Natsuya Keira

Intracellular ATP is required for mitochondrial apoptotic pathways in isolated hypoxic rat cardiac myocytes

OBJECTIVES The present study examined the possibility that intracellular ATP levels dictate whether hypoxic cardiac myocytes die by apoptosis or necrosis. BACKGROUND Although apoptosis and necrosis may appear to be distinct forms of cell death, recent studies suggest that the two may represent different outcomes of a common pathway. In ischemic myocardium, apoptosis appears early, while energy stores are presumably still available, followed only later by necrosis. METHODS Neonatal rat cardiac myocytes were exposed to continuous hypoxia, during which the intracellular ATP concentration was modulated by varying the glucose content in the medium. The form of cell death was determined at the end of the hypoxic exposure. RESULTS Under total glucose deprivation, ATP dropped precipitously and cell death occurred exclusively by necrosis as determined by nuclear staining with ethidium homodimer-1 and smearing on DNA agarose gels. However, with increasing glucose concentrations (10, 20, 50, 100 mg/dl) cellular ATP increased correspondingly, and apoptosis progressively replaced necrosis until it became the sole form of cell death, as determined by nuclear morphology, DNA fragmentation on agarose gels, and caspase-3 activation. The data showed a significantly positive correlation between myocyte ATP content and the percentage of apoptotic cells. Hypoxia resulted in lactate production and cellular acidification which stimulates apoptosis. However, acidification-induced apoptosis was also increased in an ATP-dependent fashion. Loss of mitochondrial membrane potential and cytochrome c release from the mitochondria was observed in both the apoptotic and necrotic cells. Furthermore, translocation of Bax from cytosol into mitochondria preceded these events associated with mitochondrial permeability transition. Increased lactate production and a lack of effect by the mitochondrial inhibitor oligomycin indicated that ATP was generated exclusively through glycolysis. CONCLUSIONS We demonstrate that ATP, generated through glycolysis, is a critical determinant of the form of cell death in hypoxic myocytes, independently of cellular acidification. Our data suggest that necrosis and apoptosis represent different outcomes of the same pathway. In the absence of ATP, necrosis prevails. However, the presence of ATP favors and promotes apoptosis.

Aldosterone Directly Induces Myocyte Apoptosis Through Calcineurin-Dependent Pathways

Background—Aldosterone has recently attracted considerable attention for its involvement in the pathophysiology of heart failure, in which apoptotic cell loss plays a critical role. This study examined whether aldosterone directly induces myocyte apoptosis via its specific receptors. Methods and Results—Neonatal rat cardiac myocytes were exposed to aldosterone (10−8 to 10−5 mol/L). Nuclear staining with Hoechst 33258 showed that aldosterone induced myocyte apoptosis in a dose- and time-dependent fashion. Treatment of myocytes with 10−5 mol/L aldosterone significantly increased the percentage of apoptosis (15.5±1.4%) compared with serum-deprived control (7.3±0.6%). Radio ligand binding assay revealed the existence of plasma membrane receptor with high affinity (Kd, 0.2 nmol/L) for aldosterone in cardiac myocytes but not in fibroblasts. Aldosterone rapidly (≈30 seconds) mobilized [Ca2+]i that was blocked by neomycin. Aldosterone induced dephosphorylation of the proapoptotic protein Bad, enhancement of mitochondrial permeability transition, decrease in mitochondrial membrane potential, and release of cytochrome c from the mitochondria into the cytosol with concomitant activation of caspase-3. These effects of aldosterone were inhibited by concurrent treatment with either an L-type Ca2+ channel antagonist, nifedipine, or inhibitors for the Ca2+-dependent phosphatase calcineurin, cyclosporin A and FK506. Conclusions—The present study demonstrates for the first time that the specific plasma membrane receptor (coupled with phospholipase C) for aldosterone is present on cardiac myocytes and that aldosterone accelerates the mitochondrial apoptotic pathway through activation of calcineurin and dephosphorylation of Bad, suggesting that the proapoptotic action of aldosterone may directly contribute to the progression of heart failure.

