Control Rat Hearts Research Articles

Infarction alters both the distribution and noradrenergic properties of cardiac sympathetic neurons.

Regional changes occur in the sympathetic innervation of the heart after myocardial infarction (MI), including loss of norepinephrine (NE) uptake and depletion of neuronal NE. This apparent denervation is accompanied by increased cardiac NE spillover. One potential explanation for these apparently contradictory findings is that the sympathetic neurons innervating the heart are exposed to environmental stimuli that alter neuronal function. To understand the changes that occur in the innervation of the heart after MI, immunohistochemical, biochemical, and molecular analyses were carried out in the heart and stellate ganglia of control and MI rats. Immunohistochemistry with panneuronal markers revealed extensive denervation in the left ventricle (LV) below the infarct, but sympathetic nerve fibers were retained in the base of the heart. Western blot analysis revealed that tyrosine hydroxylase (TH) expression (normalized to a panneuronal marker) was increased significantly in the base of the heart and in the stellate ganglia but decreased in the LV below the MI. NE transporter (NET) binding sites, normalized to total protein, were unchanged, except in the LV, where [3H]nisoxetine binding was decreased. TH mRNA was increased significantly in the left and right stellate ganglia after MI, while NET mRNA was not. In the base of the heart, increased TH coupled with no change in NET may explain the increase in extracellular NE observed after MI. Coupled with substantial denervation in the LV, these changes likely contribute to the onset of cardiac arrhythmias.

Regulation and role of the mitochondrial transcription factor in the diabetic rat heart.

To clarify the mechanism of abnormalities in mitochondrial expression and function in diabetic rat heart, we have studied the transcriptional activities of mitochondrial DNA using isolated intact mitochondria from the heart of either diabetic or control rats. The transcriptional activity of cardiac mitochondria isolated from diabetic rats decreased to 40% of the control level (P < 0.01). Consistently, in the heart of diabetic rats, the content of cytochrome b mRNA encoded by mitochondrial DNA was reduced to 50% of control (P < 0.01). This abnormal transcriptional activity of mitochondrial DNA could not be explained by mRNA or protein contents of mitochondrial transcription factor (mtTFA), but mtTFA binding to the promoter sequence of mitochondrial DNA, assessed by gel-shift assay, was attenuated in diabetic rats. In contrast, the mRNA expression of nuclear-encoded mitochondrial genes, such as ATP synthase-beta, was not affected by diabetes. Although O(2) consumption of the mitochondria from diabetic rats was decreased, H(2)O(2) production in these rats was increased compared with the control. Insulin treatment reversed all the abnormalities found in diabetic rats. These results clearly indicate that an impairment of binding activity of mtTFA to the promoter sequence has a key role in the abnormal mitochondrial gene expression, which might explain the mitochondrial dysfunction found in diabetic heart.

The effects of the sulfonylurea glyburide on glutathione peroxidase, superoxide dismutase and catalase activities in the heart tissue of streptozotocin-induced diabetic rat

Oxygen free radicals have been suggested to be a contributory factor in diabetes complications. The aim of this study was to examine the effects of glyburide on the antioxidant enzyme activities in the heart tissue of diabetic rats. We investigated the activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) in the hearts of both control and streptozotocin-induced diabetic rats. In the heart of diabetic rats, the activity of total superoxide dismutase decreased significantly (p < 0.005), whereas the activity of catalase and glutathione peroxidase increased to a large extent (p < 0.0001 and p = 0.05, respectively) at the end of the fourth week compared with the control group. Glyburide treatment of diabetic rats for 4 weeks corrected the changes observed in diabetic heart. In addition, blood glucose levels of untreated diabetic rats decreased following the glyburide treatment. These results demonstrate that the sulfonylurea glyburide is capable of exerting direct insulin-like effect on heart superoxide dismutase, catalase and glutathione peroxidase activities of diabetic rats in vivo.

