Keiji Okuda

Cardiac Iodine-123 Metaiodobenzylguanidine Imaging Predicts Sudden Cardiac Death Independently of Left Ventricular Ejection Fraction in Patients With Chronic Heart Failure and Left Ventricular Systolic Dysfunction: Results From a Comparative Study With Signal-Averaged Electrocardiogram, Heart Rate Variability, and QT Dispersion

OBJECTIVES We prospectively compared the predictive value of cardiac iodine-123 metaiodobenzylguanidine (MIBG) imaging for sudden cardiac death (SCD) with that of the signal-averaged electrocardiogram (SAECG), heart rate variability (HRV), and QT dispersion in patients with chronic heart failure (CHF). BACKGROUND Cardiac MIBG imaging predicts prognosis of CHF patients. However, the long-term predictive value of MIBG imaging for SCD in this population remains to be elucidated. METHODS At entry, cardiac MIBG imaging, SAECG, 24-h Holter monitoring, and standard 12-lead electrocardiography (ECG) were performed in 106 consecutive stable CHF outpatients with a radionuclide left ventricular ejection fraction (LVEF) <40%. The cardiac MIBG washout rate (WR) was obtained from MIBG imaging. Furthermore, the time and frequency domain HRV parameters were calculated from 24-h Holter recordings, and QT dispersion was measured from the 12-lead ECG. RESULTS During a follow-up period of 65 +/- 31 months, 18 of 106 patients died suddenly. A multivariate Cox analysis revealed that WR and LVEF were significantly and independently associated with SCD, whereas the SAECG, HRV parameters, or QT dispersion were not. Patients with an abnormal WR (>27%) had a significantly higher risk of SCD (adjusted hazard ratio: 4.79, 95% confidence interval: 1.55 to 14.76). Even when confined to the patients with LVEF >35%, SCD was significantly more frequently observed in the patients with than without an abnormal WR (p = 0.02). CONCLUSIONS Cardiac MIBG WR, but not SAECG, HRV, or QT dispersion, is a powerful predictor of SCD in patients with mild-to-moderate CHF, independently of LVEF.

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.

Prediction of sudden death in patients with mild-to-moderate chronic heart failure by using cardiac iodine-123 metaiodobenzylguanidine imaging

Objective: To evaluate the usefulness of cardiac iodine-123 (123I) metaiodobenzylguanidine (MIBG) imaging as a predictor of sudden death in patients with chronic heart failure (CHF). Design and setting: Prospective cohort study in a tertiary referral centre. Patients: 97 outpatients with CHF with a radionuclide left ventricular ejection fraction <40% (mean (SD) 29% (7.5%)). Interventions: At study entry, cardiac I-123 MIBG imaging was performed. The cardiac MIBG heart-to-mediastinum ratio (H/M) and washout rate (WR) were obtained from MIBG imaging. Main outcome measures: Patients were assigned to two groups based upon 27% of WR, which was the mean (2SD) control WR. 48 of 97 patients with CHF had abnormal WR (⩾27%), whereas the remaining 49 patients had normal WR (<27%). All the study patients were then followed up. Results: During the mean (SD) follow-up period of 65 (29) months, 12 (25%) patients in the abnormal WR group and 2 (4%) patients in the normal WR group died suddenly. Kaplan–Meier analysis revealed that sudden death was more often observed in patients with abnormal WR than those with normal WR (p = 0.001). On Cox regression analysis, MIBG WR, H/M on the delayed image and H/M on the early image were significantly associated with sudden death. Conclusion: Cardiac MIBG imaging would be useful for predicting sudden death in patients with CHF.

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.

