Mitsutoshi Asai

Ablation of C/EBP Homologous Protein Attenuates Endoplasmic Reticulum–Mediated Apoptosis and Cardiac Dysfunction Induced by Pressure Overload

Background— Apoptosis may contribute to the development of heart failure, but the role of apoptotic signaling initiated by the endoplasmic reticulum in this condition has not been well clarified. Methods and Results— In myocardial samples from patients with heart failure, quantitative real-time polymerase chain reaction revealed an increase in messenger RNA for C/EBP homologous protein (CHOP), a transcriptional factor that mediates endoplasmic reticulum–initiated apoptotic cell death. We performed transverse aortic constriction or sham operation on wild-type (WT) and CHOP-deficient mice. The CHOP-deficient mice showed less cardiac hypertrophy, fibrosis, and cardiac dysfunction compared with WT mice at 4 weeks after transverse aortic constriction, although the contractility of isolated cardiomyocytes from CHOP-deficient mice was not significantly different from that in the WT mice. In the hearts of CHOP-deficient mice, phosphorylation of eukaryotic translation initiation factor 2&agr;, which may reduce protein translation, was enhanced compared with WT mice. In the hearts of WT mice, CHOP-increased apoptotic cell death with activation of caspase-3 was observed at 4 weeks after transverse aortic constriction. In contrast, CHOP-deficient mice had less apoptotic cell death and lower caspase-3 activation at 4 weeks after transverse aortic constriction. Furthermore, the Bcl2/Bax ratio was decreased in WT mice, whereas this change was significantly blunted in CHOP-deficient mice. Real-time polymerase chain reaction microarray analysis revealed that CHOP could regulate several Bcl2 family members in failing hearts. Conclusions— We propose the novel concept that CHOP, which may modify protein translation and mediate endoplasmic reticulum–initiated apoptotic cell death, contributes to development of cardiac hypertrophy and failure induced by pressure overload.

Comparison of the prognostic value of cardiac iodine-123 metaiodobenzylguanidine imaging and heart rate variability in patients with chronic heart failure: a prospective study.

OBJECTIVES We sought to prospectively compare the prognostic value of cardiac iodine-123 (I-123) metaiodobenzylguanidine (MIBG) imaging with that of heart rate variability (HRV) in patients with mild-to-moderate chronic heart failure (HF). BACKGROUND Cardiac I-123 MIBG imaging, which reflects cardiac adrenergic nerve activity, provides prognostic information on chronic HF patients. Reduced HRV, indicating derangement in cardiac autonomic control, was also reported to be associated with a poor prognosis in chronic HF patients. METHODS At study entry, I-123 MIBG imaging and 24-h Holter monitoring were performed in 65 chronic HF outpatients with a radionuclide left ventricular ejection fraction <40%. The cardiac MIBG heart to mediastinum ratio (H/M) and washout rate (WR) were obtained from MIBG imaging. The time and frequency domain parameters of HRV were calculated from 24-h Holter recordings. RESULTS At a mean follow-up of 34 +/- 19 months, WR (p < 0.0001), H/M on the delayed image (p = 0.01), and normalized very-low-frequency power (n-VLFP) (p = 0.047) showed a significant association with the cardiac events (sudden death in 3 and hospitalization for worsening chronic HF in 10 patients) on univariate analysis. Multivariate analysis revealed that WR was the only independent predictor of cardiac events, although the predictive accuracy for the combination of abnormal WR and n-VLFP significantly increased, compared with that for abnormal WR (82% vs. 66%, p < 0.05). CONCLUSIONS Cardiac MIBG WR has a higher prognostic value than HRV parameters in patients with chronic HF. The combination of abnormal WR and n-VLFP would be useful to identify chronic HF patients at a higher risk of cardiac events.

