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Corticotrophin-releasing factor receptor 2β (CRFR2β) is expressed in the myocardium. In the present study we explore whether acute treatment with the neuropeptide corticotrophin-releasing factor (CRF) could induce cytoprotection against a lethal ischemic insult in the heart (isolated murine neonatal cardiac myocytes and the isolated Langendorff perfused rat heart) by activating CRFR2. In vitro, CRF offered cytoprotection when added prior to lethal simulated ischemic stress by reducing apoptotic and necrotic cell death. Ex vivo, CRF significantly reduced infarct size from 52.1±3.1% in control hearts to 35.3±3.1% (P

The aim of the present study was to determine whether induction of ischaemic heart failure by micro-embolization leads to only a single episode of myocardial injury or whether it sets up a vicious cycle of progressive myocardial damage. Acute left ventricular (LV) failure was produced in 15 closed-chest anaesthetized dogs by injection of 50 microns plastic microspheres into the left main coronary artery. The dogs showed signs of severely depressed LV function; there was a marked increase in LV end-diastolic pressure and a marked decrease in stroke volume. Myocardial lactate uptake decreased or reversed to production. Six dogs with very high LV end-diastolic pressure died during the subsequent 3 days and autopsy revealed pulmonary edema. The LV function was re-examined in four dogs at 2 and 4 weeks after embolization. Except for a modest elevation of LV end-diastolic pressure there were no haemodynamic or metabolic signs of myocardial dysfunction. Gross and light microscopic examination of the heart in dogs 8 hours to 6 weeks following microsphere injections revealed numerous small infarcts or focal areas of granulation or scar tissue throughout the entire left ventricle. At 1 to 6 weeks close to the infarcts there were scattered myocytes with strong eosinophilia and pyknosis or loss of nuclei, interpreted as myocytolysis. In two dogs killed at six weeks after the embolization there were areas of granulation tissue, similar to a recent infarction about 1 week old. Thus, in spite of apparent functional restoration there were morphological signs of repeated and progressive myocardial injury several weeks after coronary embolization.

The purpose of this study was to determine if elevated myocardial glycogen content could obviate Ca(2+) overload and subsequent myocardial injury in the setting of low oxygen and diminished exogenous substrate supplies. Isolated harp seal cardiomyocytes, recognized as having large glycogen stores, were incubated under conditions simulating ischemia (oxygen and substrate deprivation) for 1 h. Rat cardiomyocytes were used for comparison. Freshly isolated seal cardiomyocytes contained approximately 10 times more glycogen than those from rats (479 +/- 39 vs. 48 +/- 5 nmol glucose/mg dry weight (dry wt), mean +/- S.E., n = 6), and during ischemia lactate production was significantly greater in seal compared to rat cardiomyocytes (660 +/- 99 vs. 97 +/- 14 nmol/mg dry wt), while glycogen content decreased both in seal (from 479 +/- 39 to 315 +/- 58 nmol glucose/mg dry wt) and rat cardiomyocytes (from 48 +/- 5 to 18 +/- 5 nmol glucose/mg dry wt). Cellular ATP was well maintained in ischemic seal cardiomyocytes, whereas it showed a 65% decline (from 31 +/- 3 to 11 +/- 1 nmol ATP/mg dry wt) in rat cardiomyocytes. Similarly, total seal cardiomyocyte Ca(2+) content was not affected by ischemia, while Ca(2+) increased from 8.5 +/- 2.0 to 13.3 +/- 2.0 nmol/mg dry wt in ischemic rat myocytes. Rat cardiomyocytes also showed a notable decline in the percentage of rod-shaped cells in response to ischemia (from 66 +/- 4% to 30 +/- 3%), and cell morphology was unaffected in seal incubations. Addition of iodoacetate (IAA, an inhibitor of glycolysis) to seal cardiomyocytes, on top of substrate and oxygen deprivation, reduced the cellular content of ATP by 52.9 +/- 4.4% (from 25 +/- 4 to 11 +/- 2 nmol ATP/mg dry wt) and the percentage of rod-shaped myocytes from 51 +/- 3% to 28 +/- 4%, while total Ca(2+) content was unchanged by these conditions. Seal cardiomyocytes thus tolerate low oxygen conditions better than rat cardiomyocytes. This finding is most likely due to a higher glycolysis rate in seals, fueled by larger myocardial glycogen stores.

Insulin administration attenuates cardiac ischemia-reperfusion apoptosis via activation of Akt-mediated cell-survival signaling. As p70s6 kinase is a cognate Akt-mediated phosphorylation target we evaluated whether p70s6 kinase activation is a functional requirement in insulin-mediated cell survival program during post-ischemic reoxygenation. Human cardiac-derived girardi cells were subjected to 6h of simulated ischemia and 2h of reoxygenation+/-insulin treatment [0.3mU/ml]. Concurrently, cells were pre-treated with anti-sense oligodeoxynucleotides (ODNs) corresponding to the initiation start-site of human p70s6 kinase mRNA. Sense ODN and scrambled ODN were used as controls. Cell viability was measured using lactate dehydrogenase (LDH) release and propidium iodide (PI) exclusion. Insulin at reoxygenation enhanced cell viability with attenuated LDH release (or=50% , p0.001 vs. ischemic controls) and reduced PI uptake byor=30% vs. ischemic controls. The protection afforded by insulin was abolished by anti-sense ODN targeting p70s6 kinase, but not by the sense or scrambled ODNs. In parallel, insulin administration at reoxygenation significantly increased p70s6 kinase levels and activity compared with controls. P70s6 kinase activity was abolished by pre-treatment with anti-sense ODNs. Collectively, these data demonstrate that p70s6 kinase activation is a functional target of Akt following insulin-activated cytoprotection during ischemia-reoxygenation-induced injury.

