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Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in vitro model of myoblast transplantation with "patterned" or "randomly-mixed" co-culture of SkMBs and cardiomyocytes enabling subsequent electrophysiological, and arrhythmogenic evaluation. SkMBs were magnetically labeled with magnetite nanoparticles and co-cultured with neonatal rat ventricular myocytes (NRVMs) on multi-electrode arrays. SkMBs were patterned by a magnet beneath the arrays. Excitation synchronicity was evaluated by Ca(2+) imaging using a gene-encoded Ca(2+) indicator, G-CaMP2. In the monoculture of NRVMs (control), conduction was well-organized. In the randomly-mixed co-culture of NRVMs and SkMBs (random group), there was inhomogeneous conduction from multiple origins. In the "patterned" co-culture where an en bloc SKMB-layer was inserted into the NRVM-layer, excitation homogenously propagated although conduction was distorted by the SkMB-area. The 4-mm distance conduction time (CT) in the random group was significantly longer (197 ± 126 ms) than in control (17 ± 3 ms). In the patterned group, CT through NRVM-area did not change (25 ± 3 ms), although CT through the SkMB-area was significantly longer (132 ± 77 ms). The intervals between spontaneous excitation varied beat-to-beat in the random group, while regular beating was recorded in the control and patterned groups. Synchronized Ca(2+) transients of NRVMs were observed in the patterned group, whereas those in the random group were asynchronous. Patterned alignment of SkMBs is feasible with magnetic nanoparticles. Using the novel in vitro model mimicking cell transplantation, it may become possible to predict arrhythmogenicity due to heterogenous cell transplantation. J. Cell. Physiol. 231: 2249-2256, 2016. © 2016 Wiley Periodicals, Inc

BACKGROUND: Morphological observation of blood or marrow film is still described nonquantitatively. We developed a semiautomatic method for segmenting vacuoles from the cytoplasm using Photoshop (PS) and Image‐J (IJ), called PS‐IJ, and measured the relative entire cell area (rECA) and relative areas of vacuoles (rAV) in the cytoplasm of neutrophil with PS‐IJ. METHODS: Whole‐blood samples were stored at 4°C with ethylenediaminetetraacetate and in two different preserving manners (P1 and P2). Color‐tone intensity levels of neutrophil images were semiautomatically compensated using PS, and then vacuole portions were automatically segmented by IJ. The rAV and rECA were measured by counting pixels by IJ. For evaluating the accuracy in segmentations of vacuoles with PS‐IJ, the rAV/rECA ratios calculated with results from PS‐IJ were compared with those calculated with human eye and IJ (HE‐IJ). RESULTS: The rECA and rAV/ in P1 significantly (P < 0.05, P < 0.05) were enlarged and increased, but did not significantly (P = 0.46, P = 0.21) change in P2. The rAV/rECA ratios by PS‐IJ were significantly correlated (r = 0.90, P < 0.01) with those by HE‐IJ. CONCLUSION: PS‐IJ method can successfully segment vacuoles and measure the rAV and rECA, becoming a useful tool for quantitative description of morphological observation of blood and marrow film.

Antiarrhythmic effects of long-term amiodarone is supposed to be mediated in part by cardio-selective hypothyroidism, but the issue remains controversial. We examined mRNA expression of sacroplasmic reticulum Ca^2+-ATPase(SERCA2) by Northern blot analysis in rats treated with amiodarone (60mg/kg/day, 4 weeks) or dronedarone (60mg/kg/day, 4 weeks). SERCA2 mRNA level in ventricular muscle was significantly decreased in hypothyroid rats by 38±9%(p, 研究所で電子化したコンテンツを使用している。

Embryonic stem (ES) cell-derived cardiac myocytes could be valid source for cell transplantation therapy. The primary goal of the present study is to explore the efficient method to obtain cardiac cells derived from ES cells. We examined the effects of conditioned medium from cultured cardiac cells of neonatal mouse on the differentiation of ES cells toward cardiac myocytes. When Nkx2.5-GFP knock-in ES cells were incubated in the medium extracted from cultured neonatal mouse ventricular myocytes, they showed more efficient differentiation to cardiac myocytes, compared with incubation in the control medium (6.8±1.6 vs 8.6±1.9%, n=4, p, 国立情報学研究所で電子化したコンテンツを使用している。

Calpain, a Ca^-dependent cytosolic cystein protease, is as an important key protease involved in the ischemic reperfusion injury such as cardiac stunning. Myocardial stunning is a postischemic reversible contractile dysfunction of the heart. We hypothesized that overexpression of calpastatin, an endogenous calpain inhibitor, would attenuate the ischemia/reperfusion injury in the heart. Rat hearts were transfected with human calpastatin gene incorporated into adenovirus vectors. The hearts infected with adenovirus containing lacZ gene were used as controls. The hearts after transfection were heterotopically transplanted into the abdomens of recipient rats and excised on the fourth day after the surgery. The functional recovery from 30-min global ischemia was studied in the isolated hearts perfused in-vitro. The recovery of left ventricular developed pressure, max dP/dt, and min dP/dt during 60-min reperfusion was significantly greater in the calpastatin transfected group than control group heart by gene transfer. Adenovirus-mediated overexpression of calpastatin could be a novel biological storage to improve heart transplantation by minimizing ischemia/reperfusion injury., 国立情報学研究所で電子化したコンテンツを使用している。

Pluripotent embryonic stem (ES) cells differentiate to cardiac myocytes in vitro by many other previous reports demonstrated "hanging-drop" method. In this study, the number of ES cells in each hanging-drop plays an important role in the cultivation of cardiac myocytes. We examined the optimal hanging-drop size to obtain embryonic stem cell-derived cardiac cells (ESCMs) in vitro using specific labeled mouse ES cells (hCGP7) which were stably transfected with the enhanced green fluorescent protein (GFP) under transcriptional control of the cardiac myocyte-specific promoter with fluorescence-activated cell sorting (FACS) analysis. GFP-positive cells were sorted efficiently in the hanging-drop cell number of approximately 800～1600 cells/drop. The present data will be an important element to establish the optimal methods of ES cellular differentiation in vitro., 国立情報学研究所で電子化したコンテンツを使用している。