Yukio Kuramochi

Cardiac Endothelial Cells Regulate Reactive Oxygen Species-induced Cardiomyocyte Apoptosis through Neuregulin-1β/erbB4 Signaling

Neuregulin (NRG)-1β has a prosurvival effect on cardiac myocytes via the phosphatidylinositol-3-kinase/Akt pathway, but the physiological regulators of this system in the intact heart are unknown. In this study, we tested the hypothesis that reactive oxygen species regulate NRG/erbB signaling. We used isolated adult rat ventricular myocytes (ARVMs) or cardiac microvascular endothelial cells (CMECs) in monoculture, or together in coculture. H2O2 induced NRG-1β release from CMECs in a concentration-dependent manner, and conditioned medium from H2O2-treated CMEC activated ARVM erbB4. NRG-1β release occurred via proteolytic cleavage of 115-kDa transmembrane NRG-1β and was inhibited by the metalloproteinase inhibitor 1,10-phenanthroline. In myocyte monoculture, H2O2 induced erbB4-dependent, but NRG-independent, activation of Akt. To elucidate the bioactivity of CMEC-derived NRG-1β on ARVMs, we examined H2O2-induced myocyte apoptosis in co-culture using an antibody to NRG-1β. The percentages of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were significantly higher in the anti-NRG-1β group than in the control group. The change in apoptosis induced by anti-NRG-1β in co-culture was similar in magnitude to the protection of myocytes by addition of recombinant NRG-1β to ARVM monocultures. Activation of NRG/erbB paracrine signaling was also seen in the intact heart subjected to oxidative stress by ischemia-reperfusion injury. Isolated perfused mouse hearts subjected to 15 min of ischemia, followed by 30 min of reperfusion, showed complete proteolytic cleavage of 115-kDa NRG-1β, with concomitant erbB4 phosphorylation. These results demonstrate that reactive oxygen species activate NRG-1β/erbB4 paracrine signaling in the heart and suggest that this system is involved in cardiac adaptation to oxidative stress.

Heat shock protein 90 stabilization of erbB2 expression is disrupted by ATP depletion in myocytes

Heat shock protein (Hsp) 90 is a ubiquitously expressed chaperone that stabilizes expression of multiple signaling kinases involved in growth regulation, including ErbB2, Raf-1, and Akt. The chaperone activity of Hsp90 requires ATP, which binds with ∼10-fold lower affinity than ADP. This suggests that Hsp90 may be a physiological ATP sensor, regulating the stability of growth signaling cascades in relation to cellular energy charge. Here we show that lowering ATP concentration by inhibiting glycolysis or mitochondrial respiration in isolated myocytes triggers rapid dissociation of Hsp90 from ErbB2 and degradation of ErbB2 along with other client proteins. The effect of disrupting Hsp90 chaperone activity by ATP depletion was similar to the effect of the pharmacological Hsp90 inhibitor geldanamycin. ATP depletion-induced disruption of Hsp90 chaperone activity was associated with cellular resistance to growth factor activation of intracellular signaling. ErbB2 degradation was also induced by the physiological stress of β-adrenergic receptor stimulation in electrically stimulated cells. These results support a role for Hsp90 as an ATP sensor that modulates tissue growth factor responsiveness under metabolically stressed conditions and provide a novel mechanism by which cellular responsiveness to growth factor stimulation is modulated by cellular energy charge.

Inhibition of ErbB2 by receptor tyrosine kinase inhibitors causes myofibrillar structural damage without cell death in adult rat cardiomyocytes

Inhibition of ErbB2 (HER2) with monoclonal antibodies, an effective therapy in some forms of breast cancer, is associated with cardiotoxicity, the pathophysiology of which is poorly understood. Recent data suggest, that dual inhibition of ErbB1 (EGFR) and ErbB2 signaling is more efficient in cancer therapy, however, cardiac safety of this therapeutic approach is unknown. We therefore tested an ErbB1-(CGP059326) and an ErbB1/ErbB2-(PKI166) tyrosine kinase inhibitor in an in-vitro system of adult rat ventricular cardiomyocytes and assessed their effects on 1. cell viability, 2. myofibrillar structure, 3. contractile function, and 4. MAPK- and Akt-signaling alone or in combination with Doxorubicin. Neither CGP nor PKI induced cardiomyocyte necrosis or apoptosis. PKI but not CGP caused myofibrillar structural damage that was additive to that induced by Doxorubicin at clinically relevant doses. These changes were associated with an inhibition of excitation-contraction coupling. PKI but not CGP decreased p-Erk1/2, suggesting a role for this MAP-kinase signaling pathway in the maintenance of myofibrils. These data indicate that the ErbB2 signaling pathway is critical for the maintenance of myofibrillar structure and function. Clinical studies using ErbB2-targeted inhibitors for the treatment of cancer should be designed to include careful monitoring for cardiac dysfunction.

