effect on I/R-induced cell death. When subjected to IPC, WT and ASC−/− hearts were protected against I/R injury (improved function and less cell death). However, IPC did not protect NLRP3−/− hearts against I/R injury. NLRP3−/− hearts had significantly decreased cardiac IL-6 levels with a trend towards lower IL-1β levels at end reperfusion, suggesting abrogation of IPC through diminished IL-6 and/or IL-1β signaling. Subsequent experiments showed that neutralising IL-6 using an antibody against IL-6 abrogated IPC in WT hearts. However, inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) did not abrogate IPC in WT hearts. Analysis of survival kinases after IPC demonstrated decreased STAT3 expression in NLRP3−/− hearts when compared to WT hearts.ConclusionsThe data suggest that the innate immune NLRP3 protein, in an NLRP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6/STAT3 dependent mechanism.

Ischemic preconditioning preserves mitochondrial membrane potential and limits reactive oxygen species production

BackgroundMitochondrial superoxide radical (O2•¯) production increases after cardiac ischemia/reperfusion (IR). Ischemic preconditioning (IPC) preserves mitochondrial function and attenuates O2•¯ production, but the mechanism is unknown. Mitochondrial membrane potential (mΔΨ) is known to affect O2•¯ production; mitochondrial depolarization decreases O2•¯ formation. We examined the relationship between O2•¯ production and mΔΨ during IR and IPC. Materials/methodsRat hearts were subjected to Control or IPC. Mitochondria were isolated at end equilibration (End EQ), end ischemia (End I), and end reperfusion (End RP). mΔΨ was measured using a tetraphenylphosphonium electrode. Mitochondrial O2•¯ production was measured by electron paramagnetic resonance using DMPO spin trap. Cytochrome c levels were measured using high-pressure liquid chromatography. ResultsIPC preserved mΔΨ at End I (−156 ± 5 versus −131 ± 6 mV, P < 0.001) and End RP (−168 ± 2 versus −155 ± 2 mV, P < 0.05). At End RP, IPC attenuated O2•¯ production (2527 ± 221 versus 3523 ± 250 AU/mg protein, P < 0.05). IPC preserved cytochrome c levels (351 ± 14 versus 269 ± 16 picomoles/mg protein, P < 0.05) at End RP, and decreased mitochondrial cristae disruption (10% ± 4% versus 33% ± 7%, P < 0.05) and amorphous density formation (18% ± 4% versus 28% ± 1%, P < 0.05). ConclusionWe conclude that IPC preserves mΔΨ, possibly by limiting disruption of mitochondrial inner membrane. IPC also decreases mitochondrial O2•¯ production and preserves mitochondrial ultrastructure after IR. While it was previously held that slight decreases in mΔΨ decrease O2•¯ production, our results indicate that preservation of mΔΨ is associated with decreased O2•¯ and preservation of cardiac function in IPC. These findings indicate that the mechanism of IPC may not involve mΔΨ depolarization, but rather preservation of mitochondrial electrochemical potential.

Increased Mitochondrial Sensitivity To Proton Leak Is Responsible For Decreased Reactive Oxygen Species Production In Ischemic Preconditioning