Regional distribution of hyperpolarization-activated current (If) and hyperpolarization-activated cyclic nucleotide-gated channel mRNA expression in ventricular cells from control and hypertrophied rat hearts.

Hyperpolarization-activated inward current (If) and changes in the messenger RNA (mRNA) expression levels of hyperpolarization-activated cyclic nucleotide-gated channel (HCN)2 and HCN4 encoding If channels of the rat heart were studied in control and hypertrophied myocytes isolated from three ventricular regions: the septum (S), the left ventricular free wall (LV) and the right ventricular free wall (RV). Electrophysiological experiments were conducted by ruptured and perforated-patch clamp techniques and quantification of mRNA levels was carried out by quantitative reverse transcriptase polymerase chain reaction. The occurrence, density and maximal specific conductance of If were found to be significantly higher in hypertrophied ventricular myocytes isolated from S and LV than in those isolated from RV or sham-operated rats. Half-maximal activation potential, the slope of the activation curve and the threshold for activation were similar in ventricular myocytes from sham and aortic stenosed rats in the three regions studied. Isoproterenol 1 micromol l-1 increased current size by shifting current activation to more positive potentials in both sham and hypertrophied myocytes. When we studied the mRNA levels of If channel isoforms present in the ventricle, we found a significant increase of HCN2 and HCN4 mRNA levels in hypertrophied myocytes from S and LV but not in RV. We conclude that the occurrence, density and conductance of If is higher in hypertrophied than in control ventricular myocytes, S being the region where all these changes were most evident. These findings are associated with a higher expression of HCN2 and HCN4 mRNA levels in the two regions that developed hypertrophy.

Gender differences in myosin heavy chain-beta and phosphorylated phospholamban in diabetic rat hearts.

The objective of this study was to determine whether a gender difference exists in myosin heavy chain (MHC) isoform or sarcoplasmic reticulum protein levels in diabetic rat hearts. As is the case with normal rodent hearts, all four chambers of the control rat hearts expressed almost 100% MHC-alpha. In 6-wk diabetic rats, MHC-beta expression in ventricles of males was significantly greater (78 +/- 7%) than in females (50 +/- 5%). The cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) protein level was decreased and the phospholamban (PLB) protein level was increased in the left ventricle of diabetic rats, but there was no difference between male and female diabetic rats. The phosphorylated PLB level was decreased more in male than in female diabetic rats. Insulin treatment completely normalized blood glucose level, cardiac SERCA2a and PLB protein levels, and the decrease in MHC-beta levels in both male and female diabetic rats. Insulin treatment completely normalized serum insulin and almost completely normalized phosphorylation of PLB at serine 16 in male diabetic rats. Although insulin treatment completely normalized serum insulin levels in male diabetic rats, in females it only partially normalized serum insulin levels. Also, insulin treatment almost completely normalized phosphorylation of PLB at threonine 17 in female diabetic rats; however, the increase was significantly greater than that identified for insulin-treated male diabetic rats. We conclude that higher levels of MHC-beta and dephosphorylated PLB may contribute to more contractile dysfunction in male than in female diabetic rat hearts, and that phosphorylation of PLB at threonine 17 is more responsive to insulin in female diabetic rat hearts.

Evidence for rapid “metabolic switching” through lipoprotein lipase occupation of endothelial-binding sites