Coronary reperfusion improves ventricular function and survival after infarction, but the metabolic conditions at this time may not be optimal to protect the heart. The objective of this study was to evaluate if metabolic support with glucose-insulin-potassium (GIK) administered at the time of coronary reperfusion could elicit the same cardioprotection as GIK infusion during the entire ischemia/reperfusion period. Three groups of anesthetized, open-chest rats were subjected to 30 minutes of regional ischemia and 180 minutes of reperfusion. Groups 1 (controls) and 2 (GIKIR) received saline or GIK, respectively, throughout the whole experimental period, whereas a third group (GIKR) received GIK from the onset of reperfusion only. Infarct size was significantly reduced in the GIK-treated groups, compared with controls (GIKIR 44 ± 5% and GIKR 45 ± 5% vs. control 66 ± 4%; P < 0.05). Postischemic recovery of cardiac function improved when GIK was only administered during the reperfusion phase. Furthermore, infusion of GIK resulted in reduced plasma concentrations of free fatty acids and increased plasma glucose (both P < 0.05) compared with controls. This study demonstrates that glucose-insulin-potassium administration at the onset of the postischemic reperfusion period is as cardioprotective as administration of GIK during the entire ischemia/reperfusion period.

Fuel substrate utilization is highly regulated during cardiac development and with the onset of cardiac hypertrophy. Glucose and lactate are the predominant fuel substrates utilized during cardiac development. Postnatally, a switch occurs so that fatty acids become the chief energy substrate in the nonfed adult mammalian heart. A reversion back towards fetal energy metabolism occurs with the development of cardiac hypertrophy. To evaluate the role of this substrate preference switch in the development of cardiac hypertrophy, the molecular regulation directing these switches is being explored. Thus, we have begun by defining the temporal expression patterns of genes encoding key rate-controlling enzymes directing major fuel substrate metabolism during cardiac development, with pressure-overload-induced cardiac hypertrophy, and following antihypertensive therapy in spontaneously hypertensive rats. The genes encoding the fatty acid and adult enriched rate-controlling glycolytic enzymes are expressed at low levels in the fetal and neonatal rat heart. The genes encoding these enzymes are significantly and coordinately upregulated (≥ 70%) in adult rat hearts compared to the fetal expression patterns. A reciprocal and coordinate downregulation (≥ 40% reduction) of the fatty acid and adult enriched glycolytic enzyme encoding genes are observed with the induction of pressure-overload-induced hypertrophy in spontaneously hypertensive rats compared to Wistar–Furth normotensive control rats. Antihypertensive therapy with carvedilol, a vasodilating α-and β-adrenoreceptor antagonist, attenuates this reversion of the metabolic gene expression pattern towards fetal levels compared to placebo-treated littermate controls. This coordinate developmental and hypertrophy-induced regulation of genes that encode enzymes controlling both fatty acid and glycolytic catabolic pathways in the heart implicates potential mutual/overlapping regulatory signaling proteins within their gene regulatory programs. These gene regulatory pathways need to be identified and modulated in order to characterize the functional role of fuel substrate metabolism in cardiac development and with the induction of cardiac hypertrophy.

Aim: To compare the possible role of Akt and mammalian target of rapamycin (mTOR) in mediating cardioprotection against ischaemia under three different conditions: (1) During ischaemic preconditioning (IPC), (2) when insulin was given as a pretreatment agent (InsPC) and (3) when insulin was given as a reperfusion cell survival agent (InsR). Methods: Isolated perfused rat hearts were subjected to IPC (3 × 5 min) or InsPC (50 mU mL−1; 3 × 5 min), before 30 min of regional ischaemia followed by 120 min of reperfusion ± 1L-6-hydroxymethyl-chiro-inositol-2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (HIMO) (20 μm; Akt inhibitor) or rapamycin (1 nm; mTOR inhibitor). In addition, insulin (3 mU mL−1) was given at the onset of reperfusion, ±HIMO or rapamycin. Risk zone (R) and infarct size (I) were determined with Evans blue and tetrazolium staining respectively. Western blot analysis was performed on tissue from Langendorff-perfused rat hearts and cell lysates from cultured HL1 cells. Results: IPC, InsPC and InsR treatment resulted in a significant reduction in infarct size compared to controls (all P

The production of oxygen free radicals (OFR) by leucocytes was evaluated ex vivo by chemiluminescence (CL) before, during and after routine coronary artery bypass surgery (group A, n=11). The possibility of leucocyte trapping in the coronary circulation during the early reperfusion period was also investigated (group B, n=9). In group A, arterial blood samples were taken immediately before the start of surgery during anaesthesia, five minutes before and five and 30 minutes after the start of cardiopulmonary bypass (CPB), five minutes before and five and 30 minutes after the start of reperfusion of the heart, and then four and 24 hours after the end of CPB. In group B, arterial and coronary sinus blood samples were simultaneously drawn five and 30 minutes after the release of the aortic crossclamp. All blood samples were corrected for haemodilution. In group A, both CL and the level of circulating leucocytes declined during CPB. The lowest value of CL was measured 30 minutes after the start of CPB (69± 2% of baseline values) (mean±SEM). The lowest level of leucocytes was found after 30 minutes of CPB: 2.6±0.4 (109/l) vs 4.2±0.5 before surgery. Twenty-four hours after CPB, CL was increased to 170±49% and a leucocytosis was present (12.2±1.1). In group B, after five minutes of reperfusion the number of circulating leucocytes in arterial blood was 3.8±0.9 x 109/l as compared to 2.2±0.5 x 109/l in the coronary sinus (p

Polymorphonuclear granulocytes (PMN) are suggested mediators of myocardial ischemia-reperfusion injury. We have previously shown that activated PMN producing oxygen free radicals (OFR) in the coronary circulation are cardiodepressive. OFR may induce lipid peroxidation and production of eicosanoids. We have investigated the influence of cyclo-oxygenase and lipoxygenase inhibitors on the effects of activated, OFR producing PMN in the Langedorff rat heart model. Left ventricular developed pressure (VLDP) was measured by a balloon in the left ventricle. Human PMN and drugs were given into the aortic cannula for 10 min and the hearts were observed for 30 min thereafter. After infusion for 5 min OFR production in the cellular infusate was measured at the level of the aortic cannula by a chemiluminescence (CL) technique. Phorbol 12-myristate 13-acetate (PMA)-activated PMN ( n = 8), produced a CL response of 27649 ± 11048 counts (mean ± s.e.m. ), and reduced coronary flow (CF) to 53 ± 6% (mean ± s.e.m. ) and LVDP to 38 ± 9% of baseline values at the end of the observation period. Ibuprofen ( n = 6), a cyclooxygenase (CO) inhibitor, neither influenced the CL response (31915 ± 7563) of activated PMN, nor the reduction of CF and LVDP at this time. Although both BW 755C ( n = 7), a dual inhibitor of CO and lipoxygenase (LO) (CF:90 ± 4%, LVDP:99 ± 6%) and diethylcarbamazine (DCM) ( n = 8), a LO inhibitor (CF:88 ± 11%, LVDP:87 ± 4%), significantly inhibited the cardiodepressive effects of activated PMN. BW 755C alone abolished the CL response (431 ± 158 counts), whereas DCM had no effect on CL (30105 ± 1698 counts). In conclusion, the injurious effects of activated leukocytes in the coronary circulation seem to be mediated through LO products.