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.

Effects of ACE inhibition on myocardial apoptosis in an ischemia-reperfusion rat heart model.

Myocardial ischemia–reperfusion injury involves necrosis and apoptosis. The inhibition of angiotensin-converting enzyme (ACE) has been reported to suppress infarct size. In this study, it was investigated whether an ACE inhibitor affected myocardial apoptosis and apoptosis-related proteins in rats with experimental myocardial infarction. Anesthetized Sprague-Dawley rats were divided into four groups. Group I underwent 30 minutes of left coronary artery occlusion followed by 24 hours of reperfusion (control group); Group II underwent oral administration of the ACE inhibitor quinapril (10 mg/kg/day) before coronary occlusion (quinapril group); Group III underwent administration of the bradykinin B2-receptor antagonist Hoe 140 (250 &mgr;g/kg/day, subcutaneously) with quinapril (quinapril + Hoe 140 group); and Group IV underwent administration of Hoe 140 alone (Hoe 140 group). After reperfusion, myocardial infarct size was determined by triphenyltetrazolium chloride staining. Myocardial apoptosis was detected immunohistologically using terminal deoxynucleotidyl transferase–mediated nick end labeling staining and DNA electrophoresis. Myocardial caspase-3 activation was analyzed by Western blot and the expressions of Bcl-xL and Bax proteins were detected immunohistochemically. Quinapril significantly reduced the ratio of myocardial infarct size in the ischemic area at risk. In addition, quinapril significantly suppressed the incidence of apoptotic myocytes around the necrotic region (from 18.9 ± 0.8% to 8.6 ± 1.0%;P < 0.0001), the intensity of DNA ladder formation, and the activation of caspase-3. Hoe 140 attenuated these protective effects of quinapril. In the immunohistochemical study, Bax and Bcl-xL were expressed in myocytes, and ischemia–reperfusion abolished both proteins in the center region of ischemia. The Bax staining was equally observed among all groups. However, Bcl-xL staining remained in the ischemic area widely after quinapril treatment. In addition, Hoe 140 also depleted this effect of quinapril. These results suggest that inhibition of ACE reduces myocardial infarction and apoptosis via the bradykinin B2 receptor in part. The antiapoptotic effect of the ACE inhibitor is attributed to the changing expression of Bcl-xL.

The dual effects of nitric oxide synthase inhibitors on ischemia-reperfusion injury in rat hearts

Abstract.ObjectiveNitric oxide (NO) is known to act as a mediator of tissue injury as well as being a potent endogenous vasodilator. The functional and metabolic effects of NO on ischemia-reperfusion injury are still controversial. The aim of this study was to clarify the relationship between the degree of NO synthase (NOS) inhibition and the effects on ischemia-reperfusion injury.Methods and resultsLangendorff-perfused rat hearts were subjected to 30 minutes of global ischemia followed by 30 minutes of reperfusion. The recovery of left ventricular developed pressure (LVDP), creatine kinase (CK) release, and myocardial high energy phosphates were measured in hearts perfused with or without NOS inhibitors, L-NG-monomethyl arginine (L-NMMA) or NGnitro-L-arginine methylester (L-NAME). NOS inhibitors exerted different effects on the recovery of LVDP and CK release depending on the concentration. The low dose of L-NMMA improved the recovery of LVDP, decreased the CK release during reperfusion, and preserved the myocardial adenosine triphosphate content after reperfusion. In contrast, the high dose of L-NMMA had adverse effects. L-NMMA reduced NO release in coronary effluent in a dose-dependent fashion. Both effects of L-NMMA were abolished by excessive co-administration of L-arginine and the same doses of D-NG-monomethyl arginine (D-NMMA) showed no effect on ischemia-reperfusion injury. Therefore, both effects were due to NOS inhibition. In addition, L-NMMA suppressed the myocardial malondialdehyde accumulation, an indicator of oxidative stress, which might be attributed to the beneficial effects by partial NOS inhibition. On the other hand, the high dose L-NMMA significantly decreased coronary flow during aerobic perfusion and reperfusion. Therefore, it is conceivable that the vasoactive NOS inhibition contributes to the harmful effects, which might exceed the beneficial effects due to a decrease in oxidative stress.ConclusionThe present results showed that NO inhibitors had dual effects on mechanical function and energy metabolism depending on the concentration. Non-vasoactive inhibition of NOS had beneficial effects due to the suppression of oxidative injury. However, strong vasoactive inhibition of NOS exacerbated the ischemia-reperfusion injury.

