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Byline: Helmut Habazettl, Barbara W. Palmisano, Bernhard M. Graf, David L. Roerig, Zeljko J. Bosnjak, David F. Stowe Abstract: The aim of this study was to examine the effect of initial hyperkalemic reperfusion (HKR), with and without added adenosine, on coronary flow, myocardial function, and endothelium-dependent and endothelium-independent coronary vascular function. Cardioplegic arrest was induced in 40 isolated guinea pig hearts by infusing oxygenated cardioplegic (high in potassium ion) Krebs solution for 5 minutes. Hearts were then stored at room temperature for 3.5 hours. On reperfusion, hearts were divided into four groups of 10 hearts each: control, reperfusion with regular Krebs solution (4.6 mmol/L potassium chloride); base hyperkalemic reperfusion, initial reperfusion with 37A* C oxygenated, cardioplegic Krebs solution for 5 minutes; hyperkalemic reperfusion with addition of 1 mmol/L adenosine during HKR; and hyperkalemic reperfusion with addition of 5 mmol/L adenosine. Coronary reserve (adenosine bolus 2 mmol/L) and responses to acetylcholine (1 [mu]mol/L) and nitroprusside (100 [mu]mol/L) were examined before and after ischemia and reperfusion. Flow did not return to preischemic values in any group after reperfusion. Adenosine treatment during initial reperfusion increased coronary flow (percentage of baseline [+ or -] standard error of the mean) from 57% [+ or -] 4% in control and 45% [+ or -] 3% in hearts with hyperkalemic reperfusion to 79% [+ or -] 3% and 83% [+ or -] 5% in hearts with hyperkalemic reperfusion also treated with, respectively, 1 mmol/L adenosine and 5 mmol/L adenosine (p < 0.05). At 30 and 60 minutes of reperfusion, however, flow remained elevated only in the group treated with 5 mmol/L adenosine. Coronary reserve and responses to acetylcholine and nitroprusside were equivalently depressed in all groups after reperfusion. Recovery of left ventricular systolic and diastolic function was improved in all groups after hyperkalemic reperfusion (54% [+ or -] 4% of preischemic value) compared with control (39% [+ or -] 3%), and recovery was further enhanced in the group treated with 5 mmol/L adenosine (60% [+ or -] 4%). In this ex vivo model, hyperkalemic reperfusion improved myocardial function after cardioplegic arrest and the addition of 5 mmol/L adenosine improved coronary flow. Adenosine may counteract the potassium chloride-induced vasoconstriction that occurs during hyperkalemic reperfusion and may thus improve coronary flow and myocardial function. Postischemic depression of endothelium-dependent or endothelium-independent vascular functions, however, was not alleviated by hyperkalemic reperfusion with or without adenosine. (J T HORAC CARDIOVASC SURG 1996;111:74-84) Author Affiliation: Milwaukee, Wis Article History: Received 5 December 1994; Accepted 19 April 1995 Article Note: (footnote) [star] From the Anesthesiology Research Laboratory, and the Departments of Anesthesiology and Physiology, Cardiovascular Research Center, The Medical College of Wisconsin, and the Veterans Affairs Medical Center, Milwaukee, Wis., [star][star] Supported by Deutsche Forschungsgemeinschaft, grant Ha 1651/5-1 (H. H.) and VA Merit Grant 8204-04P (D. F. S.)., a Address for reprints: Helmut Habazettl, MD, Institute for Surgical Research, University of Munich, Marchioninistrasse 15, 81366 Munich, Germany., aa 12/1/65887

Preclinical investigations in animal models have consistently demonstrated neurobiological changes and life-long cognitive deficits following exposure to widely used anesthetics early in life. However, the mechanisms by which these exposures affect brain function remain poorly understood, therefore, limiting the efficacy of current diagnostic and therapeutic options in human studies. The human brain exhibits an abundant expression of long noncoding RNAs (lncRNAs). These biologically active transcripts play critical roles in a diverse array of functions, including epigenetic regulation. Changes in lncRNA expression have been linked with brain development, normal CNS processes, brain injuries, and the development of neurodegenerative diseases, and many lncRNAs are known to have brain-specific expression. Aberrant lncRNA expression has also been implicated in areas of growing importance in anesthesia-related research, including anesthetic-induced developmental neurotoxicity (AIDN), a condition defined by neurological changes occurring in patients repeatedly exposed to anesthesia, and the related condition of perioperative neurocognitive disorder (PND). In this review, we detail recent advances in PND and AIDN research and summarize the evidence supporting roles for lncRNAs in the brain under both normal and pathologic conditions. We also discuss lncRNAs that have been linked with PND and AIDN, and conclude with a discussion of the clinical potential for lncRNAs to serve as diagnostic and therapeutic targets for the prevention of these neurocognitive disorders and the challenges facing the identification and characterization of associated lncRNAs.