The effects of insulin on human beta-adrenoceptor density and isoproterenol-induced cyclic AMP (cAMP) accumulation were characterized in mononuclear leukocytes from healthy subjects. In cells equilibrated with theophylline (4 mM) at 37 degrees C, insulin (4 microU/ml) was present in periods from 1 to 35 min prior to stimulation. The basal cAMP levels were not influenced. After 1 min pretreatment with insulin, the (-)-isoproterenol concentration necessary to cause half-maximal stimulation (EC50) decreased from 260 to 170 nM (P less than 0.025) and the maximal (-)-isoproterenol response above basal increased from 44 to 63 pmol/10(6) cells (P less than 0.01). The short exposure to insulin caused an increase in the number of functional beta-adrenoceptors from 1420 to 2160 receptors/cell (P less than 0.01). The increased (-)-isoproterenol responsiveness showed a time-dependent decline. When insulin had been present for 35 min before stimulation, the EC50 value had increased to 600 nM (P less than 0.01 vs. control) and the maximal (-)-isoproterenol response above basal was reduced to 29 pmol/10(6) cells (P less than 0.01 vs. control). The receptor density decreased to the pretreatment value (1480 receptors/cell) after 35 min exposure to insulin. The present study shows that insulin modifies the beta-adrenoceptor density as well as the beta-adrenoceptor coupling to adenylate cyclase, dependent on the duration of exposure.

The authors assessed whether intracoronary injection of low osmolality contrast media induces metabolic and electrocardiographic changes consistent with myocardial ischemia in anesthetized dogs. Ioxaglate and iohexol were injected into the left main coronary artery (eight dogs) and into a carotid-coronary artery shunt (eight dogs), during free coronary flow and during 50% flow reduction. Blood samples were obtained simultaneously from a femoral artery and from a small cardiac vein draining the contrast perfused area. Contrast media had no immediate or late effects on lactate balance during free or reduced flow. Early depression of the ST segment in epicardial ECG did not reflect ischemia. The authors conclude that the two low-osmolality contrast media, iohexol and ioxaglate, did not induce ischemic changes in the myocardium.

To evaluate the significance of the JAK-STAT pathway in insulin-induced cardioprotection from reperfusion injury.In isolated perfused rat hearts subjected to insulin therapy (0.3 mU/ml) +/- AG490 (5 microM, JAK-STAT inhibitor), the phosphorylation state of STAT3 and Akt was determined after 15 min of reperfusion. Infarct size was measured after 120 min of reperfusion. Isolated cardiac myocytes from wild type (WT) and cardiac specific STAT3 deficient mice were treated with insulin at reoxygenation following simulated ischemia (SI, 26 h). Cell viability was measured after 120 min of reoxygenation following SI, whereas phosphorylation state of Akt was measured after 15 min of reoxygenation following SI.Insulin given at reperfusion led to phosphorylation of STAT3 and Akt both of which were inhibited by AG490. AG490 also blocked the insulin-dependent decrease in infarct size, supporting a role for JAK-STAT in cardioprotection. In addition, insulin protection from SI was blocked in myocytes from the STAT3 deficient mice, or in WT mice treated with AG490. Furthermore, insulin failed to phosphorylate Akt in the STAT3 deficient cardiomyocytes.Insulin-induced cardioprotection at reperfusion occurs through activation of STAT3. Inhibiting STAT3 by AG490, or STAT3 depletion in cardiac myocytes affects activation of Akt, suggesting close interaction between STAT3 and Akt in the cardioprotective signalling pathway activated by insulin treatment at reperfusion.

Publisher Summary Progress in biotechnology is a prerequisite for global health, and global health is a prerequisite for peace in the world. Therefore, biotechnology, health, and peace are strongly linked together. Unfortunately, the gap between developing and industrialized countries continues to increase. It is, therefore, immensely important that the international community becomes active in reducing this gap by promoting science and technology. There is a tremendous potential in the genomics revolution to promote health in developing countries, and thus promoting peace with justice in the world.

Carvedilol is an alpha-and beta-blocking agent with antioxidant properties. We examined if treatment with carvedilol in vivo protected the heart against ischemic injury ex vivo.Isolated hearts from treated rats (80 mg/kg/day) were subjected to 30 min regional ischemia. Hearts from non-treated animals received either no drug, 10 min carvedilol (1 microM) acute or ischemic preconditioning (IP) by 5 min ischemia +5 min reperfusion prior to regional ischemia. In separate experiments isolated hearts were subjected to 15 min global ischemia and 30 min reperfusion.Infarct size was significantly reduced by ischemic preconditioning or by chronic carvedilol treatment (9.0+/-0.9% and 7.2+/-1.9% of risk zone infarcted, respectively, vs. 33.8+/-6.4% in control hearts, mean+/-SEM, p0.05). Recovery of left ventricular developed pressure after global ischemia was not improved by carvedilol. Post-ischemic rise in left ventricular end diastolic pressure was, however, attenuated by chronic carvedilol treatment.Chronic in vivo but not acute ex vivo pretreatment with carvedilol significantly limited infarct size in isolated rat hearts.