Chemical Endoplasmic Reticulum Chaperone Alleviates Doxorubicin-Induced Cardiac Dysfunction

RATIONALE Doxorubicin is an effective chemotherapeutic agent for cancer, but its use is often limited by cardiotoxicity. Doxorubicin causes endoplasmic reticulum (ER) dilation in cardiomyocytes, and we have demonstrated that ER stress plays important roles in the pathophysiology of heart failure. OBJECTIVE We evaluated the role of ER stress in doxorubicin-induced cardiotoxicity and examined whether the chemical ER chaperone could prevent doxorubicin-induced cardiac dysfunction. METHODS AND RESULTS We confirmed that doxorubicin caused ER dilation in mouse hearts, indicating that doxorubicin may affect ER function. Doxorubicin activated an ER transmembrane stress sensor, activating transcription factor 6, in cultured cardiomyocytes and mouse hearts. However, doxorubicin suppressed the expression of genes downstream of activating transcription factor 6, including X-box binding protein 1. The decreased levels of X-box binding protein 1 resulted in a failure to induce the expression of the ER chaperone glucose-regulated protein 78 which plays a major role in adaptive responses to ER stress. In addition, doxorubicin activated caspase-12, an ER membrane-resident apoptotic molecule, which can lead to cardiomyocyte apoptosis and cardiac dysfunction. Cardiac-specific overexpression of glucose-regulated protein 78 by adeno-associated virus 9 or the administration of the chemical ER chaperone 4-phenylbutyrate attenuated caspase-12 cleavage, and alleviated cardiac apoptosis and dysfunction induced by doxorubicin. CONCLUSIONS Doxorubicin activated the ER stress-initiated apoptotic response without inducing the ER chaperone glucose-regulated protein 78, further augmenting ER stress in mouse hearts. Cardiac-specific overexpression of glucose-regulated protein 78 or the administration of the chemical ER chaperone alleviated the cardiac dysfunction induced by doxorubicin and may facilitate the safe use of doxorubicin for cancer treatment.

Ablation of IL-33 gene exacerbate myocardial remodeling in mice with heart failure induced by mechanical stress

Background and purpose: ST2 is one of the interleukin (IL)‐1 receptor family members comprising of membrane‐bound (ST2L) and soluble (sST2) isoforms. Clinical trials have revealed that serum sST2 levels predict outcome in patient with myocardial infarction or chronic heart failure (HF). Meanwhile, we and others have reported that ablation of ST2 caused exaggerated cardiac remodeling in both ischemic and non‐ischemic HF. Here, we tested whether IL‐33, the ligand for ST2, protects myocardium against HF induced by mechanical overload using ligand specific knockout (IL‐33−/−) mice. Methods and results: Transverse aortic constriction (TAC)/sham surgery were carried out in both IL‐33 and WT‐littermates. Echocardiographic measurements were performed at frequent interval during the study period. Heart was harvested for RNA and histological measurements. Following mechanical overload by TAC, myocardial mRNA expressions of Th1 cytokines, such as TNF‐&agr; were enhanced in IL‐33−/− mice than in WT mice. After 8‐weeks, IL‐33−/− mice exhibited exacerbated left ventricular hypertrophy, increased chamber dilation, reduced fractional shortening, aggravated fibrosis, inflammation, and impaired survival compared with WT littermates. Accordingly, myocardial mRNA expressions of hypertrophic (c‐Myc/BNP) molecular markers were also significantly enhanced in IL‐33−/− mice than those in WT mice. Conclusions: We report for the first time that ablation of IL‐33 directly and significantly leads to exacerbate cardiac remodeling with impaired cardiac function and survival upon mechanical stress. These data highlight the cardioprotective role of IL‐33/ST2 system in the stressed myocardium and reveal a potential therapeutic role for IL‐33 in non‐ischemic HF.

Targeted Therapy for Acute Autoimmune Myocarditis with Nano-Sized Liposomal FK506 in Rats.

Immunosuppressive agents are used for the treatment of immune-mediated myocarditis; however, the need to develop a more effective therapeutic approach remains. Nano-sized liposomes may accumulate in and selectively deliver drugs to an inflammatory lesion with enhanced vascular permeability. The aims of this study were to investigate the distribution of liposomal FK506, an immunosuppressive drug encapsulated within liposomes, and the drug’s effects on cardiac function in a rat experimental autoimmune myocarditis (EAM) model. We prepared polyethylene glycol-modified liposomal FK506 (mean diameter: 109.5 ± 4.4 nm). We induced EAM by immunization with porcine myosin and assessed the tissue distribution of the nano-sized beads and liposomal FK506 in this model. After liposomal or free FK506 was administered on days 14 and 17 after immunization, the cytokine expression in the rat hearts along with the histological findings and hemodynamic parameters were determined on day 21. Ex vivo fluorescent imaging revealed that intravenously administered fluorescent-labeled nano-sized beads had accumulated in myocarditic but not normal hearts on day 14 after immunization and thereafter. Compared to the administration of free FK506, FK506 levels were increased in both the plasma and hearts of EAM rats when liposomal FK506 was administered. The administration of liposomal FK506 markedly suppressed the expression of cytokines, such as interferon-γ and tumor necrosis factor-α, and reduced inflammation and fibrosis in the myocardium on day 21 compared to free FK506. The administration of liposomal FK506 also markedly ameliorated cardiac dysfunction on day 21 compared to free FK506. Nano-sized liposomes may be a promising drug delivery system for targeting myocarditic hearts with cardioprotective agents.

