Impaired Cholesterol Uptake Capacity in Patients with Hypertriglyceridemia and Diabetes Mellitus.

BACKGROUND: Although low high-density lipoprotein cholesterol (HDL-C) levels are a common metabolic abnormality associated with insulin resistance, their role in cardiovascular risk stratification remains controversial. Recently, we developed a simple, high-throughput, cell-free assay system to evaluate the "cholesterol uptake capacity (CUC)" as a novel concept for HDL functionality. In this study, we assessed the CUC in patients with hypertriglyceridemia and diabetes mellitus. METHODS: The CUC was measured using cryopreserved serum samples from 285 patients who underwent coronary angiography or percutaneous coronary intervention between December 2014 and May 2019 at Kobe University Hospital. RESULTS: The CUC was significantly lower in diabetic patients (n = 125) than in nondiabetic patients (93.0 vs 100.7 arbitrary units (A.U.), P = 0.002). Patients with serum triglyceride (TG) levels >150 mg/dL (n = 94) also had a significantly lower CUC (91.8 vs 100.0 A.U., P = 0.004). Furthermore, the CUC showed a significant inverse correlation with TG, hemoglobin A1c (Hb A1c), homeostasis model assessment of insulin resistance (HOMA-IR), and body mass index (BMI). Finally, the HDL-C/Apolipoprotein A1 (ApoA1) ratio, calculated as a surrogate index of HDL particle size, was significantly positively correlated with the CUC (r2 = 0.49, P < 0.001), but inversely correlated with TG levels (r2 = -0.30, P < 0.001). CONCLUSIONS: The CUC decreased in patients with hypertriglyceridemia and diabetes mellitus, and HDL particle size was a factor defining the CUC and inversely correlated with TG levels, suggesting that impaired CUC in insulin-resistant states was partially due to the shift in HDL towards smaller particles. These findings provide a better understanding of the mechanisms underlying impaired HDL functionality.

Fully automated immunoassay for cholesterol uptake capacity to assess high-density lipoprotein function and cardiovascular disease risk.

High-density lipoprotein (HDL) cholesterol efflux capacity (CEC), which is a conventional metric of HDL function, has been associated with coronary heart disease risk. However, the CEC assay requires cultured cells and takes several days to perform. We previously established a cell-free assay to evaluate cholesterol uptake capacity (CUC) as a novel measure of HDL functionality and demonstrated its utility in coronary risk stratification. To apply this concept clinically, we developed a rapid and sensitive assay system based on a chemiluminescent magnetic particle immunoassay. The system is fully automated, providing high reproducibility. Measurement of CUC in serum is completed within 20 min per sample without HDL isolation, a notably higher throughput than that of the conventional CEC assay. CUC decreased with myeloperoxidase-mediated oxidation of HDL or in the presence of N-ethylmaleimide, an inhibitor of lecithin: cholesterol acyltransferase (LCAT), whereas CUC was enhanced by the addition of recombinant LCAT. Furthermore, CUC correlated with CEC even after being normalized by ApoA1 concentration and was significantly associated with the requirement for revascularization due to the recurrence of coronary lesions. Therefore, our new assay system shows potential for the accurate measurement of CUC in serum and permits assessing cardiovascular health.

Evaluation of two semi-supervised learning methods and their combination for automatic classification of bone marrow cells.

Differential bone marrow (BM) cell counting is an important test for the diagnosis of various hematological diseases. However, it is difficult to accurately classify BM cells due to non-uniformity and the lack of reproducibility of differential counting. Therefore, automatic classification systems have been developed in which deep learning is used. These systems requires large and accurately labeled datasets for training. To overcome this, we used semi-supervised learning (SSL), in which learning proceeds while labeling. We used three methods: self-training (ST), active learning (AL), and a combination of these methods, and attempted to automatically classify 16 types of BM cell images. ST involves data verification, as in AL, before adding them to the training dataset (confirmed self-training: CST). After 25 rounds of CST, AL, and CST + AL, the initial number of training data increased from 425 to 40,518; 3682; and 47,843, respectively. Accuracies for the test data of 50 images for each cell type were 0.944, 0.941, and 0.976, respectively. Data added with CST or AL showed some imbalances between classes, while CST + AL exhibited fewer imbalances. We suggest that CST + AL, when combined with two SSL methods, is efficient in increasing training data for the development of automatic BM cells classification systems.

RGMa Signal in Macrophages Induces Neutrophil-Related Astrocytopathy in NMO.