Corticotropin-releasing factor (CRF) receptor type 2beta (CRFR2beta) is expressed in the heart. Urocortin (Ucn)-I activation of CRFR2beta is cardioprotective against ischemic reperfusion (I/R) injury by stimulation of the ERKs1/2 p42, 44. However, by binding CRF receptor type 1, Ucn-I can also activate the hypothalamic stress axis. Ucn-II/stresscopin related peptide and Ucn-III/stresscopin are two new members of the CRF/Ucn-I gene family and are selective for CRFR2beta. We propose that CRFR2beta selective Ucn-II or Ucn-III will protect cardiomyocytes and the ex vivo Langendorff perfused rat heart from I/R injury by activation of ERK1/2-p42, 44. Ucn-II is expressed in mouse cardiomyocytes, and Ucn-II or Ucn-III can bind to CRFR2beta, resulting in ERK1/2-p42, p-44 phosphorylation and cAMP stimulation. Phosphorylation of ERK1/2-p42, p-44 is regulated by the Ras/Raf-1 kinase pathway, independent of adenylate cyclase and, therefore, cAMP activation. Ucn-II and Ucn-III protect cardiomyocytes from I/R injury and reduce the percentage of infarct size:risk ratio in Langendorff perfused rat hearts exposed to regional I/R (P

The “metabolic cocktail” comprising glucose-insulin-potassium administrated at reperfusion reduces infarct size in the in vivo rat heart. We propose that insulin is the major component mediating this protection and acts via Akt prosurvival signaling. This hypothesis was studied in isolated perfused rat hearts (measuring infarct size to area of risk [%]) subjected to 35 minutes regional myocardial ischemia and 2 hours reperfusion. Insulin administered at the onset of reperfusion attenuated infarct size by ≥45% versus control hearts ( P

The metabolic cocktail of glucose-insulin-potassium (GIK) has been shown to reduce mortality in humans and reduce infarct size in the rat when administered from the onset of reperfusion following an ischemic insult. The mechanisms underlying GIK mediated cardioprotection are, however, still unclear. Recent data implicates insulin "alone" as the major protagonist of cardioprotection when administered at the time of reperfusion. We have therefore begun to investigate an insulin activated signalling pathway and the putative role of apoptosis in this insulin-induced cardioprotection. Simulated ischemia and reoxygenation were induced in rat neonatal cardiocyte experiments. The administration of insulin [0.3 mU/ml] at the moment of reoxygenation (Ins(R)) enhanced myocardial cell viablility as assessed by trypan blue exclusion compared to vehicle alone treated control myocytes (Ins(R)50+/-2%v controls 70+/-1%, P0.001). This insulin-mediated cardioprotection was due, in part to a reduction in myocyte apoptosis as measured by TUNEL (Ins(R)29+/-2%v controls 49+/-3%, P0.001) and Annexin V staining (Ins(R)34+/-2%v controls 65+/-3%, P0.001). These cardioprotective and anti-apoptotic effects of insulin were completely abolished by the tyrosine kinase inhibitor lavendustin A and by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin. Thus, we conclude that the early administration of insulin appears to be an effective modality to reduce reoxgygenation injury in cardiocytes, in part, via the attenuation of ischemia/reoxygenation-induced apoptosis. Moreover, the cardioprotective and anti-apoptotic effects of insulin are mediated via tyrosine kinase and PI3-kinase signalling pathways.

Fatty acids are the preferred physiological substrate for oxidative energy production in the heart (Neely et al., 1972, 1974). On the other hand, there is good evidence to believe that the high levels of non-esterified fatty acids observed in patients after myocardial (Kurien et al., 1966; Oliver, 1972; Opie, 1975) infarction may be harmful to jeopardized myocardium. Thus, fatty acids have been associated with increased risk of arrhythmias in both patients and experimental animals exposed to myocardial ischaemia (Mjos et al., 1974; Mjos, 1978). High levels of fatty acids have also been shown to depress recovery of mechanical function in both hypoxic rat hearts (Henderson et al., 1970) and ischaemic swine hearts (Liedtke et al., 1978).

Working rat hearts were perfused for 15 minutes at 37 °C before switching to a Langendorff perfusion (60 mm Hg aortic pressure) at 10 °C for 40 minutes of hypothermic arrest. Ventricular function was allowed to recover for 15 minutes at 37 °C by reestablishing the prehypothermic conditions. The perfusate was Krebs-Henseleit bicarbonate buffer containing 3% bovine serum albumin and either glucose (11 mmol/L) or glucose (11 mmol/L) plus palmitate (1.2 mmol/L) and gassed with 95% O 2 and 5% CO 2 . In hearts receiving glucose alone as substrate, coronary flow was maintained constant during the 40 minutes of hypothermic arrest and returned to prehypothermic rates with rewarming. Ventricular function, as estimated by peak systolic pressure and heart rate, recovered to the prehypothermic level. When palmitate was added, coronary flow decreased continuously throughout the hypothermic perfusion (22% decrease by 40 minutes), and ventricular pressure development was lower throughout the rewarming perfusion. Tissue levels of adenosine triphosphate and creatine phosphate were well maintained and long-chain acyl coenzyme A and acyl carnitine decreased during hypothermia regardless of the substrate provided. With rewarming, tissue levels of adenosine triphosphate and creatine phosphate decreased in those hearts receiving palmitate. Omission of fatty acid either during hypothermia or during the first 5 minutes of rewarming improved recovery of function. Addition of oxfenicine to inhibit fatty acid oxidation, or inhibition of Ca 2+ overload by verapamil and low perfusate Ca 2+ , prevented the effects of palmitate on ventricular function. Suppressed recovery of ventricular function after hypothermic perfusion in the presence of palmitate did not consistently correlate with low energy levels or high levels of metabolic products. Altered energy metabolism did not appear to be responsible for the fatty acid effect.

Accumulation of complement factors has been found to occur in the myocardium after infarction. We studied the possibility that the complement activation product C3a des-Arg is trapped within the coronary circulation during reperfusion of the ischemic myocardium. In 11 patients undergoing routine coronary artery bypass grafting, arterial blood was sampled before, during and after cardiopulmonary bypass. Blood was drawn from the coronary sinus concomitantly with arterial blood sampling 5 and 30 min after release of the aortic cross-clamp (n = 10). From a preoperative value of 92 +/- 13 ng/ml, C3a des-Arg rose during CPB to a maximum of 1816 +/- 393 at the end of CPB. Following reperfusion for 5 min, C3a des-Arg was 1284 +/- 232 ng/ml in arterial and 1106 +/- 100 in coronary sinus blood, a significant difference (p less than 0.05). The amount of C3a des-Arg trapped in the heart at 5-min reperfusion showed positive correlation with its arterial concentration (p less than 0.05). No significant difference was found after 30 min of reperfusion. Complement activation products trapped in the heart in the early reperfusion period may play a pathogenetic role in myocardial ischemia-reperfusion injury.