Introduction: Mitochondrial reactive oxygen species (mROS) cause significant damage to myocardium after an episode of ischemia-reperfusion (IR). Ischemic preconditioning (IPC) decreases mROS formation and protects myocardium from IR. The mechanism of decreased mROS in IPC is unknown. Proton leak (H+ leak) dissipates mitochondria membrane potential (mΔΨ) and may regulate mROS production by changing the redox status of mitochondria. We hypothesize that increased H+ leak is responsible for decreased mROS production during IPC. Methods: Rat hearts were assigned to CONTROL (30 min of equilibration (EQ), 30 min ischemia (I), and 30 min of reperfusion (RP)) or IPC (10 min EQ, two 5 min episodes of IR, 30 min I, and 30 min RP). Rate pressure product (RPP) was measured throughout. Mitochondria were isolated at end equilibration (end EQ), end ischemia (end I), and end reperfusion (end RP). Respiration was measured by polarography, and mΔΨ with a TPP electrode. H+ leak is the respiratory rate required to maintain mΔΨ of -130mV in the presence of oligomycin-A, and was titrated with FCCP (0-160nM). Complex III mROS production was measured by fluorometry using Amplex-Red at end EQ and by electron paramagnetic resonance at end I and end RP. Results: Induction of IPC increased mROS production at end EQ (59±2 vs. 43±2 nmoles H2O2/mg protein/min, p<0.05, Fig.1) but did not change H+ leak (37±9 vs. 30±10 nmoles O/mg protein/min) when compared to CONTROL. IPC attenuated the increase in H+ leak at end I seen in CONTROL (44±11 vs. 140±11 nmoles O/mg protein/min, p<0.001). At end RP, IPC decreased mROS production (2115±146 vs. 3277±333 arbitrary units/mg protein, p<0.05). There was no difference in H+ leak between IPC and CONTROL at end RP (34±9 vs. 45±9 nmoles O/mg protein/min). IPC mitochondria responded to depolarization with greater reduction in mROS production than CONTROL (Fig.1). Further depolarization led to a trend of increased mROS in both CONTROL and IPC. The inflection point occurred at a higher mΔΨ in IPC mitochondria (-160 vs. -138 mV; Fig. 1). Conclusions: Increased rate of H+ leak is not the mechanism of decreased mROS after IR. While IPC did not lead to increased H+ leak, the induction of IPC increases the sensitivity of mitochondria to changes in mΔΨ leading to greater reduction in mROS per unit of change in mΔΨ. This increased sensitivity explains the greater reduction in mROS at end RP despite a lower H+ leak. Reducing mROS formation after ischemia-reperfusion is important to improving outcomes in patients with acute coronary syndrome.

Ischemic Preconditioning Decreases Mitochondrial Proton Leak and Reactive Oxygen Species Production in the Postischemic Heart

Proton leak (H(+) leak) dissipates mitochondrial membrane potential (mΔΨ) through the re-entry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H(+) leak may affect reactive oxygen species (ROS) production. We measured H(+) leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected mΔΨ and ROS production. Isolated rat hearts (n = 6/group) were subjected to either control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2 mM). mΔΨ was measured using a tetraphenylphosphonium electrode. H(+) leak is the respiratory rate required to maintain membrane potential at -150 mV in the presence of oligomycin-A. Mitochondrial complex III ROS production was measured by fluorometry using Amplex-red. IPC improved recovery of RPP at end reperfusion (63% ± 4% versus 21% ± 2% in control-IR, P < 0.05). Ischemia-reperfusion caused increased H(+) leak (94 ± 12 versus 31 ± 1 nmol O/mg protein/min in non-ischemic control, P < 0.05). IPC attenuates these increases (55 ± 9 nmol O/mg protein/min, P < 0.05 versus control-IR). IPC reduced mitochondrial ROS production compared with control-IR (31 ± 2 versus 40 ± 3 nmol/mg protein/min, P < 0.05). As mitochondrial respiration decreased, mΔΨ and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in control-IR for all mΔΨ and respiration rates. Increasing H(+) leak is not associated with decreased ROS production. IPC decreases both the magnitude of H(+) leak and ROS production after ischemia-reperfusion.

Ischemic Postconditioning Does Not Attenuate�Ischemia-Reperfusion Injury of Rabbit Small Intestine

To determine whether ischemic postconditioning can attenuate intestinal ischemia-reperfusion (I-R) injury and has a beneficial effect on tissue blood flow during reperfusion. In vivo experimental study. New Zealand White rabbits (n=6). Rabbits were anesthetized with pentobarbital, to avoid the preconditioning effects of volatile anesthetics, and ventilated with room air. Rectal temperature, hemodynamics, and normocapnia were maintained. After celiotomy, 3 jejunal segments were isolated in each rabbit for the following groups: (1) control, (2) I-R, and (3) I-R with postconditioning. I-R was induced by a 45-minute occlusion of the segment jejunal artery followed by 2-hour reperfusion. The postconditioning segment had 4 cycles of 30-second reperfusion and 30-second reocclusion during the initial 4 minutes of reperfusion. Stable isotope-labeled microspheres were used to measure intestinal blood flow at baseline, end occlusion, and end reperfusion. At the end of reperfusion, intestine segments were harvested and the rabbits euthanatized. A semiquantitative histopathologic evaluation (0-5) was conducted by a single, blinded observer. Wet-to-dry weight ratios were calculated to assess intestinal edema. There was no significant difference in grade of necrosis, tissue wet-to-dry weight ratios, or blood flow at any time point between ischemic and postconditioning groups. Ischemic postconditioning was ineffective in this model of intestinal I-R. Further experimental studies will need to be performed before clinical application of postconditioning for intestinal ischemia.