During diabetes, impaired glucose transport and utilization by the heart switches energy production to exclusive β-oxidation of fatty acid (FA). In the current study, we examined the contribution of cardiac lipoprotein lipase (LPL) towards providing FA to the diabetic heart. Streptozotocin (STZ) caused an augmentation of LPL activity at the coronary lumen, an effect duplicated by diazoxide (DZ). With DZ, the amplification of LPL at the coronary luminal surface was determined to be exceptionally rapid. Interestingly, unlike DZ, the capability of hearts from STZ animals to maintain this amplified LPL activity was sustained in vitro. This increased enzyme in the hyperglycemic heart is likely unrelated to an increase in the number of capillary endothelial LPL-binding sites. Our data imply that binding sites for LPL in the control rat heart are only partly occupied by the enzyme and diabetes rapidly initiates filling of all of these sites. Phloridzin treatment of STZ animals normalized plasma glucose with no effect on luminal LPL suggesting that the effects of diabetes on LPL are also largely independent of changes in blood glucose. Both 2 and 8 U of insulin normalized plasma glucose in DZ-treated animals but only 8 U reversed DZ-induced augmentation of cardiac luminal LPL. Our data suggest that impaired intracellular glucose utilization allows rapid vectorial transfer of LPL to unoccupied binding sites to supply the diabetic heart with excess FA. The persistence of increased coronary luminal LPL even in a setting of normoglycemia may provide excessive FA to the diabetic heart with deleterious consequences over the long term.

Reactive hyperemia during early reperfusion as a determinant of improved functional recovery in ischemic preconditioned rat hearts

Objective Our study was undertaken to clarify the impact of the shear stress-induced reactive hyperemia (associated with reperfusion) in preconditioning-mediated protection. Methods In control rat hearts, a 40-minute preischemic perfusion (constant pressure: 70 mm Hg) period was followed by 25-minute global low-flow ischemia (constant flow: 0.3 mL/min) and 30-minute reperfusion (constant pressure). As preconditioning protocol, hearts underwent 2 cycles of 5-minute no-flow ischemia/5-minute reperfusion. Results Although coronary vasodilation in response to shear stress is severely impaired after global low-flow ischemia and reperfusion, it is fully preserved by ischemic preconditioning concomitantly with an improvement of left ventricular developed pressure. Restricting coronary peak flow to 100% of baseline at reperfusion reduced left ventricular recovery to the control level. N G-nitro- l-arginine methyl ester affects the restoration of reperfusion-reactive hyperemia and the improvement of contractile recovery afforded by ischemic preconditioning. However, if the time course of hyperemia was restored by forcibly reperfusing to 150% of baseline for 10 minutes and, therefore, by restricting final peak flow to 80% of baseline for 20 minutes, contractile function recovered to a high degree despite the presence of N G-nitro- l-arginine methyl ester. Conclusion We conclude that wall stretch and shear stress during reperfusion are necessary for the mediation phase of preconditioning.

Remodeling of cardiac fiber structure after infarction in rats quantified with diffusion tensor MRI.

Structural remodeling of myocardium after infarction plays a critical role in functional adaptation. Diffusion tensor magnetic resonance imaging (DTMRI) provides a means for rapid and nondestructive characterization of the three-dimensional fiber architecture of cardiac tissues. In this study, microscopic structural changes caused by MI were evaluated in Fischer 344 rats 4 wk after infarct surgery. DTMRI studies were performed on 15 excised, formalin-fixed rat hearts of both infarct (left anterior descending coronary artery occlusion, n = 8) and control (sham, n = 7) rats. Infarct myocardium exhibited increased water diffusivity (41% increase in trace values) and decreased diffusion anisotropy (37% decrease in relative anisotropy index). The reduced diffusion anisotropy correlated negatively with microscopic fiber disarray determined by histological analysis (R = 0.81). Transmural courses of fiber orientation angles in infarct zones were similar to those of normal myocardium. However, regional angular deviation of the diffusion tensor increased significantly in the infarct myocardium and correlated strongly with microscopic fiber disarray (R = 0.86). These results suggest that DTMRI may provide a valuable tool for defining structural remodeling in diseased myocardium at the cellular and tissue level.

Effect of chronic renal failure on cardiac contractile function, calcium cycling, and gene expression of proteins important for calcium homeostasis in the rat.

