Cytokine-induced nitric oxide production inhibits mitochondrial energy production and impairs contractile function in rat cardiac myocytes.

OBJECTIVES: The present study examined whether nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) can directly inhibit aerobic energy metabolism and impair cell function in interleukin (IL)-1beta,-stimulated cardiac myocytes. BACKGROUND: Recent reports have indicated that excessive production of NO induced by cytokines can disrupt cellular energy balance through the inhibition of mitochondrial respiration in a variety of cells. However, it is still largely uncertain whether the NO-induced energy depletion affects myocardial contractility. METHODS: Primary cultures of rat neonatal cardiac myocytes were prepared, and NO2-/NO3- (NOx) in the culture media was measured using Griess reagent. RESULTS: Treatment with IL-1beta (10 ng/ml) increased myocyte production of NOx in a time-dependent manner. The myocytes showed a concomitant significant increase in glucose consumption, a marked increase in lactate production, and a significant decrease in cellular ATP (adenosine 5'-triphosphate). These metabolic changes were blocked by co-incubation with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis. Sodium nitroprusside (SNP), a NO donor, induced similar metabolic changes in a dose-dependent manner, but 8-bromo-cyclic guanosine 3',5'-monophosphate (8-bromo-cGMP), a cGMP donor, had no effect on these parameters. The activities of the mitochondrial iron-sulfur enzymes, NADH-CoQreductase and succinate-CoQreductase, but not oligomycin-sensitive ATPase, were significantly inhibited in the IL-1beta, or SNP-treated myocytes. Both IL-1beta and SNP significantly elevated maximum diastolic potential, reduced peak calcium current (I(Ca)), and lowered contractility in the myocytes. KT5823, an inhibitor of cGMP-dependent protein kinase, did not block the electrophysiological and contractility effects. CONCLUSIONS: These data suggest that IL-1beta-induced NO production in cardiac myocytes lowers energy production and myocardial contractility through a direct attack on the mitochondria, rather than through cGMP-mediated pathways.

Cardioprotective effect of angiotensin-converting enzyme inhibition against hypoxia/reoxygenation injury in cultured rat cardiac myocytes.

BACKGROUND: Although ACE inhibitors can protect myocardium against ischemia/reperfusion injury, the mechanisms of this effect have not yet been characterized at the cellular level. The present study was designed to examine whether an ACE inhibitor, cilazaprilat, directly protects cardiac myocytes against hypoxia/reoxygenation (H/R) injury. METHODS AND RESULTS: Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 5.5 hours and subsequently reoxygenated for 1 hour. Myocyte injury was determined by the release of creatine kinase (CK). Both cilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved myocyte ATP content during H/R, whereas CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not. The protective effect of cilazaprilat was significantly inhibited by Hoe 140 (a bradykinin B2 receptor antagonist), NG-monomethyl-L-arginine monoacetate (L-NMMA) (an NO synthase inhibitor), and methylene blue (a soluble guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine (an inhibitor of inducible NO synthase), or indomethacin (a cyclooxygenase inhibitor). Cilazaprilat significantly enhanced bradykinin production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes. In addition, cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could be blunted by L-NMMA and methylene blue but not by aminoguanidine. CONCLUSIONS: The present study demonstrates that cilazaprilat can directly protect myocytes against H/R injury, primarily as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive NO synthase in hypoxic myocytes in an autocrine/paracrine fashion. NO modulates guanylate cyclase and cGMP synthesis in myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R injury.

Energy metabolism after ischemic preconditioning in streptozotocin-induced diabetic rat hearts.

OBJECTIVES: The aim of this study was to compare the cardioprotective effects of preconditioning in hearts from streptozotocin-induced diabetic rats with its effects in normal rat hearts. BACKGROUND: The protective effect of ischemic preconditioning against myocardial ischemia may come from improved energy balance. However, it is not known whether preconditioning can also afford protection to diabetic hearts. METHODS: Isolated perfused rat hearts were either subjected (preconditioned group) or not subjected (control group) to preconditioning before 30 min of sustained ischemia and 30 min of reperfusion. Preconditioning was achieved with two cycles of 5 min of ischemia followed by 5 min of reperfusion. RESULTS: In the preconditioned groups of both normal and diabetic rats, left ventricular developed pressure, high energy phosphates, mitochondrial adenosine triphosphatase and adenine nucleotide translocase activities were significantly preserved after ischemia-reperfusion; cumulative creatine kinase release was smaller during reperfusion; and myocardial lactate content was significantly lower after sustained ischemia. However, cumulative creatine kinase release was less in the preconditioned group of diabetic rats than in the preconditioned group of normal rats. Under ischemic conditions, more glycolytic metabolites were produced in the diabetic rats (control group) than in the normal rats, and preconditioning inhibited these metabolic changes to a similar extent in both groups. CONCLUSIONS: The present study demonstrates that in both normal and diabetic rats, preservation of mitochondrial oxidative phosphorylation and inhibition of glycolysis during ischemia can contribute to preconditioning-induced cardioprotection. Furthermore, our data suggest that diabetic myocardium may benefit more from preconditioning than normal myocardium, possibly as a result of the reduced production of glycolytic metabolites during sustained ischemia and the concomitant attenuation of intracellular acidosis.