Published ahead of print August 22, 2016.Mounting evidence has demonstrated that general anesthetics could induce developmental neurotoxicity, including acute widespread neuronal cell death, followed by long-term memory and learning abnormalities. Propofol is a commonly used intravenous anesthetic agent for the induction and maintenance of anesthesia and procedural and critical care sedation in children. Compared with other anesthetic drugs, little information is available on its potential contributions to neurotoxicity. Growing evidence from multiple experimental models showed a similar neurotoxic effect of propofol as observed in other anesthetic drugs, raising serious concerns regarding pediatric propofol anesthesia. The aim of this review is to summarize the current findings of propofol-induced developmental neurotoxicity. We first present the evidence of neurotoxicity from animal models, animal cell culture, and human stem cell–derived neuron culture studies. We then discuss the mechanism of propofol-induced developmental neurotoxicity, such as increased cell death in neurons and oligodendrocytes, dysregulation of neurogenesis, abnormal dendritic development, and decreases in neurotrophic factor expression. Recent findings of complex mechanisms of propofol action, including alterations in microRNAs and mitochondrial fission, are discussed as well. An understanding of the toxic effect of propofol and the underlying mechanisms may help to develop effective novel protective or therapeutic strategies for avoiding the neurotoxicity in the developing human brain.

Exposure to commonly used anesthetic agents causes widespread neuronal degeneration in the developing mammalian brain and has been shown to impair neurodevelopment in a variety of newborn vertebrate animal species. Although retrospective studies have suggested an association between anesthesia exposure in childhood and subsequent neurodevelopmental abnormalities, a causal relationship in humans has yet to be demonstrated. Unfortunately, translation of findings from bench to bedside is limited by several factors and histologic assessment in healthy children following exposure to anesthesia is not possible. Therefore, to prove that anesthesia-induced neurotoxicity occurs in humans, alternative approaches are necessary. Here we present the summary of a focus group discussion regarding the utility of biomarkers in translational studies of anesthetic neurotoxicity as part of The 2016 Pediatric Anesthesia NeuroDevelopmental Assessment (PANDA) Symposium at Columbia University Medical Center. The experts agreed that defining intermediate phenotypes using advanced neuroimaging as a biomarker is a highly feasible and reasonable modality to provide new insights into the deleterious effects of anesthetic exposure in the developing human brain and could illuminate a viable investigative path forward. Ultimately, well-defined intermediate phenotypes may allow us to fully understand the neurodevelopmental impact of anesthesia-induced neurotoxicity and permit us to develop the safest and most effective anesthetic strategies for the infants and children we care for.

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Type 1 diabetes mellitus (T1DM) is the most common type of diabetes in children and adolescents. Diabetic subjects are more likely to experience a myocardial infarction compared to nondiabetic subjects. In recent years, induced pluripotent stem cells (iPSCs) have received increasing attention from basic scientists and clinicians and hold promise for myocardial regeneration due to their unlimited proliferation potential and differentiation capacity. However, cardiomyogenesis of type 1 diabetic donor-derived iPSCs (TIDM-iPSCs) has not been investigated yet. The aim of the study was to comparatively analyze cardiomyocyte (CM) differentiation capacity of nondiabetic donor-derived iPSCs (N-iPSCs) and TIDM-iPSCs. The differentiated CMs were confirmed by both expression of cardiac-specific markers and presence of cardiac action potential. Since mitochondrial bioenergetics is vital to every aspect of CM function, extracellular acidification rates and oxygen consumption rates were measured using Seahorse extracellular flux analyzer. The results showed that N-iPSCs and T1DM-iPSCs demonstrated similar capacity of differentiation into spontaneously contracting CMs exhibiting nodal-, atrial-, or ventricular-like action potentials. Differentiation efficiency was up to 90%. In addition, the CMs differentiated from N-iPSCs and T1DM-iPSCs (N-iPSC-CMs and T1DM-iPSC-CMs, respectively) showed 1) well-regulated glucose utilization at the level of glycolysis and mitochondrial oxidative phosphorylation and 2) the ability to switch metabolic pathways independent of extracellular glucose concentration. Collectively, we demonstrate for the first time that T1DM-iPSCs can differentiate into functional CMs with well-regulated glucose utilization as shown in N-iPSCs, suggesting that T1DM-iPSC-CMs might be a promising autologous cell source for myocardial regeneration in type 1 diabetes patients.