Patients with chronic renal failure frequently develop cardiac hypertrophy and diastolic dysfunction; however, the mechanisms by which this occurs are still unclear. Male Sprague-Dawley rats were subjected to 5/6 nephrectomy and studied for their isolated myocyte function, calcium cycling, and gene expression of proteins important in calcium homeostasis after 4 wk. Comparable rats subjected to suprarenal aortic banding for the same duration were used for comparison. Rats subjected to 5/6 nephrectomy and aortic banding developed comparable hypertension; however, rats subjected to 5/6 nephrectomy experienced a greater degree of cardiac hypertrophy and downregulation of cardiac sodium potassium ATPase (Na+/K+ -ATPase) activity than rats subjected to aortic banding. Moreover, cells isolated from the 5/6 nephrectomy rat hearts displayed impaired contractile function and altered calcium cycling compared with cells isolated from control or aortic constriction rat hearts. The 5/6 nephrectomy rat heart cells displayed a prolonged time constant for calcium recovery following stimulation, which corresponded to decreases in homogenate sarcoplasmic reticulum calcium ATPase-2a (SERCA2a) activity, protein density, and mRNA for SERCA2a. In conclusion, chronic renal failure leads to alterations in cardiac gene expression, which produces alterations in cardiac calcium cycling and contractile function. These changes cannot be explained only by the observed increases in BP.

Long-term angiotensin II inhibition increases mitochondrial nitric oxide synthase and not antioxidant enzyme activities in rat heart.

To provide insight into the subcellular mechanisms involved in the improvement of cardiovascular structure and function by long-term inhibition of the renin-angiotensin system. The activities of antioxidant enzymes and mitochondrial free radical production were determined in the heart of control (C), enalapril-treated (E), and losartan-treated (L) rats to test the hypothesis of increased antioxidant enzyme activities and participation of mitochondria in the effects of chronic treatments with angiotensin II inhibitors. At 6 and 18 months of treatment, superoxide dismutases (SOD), Se-glutathione peroxidase, and catalase activities were determined in left ventricle homogenates by spectrophotometric methods and nitric oxide (NO) production in submitochondrial membranes by the oxyhemoglobin oxidation assay. The maximal rate of hydrogen peroxide (H2O2) production by submitochondrial membranes was also evaluated at 18 months by the scopoletin-horseradish peroxidase method. No significant increase was found in the antioxidant enzymes measured. At 6 months, Mn-SOD was actually decreased in E and catalase in both E and L, whereas at 18 months Se-glutathione peroxidase was decreased in L. Production of NO by submitochondrial particles was 64% higher at 6 months in E and 105% higher at 18 months in E and L. Maximal hydrogen peroxide production was lower at 18 months in both groups. Results do not support the hypothesis of an increase in antioxidant enzyme activity by long-term treatment with angiotensin II inhibitors as previously suggested and point towards a role for the NO produced by mitochondrial nitric oxide synthase (mtNOS) in the protective effect of these drugs.

Thyroid hormone regulation of cardiac bioenergetics: role of intracellular creatine.

The effect of thyroid hormone (T(3)) on the content of myocardial creatine (Cr), Cr phosphate (CrP), and high-energy adenine nucleotides and on cardiac function was examined. In the hearts of control and T(3)-treated rats perfused in vitro, while "low" and "high" contractile work was performed, T(3) treatment resulted in a approximately 50% reduction in CrP, Cr, total Cr content (Cr + CrP), and in the CrP-to-Cr ratio. In addition, there was a slight fall in myocardial content of ATP and a large rise in calculated free ADP (fADP), resulting in a significant decrease in the ATP-to-fADP ratio in the hearts of hyperthyroid compared with euthyroid rats. Moreover, there was a substantial decrease in the level of ATP in hearts of T(3)-treated rats under high work conditions. Importantly, the ratio of cardiac work to oxygen consumption was not altered by thyroid status. Treatment with T(3) also resulted in an almost threefold reduction in the content of Na(+)/Cr transporter mRNA in the ventricular myocardium and skeletal muscle but not in the brain. We conclude with the following: 1) changes in the expression of the Na(+)/Cr transporter mRNA correlate with Cr + CrP in the myocardium; 2) hearts of hyperthyroid rats contain lower levels of ATP and higher levels of fADP under both low and high work conditions but no reduction in efficiency of work output; and 3) the reduction in Cr and ATP in hearts of hyperthyroid rats may be the basis for the reduced maximal work capacity of the hyperthyroid heart.