Ischemic Preconditioning Protects Mitochondrial Respiratory Function In Complex I With Decreased Free Radical Production

Mitochondrial respiratory complexes are damaged by ischemia reperfusion injury. Oxygen free radicals (OFR) may be involved in the cardioprotective mechanism of ischemic preconditioning (IPC). The purpose of this study was to determine the effects of IPC on mitochondrial respiratory complex I activity and OFR production.Rat hearts (n=6/group) were subjected to either A) 30 minutes (min) of equilibration (EQ), 30 min of ischemia (I), and 30 min of reperfusion (R) (CONTROL) or B) 10 min of EQ, three 5 min episodes of IPC, 30 min I, and 30 min of R (IPC group). A third set of non‐ischemic control hearts were also studied (NIC). Mitochondria were isolated at end reperfusion. Respiratory complex I activity was assessed by polarography using specific substrates and inhibitors for complex I in the presence of ADP. Complex I OFR production was measured by Electron Paramagnetic Resonance (EPR) spectrometry. Complex I substrates were glutamate and malate (4.7 mM) and the inhibitor was rotenone (188 nM). Data is expressed as mean±SEM.Ischemia reperfusion impairs mitochondrial complex I activity and increases complex I OFR production. IPC preserves mitochondrial respiratory complex I activity and decreases mitochondrial free radical generation at end reperfusion.

Ventricular restraint therapy: And what of the right ventricle?

ESULTS: Post-ischemic recovery of left ventricular developed ressure (%equilibration) was significantly greater with MSCs 46.2 4.1%) and MSC conditioned media (44.2 4.0%) than with ehicle (35.3 2.7%) at end reperfusion. However, hearts treated ith siRNA VEGF deleted MSCs (29.7 1.7%) and corresponding onditioned media (28.5 5.4%) exhibited no difference from vehile. No significant difference was observed between hearts treated ith MSCs or siRNA scramble control MSCs. End diastolic pressure nd dP/dt followed a similar pattern in post ischemic functional ecovery.

Embryonic stem cells attenuate myocardial dysfunction and inflammation after surgical global ischemia via paracrine actions

Stem cell treatment may positively influence recovery and inflammation after shock by multiple mechanisms, including the paracrine release of protective growth factors. Embryonic stem cells (ESCs) are understudied and may have greater protective power than adult bone marrow stem cells (BMSCs). We hypothesized that ESC paracrine protective mechanisms in the heart (decreased injury by enhanced growth factor-mediated reduction of proinflammatory cytokines) would be superior to the paracrine protective mechanisms of the adult stem cell population in a model of surgically induced global ischemia. Adult Sprague-Dawley rat hearts were isolated and perfused via Langendorff model. Hearts were subjected to 25 min of warm global ischemia and 40 min of reperfusion and were randomly assigned into one of four groups: 1) vehicle treated; 2) BMSC or ESC preischemic treatment; 3) BMSC or ESC postischemic treatment; and 4) BMSC- or ESC-conditioned media treatment. Myocardial function was recorded, and hearts were analyzed for expression of tissue cytokines and growth factors (ELISA). Additionally, ESCs and BMSCs in culture were assessed for growth factor production (ELISA). ESC-treated hearts demonstrated significantly greater postischemic recovery of function (left ventricular developed pressure, end-diastolic pressure, and maximal positive and negative values of the first derivative of pressure) than BMSC-treated hearts or controls at end reperfusion. ESC-conditioned media (without cells) also conferred cardioprotection at end reperfusion. ESC-infused hearts demonstrated increased VEGF and IL-10 production compared with BMSC hearts. ESC hearts also exhibited decreased proinflammatory cytokine expression compared with MSC hearts. Moreover, ESCs in cell culture demonstrated greater pluripotency than MSCs. ESC paracrine protective mechanisms in surgical ischemia are superior to those of adult stem cells.

