Akiko Mano

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.

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.

Spironolactone Modulates Expressions of Cardiac Mineralocorticoid Receptor and 11β-Hydroxysteroid Dehydrogenase 2 and Prevents Ventricular Remodeling in Post-Infarct Rat Hearts

Aldosterone antagonists have been reported to prevent ventricular remodeling after myocardial infarction (MI) via their action to extracellular matrix (ECM). However, it remains largely unknown whether aldosterone antagonists attenuate myocyte loss in the remodeling process. The present study examined whether spironolactone prevents myocyte apoptosis and improves post-infarct ventricular remodeling in rats. MI was achieved by permanent occlusion of the left coronary artery. Administration of spironolactone (100 mg/kg/day) was started immediately after MI. Sprague-Dawley rats were divided into four groups: 1) sham, 2) spironolactone-treated sham, 3) untreated MI, 4) spironolactone-treated MI. Echocardiographic parameters (left ventricular [LV] diastolic dimension [LVDd], fractional shortening [%FS]), hemodynamic parameters (LV systolic pressure [LVSP], LV end-diastolic pressure [LVEDP], dP/dtmax and dP/dtmin) and collagen accumulation quantitated by Massons Trichrome staining were significantly improved in the spironolactone-treated MI group on the 14th day, compared with the untreated MI group. Moreover, the percentage of apoptotic myocytes evaluated by terminal deoxynucleotide transferase–mediated dUTP nick end labeling (TUNEL) assay was significantly lower in the spironolactone-treated MI group on the 2nd (3.54% vs. 5.79% in untreated MI group), 7th (0.65% vs. 1.37% in untreated MI group) and 14th days (0.11% vs. 0.16% in untreated MI group). Real time reverse transcription–polymerase chain reaction (RT-PCR) analysis showed that the expression of mineralocorticoid receptor (MR) mRNA and that of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) mRNA, which is known to confer aldosterone selectivity on MR, were upregulated in the untreated MI group, and that spironolactone significantly suppressed the expression of these genes. Moreover, spironolactone significantly inhibited aldosterone-induced apoptosis in cultured rat cardiac myocytes in a dose-dependent fashion. Our study demonstrates that, in addition to their effect on ECM, aldosterone antagonists inhibit myocyte apoptosis and prevent post-infarct ventricular remodeling by modulating the expression levels of MR and 11β-HSD2, which are enhanced in the remodeling heart.

Utility of left ventricular systolic torsion derived from 2-dimensional speckle-tracking echocardiography in monitoring acute cellular rejection in heart transplant recipients

BACKGROUND Reduced left ventricular torsion (LV-tor) has been reported to be associated with acute rejection in heart transplant (HTx) recipients. We investigated the utility of LV-tor analysis derived from 2-dimensional speckle-tracking echocardiography (2D-STE) for detecting allograft rejection. METHODS A total of 301 endomyocardial biopsies (EMBs), right heart catheterizations and echocardiograms were performed in 32 HTx recipients. Echocardiography was done within 3 hours from EMB or simultaneously with the procedures. The LV-tor was defined as the difference between apical and basal end-systolic rotations. The LV-tor values with and without cellular rejection were compared. In addition, we investigated whether the change in LV-tor values predicts the change in rejection grade in each patient. The baseline LV-tor value in each patient was defined as a mean value of the first 3 LV-tor measurements obtained when the patient was free from rejection. RESULTS According to the conventional International Society for Heart and Lung Transplantation criteria, 274 biopsies showed a rejection Grade of 0, 1a or 1b (Group AR(-)), whereas 27 biopsies were Grade 2 or higher (Group AR(+)). LV-tor decreased more in Group AR(+) than in Group AR(-) (9.3 ± 0.7 vs 12.2 ± 0.2 degrees, p < 0.0001). In the LV-tor measurement for each patient, the 25% reduction in LV-tor value from baseline predicted Grade 2 or higher rejection with a predictive accuracy of 92.9%. CONCLUSION LV-tor derived from 2D-STE could be of clinical value for non-invasive monitoring of acute rejection in HTx recipients.

Body mass index is a useful predictor of prognosis after left ventricular assist system implantation.

BACKGROUND The obesity paradox has recently attracted considerable interest in the study of many diseases. In this investigation we examine the relationship between body mass index (BMI) and prognosis after left ventricular assist system (LVAS) implantation. METHODS We measured the BMI of 64 patients 3 months after LVAS implantation for end-stage heart failure. The patients were classified according to BMI into Group A (BMI <16 kg/m(2)), Group B (BMI 16 to 18.4 kg/m(2)) or Group C (BMI > or =18.5 kg/m(2)). We compared the prognosis among these three groups after a mean follow-up period of 583 days. RESULTS Seven patients were weaned from their LVAS, 24 received heart transplantation, 25 died on the transplant waiting list, and 8 remain on the list. Long-term (>1 year) mortality was significantly higher in Group A than in Groups B and C (59% vs 40% and 18%, respectively; p < 0.05). The incidence of sepsis was also significantly higher in Group A than in Groups B and C (68% vs 45% and 32%, respectively; p < 0.05). After multivariate adjustment, BMI <16 kg/m(2) (hazard ratio [HR] 14.9; 95% confidence interval [CI] 2.61 to 86.0; p < 0.01) and levels of C-reactive protein (HR 1.56; 95% CI 1.15 to 2.13; p < 0.01) were independent predictors of mortality. CONCLUSIONS A lower BMI indicated a poor prognosis, as well as a higher incidence of a fatal complication, sepsis, after LVAS implantation. Control of BMI could be an effective way to improve management of patients with LVAS.

