Postischemic Recovery Of Contractile Function Research Articles

Methamphetamine use during the first or second half of pregnancy worsens cardiac ischemic injury in adult female offspring

There is growing evidence that methamphetamine use during pregnancy may produce detrimental cardiovascular effects in the adult offspring. Prior work demonstrated that chronic methamphetamine exposure throughout the gestational period causes adult female offspring to become hypersensitive to myocardial ischemic injury. The goal of the present study was to determine whether this methamphetamine-induced effect occurs early or late in the gestational period. Pregnant female rats were divided into 4 experimental groups. Groups 1 and 2 received subcutaneous injections of saline (group 1) or methamphetamine (5 mg/kg) (group 2) throughout the gestational period. Group 3 received methamphetamine injections on days 1-11 and saline on days 12-22, and group 4 received saline on days 1-11 and methamphetamine on days 12-22. Hearts were isolated from adult (8 weeks) female offspring and subjected to 30 min ischemia and 2 hours reperfusion on a Langendorff isolated heart apparatus. Contractile function was measured via an intraventricular balloon, and infarct size was measured by triphenyltetrazolium chloride staining. Infarcts were significantly larger in methamphetamine exposed offspring regardless of whether they had been exposed to methamphetamine during the first half or the second half of the gestational period. Prenatal exposure to methamphetamine had no effect on preischemic contractile function or postischemic recovery of contractile function. These data indicate that methamphetamine use during either the first half or second half of pregnancy increases susceptibility to myocardial infarction in adult female offspring. These data provide further evidence that prenatal exposure to methamphetamine may increase the risk of developing cardiovascular diseases during adulthood.

Short term methylphenidate treatment does not increase myocardial injury in the ischemic rat heart.

Methylphenidate is commonly used for the treatment of attention deficit hyperactivity disorder. The cardiovascular safety of methylphenidate has been a subject of debate with some studies indicating that methylphenidate increases the likelihood of experiencing a myocardial infarction. However, it is unknown whether methylphenidate worsens the extent of injury during an ischemic insult. The purpose of this study was to determine whether short term exposure to methylphenidate increases the extent of myocardial injury during an ischemic insult. Male and female rats received methylphenidate (5 mg/kg/day) or saline for 10 days by oral gavage. Hearts were subjected to 20 min of ischemia and 2 h of reperfusion on a Langendorff isolated heart apparatus on day 11. Cardiac contractile function was monitored via an intraventricular balloon and myocardial injury was assessed by triphenyltetrazolium chloride staining. Methylphenidate significantly increased locomotor activity in male and female rats, confirming absorption of this psychostimulant into the central nervous system. Male hearts had significantly larger infarcts than female hearts, but methylphenidate had no impact on infarct size or postischemic recovery of contractile function in hearts of either sex. These data indicate that methylphenidate does not increase the extent of injury induced by an ischemic insult.

Methylphenidate Does Not Hypersensitize the Rat Heart to Ischemic Injury

BackgroundMethylphenidate is commonly used for the treatment of attention deficit hyperactivity disorder. The cardiovascular safety of methylphenidate has been a subject of debate with some studies indicating that methylphenidate increases the likelihood of experiencing a myocardial infarction. However, it is unknown whether methylphenidate alters the extent of myocardial injury during an ischemic insult. Previous work demonstrated that a history of methamphetamine exposure increases infarct size and postischemic recovery of contractile function in the ischemic heart in a sex‐dependent manner. Methylphenidate and methamphetamine both increase synaptic concentrations of dopamine and norepinephrine, resulting in enhanced adrenergic and dopaminergic signaling. However, it is unknown whether methylphenidate also hypersensitizes the heart to ischemia. The purpose of this study was to determine whether methylphenidate increases the extent of myocardial injury during an ischemic insult.MethodsMale and female rats were treated with methylphenidate (5 mg/kg/day) or saline for 10 days. Hearts were subjected to a 20 min ischemic insult and 2 hours of reperfusion on a Langendorff isolated heart apparatus on day 11. Cardiac contractile function was monitored via an intraventricular balloon, and myocardial injury was assessed by triphenyltetrazolium chloride staining.ResultsMethylphenidate significantly increased locomotor activity in male and female rats. Male hearts had significantly larger infarcts than female hearts, but methylphenidate had no impact on infarct size or postischemic recovery of contractile function in hearts of either sex.ConclusionThese data indicate that methylphenidate does not increase myocardial sensitivity to ischemic injury.Support or Funding InformationThis work was supported by 1R15HL145546 to BRR.