Effect of cobalt on the oxidative status in heart and aorta of streptozotocin-induced diabetic rats.

Effects of cobalt on the antioxidant status of control and streptozotocin diabetic rat heart and aorta were examined at the second, fourth and sixth week of treatment. Rats were divided into four groups: control, diabetic, control treated with cobalt chloride and diabetic treated with cobalt chloride. Diabetes was induced by tail vein injection of streptozotocin (STZ). Cobalt treatment groups were given 0.5 mM of CoCl(2) in drinking water. The rats in both groups were further subdivided into three groups of six rats each. Rats in these subgroups were studied at 2-week intervals up to 6 weeks. At the end of the experiment, all animals were sacrificed by decapitation, heart and aorta samples were removed for determination of thiobarbituric acid reactive substance (TBARS) level and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities. It was found that lipid peroxidation levels and antioxidant enzyme activities were increased in the streptozotocin-induced diabetic rats at all times studied. Cobalt treatment of diabetic rats (0.5 mM in drinking water) resulted in attenuation of the increased levels of TBARS and antioxidant enzyme activities in heart and aorta. Thus, the effect of oral administration of cobalt at this dose during the early stage of experimental diabetes can be considered as a consequence of altered endogenous defence mechanisms in heart and aorta.

Energy Transfer in Acute Diabetic Rat Hearts

Hearts of rats with diabetes mellitus (DM) are characterized by energy demands exceeding their energy production, but they might also exhibit decreased vulnerability to ischemia and calcium overload. This indicates adaptation in cardiac energetics (CE), where energy transport is not rate-limiting. Aim-This study was designed to elucidate the functional significance of the DM-induced adaptation in CE by investigating the formation of mitochondrial contact sites (MiCS), facilitating the Ca-dependent/high-capacity energy transfer from mitochondria, in conjunction with testing the ischemic tolerance (IT) of hearts. After 1 week of streptozotocin-induced DM (45 mg/kg iv), the hearts of male diabetic and age-matched control rats (C) were isolated and Langendorff-perfused with either 1.6 or 2.2 mmol/L of CaCl(2). MiCS formation was assessed by cytochemical detection of mCPK octamers and was quantified stereologically as MiCS to mitochondrial surface ratio (S(S)). IT was evaluated in anesthetized open-chest animals subjected to 30-min occlusion of the LAD coronary artery followed by 4-h reperfusion, by monitoring ischemic arrhythmias and by measuring the size of infarction (tetrazolium double staining). In C hearts, increasing Ca2+ induced both positive inotropic response (dP/dt increase from 2270 +/- 220 to 2955 +/- 229, p < 0.01) and elevated MiCS formation (S(S) increase from 0.070 +/- 0.011 to 0.123 +/- 0.012, p < 0.01). In DM hearts, basic MiCS formation was already comparable with that induced by elevated Ca2+ in C hearts and could not be further stimulated by Ca2+. In C, ventricular tachycardia represented 55.4% of the total arrhythmias and occurred in 90% of the animals. In DM rats, the arrhythmia profile was similar to that in C, and the incidence of tachyarrhythmias and their severity were not enhanced (arrhythmia score: 3.18 +/- 0.4 vs. 3.30 +/- 0.3 in C). The infarct size normalized to the size of area at risk was smaller in the DM than in C hearts (52.3 +/- 5.8% vs. 69.2 +/- 2.2%, respectively; p < 0.05). Ca-signaling represents the link between energy delivery from mitochondria (via MiCS) and energy requirements of the heart. In DM hearts, energy transport via MiCS is elevated to the maximum value. This contributes to increased resistance of DM hearts to irreversible cell damage.