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.

Which factors predict the recovery of natural heart function after insertion of a left ventricular assist system

BACKGROUND Recent reports have demonstrated that use of a left ventricular assist system (LVAS) can initiate recovery of cardiac function, and subsequent weaning from the LVAS has attracted considerable interest. In this study we investigated reliable predictors of LVAS weaning. METHODS Eighty-two patients underwent LVAS implantation between April 1994 and July 2006 at our institution. Cardiac function was restored in 8 patients, who were weaned from LVAS after a mean of 5 months (Group R). Thirty-three patients remained on LVAS support for >1 year (Group N) because natural heart function did not show adequate improvement. We retrospectively evaluated the differences between these two groups. Group R was younger, and had a shorter duration of heart failure than Group N (23.4 vs 36.7 years and 13.3 vs 56.1 months, p < 0.01, respectively). Pathologic findings showed that the interstitial fibrosis score was lower in Group R (p < 0.01). Three months after LVAS insertion, B-type natriuretic peptide (BNP) and fractional shortening (FS) were more favorable (66.6 +/- 46 vs 264.5 +/- 170 pg/ml, p < 0.01, and 23 +/- 17.1 vs 12 +/- 9.1%, p < 0.05, respectively) in Group R. Furthermore, Group R received a higher dose of beta-blocker (15.4 +/- 8.4 vs 5.8 +/- 3.9 mg, p < 0.05). CONCLUSIONS Younger age, shorter history of heart failure, and less interstitial fibrosis were effective predictors of weaning from LVAS. Restoration of natural heart function was more rapid and more persistent in candidates for LVAS explantation, and presence of beta-blocker played a prominent role in improving cardiac function after LVAS implantation.

An adult patient with Kabuki syndrome presenting with Henoch-Schönlein purpura complicated with pulmonary hemorrhage

We present a case of a 33-year-old woman with Kabuki syndrome (KS) presenting with Henoch-Schönlein purpura (HSP). She was admitted to our hospital with a brain abscess in the lateral ventricle and meningitis. She had been diagnosed with KS. Skin eruptions had appeared on her lower extremities, with arthralgia, cough, and hemoptysis. She suddenly developed pulmonary hemorrhage and respiratory failure. We intubated her trachea and started mechanical ventilation in the intensive care unit (ICU). Skin biopsy revealed leukocytoclastic vasculitis with granular depositions of immunoglobulin A (IgA) in dermal vessel walls, and she was diagnosed as having HSP. Supportive management and prednisolone at 20 mg·day−1 cured the pulmonary hemorrhage and respiratory failure. On ICU day 27, she was weaned from mechanical ventilation. Pulmonary hemorrhage as a complication of HSP is rare and sometimes fatal. KS is often associated with an increased incidence of infection and congenital heart disease. Susceptibility to infection and pulmonary hypertension due to congenital heart disease in this patient may have led to the development of the pulmonary hemorrhage. Supportive care and steroid therapy appeared to be beneficial in the treatment of this patient with HSP with pulmonary hemorrhage.

Chest computed tomography of a patient revealing severe hypoxia due to amniotic fluid embolism: a case report

IntroductionAmniotic fluid embolism is one of the most severe complications in the peripartum period. Because its onset is abrupt and fulminant, it is unlikely that there will be time to examine the condition using thoracic computed tomography (CT). We report a case of life-threatening amniotic fluid embolism, where chest CT in the acute phase was obtained.Case presentationA 22-year-old Asian Japanese primiparous woman was suspected of having an amniotic fluid embolism. After a Cesarean section for cephalopelvic disproportion, her respiratory condition deteriorated. Her chest CT images were examined. CT findings revealed diffuse homogeneous ground-glass shadow in her bilateral peripheral lung fields. She was therefore transferred to our hospital. On admission to our hospitals intensive care unit, she was found to have severe hypoxemia, with SpO2 of 50% with a reservoir mask of 15 L/min oxygen. She was intubated with the support of noninvasive positive pressure ventilation. She was successfully extubated on the sixth day, and discharged from the hospital on the twentieth day.ConclusionThis is the first case report describing amniotic fluid embolism in which CT revealed an acute respiratory distress syndrome-like shadow.