Cardiac mitochondria and the sarcolemmal (sarc)KATPchannels contribute to cardioprotective signaling of anesthetic-induced preconditioning. Changes in mitochondrial bioenergetics influence the sarcolemmal ATP-sensitive K(sarcKATP) channel function, but whether this channel has impacts on mitochondria is uncertain. We used the mouse model with deleted pore-forming Kir6.2 subunit of sarcKATPchannel (Kir6.2 KO) to investigate whether the functional sarcKATPchannels are necessary for isoflurane activation of mitochondrial protective mechanisms. Ventricular cardiomyocytes were isolated from C57Bl6 wild-type (WT) and Kir6.2 KO mouse hearts. Flavoprotein autofluorescence, mitochondrial reactive oxygen species production, and mitochondrial membrane potential were monitored by laser-scanning confocal microscopy in intact cardiomyocytes. Cell survival was assessed using H2O2-induced stress. Isoflurane (0.5 mM) increased flavoprotein fluorescence to 180 ± 14 and 190 ± 15 and reactive oxygen species production to 118 ± 2 and 124 ± 6 of baseline in WT and Kir6.2 KO myocytes, respectively. Tetramethylrhodamine ethyl ester fluorescence decreased to 84 ± 6 in WT and to 86 ± 4 in Kir6.2 KO myocytes. This effect was abolished by 5HD. Pretreatment with isoflurane decreased the stress-induced cell death from 31 ± 1 to 21 ± 1 in WT and from 44 ± 2 to 35 ± 2 in Kir6.2 KO myocytes. In conclusion, Kir6.2 deletion increases the sensitivity of intact cardiomyocytes to oxidative stress, but does not alter the isoflurane-elicited protective mitochondrial mechanisms, suggesting independent roles for cardiac mitochondria and sarcKATPchannels in anesthetic-induced preconditioning by isoflurane.

We recently reported that, following induction of clumps of pluripotent H1 human embryonic stem cells (hESCs) with activin-A and Bmp4 in defined medium for 5 days, widespread differentiation of rhythmically contracting cardiomyocytes occurs within 3–4 weeks. In this study, the same approach was used to assess whether human induced pluripotent stem cells (hiPSCs), which may theoretically provide an unlimited source of patient-matched cells for transplantation therapy, can similarly undergo cardiomyocyte differentiation. Differentiation of four pluripotent cell lines (H1 and H9 hESCs and C2a and C6a hiPSCs) was compared in parallel by monitoring rhythmic contraction, morphologic differentiation, and expression of cardiomyogenic genes. Based on expression of the cardiomyogenic lineage markers MESP1, ISL1, and NKX2-5, all four cell lines were induced into the cardiomyogenic lineage. However, in contrast to the widespread appearance of striations and rhythmic contractility seen in H9 and especially in H1 hESCs, both hiPSC lines exhibited poor terminal differentiation. These findings suggest that refined modes of generating hiPSCs, as well as of inducing cardiomyogenesis in them, may be required to fulfill their potential as agents of cardiac regeneration.