To determine the protective effect during ischaemia and reperfusion of removing oxygen radicals two groups of isolated Langendorff perfused rat hearts were arrested with cardioplegic solution at 4 degrees C and kept ischaemic at 15 degrees C for 210 min before being reperfused for 60 min at 37 degrees C. To remove oxygen radicals superoxide dismutase and catalase were added to the cardioplegic solution and to the buffer during the first 30 min of reperfusion in one group, the other group serving as control. At the end of reperfusion the first derivative of left ventricular developed pressure (dP/dt), coronary flow, high energy phosphate concentrations, and ultrastructure were determined. The ultrastructure was examined using a stereological method based on point counting and the results presented as volume fractions (Vv). DP/dt after 60 min of reperfusion was 61.6(5.6)% (mean (SEM)) of the initial values in the control group and 77.6(3.4)% in the superoxide dismutase and catalase supplemented group (p less than 0.05). In the supplemented group coronary flow was significantly higher than in the control group but only in the first part of reperfusion. The concentrations of adenosine triphosphate and creatine phosphate in the control group were 9.9(1.0) and 19.6(1.8) mumol.g-1 dry weight respectively; corresponding values in the supplemented group were 14.4(2.1) and 29.4(3.6) mumol.g-1 dry weight. The morphometric examination of the ultrastructure showed no significant difference in interstitial fluid accumulation evaluated by Vv(myocyte/myocardium) measurements and there was no difference in mitochondrial alteration between the two groups. There was, however, a significant reduction in the volume of cellular oedema (Vv(cell oedema/myocyte)) in the supplemented group.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of prostaglandin E1 (PGE1) on myocardial ischaemia (as measured by epicardial ST segment changes), myocardial flow and substrate exchange has been studied in dogs. Myocardial ischaemia was induced by intermittent external clipping of a branch of the left anterior descending coronary artery. During occlusion with a continuous intravenous infusion of isoprenaline, elevated epicardial ST segments (∑ST), were raised to 46 ± 6 mV (mean ± SEM). Pretreatment with PGE1 reduced ∑ST to 34 ± 6 mV (P

The relationship of future clinical coronary heart-disease (C.H.D.) to the plasma-high-density-lipoprotein (H.D.L.)-cholesterol concentration has been examined in a 2-year case-control follow-up study of 6595 men aged 20-49 years living in the municipality of Tromso, Norway. Measurements were also made of the cholesterol concentration in lower-density (i.e., density less than 1-603 g/ml) lipoproteins, plasma-triglycerides, systolic and diastolic blood-pressures, relative body-weight, and cigarette consumption. Discriminant-function analysis showed that coronary risk was inversely related to H.D.L.-cholesterol concentration and directly related to density less than 1-063 cholesterol. These relationships were independent of each other and of the other measured variables, which showed no significant differences between the cases and controls. H.D.L. cholesterol made a three-fold greater contribution to the prediction of future C.H.D. than did density less than 1-063 cholesterol in this cohort of young men. These findings support the proposal that a low H.D.L. concentration is a common antecedent of clinical C.H.D. and is important in accelerating the progression of coronary atherosclerosis.

A model of acute ischaemic left ventricular (LV) failure is presented. In closed-chest anaesthetized dogs 50 micrometer plastic microspheres were injected repeatedly into the left main coronary artery over a period of about 40 min. The injections effected stepwise elevations of LV end-diastolic pressure (LVEDP). Thus, LVEDP could be increased to a desired level, about 20 mmHg, in a very controlled manner. All dogs developed signs of markedly depressed LV performance. Haemodynamic conditions stabilized about 60 min after embolization. The maximum LVDP/dt decreased from 2696 +/- 169 to 1823 +/- 98 mmHg . x-1, cardiac output decreased from 2.81 +/- 0.20 to 1.98 +/- 0.14 l . min-1 and mean aortic blood pressure decreased from 144 +/- 4 to 127 +/- 3 mmHg, while total peripheral resistance increased from 56 +/- 3 to 69 +/- 3 mmHg . l-1 . min. Myocardial blood flow decreased from 103 +/- 7 to 79 +/- 6 ml . min-1 . 100 g-1 and myocardial oxygen consumption decreased from 12.5 +/- 0.9 to 8.3 +/- 0.8 ml . min-1. 100 g-5. Myocardial uptake of lactate and free fatty acids decreased markedly. Electrocardiography showed signs of acute ischaemia. There were no deaths due to ventricular fibrillation. Morphological studies showed multiple small infarcts throughout the entire LV. In conclusion, repeated coronary embolization with 50 micrometers plastic microspheres, guided by the rise of LVEDP represents a simple and reproducible method for induction of uniform and stable acute LV failure.

Myocardial metabolism was studied in open-chest dogs before and during induction of myocardial ischaemia by coronary artery occlusion. Blood was sampled from a local coronary vein draining ischaemic tissue and from coronary sinus draining predominantly nonischaemic tissue. In the basal state, induction of myocardial ischaemia stimulated myocardial lipolysis as shown by release of glycerol from the ischaemic zone. During isoprenaline infusion, free fatty acids (FFA) extraction across the ischaemic myocardium was substantially increased, but no glycerol release occurred. Pretreatment with nicotinic acid or sodium salicylate markedly depressed FFA extraction across ischaemic myocardium, both during basal and isoprenaline stimulated lipolysis and nicotinic acid most likely inhibited lipolysis in the ischaemic zone. Thus, reduced severity of acute ischaemic injury by antilipolytic treatment might be due to a combination of inhibited myocardial lipolysis and reduced FFA extraction.