Effect of Chronic Continuous Normobaric Hypoxia on Functional State of Cardiac Mitochondria and Tolerance of Isolated Rat Heart to Ischemia and Reperfusion: Role of µ and delta2 Opioid Receptors.

Chronic continuous normobaric hypoxia (CNH) increases cardiac tolerance to ischemia/reperfusion injury in vivo and this effect is mediated via µ and delta2 opioid receptors (ORs) activation. CNH has also been shown to be cardioprotective in isolated rat heart. In this study, we hypothesize that this cardioprotective effect of CNH is mediated by activation of µ and delta2 ORs and preservation of mitochondrial function. Hearts from rats adapted to CNH (12 % oxygen) for 3 weeks were extracted, perfused in the Langendorff mode and subjected to 45 min of global ischemia and 30 min of reperfusion. Intervention groups were pretreated for 10 min with antagonists for different OR types: naloxone (300 nmol/l), the selective delta OR antagonist TIPP(psi) (30 nmol/l), the selective delta1 OR antagonist BNTX (1 nmol/l), the selective delta2 OR antagonist naltriben (1 nmol/l), the selective peptide µ OR antagonist CTAP (100 nmol/l) and the selective delta OR antagonist nor-binaltorphimine (3 nmol/l). Creatine kinase activity in coronary effluent and cardiac contractile function were monitored to assess cardiac injury and functional impairment. Additionally, cardiac tissue was collected to measure ATP and to isolate mitochondria to measure respiration rate and calcium retention capacity. Adaptation to CNH decreased myocardial creatine kinase release during reperfusion and improved the postischemic recovery of contractile function. Additionally, CNH improved mitochondrial state 3 and uncoupled respiration rates, ADP/O, mitochondrial transmembrane potential and calcium retention capacity and myocardial ATP level during reperfusion compared to the normoxic group. These protective effects were completely abolished by naloxone, TIPP(psi), naltriben, CTAP but not BNTX or nor-binaltorphimine. These results suggest that cardioprotection associated with adaptation to CNH is mediated by µ and delta2 opioid receptors activation and preservation of mitochondrial function.

Repeated exposure to methamphetamine induces sex-dependent hypersensitivity to ischemic injury in the adult rat heart.

BackgroundWe previously reported that adult female, but not male rats that were prenatally exposed to methamphetamine exhibit myocardial hypersensitivity to ischemic injury. However, it is unknown whether hypersensitivity to ischemic injury develops when rats are exposed to methamphetamine during adulthood. The goal of this study was to determine whether methamphetamine exposure during adulthood sensitizes the heart to ischemic injury.MethodsAdult male and female rats received daily injections of methamphetamine (5 mg/kg) or saline for 10 days. Their hearts were isolated on day 11 and subjected to a 20 min ischemic insult on a Langendorff isolated heart apparatus. Cardiac contractile function was measured by an intraventricular balloon, and infarct size was measured by triphenyltetrazolium chloride staining.ResultsHearts from methamphetamine-treated females exhibited significantly larger infarcts and suppressed postischemic recovery of contractile function compared to hearts from saline-treated females. In contrast, methamphetamine had no effect on infarct size or contractile recovery in male hearts. Subsequent experiments demonstrated that hypersensitivity to ischemic injury persisted in female hearts following a 1 month period of abstinence from methamphetamine. Myocardial protein kinase C-ε expression, Akt phosphorylation, and ERK phosphorylation were unaffected by adult exposure to methamphetamine.ConclusionsExposure of adult rats to methamphetamine sex-dependently increases the extent of myocardial injury following an ischemic insult. These data suggest that women who have a heart attack might be at risk of more extensive myocardial injury if they have a recent history of methamphetamine abuse.