Cardiovascular sensitivity to general anesthetics is highly variable among individuals in both human and animal models, but little is known about the genetic determinants of drug response to anesthetics. Recently, we reported that propofol (2,6-diisopropylphenol) causes circulatory instability in Dahl salt-sensitive SS/JRHsdMcwi (SS) rats but not in Brown Norway BN/NHsdMcwi (BN) rats and that these effects are related to genes on chromosome 13. Based on the hypothesis that propofol does target mesenteric circulation, we investigated propofol modulation of mesenteric arterial smooth muscle cells (MASMC) in SS and BN rats. The role of chromosome 13 was tested using SS-13(BN)/Mcwi and BN-13(SS)/Mcwi consomic strains with chromosome 13 substitution. Propofol (5 microM) produced a greater in situ hyperpolarization of MASMC membrane potential in SS than BN rats, and this effect was abrogated by iberiotoxin, a voltage-activated potassium (BK) channel blocker. In inside-out patches, the BK channel number, P(o), and apparent Ca(2+) sensitivity, and propofol sensitivity all were significantly greater in MASMC of SS rats. The density of whole-cell BK current was increased by propofol more in SS than BN myocytes. Immunolabeling confirmed higher expression of BK alpha subunit in MASMC of SS rats. Furthermore, the hyperpolarization produced by propofol, the BK channel properties, and propofol sensitivity were modified in MASMC of SS-13(BN)/Mcwi and BN-13(SS)/Mcwi strains toward the values observed in the background SS and BN strains. We conclude that differential function and expression of BK channels, resulting from genetic variation within chromosome 13, contribute to the enhanced propofol sensitivity in SS and BN-13(SS)/Mcwi versus BN and SS-13(BN)/Mcwi strains.

Introduction:Although anthracyclines, including doxorubicin (DOX), are among the most effective anticancer drugs to date, they cause dose-related cardiotoxicity that can lead to heart failure in a subset of patients. At present, it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). We utilized patient-specific PBMC/Neutrophils to prospectively assess patient-and tissue-specific effects of high-risk therapeutic regimens and compare the results with patient outcomes after receiving chemotherapy for breast cancer.Hypothesis:We hypothesized that the PBMC/Neutrophils could recapitulate the predisposition of the patients towards DIC.Methods:The PBMC/Neutrophils were isolated from breast cancer patients with and without cardiotoxicity, and exposed to varying concentrations of doxorubicin (DOX). The in vitroDIC in breast cancer patients specific PBMC/Neutrophils after 24 hrs was assessed via analysis of: (a) Cytotoxicity determined via Alamar Blue cell viability assay kit; (b) Cytotoxicity determined using ATP based luminescence assay; (c) Oxygen consumption rate (OCR); and (d) Genome wide transcriptomic analysis of PBMCs.Results:Patient specific PBMC/Neutrophils recapitulate the predilection of the patients towards DIC. The PBMC/Neutrophils derived from breast cancer patients who experienced clinical doxorubicin-induced cardiotoxicity were more sensitive to doxorubicin toxicity depicting: (1) An increased response to DOX with neutrophils being more sensitive to DOX at lower concentrations than PBMCs; (2) Decreased ATP production indicating lower cellular viability; and (3) Lower OCR associated with basal mitochondrial respiration, ATP production, maximal respiration and spare respiratory capacity (representing mitochondrial function), as compared to breast cancer patients without cardiotoxicity.Conclusions:Together, our data indicate that this in vitroapproach may represent a fast, cost-effective, and high throughput means to assess the future cardiotoxic effects of chemotherapy drugs. Furthermore, it offers the opportunity to understand the on-target and off-target molecular mechanisms to optimize the efficacy and safety of the cancer drugs.

The heart is a passively filling pump in a circulatory system that is connected in series with distensible blood vessels. Therefore, systemic blood pressure and tissue perfusion depend upon adequate peripheral vascular tone as well as myocardial function. Likewise, pharmacologic agents that alter circulatory stability can affect one or both of these components. The generalized depressor effects of general anesthetics have been well known clinically for over 50 years. Moreover, there are many similarities in basic cellular regulatory mechanisms among the different tissue types, and general anesthetics are well known to distribute freely among the perfusion-rich tissues (eg, central nervous system, cardiovascular system, and renal system). Therefore, it is likely that the hemodynamic depression resulting from the systemic administration of anesthetics results from actions on regulatory mechanisms of the peripheral vasculature as well as on the heart. The peripheral vasculature is regulated by extrinsic neural, endothelial, and humoral mechanisms, which interact with each other as well as with intrinsic membrane and intracellular systems within the vascular smooth muscle cell. Different general anesthetics have been found to act on specific mechanisms at each of these levels. However, the large number and complexity of these known mechanisms, as well as the many anesthetic agents, has made it extremely difficult to determine which are significant in terms of the meaningful mechanisms that are responsible for anesthetic action, major side effects, or both. Current knowledge about the effects of general anesthetics on both the extrinsic intrinsic regulatory mechanisms of peripheral vascular control is reviewed.

