Masaya Shimojima

Impaired respiratory function in MELAS‐induced pluripotent stem cells with high heteroplasmy levels

Mitochondrial diseases are heterogeneous disorders, caused by mitochondrial dysfunction. Mitochondria are not regulated solely by nuclear genomic DNA but by mitochondrial DNA. It is difficult to develop effective therapies for mitochondrial disease because of the lack of mitochondrial disease models. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke‐like episodes (MELAS) is one of the major mitochondrial diseases. The aim of this study was to generate MELAS‐specific induced pluripotent stem cells (iPSCs) and to demonstrate that MELAS‐iPSCs can be models for mitochondrial disease. We successfully established iPSCs from the primary MELAS‐fibroblasts carrying 77.7% of m.3243A>G heteroplasmy. MELAS‐iPSC lines ranged from 3.6% to 99.4% of m.3243A>G heteroplasmy levels. The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS‐iPSC‐derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS‐iPSC‐derived fibroblasts with low heteroplasmy levels showed normal complex I activity. Our data indicate that MELAS‐iPSCs can be models for MELAS but we should carefully select MELAS‐iPSCs with appropriate heteroplasmy levels and respiratory functions for mitochondrial disease modeling.

Embryonic type Na + channel β-subunit, SCN3B masks the disease phenotype of Brugada syndrome

SCN5A is abundant in heart and has a major role in INa. Loss-of-function mutation in SCN5A results in Brugada syndrome (BrS), which causes sudden death in adults. It remains unclear why disease phenotype does not manifest in the young even though mutated SCN5A is expressed in the young. The aim of the present study is to elucidate the timing of the disease manifestation in BrS. A gain-of-function mutation in SCN5A also results in Long QT syndrome type 3 (LQTS3), leading to sudden death in the young. Induced pluripotent stem cells (iPSCs) were generated from a patient with a mixed phenotype of LQTS3 and BrS with the E1784K SCN5A mutation. Here we show that electrophysiological analysis revealed that LQTS3/BrS iPSC-derived cardiomyocytes recapitulate the phenotype of LQTS3 but not BrS. Each β-subunit of the sodium channel is differentially expressed in embryonic and adult hearts. SCN3B is highly expressed in embryonic hearts and iPSC-derived cardiomyocytes. A heterologous expression system revealed that INa of mutated SCN5A is decreased and SCN3B augmented INa of mutated SCN5A. Knockdown of SCN3B in LQTS3/BrS iPSC-derived cardiomyocytes successfully unmasked the phenotype of BrS. Isogenic control of LQTS3/BrS (corrected-LQTS3/BrS) iPSC-derived cardiomyocytes gained the normal electrophysiological properties.

Age-specific differences in prognostic significance of rhythm conversion from initial non-shockable to shockable rhythm and subsequent shock delivery in out-of-hospital cardiac arrest☆

BACKGROUND Early rhythm conversion from an initial non-shockable to a shockable rhythm and subsequent shock delivery in patients with out-of-hospital cardiac arrest (OHCA) has been associated with favourable neurological outcome (Cerebral Performance Category score 1 or 2; CPC 1-2). We hypothesized that the prognostic significance of rhythm conversion and subsequent shock delivery differs by age and time from initiation of cardiopulmonary resuscitation (CPR) by emergency medical service (EMS) providers to first defibrillation (shock delivery time). METHODS We analysed 430,443 OHCA patients with an initial non-shockable rhythm using a prospective Japanese Utstein-style database from 2011 to 2014. The primary endpoint was 1-month CPC 1-2. RESULTS Multivariate logistic regression revealed that rhythm conversion and subsequent shock delivery is positively associated with 1-month CPC 1-2: the adjusted odds ratio was 6.09 (95% confidence interval: 3.65-9.75) for shock delivery time <10min and 3.34 (2.58-4.27) for 10-19min in patients aged 18-64 years, and 3.16 (1.45-6.09) for <10min and 2.17 (1.51-3.03) for 10-19min in patients aged 65-74 years. However, it is negatively associated with 1-month CPC 1-2 for shock delivery time of 20-59min in patients aged 75-84 years (0.55; 0.27-0.98) and ≥85 years (0.17; 0.03-0.53). CONCLUSIONS Early rhythm conversion from an initial non-shockable to a shockable rhythm and subsequent shock delivery is associated with increased odds of 1-month CPC 1-2 in OHCA patients aged 18-74 years but not in those aged ≥75 years.