Myocardial stretch induced by increased left ventricular diastolic pressure preconditions isolated perfused hearts of normotensive and spontaneously hypertensive rats.

OBJECTIVE: The aim of our study was to determine whether myocardial stretch (non-ischemic stress) could precondition isolated perfused hearts of both normotensive Wister-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: The perfused hearts in Langendorff mode were subjected to 30 min of global no-flow ischemia followed by 30 min of reperfusion. Left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) were measured. In the control group, LVEDP was set at 10 mmHg. In the stretch group, LVEDP was increased to 30 or 60 mmHg for 5 min before 30 min of ischemia. In the ischemic preconditioning group, the hearts were exposed to two cycles of a 5-min period of ischemia before 30 min of ischemia. Myocardial lactate contents were measured at the baseline and at the end of the 60 mmHg stretch. RESULTS: Hemodynamic parameters of LVDP and LVEDP at 30 min of reperfusion improved in the stretch group (LVEDP of 60 mmHg) and the ischemic preconditioning group. Coronary flow did not decrease during the stretch. Recovery of the coronary flow during reperfusion was better in the stretch and ischemic preconditioning groups. Postischemic contractile function was better in WKY rats than in SHR. Myocardial lactate contents at the end of 60 mmHg stretch were negligible. CONCLUSIONS: Myocardial stretch induced by increasing LVEDP preconditioned isolated perfused hearts of both WKY rats and SHR, via mechanisms not involving myocardial ischemia during stretch.

Effects of glibenclamide and nicorandil in post-ischaemic contractile dysfunction of perfused hearts in normotensive and spontaneously hypertensive rats.

OBJECTIVE: We have demonstrated previously that nicorandil, an ATP-sensitive potassium channel opener, improved post-ischaemic contractile dysfunction of perfused hearts in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats dose-dependently. This study aimed to characterize the effect of glibenclamide, an ATP-sensitive potassium channel blocker, and nicorandil in post-ischaemic contractile dysfunction of SHR and WKY rats. METHODS: The perfused hearts were subjected to 30 min of global ischaemia and then 30 min of reperfusion. Administration of 10 or 50 mumol/l glibenclamide or of a combination of glibenclamide and 300 mumol/l nicorandil was performed for 10 min before the ischaemia. The left ventricular developed pressure and end-diastolic pressure were measured. RESULTS: Postischaemic contractile function was better in WKY rats than it was in SHR. Neither glibenclamide nor a combination of glibenclamide and nicorandil influenced the postischaemic contractile function or increased the incidence of reperfusion arrhythmias. The recoveries of coronary flow and heart rate after reperfusion were poor and the incidence of reperfusion arrhythmias was low in SHR. CONCLUSIONS: These results suggest that nicorandil improves postischaemic contractile dysfunction via a mechanism involving ATP-sensitive potassium channel opening both in SHR and in WKY rats. The hypertensive hearts were more susceptible to cardiac reperfusion dysfunction, compared with normal hearts.

Effect of ischemic preconditioning on mitochondrial oxidative phosphorylation and high energy phosphates in rat hearts.