Silent Myocardial ischemia in Kawasaki disease : Evaluation of percutaneous transluminal coronary angioplasty by dobutamine stress testing

BACKGROUND Myocardial ischemia and myocardial infarction are the most serious complications of coronary artery lesions in children with Kawasaki disease (KD). Therefore, early detection and treatment of myocardial ischemia in patients with KD is essential. We studied the effectiveness of percutaneous transluminal coronary angioplasty (PTCA) in patients with silent myocardial ischemia detected by dobutamine stress 99mTc myocardial scintigraphy (TMS), body surface mapping (BMS), and signal-averaged ECG late potentials (ELP). METHODS AND RESULTS Eight of 76 asymptomatic patients with a coronary stenosis >25% and a positive dobutamine stress test were considered to have silent myocardial ischemia. All eight patients had >95% stenoses demonstrated by coronary angiography (CAG) just before PTCA. After PTCA, CAG showed that all of the coronary artery stenoses had been reduced to <50%. Additionally, intravascular ultrasonography (IVUS) performed in five patients before and after PTCA demonstrated adequate dilation of the coronary stenosis after PTCA. All eight patients underwent dobutamine stress TMS, BMS, and ELP 2 to 3 months after PTCA, which demonstrated no regions of myocardial ischemia. Approximately 6 months later, CAG was performed in all eight patients, and only one patient had developed restenosis. CONCLUSIONS PTCA effectively dilates stenotic coronary arteries in children with KD. Moreover, dobutamine stress TMS, BMS, and ELP are useful for detecting silent myocardial ischemia and estimating the effectiveness of PTCA. Furthermore, IVUS is useful for evaluating the severity of coronary artery lesions before and after PTCA in patients with KD.

Hemodynamic factors of thrombus formation in coronary aneurysms associated with Kawasaki disease

Abstract Background: We studied the mechanism of thrombus formation in coronary aneurysms based on rheological findings.

Cardiomyocyte regeneration from circulating bone marrow cells in mice.

We investigated the role of circulating bone marrow cells (BMC) in cardiomyocyte regeneration. BMC, isolated from transgenic mice expressing enhanced green fluorescent protein (GFP), were transplanted into lethally irradiated C57BL6 mice. Five weeks after bone marrow transplantation (BMT), flow cytometric analysis for GFP-positive cells confirmed reconstitution of transplanted bone marrow. Bone marrow transplant mice subsequently underwent left coronary artery ligation (myocardial infarction) or sham-operation, and were killed at 1 mo or 3 mo after operation. Infarct size was similar in bone marrow transplant mice at 1 mo (47.1 ± 5.9%) and at 3 mo (45.3 ± 7.8%), and echocardiography at 2 and 8 wk revealed decreasing left ventricular function. In infarcted heart, GFP-positive cells that expressed desmin and troponin T-C were identified by confocal microscopy. GFP and troponin T-C double-positive cells were predominantly in the peri-infarcted region (1 mo, 365 ± 45 cells/50 sections; 3 mo: 458 ± 100 cells/50 sections; p < 0.05 versus noninfarct, infarct, and sham-operated regions). Furthermore, BMC mobilization and differentiation into cardiomyocytes was found to be complete within 1 mo after myocardial infarction. These results demonstrate that circulating BMC undergo mobilization and differentiation in cardiac cells after myocardial infarction. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon.