Prognostic significance of cardiac 123I metaiodobenzylguanidine imaging for mortality and morbidity in patients with chronic heart failure: a prospective study

OBJECTIVE To determine whether cardiac iodine-123 metaiodobenzylguanidine (123I MIBG) imaging is useful in predicting the prognosis of patients with chronic heart failure. DESIGN Cardiac 123I MIBG imaging was done on entry to the study. The cardiac MIBG washout rate was calculated from anterior chest view images obtained 20 and 200 minutes after injection of the isotope. Study patients were divided into two groups with washout rates above and below 27% (the mean value + 2 SD obtained in 20 normal subjects), and were then followed up. SETTING Tertiary referral centre. PATIENTS 79 patients with chronic heart failure in whom the left ventricular ejection fraction was less than 40%. RESULTS There were 37 patients in group 1 (washout rate of ⩾ 27%) and 42 in group 2 (< 27%). During a follow up period of between 1 and 52 months, eight patients died suddenly and five died of worsening heart failure in group 1, while none died in group 2; 13 patients in group 1 and four in group 2 were admitted to hospital for progressive heart failure. Kaplan–Meier analysis showed that group 1 had a significantly higher mortality and morbidity (p = 0.001 and p < 0.001, respectively) than group 2. CONCLUSIONS Cardiac 123I MIBG washout rate seems to be a good predictor of prognosis in patients with chronic heart failure.

Overexpression of endoplasmic reticulum-resident chaperone attenuates cardiomyocyte death induced by proteasome inhibition

AIMS Proteasome inhibitors are a novel class of anticancer agents that induce tumour cell death via endoplasmic reticulum (ER) stress. Since ER stress is involved in the development of heart failure, we investigated the role of ER-initiated cardiomyocyte death by proteasome inhibition. METHODS AND RESULTS Rat neonatal cardiomyocytes were used in this study. Proteasome activity was assayed using proteasome peptidase substrates. Cell viability and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide and flow cytometry, respectively. Western blot analysis, real-time polymerase chain reaction (PCR) and reverse transcriptional PCR were used to detect the expression of protein and messenger ribonucleic acid (RNA). The location of overexpressed glucose-regulated protein (GRP) 78 was observed by confocal fluorescence microscopy. Proteasome inhibition induced cardiomyocyte death and activated ER stress-induced transcriptional factor ATF6, but not XBP1 (X-box binding protein 1), without up-regulating ER chaperones. ER-initiated apoptosis signalling, including cytosine-cytosine-adenine-adenine-thymine enhancer-binding protein (C/EBP) homologous protein (CHOP), c-Jun-N-terminal kinase (JNK), and caspase-12, was activated by proteasome inhibition. Short interference RNA targeting CHOP, but not the blockage of caspase-12 or JNK pathway, attenuated cardiomyocyte death. Overexpression of GRP78 suppressed both CHOP expression and cardiomyocyte death by proteasome inhibition. CONCLUSION These findings demonstrate that proteasome inhibition induces ER-initiated cardiomyocyte death via CHOP-dependent pathways without compensatory up-regulation of ER chaperones. Supplement and/or pharmacological induction of GRP78 can attenuate cardiac damage by proteasome inhibition.

PKA rapidly enhances proteasome assembly and activity in in vivo canine hearts.

Proteasome regulates diverse cellular functions by eliminating ubiquitinated proteins. Protein kinase A (PKA) is a key regulator of proteasome activity. However, it remains unknown how PKA regulates proteasome activity and whether it controls proteasome activity in in vivo hearts. Both the in vitro peptidase assay and the in-gel peptidase assays showed that the treatment with PKA for 30 min dose-dependently activated purified 26S proteasome. Simultaneously, PKA treatment enhanced phosphorylation and assembly of purified 26S proteasome evaluated by non-reducing native polyacrylamide gel electrophoresis, either of which was blunted by the pretreatment with a PKA inhibitor, H-89. In in vivo canine hearts, proteasome assembly and activity were enhanced 30 min after the exogenous or endogenous stimulation of PKA by the intracoronary administration of isoproterenol or forskolin for 30 min or by ischemic preconditioning (IP) with 4 times of repeated 5 min of ischemia. The intracoronary administration of H-89 blunted the enhancement of proteasome assembly and activity by IP. Myocardial proteasome activity at the end of ischemia was decreased compared with the control, however, it did not differ from the control in dogs with IP. IP decreased the accumulation of ubiquitinated proteins in the canine ischemia/reperfusion myocardium, which was blunted by the intracoronary administration of a proteasome inhibitor, epoxomicin. However, proteasome activation by IP was not involved in its infarct size-limiting effects. These findings indicate that PKA rapidly enhances proteasome assembly and activity in in vivo hearts. Further investigation will be needed to clarify pathophysiological roles of PKA-mediated proteasome activation in ischemia/reperfusion hearts.