The ability of ischemic preconditioning (IP) to protect the myocardium against prolonged ischemia may derive from improved energy balance. We therefore examined myocardial energy metabolism and mitochondrial oxidative phosphorylation in isolated perfused rat hearts which were either subjected (IP group), or not subjected (control group), to preconditioning prior to 30 min sustained ischemia and 30 min reperfusion. Preconditioning was achieved with two cycles of 5 min ischemia followed by 5 min reperfusion. Recovery of myocardial function was significantly greater, and creatine kinase release was significantly lower, in the IP group. Although ATP hydrolysis during the sustained ischemia remained unchanged in both groups, greater preservation of high energy phosphate (eg. ATP and CP) was observed in the IP group after reperfusion. CP content immediately after preconditioning greatly exceeded pre-ischemic values. Lactate production during the sustained ischemia was significantly lower in the IP group, suggesting a decrease in anaerobic glycolysis and a probable attenuation of intracellular acidosis. Oligomycin-sensitive mitochondrial ATPase activity in the control group was significantly decreased both after the sustained ischemia and the reperfusion, but in the IP group it did not change after the preconditioning, sustained ischemia, or reperfusion. Although atractyloside-inhibitable adenine nucleotide translocase activity was markedly decreased during sustained ischemia in both groups, its activity was significantly higher after reperfusion in the IP group. These data suggest that (1) mitochondrial ATPase contributes only slightly to ATP depletion during sustained ischemia, (2) both the CP overshoot phenomenon and the decrease in anaerobic glycolysis can be attributable to cardioprotection during the sustained ischemia, and (3) the preservation of ATPase and adenine nucleotide translocase activities may be a possible explanation for the restoration of high energy phosphates after sustained ischemia-reperfusion injury in the preconditioned hearts of rats.

Effect of doxorubicin on postrest contraction in isolated rat hearts.

Clinical use of doxorubicin is limited by its cardiotoxicity. In doxorubicin-induced cardiomyopathy, vacuolization of the sarcoplasmic reticulum (SR) has been reported. We investigated whether doxorubicin had a direct action on the sarcoplasmic reticulum (SR) in isolated perfused rat hearts. The left and right atria were trimmed to maintain heart rate (HR) <200 beats/min. Postrest contractions, which are believed to be due primarily to Ca2+ release from the SR, were evoked with a programmable stimulator after variable rest intervals. The amplitude of the postrest contractions increased markedly as the rest interval increased. Doxorubicin (0.1 mM) significantly suppressed this potentiation of the postrest contractions. Furthermore, doxorubicin slowly induced aftercontractions and an increase in left ventricular diastolic pressure (LVDP), phenomena that are usually associated with ouabain intoxication. We conclude that doxorubicin-induced cardiomyopathy may be due to SR dysfunction, leading to intracellular Ca2+ overload.

Effect of nicorandil on cardiac dysfunction during reperfusion in normotensive and spontaneously hypertensive rats.

The cardioprotective effect of nicorandil, an opener of ATP-sensitive potassium channels, was studied in the isolated perfused hearts of the spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto (WKY) rat. The hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. Controls received no drug. In the nicorandil group, the hearts were treated with 0.03 to 0.3 mmol/L nicorandil for 15 min before ischemia. Left ventricular developed pressure (LVDP) and end diastolic pressure (LVEDP) at 30 min of reperfusion were significantly lower and larger, respectively, in SHR than in WKY rats. Nicorandil improved LVDP and decreased LVEDP at 30 min of reperfusion in both SHR and WKY rats dose-dependently. The hypertensive heart in the early stage is already susceptible to reperfusion-cardiac dysfunction. Nicorandil has a beneficial effect on the post-ischemic dysfunction in both SHR and WKY rats.

Effects of ischemic preconditioning on the release of cardiac troponin T in isolated rat hearts.

The aim of this study was to examine the effect of ischemic preconditioning on the releases of cardiac troponin T (TnT) during reperfusion in isolated rat hearts. Experiments were done on 22 rat hearts, which were perfused according to the method of Langendorff and were divided into the control group (n = 14) and the preconditioning group (n = 8). Double 5 min of ischemia each followed by 5 min reflow were applied as ischemic preconditioning. After 20 min of global ischemia, the releases of TnT, creatine kinase (CK), and lactate dehydrogenase (LD) in coronary effluent and the left ventricular developed pressure (LVP) were measured during 60 min of reperfusion. Ischemic preconditioning significantly suppressed the amounts of TnT released during reperfusion, as with those of CK and LD, and also improved contractile dysfunction (nine hearts in which ventricular fibrillation was sustained were excluded from the evaluation for hemodynamics), though the release kinetics of TnT was different from that of CK and LD. There were good inverse relationships between the LVP and the total amounts of TnT released during reperfusion period (sigma TnT) or TnT levels at 60 min of reperfusion. Cardiac TnT can be used as a useful biochemical marker for hemodynamics and myocardial damage after reperfusion.

Effects of repeated ischemia on release kinetics of troponin T, creatine kinase, and lactate dehydrogenase in coronary effluent from isolated rat hearts.