Summary. The study was carried out to examine whether high plasma concentrations of free fatty acids (FFA) alone have cardiac electrophysiological effects, and whether the electrophysiological effects of isoprenaline are dependent on elevated plasma FFA. Experiments were performed in sixteen intact, healthy, pentobarbital anaesthetized dogs. Atrial, atrioventricular nodal and ventricular conduction times and refractoriness, and sinus node function were studied by His bundle electrography combined with programmed electrical stimulation. Elevation of plasma FFA by Intralipid and heparin from a median value of 363 to 1780 u-moM-1 did not induce any changes in cardiac electrophysiology. Intravenous infusion of isoprenaline (0.15 μg-min_1-kg-1) increased median plasma FFA from 410 to 2254 μmol-l-1. This rise in plasma FFA could be prevented by administration of nicotinic acid. The electrophysiological effects of isoprenaline were similar before and after inhibition of lipolysis. The study indicates that the cardiac electrophysiological effects of isoprenaline in the intact, healthy heart are independent of changes in plasma FFA concentrations.

Summary10 healthy male volunteers fasted for 72 hours. Their plasma concentration of free fatty acid increased more than two-fold, to 1.8 mmol/1. The number of reversible venous “in vivo” platelet aggregates increased significantly (p

The present study was undertaken to examine the effects of changes in PaCO2 and pHa on myocardial blood flow and central hemodynamics during acute ischemic left ventricular failure. Six closed-chest dogs anesthetized with pentobarbital were hyperventilated, and CO2 was added to the inspiratory gas to induce: a) normocapnia, b) hypocapnia, c) hypercapnia, and d) hypercapnia with sodium carbonate given to correct pH. Embolization of the left coronary artery with 50-microns microspheres resulted in deterioration of left ventricular function, as indicated by increased left ventricular end-diastolic pressure and mean pulmonary arterial pressure, while cardiac output decreased. During hypocapnia with left ventricular failure, the central hemodynamics remained unchanged, while a minor but nonsignificant decrease in myocardial blood flow was observed. Hypercapnia aggravated the heart failure, as indicated by increased left ventricular end-diastolic pressure, mean right atrial pressure, and mean pulmonary arterial pressure; however, the pump function of the heart was unchanged, as demonstrated by the unaltered cardiac output, heart rate, and mean aortic blood pressure. The changes in the central hemodynamics were reversed when pH was normalized during hypercapnia. Thus, in the present study pH, and not PaCO2, was responsible for the hemodynamic deterioration observed during hypercapnia in the failing heart.

We have previously demonstrated a 40% increase in myocardial blood flow (MBF) during hypercapnia but no significant decrease of MBF during hypocapnia. The present study was undertaken to evaluate if epinephrine infusion, which increases both myocardial oxygen consumption [Formula: see text] and myocardial performance, might influence the effects of hypocapnia and hypercapnia on MBF. Induction of hypocapnia was performed by hyperventilation in closed-chest dogs anesthetized with pentobarbital. By adding carbon dioxide to the inspiratory gas, normocapnia and hypercapnia were created. Epinephrine infusion (0.8 μg∙kg−1∙min−1) increased MBF and cardiac output (CO) by 90 and 140%, respectively, while [Formula: see text] was increased by 45%. Epinephrine had a direct coronary vasodilating effect in excess of myocardial needs evidenced by increased oxygen content of the coronary sinus blood. During epinephrine infusion, induction of hypocapnia effected no change of MBF, while myocardial oxygen extraction increased significantly. Although oxygen saturation [Formula: see text] and [Formula: see text] in the coronary sinus blood decreased, these values remained well above those observed with hypocapnia without epinephrine infusion, thereby excluding impaired oxygen supply to the heart. Hypercapnia induced an increase of MBF by nearly 40% despite the coronary vasodilatation already induced by epinephrine infusion.

This study describes the effect of oxygen radicals on the ultrastructure of the isolated Langendorff-perfused rat heart. Oxygen radicals were enzymatically generated by xanthine oxidase (0.025 U/ml) and hypoxanthine (0.96 mM). Hearts were perfusion-fixed for electron microscopy and stereological technique was performed to obtain estimates of volume fractions (Vv) of different tissue components. Perfusion with oxygen radicals resulted in areas with severely damaged myocardial cells. These changes included swelling and cristolysis of mitochondria, disruption of filaments, development of intracellular edema and focal disruption of the sarcolemma. Stereological examination revealed few alterations after 5 min perfusion with oxygen radicals. After 10 min perfusion with oxygen radicals, however, the Vv (myocyte/myocardium) increased from 0.542 +/- 0.042 (mean +/- S.D.) to 0.663 +/- 0.144, and this paralleled the development of Vv (cellular edema/myocyte) being 0.047 +/- 0.028. Vv (capillary wall/capillary) increased from 0.215 +/- 0.046 to 0.411 +/- 0.123 indicating endothelial swelling. Although the mitochondria appeared swollen, Vv (mitochondria/myocyte) remained constant. The effect of a 35 min recovery period on the ultrastructure was minor. The application of SOD and catalase together with xanthine oxidase and hypoxanthine reduced the observed changes significantly, thus proving the participation of oxygen radicals. This study confirms that oxygen radicals can induce major alterations in myocardial ultrastructure.

Ischemia-reperfusion activates polymorphonuclear leukocytes (PMN). Depletion of PMN has been shown to reduce the size of experimental myocardial infarction. We have studied whether PMN activated by phorbol myristate acetate (PMA) would depress function of the isolated rat heart, and if this effect was mediated by oxygen free radicals (OFR). Cells and/or drugs were added to the perfusate into the aortic cannula for 10 min, followed by a 30 min recovery period. Oxygen free radicals formation was verified by chemiluminescence (CL). PMA-activated PMN (n = 13) caused CL response of 27,493 +/- 5113 counts (mean +/- S.E.M.) and reduced left ventricular developed pressure (LVDP) to 30 +/- 9% and coronary flow (CF) to 49 +/- 7% of the baseline value at the end of the observation period. Addition of super-oxide dismutase (SOD) and catalase (CAT) (n = 11) reduced the CL response to 5623 +/- 806 counts, but did not influence either LVDP (36 +/- 15%) or CF (51 +/- 18%). Addition of thiourea (TU) to the activated cell suspension (n = 8) further reduced the CL response (3663 +/- 474 counts), and LVDP was 86 +/- 5% and CF was 87 +/- 3%. When TU + SOD + CAT was mixed with PMN + PMA (n = 11), the CL was almost abolished (117 +/- 21 counts) and LVDP was 73 +/- 8% and CF was 94 +/- 6%. When CF was reduced (n = 7) alike the CF reduction in the hearts receiving PMA + PMA, LVDP was not significantly changed at the end of the observation period (75 +/- 6%). Unactivated PMN (n = 8) caused minor response of LVDP and CF, similar to PMN + PMA + TU and PMN + PMA + SOD + CAT + TU. PMA alone (n = 8) was cardiotoxic and caused changes similar to PMN + PMA. This effect was not inhibited by scavengers (n = 6). The supernatant of the PMN + PMA suspension (n = 7) did not impair cardiac function, suggesting that no free PMA was available after mixing with PMN. We conclude that activated PMN in the coronary circulation depressed cardiac function and increased vascular resistance due to OFR production.