Prenatal methamphetamine differentially alters myocardial sensitivity to ischemic injury in male and female adult hearts.

Methamphetamine is one of the most common illicit drugs abused during pregnancy. The neurological effects of prenatal methamphetamine are well known. However, few studies have investigated the potential effects of prenatal methamphetamine on adult cardiovascular function. Previous work demonstrated that prenatal cocaine exposure increases sensitivity of the adult heart to ischemic injury. Methamphetamine and cocaine have different mechanisms of action, but both drugs exert their effects by increasing dopaminergic and adrenergic receptor stimulation. Thus the goal of this study was to determine whether prenatal methamphetamine also worsens ischemic injury in the adult heart. Pregnant rats were injected with methamphetamine (5 mg·kg(-1)·day(-1)) or saline throughout pregnancy. When pups reached 8 wk of age, their hearts were subjected to ischemia and reperfusion by means of a Langendorff isolated heart system. Prenatal methamphetamine had no significant effect on infarct size, preischemic contractile function, or postischemic recovery of contractile function in male hearts. However, methamphetamine-treated female hearts exhibited significantly larger infarcts and significantly elevated end-diastolic pressure during recovery from ischemia. Methamphetamine significantly reduced protein kinase Cε expression and Akt phosphorylation in female hearts but had no effect on these cardioprotective proteins in male hearts. These data indicate that prenatal methamphetamine differentially affects male and female sensitivity to myocardial ischemic injury and alters cardioprotective signaling proteins in the adult heart.

Effects of the AT1 receptor blocker candesartan on myocardial ischemia/reperfusion in isolated rat hearts.

We sought to investigate the effects of the angiotension II receptor blocker candesartan on ischemia-reperfusion injury using a cardioplegia arrested isolated rat heart model. Ischemia-reperfusion injury was induced in isolated rat hearts with 40 minutes of global ischemia followed by a 30-minute reperfusion protocol. Throughout the experiment, constant pressure perfusion was achieved using a Langendorff apparatus. Cardioplegic solution alone, and in combination with candesartan, was administered before ischemia and 20 minutes after ischemia. Post-ischemic recovery of contractile function, left ventricular developed pressure, left ventricular end-diastolic pressure and contraction and relaxation rates were evaluated. In the control group, left ventricular developed pressure, rate pressure product, contraction and relaxation rates and coronary flow significantly decreased but coronary resistance increased following reperfusion. With the administration of candesartan alone, parameters did not differ compared to controls. Contractile parameters improved in the group that received candesartan in combination with the cardioplegia compared to the group that received cardioplegia alone; however, the difference between these two groups was insignificant. In this study, the addition of candesartan to a cardioplegic arrest protocol routinely performed during cardiac surgery did not provide a significant advantage in protection against ischemia-reperfusion injury compared with the administration of cardioplegic solution alone.

Mitigation of postischemic cardiac contractile dysfunction by CaMKII inhibition: effects on programmed necrotic and apoptotic cell death

While Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been suggested to be an important protein regulating heart function upon ischemia/reperfusion (I/R), the mechanisms responsible are not fully known. Furthermore, it is not known whether CaMKII activation can modulate necroptosis, a recently described form of programmed cell death. In order to investigate these issues, Langendroff-perfused rat hearts were subjected to global ischemia and reperfusion, and CaMKII inhibition was achieved by adding the CaMKII inhibitor KN-93 (0.5μmol/dm(3)) to the perfusion solution before the induction of ischemia. Immunoblotting was used to detect changes in expression of proteins modulating both necroptotic and apoptotic cell death. CaMKII inhibition normalized I/R induced increases in expression of necroptotic RIP1 and caspase-8 along with proteins of the intrinsic apoptotic pathway, namely cytochrome c and caspase-9. In addition, it increased the Bcl-2/Bax ratio and reduced caspase-3 and cleaved PARP1 content suggesting reduction of cell death. These changes coexisted with improvement of postischemic contractile function. On the other hand, there was no correlation between levels of pT287-CaMKIIδ and LVDP recovery after I/R. These results demonstrate for the first time that CaMKII inhibition may mitigate cardiac contractile dysfunction, at least partially, by limiting the contents of not only apoptotic, but also necroptotic proteins. Phosphorylation of CaMKII seems unlikely to determine the degree of postischemic recovery of contractile function.