Impact of intraoperative myocardial tissue acidosis on postoperative adverse outcomes and cost of care for patients undergoing prolonged aortic clamping during cardiopulmonary bypass

Background This study examined the impact of intraoperative myocardial acidosis and adverse postoperative outcomes on the cost of cardiac surgical care. Methods Myocardial tissue pH corrected to 37°C (pH 37C) was measured in 162 patients with cross-clamp (XC) duration of 119 minutes or longer. Perioperative data and outcomes were collected prospectively. The Veterans Affairs cost accounting system was used to determine the cost of care in a subset of 57 patients. Results Long XC duration was associated with significantly increased acidosis and adverse postoperative outcomes. The cost of care for patients with adverse outcomes was increased by 110% ( P < .0001). Patients with acidosis at the end of reperfusion had significantly ( P = .0470) increased costs of care. End reperfusion of myocardial tissue pH 37C of less than 7.0, diabetes mellitus, and body surface area were significant determinants of postoperative adverse outcomes. Conclusions Intraoperative myocardial acidosis is a determinant of postoperative adverse outcomes and cost in cardiac surgery. Reducing XC duration and improving intraoperative myocardial protection should improve outcomes and reduce cost.

Estradiol-Treated Mesenchymal Stem Cells Improve Myocardial Recovery After Ischemia

Stem cell therapy is a promising treatment modality for injured cardiac tissue. A novel mechanism for this cardioprotection may include paracrine actions. Our lab has recently shown that gender differences exist in mesenchymal stem cell (MSC) paracrine function. Estrogen is implicated in the cardioprotection found in females. It remains unknown whether 17beta-estradiol (E2) affects MSC paracrine function and whether E2-treated MSCs may better protect injured cardiac tissue. We hypothesize that E2-exposed MSCs infused into hearts prior to ischemia may demonstrate increased vascular endothelial growth factor (VEGF) production and greater protection of myocardial function compared to untreated MSCs. Untreated and E2-treated MSCs were isolated, cultured, and plated and supernatants were harvested for VEGF assay (enzyme-linked immunosorbent assay). Adult male Sprague-Dawley rat hearts (n = 13) were isolated and perfused via Langendorff model and subjected to 15 min equilibration, 25 min warm global ischemia, and 40 min reperfusion. Hearts were randomly assigned to perfusate vehicle, untreated male MSC, or E2-treated male MSC. Transcoronary delivery of 1 million MSCs was performed immediately prior to ischemia in experimental hearts. E2-treated MSCs provoked significantly more VEGF production than untreated MSCs (933.2 +/- 64.9 versus 595.8 +/- 10.7 pg/mL). Postischemic recovery of left ventricular developed pressure was significantly greater in hearts infused with E2-treated MSCs (66.9 +/- 3.3%) than untreated MSCs (48.7 +/- 3.7%) and vehicle (28.9 +/- 4.6%) at end reperfusion. There was also greater recovery of the end diastolic pressure with E2-treated MSCs than untreated MSCs and vehicle. Preischemic infusion of MSCs protects myocardial function and viability. E2-treated MSCs may enhance this paracrine protection, which suggests that ex vivo modification of MSCs may improve therapeutic outcome.

Vascular Endothelial Growth Factor Improves Myocardial Functional Recovery Following Ischemia/Reperfusion Injury

Vascular endothelial growth factor (VEGF) is a central growth and survival factor for both the endothelium and the myocardium. Recent evidence also suggests that VEGF may play a critical role in stem-cell-mediated paracrine cardioprotection. However, the acute effect of exogenous VEGF on myocardium after ischemia, indeed whether isolated VEGF alone may be a clinically useful therapeutic modality, remains unknown. We hypothesize that infusion of exogenous VEGF immediately prior to ischemia will improve myocardial functional recovery. Adult male Sprague Dawley rat hearts were isolated and perfused via Langendorff model. All hearts were subject to 15-min equilibration, 25-min warm global ischemia, and 40-min reperfusion. Experimental hearts received a VEGF infusion of 3 x physiological (13 nM, n = 4), 5x physiological (20 nM, n = 4), or 10x physiological (40 nM, n = 5) immediately prior to ischemia. Controls (n = 5) were infused with perfusate vehicle. Functional indices (left ventricular developed pressure), end diastolic pressure, +/-dP/dt were continuously recorded. End diastolic pressure (mmHg) was elevated in response to ischemia/reperfusion. However, hearts infused with 10x VEGF demonstrated significantly (P < 0.05, analysis of variance and Bonferroni's) decreased end diastolic pressure throughout reperfusion compared to control (49.82 +/- 10.35 mmHg versus 80.73 +/- 6.08 mmHg at end reperfusion). 10x VEGF-treated hearts also exhibited significantly (P < 0.05, analysis of variance and Bonferroni's) greater recovery of left ventricular developed pressure (69.97 +/- 9.69% versus 39.74 +/- 7.01% of equilibration), +dP/dt, and -dP/dt at end reperfusion. However, neither 3 x nor 5x VEGF improved recovery after ischemia. VEGF also did not influence coronary flow after ischemia. This is the first demonstration that exogenous VEGF administration acutely improves myocardial functional recovery after ischemia. These findings may help elucidate the role of VEGF in acute stem-cell-mediated paracrine effects and suggests that isolated VEGF may be of therapeutic value.