Determination of Early and Late Endothelial Progenitor Cells in Peripheral Circulation and Their Clinical Association with Coronary Artery Disease

The clinical implications of early and late endothelial progenitor cells (EPCs) in coronary artery disease (CAD) remain unclear. We investigated endothelial dysfunction in CAD by simultaneously examining early and late EPC colony formation and gene expression of specific surface markers in EPCs. EPCs were extracted from a total of 83 subjects with (n = 47) and without (n = 36) CAD. Early and late EPC colonies were formed from mononuclear cells extracted from peripheral blood. We found that fewer early EPC colonies were produced in the CAD group (7.2 ± 3.l/well) than those in the control group (12.4 ± 1.4/well, p < 0.05), and more late EPC colonies were produced in the CAD group (0.8 ± 0.2/well) than those in the control group (0.25 ± 0.02/well, p < 0.05). In the CAD group, the relative expression of CD31 and KDR of early and late EPCs was lower than in the control group. These results demonstrate that CAD patients could have increased late EPC density and that early and late EPCs in CAD patients exhibited immature endothelial characteristics. We suggest that changes in EPC colony count and gene expression of endothelial markers may have relation with development of CAD.

Emerin plays a crucial role in nuclear invagination and in the nuclear calcium transient

Alteration of the nuclear Ca2+ transient is an early event in cardiac remodeling. Regulation of the nuclear Ca2+ transient is partly independent of the cytosolic Ca2+ transient in cardiomyocytes. One nuclear membrane protein, emerin, is encoded by EMD, and an EMD mutation causes Emery-Dreifuss muscular dystrophy (EDMD). It remains unclear whether emerin is involved in nuclear Ca2+ homeostasis. The aim of this study is to elucidate the role of emerin in rat cardiomyocytes by means of hypertrophic stimuli and in EDMD induced pluripotent stem (iPS) cell-derived cardiomyocytes in terms of nuclear structure and the Ca2+ transient. The cardiac hypertrophic stimuli increased the nuclear area, decreased nuclear invagination, and increased the half-decay time of the nuclear Ca2+ transient in cardiomyocytes. Emd knockdown cardiomyocytes showed similar properties after hypertrophic stimuli. The EDMD-iPS cell-derived cardiomyocytes showed increased nuclear area, decreased nuclear invagination, and increased half-decay time of the nuclear Ca2+ transient. An autopsied heart from a patient with EDMD also showed increased nuclear area and decreased nuclear invagination. These data suggest that Emerin plays a crucial role in nuclear structure and in the nuclear Ca2+ transient. Thus, emerin and the nuclear Ca2+ transient are possible therapeutic targets in heart failure and EDMD.

Impact of Enhanced Production of Endogenous Heme Oxygenase-1 by Pitavastatin on Survival and Functional Activities of Bone Marrow–derived Mesenchymal Stem Cells

Abstract: Although mesenchymal stem cells (MSCs) have a therapeutic potential for the repair of tissue injuries, their poor viability in damaged tissue limits their effectiveness. Statins can induce an increased production of heme oxygenase-1 (HO-1), which may prevent this detrimental effect in MSCs. We investigated the protective effect of statin-induced overexpression of HO-1 by examining changes in gene expression and function in MSCs after pitavastatin treatment. The relative expression of the HO-1 and endothelial nitric oxide synthase genes in MSCs was significantly increased after treatment with pitavastatin (PitaMSCs). Immunocytological analysis showed that PitaMSCs also stained with phospho-Akt. After exposure to oxidative stress, PitaMSCs showed increased resistance to induced cell death compared with control MSCs. Under serum starvation conditions, MSCs treated with 1 &mgr;M pitavastatin showed enhanced cell proliferation and a marked increase in vascular endothelial growth factor production compared with control MSCs. Interestingly, PitaMSCs showed enhanced tube formation under both normoxia and hypoxia. These results demonstrate that pitavastatin can enhance endogenous HO-1 expression in MSCs, which may protect the cells into the environment of oxidative stress with partial activation of endothelial nitric oxide synthase and Akt phosphorylation.

Duration of cardiopulmonary resuscitation in patients without prehospital return of spontaneous circulation after out-of-hospital cardiac arrest: results from a severity stratification analysis