Lecithin:cholesterol acyltransferase (LCAT) in rats treated with 4-amino-pyrazolo-pyrimidine (4-APP) has been studied. Treatment with 4-APP decreased the plasma concentration of triglycerides, cholesterol and cholesteryl ester concomitantly with a decreased rate of cholesterol esterification in plasma. The reduced cholesterol esterification was due both to an effect on the enzyme and on the lipoprotein substrate. Plasma prothrombin decreased to the same extent as that of LCAT, indicating that the primary effect of 4-APP is upon protein synthesis and secretion from the liver.

Summary. The relative importance of pCO2 versus pH in regulating myocardial blood-flow (MBF) is not settled. Therefore, the influence of hypocapnia, hypercapnia and sodium carbonate infusion, on MBF and myocardial metabolism, has been investigated in 10 closed-chest pentobarbital anaesthetized dogs. The animals were hyperventilated, and CO2 was added to the inspiratory gas to induce normocapnia and hypercapnia. A mass spectrograph continuously measured the ventilatory gas components, and MBF was measured by the hydrogen desaturation technique with a catheter positioned in the coronary sinus. During the experiments, there were no significant alterations in heart rate, mean aortic blood-pressure, myocardial oxygen consumption or uptake of glucose and free fatty acids. During hypocapnia MBF was insignificantly reduced, while myocardial oxygen extraction increased significantly. During hypercapnia, however, MBF increased more than 40%. This increase in MBF was abolished following an infusion of sodium carbonate. Thus, in the present study, increased MBF, observed during hypercapnia, was due to the reduction in pH and not to the increase in pCO2.

Myocardial oxygen consumption (MVO2) is influenced by the substrate supply to the heart. Utilization of free fatty acids increases MVO2, and catecholamines sensitize the heart to the oxygen-wasting effect of free fatty acids. Alteration of myocardial metabolism from mainly free fatty acid to carbohydrate oxidation reduces the extent of myocardial ischemic injury. Within the ischemic myocardium, lipolysis is stimulated with breakdown of endogenous triglycerides to fatty free acids and glycerol. Antilipolytic agents seem to have a combined effect on myocardial metabolism partly through inhibition of lipolysis in adipose tissue with reduction of free fatty acid mobilization to plasma, and partly through a local inhibition of lipolysis in the ischemic myocardium. In patients with high sympathoadrenal activity, for example, patients with acute myocardial ischemia in unstable ischemic heart disease, elevation of free fatty acids might effect a critical increase in both myocardial oxygen requirement and infarct size.

The relationship between HDL cholesterol and relative body weight, cigarette smoking and leisure time physical activity were examined in 7 338 healthy men and 6 768 healthy women aged 20–53 years, living in Finnmark County, Northern Norway. The independent effects of the three factors were examined by an analysis of covariance. In both sexes a strong and almost linear negative association was observed between relative body weight and HDL cholesterol. HDL cholesterol was lower in smokers than in non-smokers, the difference being more pronounced in females than in males. A weak but significant positive association was observed between leisure time physical activity and HDL cholesterol in both sexes.

Oxfenicine inhibits myocardial metabolism of nonesterified fatty acids (NEFA). The purpose of the present study was to examine the effects of oxfenicine on triglyceride accumulation and the development of histologically visible lipid droplets. The beta-agonist isoproterenol was used to induce elevated arterial NEFA. Four groups of rats were used in the experiment (12 to 14 rats in each group), and each group received two subcutaneous injections, the second injection 25 min after the first, of oxfenicine-isoproterenol, oxfenicine-saline, saline-isoproterenol and saline twice, respectively. One hour after the second injection, the rats were anesthetized, and the hearts from six rats from each group were quickly removed and frozen for later analysis of triglyceride content. From the remaining rats blood samples were drawn for NEFA analysis, and biopsies were taken from the left ventricular wall before the hearts were frozen in liquid nitrogen and prepared for analysis of esters of carnitine and CoA. Quantitative morphometric techniques were used to determine the fractional volume of lipid droplets in myocardial biopsies. Our results show a marked increase in the triglyceride and lipid droplet content in all groups receiving oxfenicine or isoproterenol. The effect was most pronounced after treatment with both drugs. The close association between the increase in triglyceride and lipid droplet supports the notion that the lipid droplets are composed of triglycerides. Our finding that oxfenicine induces lipid droplet accumulation independent of NEFA increase supports the hypothesis that oxfenicine exerts its effect by inhibiting carnitine acyl transferase.

Ten healthy male student in regular sinus rhythm fasted for 66 hours. Their overnight fasting plasma concentration of free fatty acids (FFA) was 455 +/- 104 micro mol/1 (mean +/- S.E.M., n=7), the reference value of our laboratory, measured in another normal population of young men, being 344 +/- 28 micro mol/1 (n=10). After 42 and 66 hours of fasting, the plasma concentration of FFA rose to 1198 +/- 181 (p less than 0.01, n=10) and 1471 +/- 89 micro mol/1 (p less than 0.001, n=10), respectively. During the last 24 hours of fasting, the heart rate rhythm was monitored continuously by means of a Holter recorder and computer. No arrhythmias were observed, indicating that elevated plasma concentrations of FFA, exceeding those reported in patients with acute myocardial infarction, are well tolerated by the healthy human myocardium.