In the adult mesenchymal stem cell population, source gender is a biologically relevant aspect of protective power

Acute treatment with bone marrow mesenchymal stem cells (MSC) reduces myocardial infarct size by multiple mechanisms, including the paracrine release of protective growth factors. Female MSCs produce more growth factor when stressed; therefore, we hypothesized that myocardial protection provoked by female MSCs would be greater than that elicited by male MSCs. Hearts were subjected to 25 min of warm global ischemia, 40 min of reperfusion, and randomly assigned into one of three groups: (1) vehicle treated; (2) male MSC treated; and (3) female MSC treated. Myocardial function was continuously recorded and in separate experiments, male and female MSC growth factor production was assessed by ELISA. All indices of functional recovery were significantly higher in the stem cell infused rat heart compared with control hearts. Interestingly, female MSC treated rat hearts demonstrated significantly greater recovery of left ventricular developed pressure, +dP/dT, and -dP/dT than male MSC treated hearts at end reperfusion. In addition, male MSCs produced significantly greater tumor necrosis factor alpha, and significantly less vascular endothelial growth factor than female MSCs. This study is the first to demonstrate that, in the adult mesenchymal population, source gender is a biologically relevant aspect of ultimate stem cell function in the heart.

Postischemic Infusion of 17-β-Estradiol Protects Myocardial Function and Viability

Females demonstrate improved cardiac recovery after ischemia/reperfusion injury compared with males. Attenuation of myocardial dysfunction with preischemic estradiol suggests that estrogen may be an important mediator of this cardioprotection. However, it remains unclear whether post-injury estradiol may have clinical potential in the treatment of acute myocardial infarction. We hypothesize that postischemic administration of 17beta-estradiol will decrease myocardial ischemia/reperfusion injury and improve left ventricular cardiac function. Adult male Sprague Dawley rat hearts (n = 20) (Harlan, Indianapolis, IN) were isolated, perfused with Krebs-Henseleit solution via Langendorff model, and subjected to 15 min of equilibration, 25 min of warm ischemia, and 40 min reperfusion. Experimental hearts received postischemic 17beta-estradiol infusion, 1 nm (n = 4), 10 nm (n = 4), 25 nm (n = 4), or 50 nm (n = 4), throughout reperfusion. Control hearts (n = 4) were infused with perfusate vehicle. Postischemic recovery of left ventricular developed pressure was significantly greater with 1 nm (51.6% +/- 7.4%) and 10 nm estradiol (47.7% +/- 8.6%) than with vehicle (37.8% +/- 9.7%) at end reperfusion. There was also greater recovery of the end diastolic pressure with 1 nm (47.8 +/- 4.0 mmHg) and 10 nm estradiol (54.0 +/- 4.0) compared with vehicle (75.3 +/- 7.5). Further, 1 nm and 10 nm estrogen preserved coronary flow after ischemia and decreased coronary effluent lactated dehydrogenase compared with controls. Estrogen at 25 nm and 50 nm did not provide additional benefit in terms of functional recovery. Estrogen at all concentrations increased extracellular signal-regulated protein kinase phosphorylation. Postischemic infusion of 17beta-estradiol protects myocardial function and viability. The attractive potential for the clinical application of postischemic estrogen therapy warrants further study to elucidate the mechanistic pathways and differences between males and females.