Chronic myocardial hypoxia results in elevated nitric oxide (NO) production and increased current through the sarcolemmal KATPchannel. We hypothesized these two processes are related and determined whether NO alters the electrophysiology of Purkinje fibers obtained from rabbits (n=12/group) raised in a normoxic (FIO2=0.21) or hypoxic (FIO2=0.12) environment from birth to 9 days of age. Action potential duration (APD)90was shorter (112±3 ms v126±3 ms) and maximum diastolic potential (MDP) was more negative (−84±2 mV v−80±1 mV) in hypoxic hearts compared with normoxic controls. In normoxic hearts the NO donors, S-nitrosoglutathione (GSNO) 50 μMand spermine NONOate (50μM) shortened APD90and increased MDP to levels present in chronically hypoxic hearts. This effect was completely abolished by the KATPchannel blocker glibenclamide (3 μM) and by a nitric oxide trap, Carboxy-PTIO (100 μM). The NO carrier glutathione (50 μM) and decomposed spermine NONOate had no effect on APD90or MDP. GSNO had no effect in hypoxic hearts; however, when GSNO was combined with glibenclamide APD90increased, and MDP decreased to normoxic values. 8-Bromo cGMP (100 μM) shortened APD90and increased MDP to levels present in chronically hypoxic hearts. This effect was abolished by glibenclamide. A soluble guanylyl cyclase inhibitor, ODQ (10 μM), had no effect on action potentials in normoxic hearts but in hypoxic hearts resulted in an increase in APD90to levels present in normoxic hearts and a decrease in MDP. The effect of ODQ could not be reversed by GSNO. We conclude that NO activates the sarcolemmal KATPchannel in normoxic and chronically hypoxic hearts by a cyclic GMP-dependent mechanism.

Laser Doppler flowmetry was utilized to investigate whether nitric oxide (NO) plays a role in the cerebrocortical hyperemic effect of halothane in rats. A particular objective was to elucidate whether the increased vascular tone or the removal of basal NO secondary to NO synthase inhibition influenced the response to halothane. The animals were anesthetized with i.p. pentobarbital for surgery and 90 min later were ventilated with 1.0 minimum alveolar concentration (MAC) halothane for 1 h to achieve a steady-state baseline. The control group was infused with either 1 ml of saline or 20 mg/kg of D-NAME, and the treatment group received 20 mg/kg of L-NAME intravenously. In a subset of the treatment group, we restored baseline flow and vascular tone using i.v. sodium nitroprusside (SNP). Mean arterial pressure (MAP) was maintained constant with an infusion of phenylephrine (0.5–5 μg/ kg/min). Then, 30 to 45 min later, inspired halothane was raised to 1.7 MAC in each group, and the increase in laser Doppler flow (LDF) was measured. On increasing halothane MAC in the control group, LDF increased by 28 ± 4. L-NAME increased MAP by 21 ± 4 and reduced baseline LDF by 26 ± 2. In the L-NAME-only treated group, 1.7 MAC halothane increased LDF by 12 ± 3, significantly less than control. The decrease in cerebrovascular resistance induced by increasing inspired halothane MAC was similar in the control group and in the L-NAME treated group at 23 ± 6 and 22 ± 7, respectively. In the L-NAME + SNP group, increasing halothane MAC decreased cerebrovascular resistance by 30 ± 2 and increased LDF by 37 ± 2, not statistically different from the control group. Thus cerebrocortical vasodilation in response to increasing halothane MAC from 1.0 to 1.7 MAC was not attenuated by L-NAME when baseline flow and tone were restored with an NO donor to control levels. These data suggest that (a) NO is not an obligatory mediator of halothane-induced cerebrocortical vasodilatation, (b) altered vascular tone and NO level after NO synthase inhibition may influence the cerebral vasodilator response to halothane, and (c) NO may have a permissive role in halothane-induced cerebral vasodilatation.