Diabetic alterations in cardiac sarcoplasmic reticulum Ca 2+-ATPase and phospholamban protein expression

Diabetic cardiomyopathy has been suggested to be caused by abnormal intracellular Ca 2+ homeostasis in the myocardium, which is partly due to a defect in calcium transport by the cardiac sarcoplasmic reticulum (SR). In the present study, the underlying mechanism for this functional derangement was investigated with respect to SR Ca 2+-ATPase and phospholamban (the inhibitor of SR Ca 2+-ATPase). The maximal Ca 2+ uptake and the affinity of Ca 2+-ATPase for Ca 2+ were decreased, and exogenous phosphorylation level of phospholamban was higher in streptozotocin-induced diabetic rat SR. Levels of both mRNA and protein of phospholamban were significantly increased in the diabetic hearts, whereas those of SR Ca 2+-ATPase were significantly decreased. Consequently, the relative phospholamban/Ca 2+-ATPase ratio was 1.88 in the diabetic hearts, and these changes were correlated with changes in the rates of SR Ca 2+ uptake. However, phosphatase pretreatment of phospholamban for dephosphorylation of the sites phosphorylated in vivo did not change the levels of subsequent phospholamban phosphorylation in either control or diabetic rat hearts. The above data indicated that the increased phospholamban phosphorylation was not due to autonomic dysfunction but possibly due to increased phospholamban expression. These findings suggest that reduction of the SR Ca 2+-ATPase level would contribute to decreased rates of SR Ca 2+ uptake and that this function is further impaired by the enhanced inhibition by phospholamban due to its increased expression in the diabetic heart.

Myocardial Contractile Responsiveness to Endothelin-1 in the Post-infarction Rat Model of Heart Failure: Effects of Chronic Quinapril

Cardiac endothelin-1 (ET-1) levels and ET receptor expression are increased in congestive heart failure (CHF). In order to determine whether this results in increased responsiveness of ET-A or ET-B receptors to ET-1, we evaluated the contractile effects of ET-1 in isolated papillary muscles isolated from hearts of control rats and from rats 4 weeks post myocardial infarction (MI) having received no therapy or chronic quinapril therapy. The ET-1 dose-response was biphasic in normal muscles. The use of the selective ET-A receptor antagonist BQ123 and the selective ET-B receptor antagonist BQ788 revealed that the initial decrease in tension was the result of ET-B receptor stimulation. Blockade of nitric oxide (NO) production with L-NAME abolished the initial decrease in tension. MI resulted in CHF that was partially reversed by quinapril. In MI, the positive inotropic effects of ET-1 were enhanced due to the loss of the initial ET-B receptor mediated decrease in tension, as well as an increase in the positive inotropic effects of ET-A receptors. This was associated with an increase in ET-A and ET-B receptor mRNA and a decrease in cardiac ecNOS protein. Four weeks of therapy with quinapril attenuated the positive inotropic effects of ET-1 and prevented the increase in ET-A receptor mRNA. Although quinapril did not restore the effects of ET-B receptor stimulation or prevent the increase in ET-B mRNA, it did restore cardiac ecNOS protein expression. Thus, the inotropic response to ET-1 is biphasic due to an overall positive inotropic effect of ET-A receptor stimulation and an ET-B receptor mediated decrease in contractility at low ET-1 concentrations which appears to be mediated by cardiac ecNOS (NO). In post-MI CHF, responsiveness to ET-A receptors increases and the ET-B mediated negative inotropic response is lost despite an increase in both receptor subtypes. Quinapril therapy attenuates these effects and normalises cardiac ecNOS protein.