BACKGROUND The relationship between duration of cardiopulmonary resuscitation (CPR) and post-arrest outcomes based on severity stratification in out-of-hospital cardiac arrest (OHCA) patients without prehospital return of spontaneous circulation (ROSC) remains unclear. METHODS We analysed 420,959 adult patients without prehospital ROSC in the All-Japan OHCA registry for 4 years. Prehospital CPR duration was defined as the time from CPR initiation by emergency medical service (EMS) providers to hospital arrival. The primary outcome was 1-month neurologically intact survival (cerebral performance category 1 or 2, CPC 1-2). RESULTS The rate of overall 1-month CPC 1-2 was 0.45% (1899/420,959). Using recursive partitioning analysis to predict 1-month CPC 1-2, we stratified patients into 4 groups with 3 predictors: patients aged <75 years with initial shockable rhythm (1-month CPC 1-2 rate, 6.15%), those aged ≥75 years with initial shockable rhythm (1.32%), those with EMS-witnessed arrest and initial non-shockable rhythm (1.62%), and those with EMS-unwitnessed arrest and initial non-shockable rhythm (0.15%). Prehospital CPR duration was negatively associated with 1-month CPC 1-2 (adjusted odds ratio 0.94 per 1-min increment; 95% confidence interval 0.94-0.95). Prehospital CPR durations beyond which the dynamic probability of 1-month CPC 1-2 decreased to <1% were 26 min, 10 min, 7 min, and at all times in above-mentioned stratification, respectively. CONCLUSIONS In OHCA patients without prehospital ROSC, those aged <75 years with initial shockable rhythm had acceptable 1-month CPC 1-2 rate. However, CPR efforts lasting 26 min or over before hospital arrival could be futile.

Age-Specific Differences in the Duration of Prehospital Cardiopulmonary Resuscitation Administered by Emergency Medical Service Providers Necessary to Achieve Favorable Neurological Outcome After Out-of-Hospital Cardiac Arrest

BACKGROUND The appropriate duration of prehospital cardiopulmonary resuscitation (CPR)administered by emergency medical service (EMS) providers for patients with out-of-hospital cardiac arrest (OHCA) necessary to achieve 1-month survival with favorable neurological outcome (Cerebral Performance Category 1 or 2, CPC 1-2) is unclear and could differ by age.Methods and Results:We analyzed the records of 35,709 adult OHCA patients with return of spontaneous circulation (ROSC) before hospital arrival in a prospectively recorded Japanese registry between 2011 and 2014. The CPR duration was defined as the time from CPR initiation by EMS providers to prehospital ROSC. The rate of 1-month CPC 1-2 was 21.4% (7,650/35,709). The CPR duration was independently and inversely associated with 1-month CPC 1-2 (adjusted odds ratio, 0.93 per 1-min increment; 95% confidence interval, 0.93-0.94). The CPR duration increased with age (P<0.001). However, the CPR duration beyond which the proportion of OHCA patients with 1-month CPC 1-2 decreased to <1% declined with age: 28 min for patients aged 18-64 years, 25 min for 65-74 years, 23 min for 75-84 years, 20 min for 85-94 years, and 18 min for ≥95 years. CONCLUSIONS In patients who achieved prehospital ROSC after OHCA, the duration of CPR administered by EMS providers necessary to achieve 1-month CPC 1-2 varied by age.

Pericarditis-complicated takotsubo cardiomyopathy in a patient with rheumatoid arthritis

A 64-year-old woman with medication-controlled rheumatoid arthritis (RA) was admitted to our hospital complaining of chest pains. An electrocardiogram showed elevated ST-segments in the inferior leads, and inverted T-waves in the left precordial leads. Left ventriculography demonstrated apical ballooning, and cardiac magnetic resonance imaging demonstrated apical ballooning of the left ventricle, and moderate pericardial effusion. The patient was diagnosed with takotsubo cardiomyopathy (TTC), complicated by pericarditis. In the literature, autoimmune disorders have been associated with TTC. We suggest that this patients pericardial effusion was caused by TTC, and that her coexisting RA might have played a role in the etiology of the significant pericardial fluid accumulation.

Enhanced Device for Cell Delivery to the Myocardium: Validation in Swine Hearts

Background: Endocardial infusion is a minimally invasive procedure for cell delivery with good selectivity to the target region. However, certain limitations to current devices could affect the precision of the procedure and the therapeutic outcome. Therefore, we developed an enhanced device for transendocardial cell infusion. Methods and Results: Our device is based on an electrode-guided transendocardial bidirectional 75 cm long catheter and 0.5 mm diameter inner needle. The key advantages of our device are the slender catheter diameter (7 Fr), consistent needle tip length, regulation of the catheter angle and independence between the needle and catheter. Mesenchymal stem cells (MSCs) were obtained from the inguinal adipose tissue of six healthy swine and propagated through 2-3 passages. Using the catheter, pre-labeled MSCs were infused autogenously into the swine hearts. The MSCs-infused myocardial regions were harvested on the infusion day (day 0) or 2 days later, and histological analysis was performed. The MSCs were successfully infused into all six swine myocardia and distributed along the hole made by the needle. The spread area of MSCs was larger at 2 days after infusion than at day 0 (1.38 ± 0.26 vs. 0.51 ± 0.17 mm2/infusion, p=0.013). No complications occurred during the procedure, such as cardiac tamponade or arrhythmia. Conclusion: These results demonstrate that our enhanced device could be useful for delivering cells into the myocardium.