Creatine Phosphate: Pharmacological and Clinical Perspectives

Since the 1970s, extensive experimental and clinical research has demonstrated that relevant reductions of creatine phosphate (CrP) or phosphocreatine availability occur in a wide spectrum of pathophysiological situations. A decrease in intracellular concentrations of creatine (Cr) and CrP results in a hypodynamic state of cardiac and skeletal muscle pathology. Many experimental and clinical studies have evaluated the possibility to improve cardiac and skeletal muscle performance by exogenous administration of CrP. Furthermore, many experimental studies have shown that CrP may play two important roles in the regulation of muscle energetics and work. First, CrP maintains local adenosine triphosphate pools and stabilizes cellular membranes due to electrostatic interactions with phospholipids. The second mechanism decreases the production of lysophosphoglycerides in hypoxic hearts, protects the sarcolemma of cardiac cells against ischemic damage, decreases the frequency of arrhythmias, and increases post-ischemic recovery of contractile function. Recent research on CrP has demonstrated positive therapeutic results in various clinical applications. These benefits have been applied in several pathological conditions, such as heart failure, acute myocardial ischemia, chronic ischemic heart disease, cardiac surgery, skeletal muscle hypotonotrophy, and cerebral ischemia. This review describes the CrP shuttle, pathophysiological basis of the supplementation of CrP, and its therapeutic effects in multiple clinical conditions. The major aim is to summarize results of the intense research carried out over 40 years to provide evidence to support the adjunctive use of CrP in many pathological conditions that may target cellular energy impairment; thus, increasing energy metabolism.

Inhibition of Gαi2 – RGS Protein Interactions Protects the Heart from Ischemic Injury

Signaling through Gαi-coupled receptors protects the heart from ischemic injury. The activity of Gαi is modulated by regulator of G protein signaling (RGS) proteins that terminate Gαi signaling by catalyzing the hydrolysis of Gαi-bound GTP. We used knock-in mice in which interactions between Gαi2 and RGS proteins are disrupted by a Gαi2G184S (GS) point mutation to determine whether interactions between Gαi2 and endogenous RGS proteins modulate Gαi-mediated protection from ischemic injury. Hearts isolated from wildtype (+/+) mice and mice homozygous (GS/GS) or heterozygous (GS/+) for Gαi2G184S were subjected to 30 minutes ischemia and 2 hours reperfusion using the Langendorff method. Infarcts in GS/GS and GS/+ hearts were significantly smaller than those of +/+ hearts. Expression of Gαi2G184S had no effect on preischemic contractile function. However, postischemic recovery of contractile function was significantly enhanced in GS/GS and GS/+ hearts compared to +/+ hearts. The cardioprotective phenotype of GS/+ and GS/GS hearts was reversed by 5-hydroxydecanoate indicating that inhibition of RGS- Gαi2 interactions protects the heart through a mechanism that requires mitochondrial K+ATP channels. These data suggest that RGS proteins may provide novel therapeutic targets to protect the heart from ischemic injury. (Supported by NIH R01 GM39561; AFPE, and the Bower, Bennet and Bennet Endowed Research Award).

Systems bioenergetics of creatine kinase networks: physiological roles of creatine and phosphocreatine in regulation of cardiac cell function