We studied the release kinetics of cardiac troponin T (TnT) from coronary effluent in a re-stenosis model of 13 isolated rat hearts. After a 20-min period of global ischemia, we reperfused the hearts for 60 min according to the method of Langendorff. A second period of global ischemia was then induced for 5 min (protocol A) or 20 min (protocol B), followed by a second 60-min period of reperfusion. Coronary flow was measured by a timed collection of the coronary effluent. Levels of TnT in the effluent were compared to those of creatine kinase (CK) and lactate dehydrogenase (LD). Levels of TnT increased after the second global ischemia, but no differences were found in the released levels of TnT between protocols A and B. However, the amounts of CK and LD released in protocol B were much greater than those released in protocol A. These studies indicate that the release kinetics of TnT are different from that of CK and LD during reperfusion. It appears that after the initial ischemic damage to TnT, subsequent ischemia causes damage to TnT regardless of the duration of the insult, whereas the damage to sarcolemma is dependent on the duration of the ischemia.

Release kinetics and correlation with hemodynamic dysfunction of cardiac troponin T in coronary effluent from isolated rat hearts during reperfusion.

Previously, we reported that cardiac troponin T (TnT) can be detected and measured in coronary effluent from isolated rat hearts during hypoxia. The present study was designed to evaluate the release kinetics of TnT from post-ischemic rat hearts. Using the Langendorff technique, the hearts were reperfused for 4 h after 20 min or 60 min of global ischemia. Coronary flow was measured by timing the collection of the coronary perfusate that dripped from the hearts, and left ventricular pressure (LVP) was monitored continuously during the experiments. The amount of TnT released in 1 min was compared with the release of creatine kinase (CK) and lactate dehydrogenase (LD). The release kinetics of CK and LD showed a monophasic pattern and the levels at 4 h after reperfusion returned to baseline levels. By contrast, the release kinetics of TnT showed a small peak followed by a larger and more sustained peak. There were good negative correlations between developed pressure of LVP and both sigma TnT and the amount of TnT released within 1 min at 4 h after reperfusion. These results indicate that the release kinetics of TnT is different from that of CK and LD during reperfusion, and further that cardiac TnT is a useful indicator of myocardial cell damage and can be used to evaluate the degree of myocardial cell damage in both the early and late phase of acute myocardial infarction.

The benefit of head rotation on pharyngoesophageal dysphagia.

This study examined the effect of head rotation on the mechanics of swallowing in healthy subjects, as well as the effects of this postural change on the oropharyngeal swallow of five patients with lateral medullary syndrome (LMS). Videofluoroscopic studies of swallowing in the normal subjects revealed that head rotation to either side increased upper esophageal sphincter (UES) opening diameter by an average of 2mm without affecting the period of UES opening or the oropharyngeal transit time. Maximal rotation of the head to the right or left caused the bolus to lateralize away from the direction of rotation, and also caused a significant (18mmHg or 35%) fall in UES pressure. In the face forward position, the LMS patients exhibited barium residue in the pharynx and pyriform sinuses, as well as diminished UES opening diameter. The fraction of the bolus swallowed and the UES opening diameter increased significantly with the head turned toward the paretic side in the LMS patients. We conclude that head rotation can improve swallowing in patients with unilateral oropharyngeal dysphagia. Two potentially beneficial effects were observed: (1) functional exclusion of the relatively flaccid, weakened pharyngeal wall, and (2) reduced UES tone. Which of these mechanisms is operative probably depends on the dominant mechanisms of dysphagia. In individuals with substantial impairment of UES opening, head turning reduces the resistance of the sphincter that must be overcome by pharyngeal contraction. In individuals with a flaccid hemipharynx, which dissipates pharyngeal pressure, head rotation excludes these structures from the bolus path and allows pharyngeal pressure to be directed at the UES.

Preliminary observations on the effects of age on oropharyngeal deglutition.

Swallows of 4 bolus volumes (1, 5, 10, 20 ml) were examined in three groups of subjects: 6 subjects 20-29 years of age, 12 subjects 30-59 years of age, and 6 subjects 60-79 years of age. A simultaneous manometric and videofluoroscopic data collection protocol permitted measurement of bolus transit, temporal aspects of the oropharyngeal swallow, and pharyngeal peristalsis. Statistically significant effects of increasing bolus volume were oral transit of the bolus head (decreased) and duration of cricopharyngeal opening (increased). Five measures were significantly changed with increasing age: duration of pharyngeal swallow delay (increased), duration of pharyngeal swallow response (decreased), duration of cricopharyngeal opening (decreased), peristaltic amplitude (decreased), and peristaltic velocity (decreased).