8-Arginine vasopressin (AVP) is a powerful coronary vasoconstrictor as well as peripheral vasoconstrictor, but AVP also is reported to have negative cardiac inotropic and chronotropic effects in vitro and in vivo. Our aim was to examine the direct effects of coronary vasoconstriction by AVP on cardiac function and metabolism in isolated guinea pig hearts perfused either at a constant perfusion pressure (CPP) of 55 mm Hg or at a constant coronary flow (CCF) equal to the initial natural flow at constant pressure. Coronary vasoconstriction was elicited by perfusing hearts with increasing concentrations of AVP in random order. Variables assessed were atrial heart rate (HR), atrioventricular (AV) conduction time, left ventricular pressure (LVP), coronary flow, inflow and outflow O2tensions, O2delivery (DO2), oxygen consumption (MVO2), percentage oxygen extraction (%O2E) and cardiac efficiency (HR.LVP/MVO2). We found that AVP increased coronary vascular resistance more at CCF than at CPP. The decrease in coronary flow, as a function of AVP at CPP, produced concentration-dependent decreases in heart rate, LVP, and MVO2, a decrease in DO2/MVO2, increases in AV conduction time and%O2E, and no significant change in cardiac efficiency. In contrast, the increase in perfusion pressure as a function of AVP at CCF caused no change in HR and AV conduction time, much smaller decreases in LVP and DO2/MVO2, a smaller increase in%O2E, an increase rather than a decrease in MVO2, and a decrease in cardiac efficiency. Our results indicate that larger decreases in HR, LVP, MVO2, and DO2/MVO2, and the larger increases in AV conduction time and%O2E with the AVP-induced decrease in CF at CPP are consistent with myocardial depression resulting from reduced global perfusion. However, cardiac efficiency was maintained at CPP because the decreased HR and LVP product (cardiac work) matched the decrease in MVO2. At CCF, AVP did not directly produce myocardial depression, but the small time-dependent decrease in LVP over time was not matched by the increase in MVO2, so that cardiac efficiency was not maintained. The demonstration of an increase in MVO2despite no change or a decrease in cardiac work by coronary vasoconstriction with AVP at CCF, but not at CPP, suggests that cardiac O2use is dependent more on maintenance of CF, despite increased resistance to perfusion, rather than on maintenance of perfusion pressure. Our data agree that Gregg's phenomenon results from a hydraulic effect to distend coronary vasculature because when flow is not allowed to decrease during vasoconstriction, MVO2increases even though HR is unchanged and LVP is slightly decreased. This is supported by the finding that AVP does not increase coronary vascular resistance during CCF as much as during CPP, so that O2supply is better maintained to match MVO2.

Increased tolerance to ischemia exhibited in chronically hypoxic immature rabbit hearts is associated with increased activation of ATP-sensitive potassium (KATP) channels. We determined whether exposure to hypoxia from birth alters the electrophysiological characteristics of Purkinje fibers obtained from rabbits (n=12/group) which were raised in a normoxic (F1O2=0.21) or hypoxic (F1O2=0.12) environment from birth to 9 days of age and the involvement of the KATPchannel. The endocardial surface was exposed and impaled with microelectrodes to record action potential characteristics from Purkinje fibers under control conditions and following exposure to glibenclamide (3μm). Action potential durations (APD)90in Purkinje fibers were significantly shorter in hypoxic hearts compared with normoxic controls (110±5 msv121±4 ms). Glibenclamide increased APD90in hypoxic hearts (120±4 ms) to values similar to those observed in normoxic controls (121±4 ms). Glibenclamide had no effect on APD90in normoxic hearts. Maximum diastolic potenital was more negative in hypoxic hearts and this effect was attenuated by glibenclamide. We conclude that chronic myocardial hypoxia results in a shorter APD as compared with normoxic controls by enhanced activation of KATPchannels.