Anti-arrhythmic and electrophysiological effects of the endothelin receptor antagonists, BQ-123 and PD161721

The effects of the endothelin ET A, (BQ-123) and endothelin ET A/B (PD161721) receptor antagonists were investigated on ischaemia-induced arrhythmias and on the maximum following frequency. The study was carried out in Langendorff perfused rat hearts subjected to coronary artery occlusion in which the severity of arrhythmias, coronary perfusion pressure and heart rate were measured. The % incidence of ischaemia-induced irreversible ventricular fibrillation (ventricular fibrillation) was reduced significantly from 58%, in control rat hearts, to 0% (at 10 −7 and 10 −6 M PD161721 and 10 −6 M BQ-123 P<0.05). Maximum following frequency was measured in guinea-pig isolated atria. In the presence of normal extracellular [K +], BQ-123 and PD161721, at 10 −6 M, significantly decreased the maximum following frequency from 9.0±0.7 to 7.2±0.4 and from 8.3±0.4 to 6.7±0.3 Hz, respectively ( P<0.05). These effects were not potentiated by raising the extracellular [K +] with the exception of 10 −9 M PD161721. In contrast, lignocaine's ability to reduce the maximum following frequency was greater in elevated (e.g. at 1.7×10 −4 M from 8.4±0.3 to 2.5±0.6 Hz) than in normal [K +] (from 9.0±0.3 to 4.9±0.5 Hz). In conclusion, both BQ-123 and PD161721 had an anti-fibrillatory effect in isolated rat hearts that may be due, at least in part, to an ability to reduce the maximum following frequency. This latter effect is unlikely to be due to Na + channel blockade since it was not markedly potentiated by elevation of extracellular [K +].

Menadione mimics the infarct-limiting effect of preconditioning in isolated rat hearts.

The role of mitochondrial free radicals in the cardioprotective effect of ischemic preconditioning was examined in isolated buffer-perfused rat hearts. Infarct size in control rat hearts subjected to 30 min of regional ischemia and 120 min of reperfusion was 32.6 +/- 3.4% of the risk zone. Ischemic preconditioning (3 cycles of 5-min global ischemia/5-min reperfusion) before the same regional ischemia and reperfusion protocol significantly reduced infarct size to 2.6 +/- 0.8% of the risk zone. Perfusion with menadione (3.0 microM), a generator of mitochondrial free radicals, in lieu of preconditioning ischemia significantly reduced infarction to 10.9 +/- 2.7%. N-2-mercaptopropionylglycine (1.0 mM), a free radical scavenger, blocked the protection of menadione, significantly increasing infarction to 23.5 +/- 1.1%. Myxothiazol (0.6 microM), a site III mitochondrial inhibitor, blocked the protection of menadione and significantly increased infarction to 25.2 +/- 3.8%. The infarct-limiting effect of menadione was attenuated to 19.7 +/- 1.5% of the risk zone by 10 microM SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Furthermore, menadione significantly increased p38 MAPK phosphorylation to a level 5.6-fold over basal. These results indicate that free radicals that originate within mitochondria can activate p38 MAPK and protect hearts against infarction.

Na+ effects on mitochondrial respiration and oxidative phosphorylation in diabetic hearts.

Intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control. We hypothesized that the increase in Na+i activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to loss of intramitochondrial Ca2+, with a subsequent alteration (generally depression) in bioenergetic function. To further evaluate this hypothesis, mitochondria were isolated from hearts of control and streptozotocin-induced (4 weeks) diabetic rats. Respiratory function and ATP synthesis were studied using routine polarography and 31P-NMR methods, respectively. While addition of Na+ (1-10 mM) decreased State 3 respiration and rate of oxidative phosphorylation in both diabetic and control mitochondria, the decreases were significantly greater for diabetic than for control. The Na+ effect was reversed by providing different levels of extramitochondrial Ca2+ (larger Ca2+ levels were needed to reverse the Na+ depressant effect in diabetes mellitus than in control) and by inhibiting the Na+/Ca2+ exchanger function with diltiazem (a specific blocker of Na+/Ca2+ exchange that prevents Ca2+ from leaving the mitochondrial matrix). On the other hand, the Na+ depressant effect was enhanced by Ruthenium Red (RR, a blocker of mitochondrial Ca2+ uptake, which decreases intramitochondrial Ca2+). The RR effect on Na+ depression of mitochondrial bioenergetic function was larger in diabetic than control. These findings suggest that intramitochondrial Ca2+ levels could be lower in diabetic than control and that the Na+ depressant effect has some relation to lowered intramitochondrial Ca2+. Conjoint experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels in diabetic rats, but produced smaller changes in control. These data support our hypothesis that in diabetic cardiomyocytes, increased Na+ leads to abnormalities of oxidative processes and subsequent decrease in ATP levels, and that these changes are related to Na+ induced depletion of intramitochondrial Ca2+.