Selective mitochondrial adenosine triphosphate–sensitive potassium channel activation is sufficient to precondition human myocardium

Objectives: Recently, the mitochondrial adenosine triphosphate–sensitive potassium channel has been suggested to be the final common effector of myocardial preconditioning. The purpose of this study is to determine whether selective mitochondrial adenosine triphosphate–sensitive potassium channel activation alone can precondition human myocardium from an ischemia/reperfusion insult. Methods: Isolated human right atrial trabeculae were placed in tissue baths, paced, and subjected to 30 minutes of normothermic hypoxia (ischemia) followed by 45 minutes of reoxygenation (reperfusion). Trabeculae were preconditioned with a selective mitochondrial adenosine triphosphate–sensitive potassium channel opener (diazoxide 30 μmol/L) or a nonselective purinergic agonist, adenosine (125 μmol/L), for 5 minutes (adenosine) followed by a 10-minute washout period. Developed force at end reperfusion (mean ± standard error) was compared with baseline, and tissue creatine kinase and adenosine triphosphate levels were measured after ischemia/reperfusion. Results: Trabeculae subjected to ischemia/reperfusion exhibited 30% ± 2% of baseline developed force, whereas trabeculae subjected to selective adenosine triphosphate–sensitive potassium channel opening (diazoxide) and nonselective purinergic agonist (adenosine) recovered to 55% ± 7% and 46% ± 3% of baseline developed force, respectively. Tissue creatine kinase activity was preserved in both the diazoxide- and adenosine-treated trabeculae (5.4 ± 12 and 5.4 ± 14 μmol/L per gram wet tissue) compared with ischemia/reperfusion (1.8 ± 0.2 U/mg wet tissue). Adenosine triphosphate levels at end reperfusion were also increased in the trabeculae treated with selective (diazoxide) and nonselective (adenosine) adenosine triphosphate–sensitive potassium channel opener (4.1 ± 0.01 and 4.4 ± 0.2 μmol/L per gram wet tissue) compared with trabeculae subjected to ischemia/reperfusion (1.5 ± 0.1 μmol/L per gram wet tissue). Conclusions: These results suggest that selective mitochondrial adenosine triphosphate–sensitive potassium channel activation preconditions human myocardium and the protection conferred is equal to that of adenosine preconditioning. Targeted openers of mitochondrial adenosine triphosphate– sensitive potassium channels promote constructive protection of myocellular energy levels, contractile function, and cellular viability in human myocardium after ischemia/reperfusion. (J Thorac Cardiovasc Surg 2000;120:387-92)

Transportation or Noise Is Associated with Tolerance to Myocardial Ischemia and Reperfusion Injury

Myocardial stress can result in myocellular phenotypic changes including enhanced activity of antioxidant enzyme systems. Accordingly, endogenous tissue antioxidant enzyme activity has been associated with resistance to cardiac ischemia and reperfusion injury. The present study was designed to determine if environmental perturbations could alter myocardial antioxidant enzyme (catalase) activity and function after ischemia. Isolated perfused rat hearts (Langendorff apparatus, 37°C) were subjected to 20 min global ischemia (37°C) and 40 min reperfusion. Rats studied immediately following shipment had increased myocardial catalase activity (1330 ± 3.5 U/g, P < 0.05 vs quarantined control) and increased resistance to ischemia and reperfusion injury (end reperfusion developed pressure, DP 55 ± 4.0 mm Hg, P < 0.05 vs quarantined control). However, control rats that were quarantined for 4 weeks exhibited a progressive decrease in catalase activity (760 ± 10 U/g) for 3 weeks of quarantine. There was a concurrent decrease in resistance to myocardial ischemia and reperfusion injury (DP 40 ± 3.6 mm Hg). Similarly, quarantined rats subjected to construction-related noise levels in excess of 90 dB (A scale) had increased myocardial catalase activity (1140 ± 3.3 U/g, P < 0.05) and functional tolerance to ischemia and reperfusion (DP 66 ± 3.3 mm Hg, P < 0.05). Finally, rats experiencing 90-dB noise levels for 2 days exhibited increased myocardial catalase activity (1125 ± 30 U/g, P < 0.05) and myocardial ischemia and reperfusion injury tolerance (DP 62 ± 1.7 mm Hg, P < 0.05). We conclude that variations in environmental conditions can relate to changes in antioxidant defense mechanisms and tolerance to myocardial ischemia and reperfusion injury in the rat.