Physiological role of creatine (Cr) became first evident in the experiments of Belitzer and Tsybakova in 1939, who showed that oxygen consumption in a well-washed skeletal muscle homogenate increases strongly in the presence of creatine and with this results in phosphocreatine (PCr) production with PCr/O(2) ratio of about 5-6. This was the beginning of quantitative analysis in bioenergetics. It was also observed in many physiological experiments that the contractile force changes in parallel with the alteration in the PCr content. On the other hand, it was shown that when heart function is governed by Frank-Starling law, work performance and oxygen consumption rate increase in parallel without any changes in PCr and ATP tissue contents (metabolic homeostasis). Studies of cellular mechanisms of all these important phenomena helped in shaping new approach to bioenergetics, Molecular System Bioenergetics, a part of Systems Biology. This approach takes into consideration intracellular interactions that lead to novel mechanisms of regulation of energy fluxes. In particular, interactions between mitochondria and cytoskeleton resulting in selective restriction of permeability of outer mitochondrial membrane anion channel (VDAC) for adenine nucleotides and thus their recycling in mitochondria coupled to effective synthesis of PCr by mitochondrial creatine kinase, MtCK. Therefore, Cr concentration and the PCr/Cr ratio became important kinetic parameters in the regulation of respiration and energy fluxes in muscle cells. Decrease in the intracellular contents of Cr and PCr results in a hypodynamic state of muscle and muscle pathology. Many experimental studies have revealed that PCr may play two important roles in the regulation of muscle energetics: first by maintaining local ATP pools via compartmentalized creatine kinase reactions, and secondly by stabilizing cellular membranes due to electrostatic interactions with phospholipids. The second mechanism decreases the production of lysophosphoglycerides in hypoxic heart, protects the cardiac cells sarcolemma against ischemic damage, decreases the frequency of arrhythmias and increases the post-ischemic recovery of contractile function. PCr is used as a pharmacological product Neoton in cardiac surgery as one of the components of cardioplegic solutions for protection of the heart against intraoperational injury and injected intravenously in acute myocardial ischemic conditions for improving the hemodynamic response and clinical conditions of patients with heart failure.

Exercise training improves functional post-ischemic recovery in senescent heart

The incidence of ischemic cardiac diseases increases with age and elderly subjects are more vulnerable to myocardial infarction. Endurance exercise (e.g. treadmill training) provides cardioprotection against an ischemia and reperfusion (IR) event in adult heart but such a potential beneficial effect of regular exercise has never been evaluated during aging. Therefore, this study investigated the effects of moderate running training on post-ischemic recovery of contractile function and coronary perfusion in senescent myocardium. Isolated hearts of sedentary (24 mo-sedentary; n = 10) and trained senescent (24 mo-trained; n = 11; moderate running: 1 h/day, 5 days/week for 12 weeks) rats were submitted to 45 min low-flow ischemia (15% of initial coronary flow (CF)) followed by 30 min reperfusion. Active tension (AT) and CF were recorded at baseline and after 1, 3, 5, 10, 15, 20, 25 and 30 min of reperfusion. Left ventricular protein carbonylation, and both heat-shock-protein 70 (HSP70) and endothelial nitric oxide synthase (eNOS) contents were determined by Oxyblotting and Western blotting, respectively. Regular physical exercise improves impairment of functional post-ischemic recovery (AT and CF) of aged hearts during reperfusion and this cardioprotection is associated to limited protein oxidation and increased HSP70 and eNOS myocardial contents.

Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.

Role of high molecular weight isoforms of fibroblast growth factor-2 (FGF-2) in cardiac ischemia–reperfusion injury (I/R)

Our lab showed that FGF2 is an important cardioprotective molecule in I/R injury. Different FGF2 isoforms (low [LMW] and high [HMW] molecular weight isoforms) from different translation start sites exist and are differentiately localized, indicating possible unique biological functions. Our lab showed that FGF2 LMW isoform plays a role in protecting the heart from myocardial dysfunction via inhibition of JNK signaling and ultimately apoptosis. The biological actions of the HMW isoforms in I/R injury remain to be elucidated. To delineate the role of FGF2 HMW isoforms in cardioprotection, murine hearts that are deficient of HMW isoforms (FGF2 HMWKO) or that overexpress the human 24 kDa HMW isoform (24 kDa FGF2 Tg) were subjected to 60 min of global, low-flow ischemia and 120 min of reperfusion. Post-ischemic recovery of contractile function was markedly reduced in the 24 kDa transgenic vs. non-transgenic hearts (p<0.05). Conversely, there was a significant improvement in post-ischemic recovery of contractile function in FGF2 HMWKO vs. wildtype (Wt) hearts (p<0.05). There was a significant decrease in creatine kinase release in FGF2 HMWKO vs. Wt hearts. These results provide molecular evidence that the HMW isoform(s) of FGF2 plays a deleterious role in restoring cardiac function and reducing myocardial cell death following I/R injury. These findings begin to delineate the differential roles of LMW and HMW isoforms in I/R injury and cardioprotection, and may lead to the development of novel therapeutic FGF2 interventions in patients susceptible to or with ischemic heart disease.