X-box binding protein 1 regulates brain natriuretic peptide through a novel AP1/CRE-like element in cardiomyocytes

The unfolded protein response (UPR) is triggered to assist protein folding when endoplasmic reticulum (ER) function is impaired. Recent studies demonstrated that ER stress can also induce cell-specific genes. In this study, we examined whether X-box binding protein 1 (XBP1), a major UPR-linked transcriptional factor, regulates the expression of brain natriuretic peptide (BNP) in cardiomyocytes. In samples from failing human hearts, extensive splicing of XBP1 was observed along with increased expression of glucose-regulated protein of 78 kDa (GRP78), a target of spliced XBP1 (sXBP1), suggesting that the UPR was induced in heart failure in humans. Interestingly, quantitative real-time PCR revealed a positive correlation between cardiac expression of GRP78 and BNP, leading us to test the hypothesis that sXBP1 regulates BNP as well as GRP78 in cardiomyocytes. A pharmacological ER stressor caused a dose-dependent increase in the expression of sXBP1 and BNP by cultured cardiomyocytes. Short interfering RNA targeting XBP1 suppressed the induction of BNP expression by a pharmacological ER stressor or norepinephrine, which was rescued by the adenovirus-mediated overexpression of sXBP1. The promoter assay with overexpression of sXBP1 or norepinephrine showed that the proximal AP1/CRE-like element in the promoter region of BNP was critical for transcriptional regulation of BNP by sXBP1. Direct binding of sXBP1 to this element was confirmed by the chromatin immunoprecipitation assay. These findings suggest that ER stress observed in failing hearts regulates cardiac BNP expression through a novel promoter region of the AP1/CRE-like element.

Detection of disrupted plaques by coronary CT: comparison with angioscopy

Background Disrupted plaques are the major cause of acute coronary syndrome (ACS). Although the detection of vulnerable plaques by coronary CT (CCT) has been examined and reported, there has been no report on the detection of disrupted plaques by CCT. Objectives To test the ability of CCT to detect disrupted coronary plaques. Methods 32 consecutive patients with suspected ischaemic heart disease who underwent successful coronary angioscopic examination and CCT were analysed. Yellow plaques of colour grade 1−3 and disrupted yellow plaques were examined by angioscopy. CCT findings (low attenuation, positive remodelling and ring-like enhancement) were examined for each site of yellow plaques. Results In the 32 patients, 65 yellow plaques were detected. Higher-colour-grade yellow plaques and disrupted yellow plaques had a significantly higher incidence of CCT findings: low attenuation (grade 1 vs grade 2 vs grade 3, 18% vs 59% vs 69%; non-disrupted vs disrupted, 36% vs 66%), positive remodelling (24% vs 59% vs 75%; 33% vs 75%), and ring-like enhancement (0% vs 19% vs 25%; 6% vs 44%). Positive and negative predictive values for ring-like enhancement to detect disrupted plaque were 88% and 63%, respectively; those for the combined CCT findings (low attenuation, positive remodelling and ring-like enhancement) to detect disrupted plaque were 90% and 58%, respectively. Conclusion CCT findings were associated with disrupted plaques confirmed by angioscopy. Ring-like enhancement had a high positive predictive value for detecting disrupted plaque.

Frequency and Healing of Nonculprit Coronary Artery Plaque Disruptions in Patients With Acute Myocardial Infarction