Rapid electrical stimulation induces early activation of kinase signal transduction pathways and apoptosis in adult rat ventricular myocytes

Chronic tachycardia in patients and rapid pacing in animal models induce myocardial dysfunction and initiate a cascade of compensatory adaptations that are ultimately unsustainable, leading to ventricular enlargement and failure. The molecular pathogenesis during the early stages of tachycardia‐induced cardiomyopathy, however, remains unclear. We utilized our previously reported cell culture pacing system to directly assess phosphatidylinositol‐3‐kinase (PI3K)/Akt and mitogen‐activated protein kinase (MAPK) signalling of adult rat ventricular myocytes (ARVM) in response to rapid electrical stimulation. Freshly isolated ARVMs were maintained quiescent (0 Hz), or continuously stimulated at 5 (normofrequency) and 8 Hz (rapid frequency). Pacing resulted in an increase in mitochondrial respiration, assessed by mitochondrial uptake of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) at 48 h. Rapid pacing at 8 Hz significantly increased cell injury and death as assessed by Trypan Blue uptake, creatine phosphokinase release, and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assay. Pacing at 5 Hz induced early, but weak, activation of Akt and protein kinase 38 (p38). Rapid pacing further augmented the early activation of Akt and p38, and induced extracellular signal‐related kinase (Erk) and c‐jun amino terminal kinase (JNK) activation. Incubation of ARVM with PI3K inhibitor LY294002 resulted in a twofold increase of TUNEL‐positive cells under all pacing conditions examined. In conclusion, rapid pacing has immediate and detrimental consequences for cardiomyocyte survival, with pro‐apoptotic pathways (e.g. JNK, p38) able to overwhelm antiapoptotic signalling (PI3K/Akt, Erk). The rapid pacing methodology described in this report will be particularly useful in determination of cell signalling pathways associated with tachycardia‐induced cardiomyopathy.

Feasibility of Percutaneous Transluminal Coronary Angioplasty to Patients with Kawasaki Disease as an Early Management Strategy

Abstract. We successfully performed percutaneous transluminal coronary angioplasty (PTCA) in three infants with Kawasaki disease ages 11 to 29 months. The time from the onset of disease to PTCA ranged from 6 to 21 months. On period reevaluation 12 to 40 months after PTCA, they had no evidence of myocardial ischemia or restenosis. As an early management strategy, PTCA could be a good palliation to control myocardial ischemia associated with Kawasaki disease. From our study and a literature review, we suggest that PTCA may be more effective if it is performed earlier, even in young patients.

Longitudinal estimation of signal‐averaged electrocardiograms in patients with Kawasaki disease

Background : Myocarditis associated with Kawasaki (KD) disease is prominent, but rarely detected by conventional methods. The hypothesis of this study is to see if signal‐averaged electrocardiogram can detect myocarditis with KD.

Coronary Artery Bypass Grafting by Utilizing Only Arterial Grafts in Patients with Coronary Artery Aneurysm after Kawasaki Disease

Recent studies have reported the efficacy of coronary arterial bypass grafts (CABG) for obstructive lesions of coronary arteries in Kawasaki disease; however, the efficacy in coronary aneurysms is still remains controversial. Recently we studied the coronary flow velocity and perfusion pressure of coronary aneurysms. The results of that study suggested that the stagnation of flow and the reduction of shear stress in large aneurysms could initiate thrombus formation. Thus, we have adopting CABG for patients with large coronary aneurysm to avoid future ischemic damage to the distal myocardium. Furthermore, to achieve long term patency, revascularization was performed using only arterial grafts. Thirteen patients (mean 11.9 years old) with Kawasaki disease underwent CABG in the last 10 years at our hospital. In these patients coronary aneurysms were located at the LMT in 4, LAD in 10, LCX in 4 and RCA in 9. As the arterial grafts, the left internal thoracic artery was used for all patients (8 were to LAD only, 5 were to the diagonal branch and LAD as a sequential graft), whereas the right internal thoracic artery and the right gastroepiploic artery were used for 3 and 1 patients respectively. All the patients survived from the operation without major morbidity. Coronary angiogram showed 100% patency and satisfactory growth of the grafts. Moreover, six of the large aneurysms showed complete degradation during the follow-up period. As a conclusion, we successfully applied CABG using only arterial grafts for the aneurysm as well as the stenosis of coronary arteries without any mortality and morbidity. The long term patency and the growth of the grafts were satisfactory. We consider that CABG with arterial grafts improve the patients quality of life.