The cardioprotective effect of the low molecular weight isoform of fibroblast growth factor-2: The role of JNK signaling

Our laboratory showed that overexpression of fibroblast growth factor-2 (FGF2) protected the heart against ischemia–reperfusion injury. FGF2 has different protein isoforms (low [LMW] and high [HMW] molecular weight isoforms) produced from alternative translation start sites. However, which FGF2 isoform(s) mediates this cardioprotection, and which signaling pathway (i.e., mitogen-activated protein kinase (MAPK)) elicits FGF2 isoform-induced cardioprotection remains to be elucidated. Methods and results: Wildtype, Fgf2 KO (absence of all FGF2 isoforms) and FGF2 LMWKO (absence of LMW isoform) hearts were subjected to an ex vivo work-performing heart ischemic model of 60 min ischemia and 120 min reperfusion. There was a significant decrease in the recovery of post-ischemic contractile function (p<0.05) in Fgf2 KO and FGF2 LMWKO mouse hearts compared to wildtype hearts. Following ischemia–reperfusion injury, MKK4/7, JNK, and c-Jun were significantly phosphorylated (i.e., activated), and the levels of TUNEL-positive nuclei and caspase 3 cleavage were significantly increased in vehicle-treated Fgf2 KO and FGF2 LMWKO compared to wildtype hearts (p<0.05). A novel JNK pathway inhibitor, CEP11004 (50 nM), significantly restored the post-ischemic contractile function and reduced myocardial cell death, as measured by CK release and apoptotic markers, compared to DMSO-treated cohorts (p<0.05). Overall, our data indicate that the LMW isoform has an important role in restoring cardiac function after ischemia–reperfusion (I/R) injury. These results provide unequivocal evidence that inhibition of JNK signaling is involved in FGF2 LMW isoform-mediated cardioprotection and that the potential mechanism may be through inhibition of the apoptotic process.

High dietary sucrose triggers hyperinsulinemia, increases myocardial β-oxidation, reduces glycolytic flux and delays post-ischemic contractile recovery

Although the causal relationship between insulin resistance (IR) and hypertension is not fully resolved, the importance of IR in cardiovascular dysfunction is recognized. As IR may follow excess sucrose or fructose diet, the aim of this study was to test whether dietary starch substitution with sucrose results in myocardial dysfunction in energy substrate utilization and contractility during normoxic and post-ischemic conditions. Forty-eight male Wistar rats were randomly allocated to three diets, differing only in their starch to sucrose (S) ratio (13, 2 and 0 for the Low S, Middle S and High S groups, respectively), for 3 weeks. Developed pressure and rate x pressure product (RPP) were determined in Langendorff mode-perfused hearts. After 30 min stabilization, hearts were subjected to 25 min of total normothermic global ischemia, followed by 45-min reperfusion. Oxygen consumption, beta-oxidation rate (using 1-13C hexanoate and Isotopic Ratio Mass Spectrometry of CO2 produced in the coronary effluent) and flux of non-oxidative glycolysis were also evaluated. Although fasting plasma glucose levels were not affected by increased dietary sucrose, high sucrose intake resulted in increased plasma insulin levels, without significant rise in plasma triglyceride and free fatty acid concentrations. Sucrose-rich diet reduced pre-ischemic baseline measures of heart rate, RPP and non-oxidative glycolysis. During reperfusion, post-ischemic recovery of RPP was impaired in the Middle S and High S groups, as compared to Low S, mainly due to delayed recovery of developed pressure, which by 45 min of reperfusion eventually resumed levels matching Low S. At the start of reperfusion, delayed post-ischemic recovery of contractile function was accompanied by: (i) reduced lactate production; (ii) decreased lactate to pyruvate ratio; (iii) increased beta-oxidation; and (iv) depressed metabolic efficiency. In conclusion, sucrose rich-diet increased plasma insulin levels, in intact rat, and increased cardiac beta-oxidation and coronary flow-rate, but reduced glycolytic flux and contractility during normoxic baseline function of isolated perfused hearts. Sucrose rich-diet impaired early post-ischemic recovery of isolated heart cardiac mechanical function and further augmented cardiac beta-oxidation but reduced glycolytic and lactate flux.