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 at 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.

Subacutely progressed extensive aortic dissection complicated with catheter-induced dissection in left main coronary artery.

A 64-year-old man complaining of resting angina underwent emergent coronary angiogram and significant stenosis in the mid-left anterior descending artery was discovered. Although deployment of the drug-eluting Cypher stent relieved the stenosis, the guiding catheter accidentally induced coronary dissection in the left main coronary artery (LMCA). Then, deployment of another Cypher stent at the lesion successfully managed the complication. 20 days later, although asymptomatic, extensive aortic dissection was detected from the coronary sinus of Valsalva to the femoral artery. 64-Row multidetector computed tomography demonstrated that the dissection originated from the LMCA and retrogradely expanded to the aorta. This type of dissection is a rare complication related to coronary intervention and even in such a clinical setting, asymptomatic delayed progression of retrograde aortic dissection has not previously been reported to our knowledge.

Reverse bent wiring with Crusade catheter can be a useful technique for penetrating an abrupt-type entry of coronary occlusion at branching ostium.

We encountered a case of chronic total in-stent occlusion which involving an abrupt-type entry at an obtuse marginal branching ostium. It is usually difficult to antegradely penetrate this kind of proximal fibrous cap. Therefore, we adopted a reverse bent wiring technique with a Crusade catheter and successfully completed all procedures. This technique is very simple but can be very effective in specific situations in daily percutaneous coronary intervention (PCI). Many PCI operators may empirically adopt this kind of wire manipulation technique. However, this kind of technique has never been officially reported to our knowledge.

Clinical characteristics of silent myocardial ischemia diagnosed with adenosine stress 99mTc-tetrofosmin myocardial scintigraphy in Japanese patients with acute cerebral infarction

It is well known that silent myocardial ischemia (SMI) often complicates patients with cerebral infarction and that stroke patients often die of ischemic heart disease. Therefore, it is considered important to treat myocardial ischemia in stroke patients. This study investigated SMI complicating Japanese patients with fresh stroke, using 99mTc-tetrofosmin myocardial scintigraphy with pharmacologic stress testing to elucidate their clinical manifestations. This study included 41 patients (26 men, mean age 76.0 ± 10.7 years) with acute cerebral infarction and no history of coronary artery disease. All patients underwent 99mTc-tetrofosmin myocardial scintigraphy with intravenous administration of adenosine to diagnose SMI. Of the 41 patients, myocardial ischemia was confirmed in 17 patients (41.5%). Atherosclerotic etiology was the major cause of stroke in the ischemia(+) group and embolic origin was the major cause in the ischemia(−) group. Patients with myocardial ischemia had a higher incidence of diabetes mellitus (52.9 vs 20.8%; P = 0.0323) and more than two conventional cardiovascular risk factors (64.7 vs 25.0%; P = 0.0110) compared with the nonischemic patients. Infarction subtype of atherosclerotic origin was an independent positive predictor of asymptomatic myocardial ischemia in patients with stroke. These findings indicate that the prevalence of asymptomatic myocardial ischemia is relatively high, especially in patients with stroke of atherosclerotic origin. Therefore, it is beneficial for us to narrow the target population who are at the highest risk when screening for SMI in Japanese patients with acute cerebral infarction.