Summary:

The function of isolated guinea pig hearts treated with 2,3 butanedione monoxime (BDM) before, during, and initially after 22 h of hypothermic perfusion was examined during 4 h of normothermic reperfusion. BDM is a vasodilatory and negative inotropic agent that reversibly decreases sensitivity of contractile proteins to Ca2+. Also examined were the effects of adenosine (ADE) and nitroprusside (NP) in improving coronary flow (CF) and contractile function when given with BDM during rewarming and during the initial period of normothermic reperfusion. Isovolumetric left ventricular pressure (LVP), CF, and percentage of O2extraction (O2E) were measured in Krebs-Ringer-perfused hearts divided into three groups of 11 hearts each: drug-free controls (0 BDM); 10 mM BDM alone; and 10 mMBDM + 10 μMADE + 100 μMNP. BDM was given 20 min before hypothermia, during hypothermia (3.8° μ 0.1°C) for 22 h, and for 30 min after rewarming to 37° μ 0.1°C; ADE was given with NP for only 20 min before and during rewarming and for 30 min after rewarming. Hearts were perfused at low constant flow with oxygenated Krebs' solution during hypothermia. After 2.5-h normothermic reperfusion, LVP (initial controls 108 μ 6 mm Hg) increased more with BDM +ADE + NP (80 μ 4 of control) than with BDM alone (62 μ 3) or without BDM (28 μ 5). CF (controls 6.0 μ 0.5 ml/g/min) decreased less with BDM + ADE + NP (77 μ 4 of control) than with BDM alone (60 μ 5) or without BDM (53 μ 6). O2E (controls 59 μ 4) was similar in the 0 BDM (76 μ 3), BDM (74 μ 2), and BDM + ADE + NP (79 μ 2) groups. These results indicate that BDM given before, during, and initially after normothermic reperfusion restores cardiac function better after prolonged hypothermic perfusion and that ADE plus NP, given during the initial rewarming period with BDM, restores function better than does BDM given alone. Temporary use of ADE and NP with BDM may prove helpful in restoring vascular compliance and cardiac contractility in the early posttrans-plant period after prolonged hypothermic perfusion.

Introduction:Diabetes exacerbates myocardial ischemia/reperfusion injury (IRI) and impairs mitochondrial function. The inhibitors of histone deacetylase (HDAC) are recently shown to attenuate myocardial IRI and maintain mitochondrial homeostasis in non-diabetic animals. It remains unknown how genetic disruption of HADC6 affects myocardial IRI and mitochondrial respiratory chain complexes in diabetes.Hypothesis:We hypothesized that genetic deletion of HDAC6 will benefit the heart by preserving mitochondrial respiratory complex I during IRI in diabetic mice.Methods:HDAC6 knockout and C57BL/6 wild-type mice at 6-8 weeks of age were administered 50 mg/kg/day streptozotocin for 5 days to induce type 1 diabetes or citrate buffer as control. The geometry and function of the left ventricle were evaluated using an echocardiography. Mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion ex vivo. Mitochondrial nicotinamide adenine dinucleotide fluorescence was kinetically determined in Langendorff-perfused hearts. Complex I activity was spectrophotometrically measured.Results:Fasting blood glucose concentrations were significantly increased in both HDAC6-/-and C57BL/6 mice 6 weeks after streptozotocin injection. There were no significant differences in left ventricular thickness, fractional shortening, and the ratio of mitral E/A (P>0.05, n=10 mice/group) among diabetic C57BL/6, non-diabetic C57BL/6, diabetic HDAC6-/-, and non-diabetic HDAC6-/-groups at baseline and 6 weeks after streptozotocin injection. Compared with non-diabetic controls, diabetic C57BL/6 mice undergoing IRI showed significant decreases in ?dP/dt 60 to 120 min after reperfusion and complex I activity and increases in left ventricular end-diastolic pressure and nicotinamide adenine dinucleotide levels 5 min after ischemia. These adverse changes in diabetic C57BL/6 mice were significantly attenuated during IRI in diabetic HDAC6-/-mice (P<0.05, n=8-10 mice/group).Conclusions:Genetic disruption of HDAC6 protects the heart by preserving mitochondrial respiratory complex I during IRI in diabetic mice. Our results suggest the therapeutic potential of HDAC6 silencing in ischemic diabetic hearts.