Role of the α2-isoform of AMP-activated protein kinase in the metabolic response of the heart to no-flow ischemia

AMP-activated protein kinase (AMPK) is a major sensor and regulator of the energetic state of the cell. Little is known about the specific role of AMPKalpha(2), the major AMPK isoform in the heart, in response to global ischemia. We used AMPKalpha(2)-knockout (AMPKalpha(2)(-/-)) mice to evaluate the consequences of AMPKalpha(2) deletion during normoxia and ischemia, with glucose as the sole substrate. Hemodynamic measurements from echocardiography of hearts from AMPKalpha(2)(-/-) mice during normoxia showed no significant modification compared with wild-type animals. In contrast, the response of hearts from AMPKalpha(2)(-/-) mice to no-flow ischemia was characterized by a more rapid onset of ischemia-induced contracture. This ischemic contracture was associated with a decrease in ATP content, lactate production, glycogen content, and AMPKbeta(2) content. Hearts from AMPKalpha(2)(-/-) mice were also characterized by a decreased phosphorylation state of acetyl-CoA carboxylase during normoxia and ischemia. Despite an apparent worse metabolic adaptation during ischemia, the absence of AMPKalpha(2) does not exacerbate impairment of the recovery of postischemic contractile function. In conclusion, AMPKalpha(2) is required for the metabolic response of the heart to no-flow ischemia. The remaining AMPKalpha(1) cannot compensate for the absence of AMPKalpha(2).

Peroxisome proliferator-activated receptor alpha agonist, clofibrate, has profound influence on myocardial fatty acid composition

The hypolipidemic fibrates have been identified as agonists of the peroxisome proliferator-activated receptor α (PPARα), which plays a critical role in the regulation of cardiac fatty acid metabolism. Despite the widespread clinical use of fibrates, their role in myocardial oxidative stress and fatty acid composition is less known. In this study, male Sprague–Dawley rats were treated with either vehicle (olive oil, 1 ml/kg) or clofibrate (300 mg/kg day i.p.) for 1–14 days. Lipid peroxidation in heart homogenate was determined by thiobarbituric acid reactive substance (TBARS) assay. Results show that hearts from clofibrate-treated rats are more susceptible to FeSO 4-induced TBARS production. The antioxidants including catalase and glutathione-related enzymes were marginally affected. We demonstrated that myocardial fatty acid composition was dramatically altered by clofibrate treatment. In hearts from clofibrate-treated rats, the principal n − 6 polyunsaturated fatty acids (PUFAs), linoleic acid (C18:2 n − 6) and arachidonic acid (C20:4 n − 6), was significantly reduced, while the content of the principal n − 3 PUFA, docosahexaenoic acid (C22:6 n − 3), was markedly increased. The overall effect was to reduce n − 6/ n − 3 ratio and increase the unsaturation extent of myocardial fatty acids. Functional study showed that hearts from clofibrate-treated rats had an improved recovery of post-ischemic contractile function and reduced ischemia/reperfusion (I/R)-induced infarct size. The data shows that clofibrate has a profound impact on cardiac fatty acid composition, which may contribute to its cardioprotective effect.

Mechanisms of Erythropoietin-Mediated Cardioprotection during Ischemia-Reperfusion Injury: Role of Protein Kinase C Signaling.