Decreased mortality associated with statin treatment in patients with acute myocardial infarction and lymphotoxin-alpha C804A polymorphism.

AIMS We previously reported the association of single nucleotide polymorphisms in the lymphotoxin alpha (LTα) gene with susceptibility to acute myocardial infarction (AMI) and increased mortality after discharge. In the present study, we investigated whether the adverse effect of LTα C804A polymorphism on mortality could be pharmacologically modified by statin treatment after AMI. METHODS AND RESULTS We conducted a multicenter study that included 3486 post-AMI patients between 1998 and 2008. During a median follow-up period of 1775 days, 247 deaths were recorded. The mortality rate was significantly higher in LTα 804A allele carriers compared to non-804A allele carriers (7.9% vs. 5.7%, p = 0.011). The LTα 804A allele was significantly associated with increased mortality for post-AMI patients not receiving statins (hazard ratio [HR]: 1.48, 95% confidence interval [CI]: 1.03-2.12, p = 0.034), but not for those receiving statins (HR: 1.22, 95% CI: 0.70-2.10, p = 0.486). In-vitro experimental analyses demonstrated that the LTα 804A polymorphic protein, 26Asn-LTα3, induced monocyte-endothelial interaction and endoplasmic reticulum (ER) stress in cardiomyocytes more strongly than the LTα3 804C polymorphic protein 26Thr-LTα3. However, the effects of both LTα3 proteins were decreased and became comparable by the pretreatment of cells with pravastatin. CONCLUSION LTα C804A polymorphism was associated with an increased risk of mortality for AMI patients, although this effect was masked in patients treated with statins. This finding is supported by the observed attenuation of 26Asn-LTα3-mediated monocyte-endothelial interaction and ER stress in cardiomyocytes treated with pravastatin. LTα C804A polymorphism may have potential as a novel therapeutic target for secondary prevention after AMI.

Atorvastatin therapy associated with improvement in left ventricular remodeling in a case of idiopathic dilated cardiomyopathy.

Statins have pleiotropic effects such as anti-inflammatory and vascular protective effects that would be beneficial for patients with chronic heart failure. This report describes a patient with idiopathic dilated cardiomyopathy and a long-standing history of heart failure that was treated with atorvastatin in addition to conventional therapy that included beta-blockers. Atorvastatin therapy for 12 months was associated with an improvement in cardiac function and improved left ventricular remodeling and peak oxygen consumption. This result suggests that statin therapy may be a potential novel treatment strategy for patients with chronic heart failure.

Development of a novel one-step production system for injectable liposomes under GMP

Abstract There are few methods available for injectable liposome production under good manufacturing practices (GMP). Injectable liposome production processes under GMP generally consist of liposome formation, size homogenization, organic solvent removal, liposome concentration control and sterilization. However, these complicated and separate processes make it difficult to maintain scalability, reproducibility and sterility. To overcome these limitations, we developed a novel one-step in-line closed liposome production system that integrated all production processes by combining the in-line thermal mixing device with modified counterflow dialysis. To validate the system, we produced liposomal cyclosporine A (Lipo-CsA) and lyophilized the liposomes. The three independent pilot batches were highly reproducible and passed the quality specifications for injectable drugs, demonstrating that this system could be used under GMP. The accelerated stability test suggested that the liposomes would be stable in long-term storage. This one-step system facilitates a fully automated and unattended production of injectable liposomes under GMP.