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Objective: The effect of the nitric oxide donor, SIN-1, in proximal and distal coronary arteries with normal endothelium was characterised before and after inhibition of NO synthesis with L-nitroarginine methyl ester (L-NAME). The effect of reperfusion injury in vivo in similar vessels on the response to SIN-1 was also assessed. Methods: In vitro reactivity of preconstricted coronary arterial rings was studied in control dogs (group 1), and dogs in which the left circumflex coronary artery was subjected in vivo to four acute occlusions of 5 min duration, with three intervening reperfusion periods of 5 min and a final reperfusion period of 60 min (group 2). The effects of acetylcholine and SIN-1 on the tone of left circumflex and left anterior descending coronary vascular rings were examined before and after treatment with L-NAME. Results: Proximal [1851(SEM 82) µm] and distal [477(19) µm] vessels were studied. In control dogs (group 1) acetylcholine caused relaxation in proximal and distal coronary arteries (p>0.05). No difference in responsiveness of left circumflex or left anterior descending coronary arteries was observed in the control group. In group 2 the response to acetylcholine was significantly (p < 0.05) attenuated in left circumflex coronary arteries exposed to ischaemia and reperfusion compared with left anterior descending control rings from the same heart. Proximal vessels in group 1 and group 2 showed greater sensitivity to the vasodilator effects of SIN-1 than distal vessels. Proximal left circumflex vessels exposed to ischaemia and reperfusion showed enhanced sensitivity to the relaxant effects of SIN-1 compared to control proximal vessels obtained from the same hearts. Reperfusion was not associated with any alteration in sensitivity of distal vessels to SIN-1. Similarly, inhibition of the synthesis of endothelium derived relaxing factor (EDRF) by L-NAME resulted in an enhanced response to SIN-1 in proximal vessels only. Conclusions: Endothelium dependent vasodilatation is attenuated by ischaemia and reperfusion in both proximal and distal coronary arteries of the size studied. The response to direct nitric oxide donation (bypassing vascular endothelial synthesis of EDRF) is inhibited by a basal endothelial process present in proximal coronary arteries only. This inhibition is abolished following reperfusion injury or inhibition of NO synthesis. Cardiovascular Research 1993;27:1444-1448

Objective: The aims were to validate the use of a direct reading NO electrode, to compare the effects of diverse acting drugs on altering coronary flow (CF) and NO release, and to examine the effects of altered perfusion pressure on flow-induced changes in NO concentration [NO] in the hemoglobin free effluent of guinea pig isolated hearts. Methods: Hearts were isolated and perfused initially at a constant perfusion pressure (55 mmHg) with a modified Krebs–Ringers solution equilibrated with 97% O2 and 3% CO2 at 37°C. Heart rate, left ventricular pressure, CF, and effluent pH, pCO2, pO2, and NO generated current were monitored continuously on-line. Effluent was sampled for l-citrulline. Percent O2 extraction and O2 consumption were calculated. [NO] was quantitated with a sensitive amperometric sensor (sensitivity ≥1 nmol/l≈3 pA) and a selective gas permeable membrane. Results: The electrode was not sensitive to changes in solution pO2, flow, or pressure. The electrode was sensitive to pCO2 (−0.50 nmol/l/mmHg) and temperature (+24.5 nmol/l/°C), so coronary effluent pCO2 was measured to compensate for a small decrease in pCO2 that occurred with an increase in coronary flow, and effluent temperature was rigidly controlled. Serotonin, bradykinin, and nitroprusside increased NO release along with CF, whereas nifedipine, butanedione monoxime, zaprinast, and bimakalim comparably increased CF but did not increase [NO] or NO release. Increases in CF (ml/g/min) and NO release (pmol/g/min), respectively, were 5.0±1 and 100±17 for 1 μmol/l serotonin, 7.5±1 and 148±18 for 100 nmol/l bradykinin, and 7.8±1 and 173±28 for 100 μmol/l nitroprusside. The increases in effluent NO by bradykinin were proportional to the increases in l-citrulline. Tetraethylammonium decreased CF, but did not change NO release, indomethacin changed neither CF nor NO release, and NG-nitro-l-arginine methyl ester (l-NAME) reduced CF by 2.6±1 ml/g/min and NO release by 25±8 pmol/g/min. An increase of CF of 8.0±0.3 ml/g/min, produced by increasing perfusion pressure from 25 to 90 mmHg, increased [NO] by 30±4 nmol/l; l-NAME but did not reduce the pressure-induced increase in CF, but reduced the increase in [NO] to 10±5 nmol/l. Conclusions: This study demonstrates in intact hearts real-time release of NO by several vasodilator drugs and by pressure-induced increases in flow (shear stress) and attenuation of these effects by l-NAME.