The biologic effects of erythropoietin (EPO) are mediated by its cellular receptor EPOR, a member of the cytokine receptor superfamily. EPOR expression in non-hematopoietic cells is associated with novel biologic effects for EPO in diverse organ systems. We recently demonstrated functional EPOR expression in adult rat cardiac myocytes and found that recombinant EPO exerts a rapid cardioprotective effect during ischemia-reperfusion injury of the isolated, perfused heart. Here we investigated the mechanisms of the cardioprotective effect of EPO using Langendorff-perfused rat hearts while left-ventricular-developed pressure (LVDP) was measured continuously to assess contractile function. Hearts were treated directly with EPO in the presence or absence of inhibitors of specific signal transduction pathways prior to normothermic global ischemia followed by reperfusion. Post-ischemic recovery of contractile function was determined by measuring LVDP at the end of reperfusion and expressed as a percentage of the baseline pre-treatment measurement. We investigated EPO-mediated activation of signal transduction pathways in the isolated, perfused heart and observed phosphorylation of p44/p42 MAP kinases ERK 1/2 (Thr202/Tyr204) and protein kinase B/Akt (Ser473), a downstream target of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Furthermore, EPO treatment of the isolated, perfused heart was associated with translocation of protein kinase C (PKC) ε and δ isoforms to the membrane fraction. We investigated the role of specific signaling pathways in EPO-mediated cardioprotection by employing inhibitors targeting PI3K, PKC and MAP kinase kinase (MEK1). PI3K inhibitors LY294002 and wortmannin attenuated EPO-induced phosphorylation of Akt but had no effect on EPO-mediated cardioprotection. MEK1 inhibitor U0126 had no effect on EPO-mediated cardioprotection. The PKC catalytic inhibitor chelerythrine (chel) significantly inhibited EPO-mediated improvement in post-ischemic recovery of LVDP (figure 1). Hearts pre-treated with EPO exhibited significantly improved post-ischemic recovery of LVDP compared to control hearts (mean±SE: 72±3 in EPO-treated versus 35±3% in control hearts, P<0.05 by ANOVA and Bonferroni post-hoc test, n=10 experiments each group) and the protective effect of EPO was significantly inhibited in chel-treated hearts (52±4% in EPO+chel versus 72±3% in EPO-treated hearts, P<0.05, n=10). As a control, treatment of the hearts with chelerythrine alone had no significant effect on LVDP (49±4%) compared to control hearts. These data demonstrate that EPO-mediated activation of the PKC signaling pathway is required for the cardioprotective effect of EPO during ischemia-reperfusion injury. [Display omitted]

Oxygen delivery modulates intracellular hydrogen peroxide generation during initial reperfusion of the isolated ischemic rat heart

Study objectives: Reactive oxygen species (ROS) generated during reperfusion of the ischemic heart play a major role in myocyte injury and may be dependent on changes in O₂ delivery (DO₂). We hypothesize that during low-flow reperfusion (eg, cardiopulmonary resuscitation after cardiac arrest), DO₂ modulates ROS formation and affects contractile recovery in the postischemic period. Methods: Isolated perfused rat hearts were loaded with 25 μM of dihydrofluorescein-diacetate (HFLUOR) and then subjected to 5 minutes of global ischemia. Controlled reperfusion consisted of 5 minutes of variable DO₂, ranging from 437.8 to 70.7 mL O₂/min before restoration of full baseline perfusion. Intracellular ROS was quantitated using surface fluorometry at the left ventricle to measure the oxidation of HFLUOR to fluorescein, reflecting H₂O₂ generation. Cellular NADH changes and contractile function were simultaneously measured. Results: At reperfusion, a burst of H₂O₂ was observed, which was significantly reduced by lowering DO₂ (354.4, 22.8 to 84.7; 4.2 mL O₂/min) during 5 minutes of controlled reperfusion (n=8/group, 0.47, 0.05 AU, versus 0.88; 0.14 AU, <i>P</i>=.035). However, this reduction did not correlate with improved functional recovery. Hearts reperfused with transient lower DO₂ before full reperfusion had a lower percentage of recovery of left ventricular–developed pressure (39.2%, 7.6% versus 73.5%; 6.3, <i>P</i><.01), and rate-pressure product (84.8%, 13.7 versus 27.4%; 4.9, <i>P</i><.01). Conclusion: Controlled reperfusion using an initial lower DO₂ reduces the intracellular ROS burst but does not improve recovery of postischemic contractile function.