The role of K+ATP channels in the control of pre- and post-ischemic left ventricular developed pressure in septic rat hearts

Myocardial function is impaired 24 h after the induction of sepsis, however, recovery of left ventricular (LV) function after 35 min of global ischemia is complete. The mechanisms by which this protection occurs are unknown. Ischemic preconditioning, another form of myocardial protection from ischemia/reperfusion (I/R) injury, has been shown to be modulated by ATP-sensitive potassium (K+ATP) channels. To investigate the role of K+ATP channels in the regulation of coronary flow (CF) and protection from I/R injury in septic rat hearts, we assessed the effects of the K+ATP channel antagonist glibenclamide (GLIB) and the agonist cromakalim (CROM) on pre- and post-ischemic CF and left ventricular developed pressure (LVDP). Although GLIB decreased pre-ischemic CF in both control and septic rat hearts, LVDP was unaffected. After I/R, CF was decreased in GLIB-treated control and septic rat hearts and LVDP was more severely depressed in control rat hearts than in septic rat hearts. CROM increased pre-ischemic CF in the septic group although LVDP was unaltered in both groups. After I/R, control rat heart CF was depressed but LVDP completely recovered. Post-ischemic CF in septic rat hearts was elevated compared with vehicle-treated septic rat hearts, but the recovery of LVDP was not improved. These results suggest that K+ATP channels modulate CF in septic rat hearts, but do not mediate cardioprotection as observed in control rat hearts.

Free fatty acids, but not ketone bodies, protect diabetic rat hearts during low-flow ischemia.

To determine whether the effects of fatty acids on the diabetic heart during ischemia involve altered glycolytic ATP and proton production, we measured energetics and intracellular pH (pH(i)) by using (31)P NMR spectroscopy plus [2-(3)H]glucose uptake in isolated rat hearts. Hearts from 7-wk streptozotocin diabetic and control rats, perfused with buffer containing 11 mM glucose, with or without 1.2 mM palmitate or the ketone bodies, 4 mM beta-hydroxybutyrate plus 1 mM acetoacetate, were subjected to 32 min of low-flow (0.3 ml x g wet wt(-1) x min(-1)) ischemia, followed by 32 min of reperfusion. In control rat hearts, neither palmitate nor ketone bodies altered the recovery of contractile function. Diabetic rat hearts perfused with glucose alone or with ketone bodies, had functional recoveries 50% lower than those of the control hearts, but palmitate restored recovery to control levels. In a parallel group with the functional recoveries, palmitate prevented the 54% faster loss of ATP in the diabetic, glucose-perfused rat hearts during ischemia, but had no effect on the rate of ATP depletion in control hearts. Palmitate decreased total glucose uptake in control rat hearts during low-flow ischemia, from 106 +/- 17 to 52 +/- 12 micromol/g wet wt, but did not alter the total glucose uptake in the diabetic rat hearts, which was 42 +/- 5 micromol/g wet wt. Recovery of contractile function was unrelated to pH(i) during ischemia; the glucose-perfused control and palmitate-perfused diabetic hearts had end-ischemic pH(i) values that were significantly different at 6.36 +/- 0.04 and 6.60 +/- 0.02, respectively, but had similar functional recoveries, whereas the glucose-perfused diabetic hearts had significantly lower functional recoveries, but their pH(i) was 6.49 +/- 0.04. We conclude that fatty acids, but not ketone bodies, protect the diabetic heart by decreasing ATP depletion, with neither having detrimental effects on the normal rat heart during low-flow ischemia.