Haemodynamic and metabolic effects of elevated plasma concentrations of free fatty acids (FFA) were studied during acute ischaemic left ventricular failure in closed-chest anaesthetized dogs. Embolization of the left main coronary artery with 50 micron plastic microspheres induced severe depression of left ventricular performance as indicated by a marked increase in left ventricular end-diastolic pressure (LVEDP), and marked reductions in LVdP/dtmax, cardiac output and myocardial oxygen consumption (MVO2). When stable conditions were reached, eight dogs received a triglyceride emulsion and heparin to raise plasma FFA. This was associated with increased MVO2 and further elevation of LVEDP. In two dogs receiving the triglyceride emulsion but no heparin, plasma FFA was not elevated, and MVO2 and LVEDP were unchanged. In conclusion, elevation of plasma FFA was associated with increased myocardial oxygen requirement and further depression of LV function in dogs with acute ischaemic LV failure.

Haemodynamic and metabolic effects of ventilation with positive end-expiratory pressure (PEEP) were studied in closed-chest dogs anaesthetized with sodium pentobarbital during normal cardiac function and during acute left ventricular (LV) failure. LV failure was induced by embolizing the left coronary bed with 50 micron plastic microspheres causing a marked depression of LV function. End-expiratory pressure was set to 0, 5, 10 and 15 cm H2O both before and after coronary embolization. During normal cardiac function PEEP above 5 cm H2O depressed cardiac output (CO) significantly. However, following coronary embolization after which LV function was seriously impaired, CO was maintained as PEEP was applied. This is attributed to reduced sensitivity to the LV pre-load reductions induced by PEEP during LV failure. PEEP reduced MBF both during normal and impaired LV function. This did not result in ischaemic myocardial metabolism assessed by lactate extraction either in normal hearts or following coronary embolization. The reduced MBF was, however, associated with reduced MVO2 both during normal cardiac function and during LV failure. It is suggested that the reduced MBF is mainly due to reduced myocardial oxygen demand probably caused by reduced LV wall tension during PEEP ventilation.

Publisher Summary The severity of an acute myocardial ischemic injury is determined by factors that alter the oxygen requirement of the heart relative to oxygen supply. Plasma fatty acid concentration is an important determinant of myocardial oxygen consumption. This chapter describes an investigation to study whether the induction of myocardial ischemia per se might stimulate myocardial lipolytic activity with breakdown of endogenous triglycerides in the in vivo situation. If the induction of myocardial ischemia per se might stimulate myocardial lipolytic activity with breakdown of endogenous triglycerides in the in vivo situation, then a part of the beneficial effect of antilipolytic agents on the severity of myocardial ischemia might be because of the inhibition of myocardial lipolysis with reduced local free fatty acid (FFA) release to the ischemic myocardium in addition to reduced extraction of FFA from plasma. Another purpose of the investigation was to study the possible mechanisms of action of the antilipolytic agents, namely, nicotinic acid and sodium salicylate, both of which have shown to reduce an acute myocardial ischemic injury.

The effect of hypoxia on myocardial lipolysis (glycerol release) was investigated in freshly isolated, calcium-tolerant rat ventricular myocytes. Hypoxia was produced by gassing the incubation medium (Joklik-minimum essential medium, supplemented with 1.2 mM MgSO4, 1 mM DL-carnitine, 1.5 mM CaCl2 and 0.6 mM palmitate bound to 0.15 mM fatty acid free bovine serum albumin) with 95% N2–5% CO2. Control (normoxic) incubations were carried out under air-5% CO2 atmosphere. Basal glycerol release increased from 46.6 ± 3.0nmol/106 cells · 30 min in normoxia to 64.5 ± 4.3 nmol/106 cells · 30 min in hypoxia (p < 0.05). Addition of isoprenaline (10 μM) resulted in a significant (p < 0.05) stimulation of the glycerol release both in normoxia and in hypoxia, but the enhancement above basal rates was apparently lower in hypoxia (8.7 ± 2.5 nmol/106 cells · 30 min) than in normoxia (12.2 ± 2.7nmol/106 cells · 30 min). Furthermore, whereas the isoprenaline-induced rise in lipolysis both in normoxia and hypoxia was prevented by inclusion of propranolol (10 μM), propranolol did not affect the hypoxia-induced increase in lipolysis. Thus, the above findings suggest that myocardial lipolysis may be stimulated by local non-adrenergic mechanisms during hypoxia.

A family study of serum high density lipoprotein (HDL) cholesterol and total serum cholesterol concentration has been undertaken, and the relation to age, sex, cigarette smoking, physical activity and familial occurrence of myocardial infarction (MI) was examined. HDL cholesterol was determined in 251 females and 194 males and total serum cholesterol in 677 females and 657 males, all aged 0-49 years. With respect to HDL cholesterol, significant sex differences were observed both in absolute level and in age-related change. A negative correlation between HDL cholesterol and total serum cholesterol was observed in all age groups except females aged 0-19 years, supporting the hypothesis of HDL as a ""clearing'' lipoprotein. HDL cholesterol showed a positive correlation only in pairs of first-degree relatives involving the mother and in sib-sib paris of the same sex. On the other hand, for serum cholesterol a positive correlation was found among all family members, although significantly higher between first-degree relatives than between spouses. No relation was found between cigarette smoking, physical activity of familial occurrence of MI and the HDL cholesterol or total serum cholesterol concentrations. In accordance with the ""HDL hypothesis'', the present finding could partly explain the higher incidence of ischaemic heart disease (IHD) in males than in females, and partly also the high risk which is transmitted from women with IHD to their first-degree relatives.

Glucose metabolism in the healthy heart is stimulated by dichloroacetate (DCA). The possibility has been examined in dogs that DCA, by increasing glucose utilization, might limit the severity of acute myocardial ischaemic injury. Intravenous administration of DCA reduced the degree of epicardial ST-segment elevation induced by subsequent coronary occlusion, both under basal conditions and during isoprenaline infusion. A similar result was obtained when DCA was given during an established coronary occlusion. This effect could not be explained by changes in mean aortic blood pressure, heart rate, or regional myocardial blood flow as measured by radioactive microspheres. Measurements in arterial and coronary sinus blood demonstrated an increase in the extraction of glucose and a decrease in that of FFA by the heart. Glucose extraction also tended to be increased in the ischaemic zone, as shown by the differences in the concentrations of these substrates between arterial blood and blood obtained from the local vein draining that zone. Lactate release by the ischaemic zone was markedly reduced.