The pathophysiology of plaque disruption and healing in nonculprit segments has not been clarified. Therefore, we investigated the frequency of plaque disruptions in nonculprit segments and whether those plaques are stabilized during follow-ups in patients with acute myocardial infarction (AMI) by serial angioscopic observations. Analyzed were 13 consecutive patients with AMI in whom infarct-related arteries were serially observed by angioscopy immediately after reperfusion and at 1- and 6-month follow-ups. Color of plaques was graded as 0 (white), 1 (slight yellow), 2 (yellow), or 3 (intensive yellow). Plaques with thrombus were defined as disrupted. Although number of nonculprit yellow plaques did not change from immediately after reperfusion to 6 months, the maximum color grade of those plaques and incidence of disrupted plaques in nonculprit segments (immediate vs 1 month vs 6 months 31% vs 8% vs 0%) decreased significantly by 6 months. Plaque stabilization as shown by disappearance of thrombus was significantly associated with plaque regression as shown by a decrease of maximum yellow color grade in nonculprit segments. In conclusion, patients with AMI frequently had disrupted and actively thrombogenic yellow plaques in nonculprit segments of the culprit vessel, and those plaques healed with decreases of yellow color grade and thrombogenicity during 6-months follow-up. Plaque disruption and healing occur not only at the culprit lesion but may be a pan-coronary process in patients with AMI.

Ruptured plaque and large plaque burden are risks of distal embolisation during percutaneous coronary intervention: evaluation by angioscopy and virtual histology intravascular ultrasound imaging.

AIMS Slow flow and no flow phenomena have been associated with distal embolisation, especially of plaque debris, and with unfavourable clinical outcomes. However, patients at high risk of distal embolisation for whom distal protection might be beneficial have not been adequately identified. We examined the frequency of distal embolisation and its predicting factors, including both ACS and non-ACS patients. METHODS AND RESULTS Consecutive patients (n=98) with or without ACS who had received PCI with a filter-type distal protection device and successful angioscopic and VH-IVUS examination were prospectively enrolled. The presence of yellow plaque and plaque rupture was evaluated by angioscopy. Tissue classification and plaque burden was evaluated by VH-IVUS. Distal embolisation was evaluated by pathological examination of material collected in the filter. Distal embolisation of plaque debris was more frequently detected in patients with ACS (48% vs. 25%, p=0.02), in those with ruptured plaque (86% vs. 13%, p<0.001), in those with large (>75%) plaque burden (50% vs. 23%, p=0.006), and in those with grade 2/3 yellow plaque (52% vs. 7%, p<0.001), as compared to those without it. CONCLUSIONS The presence of ruptured yellow plaque and of large plaque burden, rather than the setting of ACS, was highly predictive of distal embolisation of plaque debris.

Thrombogenic potential of whole blood is higher in patients with acute coronary syndrome than in patients with stable coronary diseases.

INTRODUCTION Although thrombogenic potential of blood may play an important role for the onset of acute coronary syndrome (ACS), there is no established way to evaluate it by single parameter. We compared the thrombogenic potential of whole blood between patients with ACS and those with stable coronary diseases using single comprehensive parameter. MATERIALS AND METHODS Consecutive patients with ACS (n=146) and those with stable coronary heart diseases (control, n=92) were prospectively examined. Thrombogenic potential of whole blood was evaluated by blood vulnerability index measured by Micro-Channel Array Flow Analyzer (MC-FAN). RESULTS Blood vulnerability index was higher in ACS than in control patients (5099±2278 vs. 2071±389, p<0.0001), higher in acute MI than in unstable angina patients (5693±2146 vs. 3524±1841, p<0.0001), and higher in ACS patients with initial TIMI 0/1 flow grade than in those with TIMI 2/3 flow grade (6061±1936 vs. 2560±1301, p<0.0001). Furthermore, blood vulnerability index decreased from acute to chronic stage in acute MI patients. Multivariate logistic regression analysis revealed that high blood vulnerability index, high LDL cholesterol, high CRP, no use of aspirin, and no use of β-blocker were the independent contributors for the onset of ACS. CONCLUSION High thrombogenic potential of whole blood evaluated by blood vulnerability index was significantly associated with ACS and was reduced from acute to chronic stage in acute MI. CONDENSED ABSTRACT Thrombogenic potential of whole blood was evaluated by blood vulnerability index measured comprehensively by Micro-Channel Array Flow Analyzer (MC-FAN) in consecutive patients with ACS (n=146) or stable coronary diseases (control, n=92) prospectively. Blood vulnerability index was significantly higher in ACS patients, especially in acute MI and poor initial TIMI flow grade patients, compared with control patients; and blood vulnerability index was reduced from acute to chronic stage in acute MI patients.