OBJECTIVE: Repulsive guidance molecule-a (RGMa) is a glycosylphosphatidylinositol-linked glycoprotein which has multiple functions including axon growth inhibition and immune regulation. However, its role in the pathophysiology of neuromyelitis optica (NMO) is poorly understood. Perivascular astrocytopathy, which is induced by the leakage of aquaporin-4 (AQP4)-specific IgG into the central nervous system parenchyma, is a key feature of NMO pathology. We investigated the RGMa involvement in the pathology of NMO astrocytopathy, and tested a therapeutic potential of humanized anti-RGMa monoclonal antibody (RGMa-mAb). METHODS: Using a clinically relevant NMO rat model, we evaluated the therapeutic effect of a RGMa-mAb by behavioral testing, immunohistochemistry, and gene expression assay. We further performed in vitro experiments to address the RGMa-signaling in macrophages. RESULTS: In both NMO rats and an NMO-autopsied sample, RGMa was expressed by the spared neurons and astrocytes, whereas its receptor neogenin was expressed by infiltrating macrophages. AQP4-IgG-induced astrocytopathy and clinical exacerbation in NMO rats were ameliorated by RGMa-mAb treatment. RGMa-mAb treatment significantly suppressed neutrophil infiltration, and decreased the expression of neutrophil chemoattractants. Interestingly, neogenin-expressing macrophages accumulated in the lesion expressed CXCL2, a strong neutrophil chemoattractant, and further analysis revealed that RGMa directly regulated CXCL2 expression in macrophages. Finally, we found that our NMO rats developed neuropathic pain, and RGMa-mAb treatment effectively ameliorated the severity of neuropathic pain. INTERPRETATION: RGMa signaling in infiltrated macrophages is a critical driver of neutrophil-related astrocytopathy in NMO lesions, and RGMa-mAb may provide an efficient therapeutic strategy for NMO-associated neuropathic pain and motor deficits in patients with NMO. ANN NEUROL 2022;91:532-547.

Deep Learning-Based Nuclear Lobe Count Method for Differential Count of Neutrophils.

Differentiating neutrophils based on the count of nuclear lobulation is useful for diagnosing various hematological disorders, including megaloblastic anemia, myelodysplastic syndrome, and sepsis. It has been reported that one-fifth of sepsis-infected patients worldwide died between 1990 and 2017. Notably, fewer nuclear-lobed and stab-formed neutrophils develop in the peripheral blood during sepsis. This abnormality can serve as an early diagnostic criterion. However, testing this feature is a complex and time-consuming task that is rife with human error. For this reason, we apply deep learning to automatically differentiate neutrophil and nuclear lobulation counts and report the world's first small-scale pilot. Blood films are prepared using venous peripheral blood taken from four healthy volunteers and are stained with May-Grünwald Giemsa stain. Six-hundred 360 × 363-pixel images of neutrophils having five different nuclear lobulations are automatically captured by Cellavision DM-96, an automatic digital microscope camera. Images are input to an original architecture with five convolutional layers built on a deep learning neural-network platform by Sony, Neural Network Console. The deep learning system distinguishes the four groups (i.e., band-formed, two-, three-, and four- and five- segmented) of neutrophils with up to 99% accuracy, suggesting that neutrophils can be automatically differentiated based on their count of segmented nuclei using deep learning.

Scaffold-Mediated Developmental Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Are Preserved After External Support Removal.

Human induced pluripotent stem (hiPS) cells have been used as a cell source for regenerative therapy and disease modeling. The purity of hiPS-cardiomyocytes (hiPS-CMs) has markedly improved with advancements in cell culture and differentiation protocols. However, the morphological features and molecular properties of the relatively immature cells are still unclear, which has hampered their clinical application. The aim of the present study was to investigate the extent to which topographic substrates actively influence hiPS-CMs. hiPS-CMs were seeded on randomized oriented fiber substrate (random), anisotropic aligned fiber substrate (align), and flat non-scaffold substrate (flat). After culturing for one week, the hiPS-CMs on the aligned patterns showed more mature-like properties, including elongated rod shape, shorter duration of action potential, accelerated conduction velocity, and elevated cardiac gene expression. Subsequently, to determine whether this development was irreversible or was altered after withdrawal of the structural support, the hiPS-CMs were harvested from the three different patterns and reseeded on the non-scaffold (flat) pattern. After culturing for one more week, the improvements in morphological and functional properties diminished, although hiPS-CMs pre-cultured on the aligned pattern retained the molecular features of development, which were even more significant as compared to that observed during the pre-culture stage. Our results suggested that the anisotropic fiber substrate can induce the formation of geometrical mimic-oriented heart tissue in a short time. Although the morphological and electrophysiological properties of hiPS-CMs obtained via facilitated maturation somehow rely on the existence of an exterior scaffold, the molecular developmental features were preserved even in the absence of the external support, which might persist throughout hiPS-CM development.

Association of cholesterol uptake capacity, a novel indicator for HDL functionality, and coronary plaque properties: An optical coherence tomography-based observational study.

BACKGROUND: Cholesterol efflux from atherosclerotic lesion is a key function of high-density lipoprotein (HDL). Recently, we established a simple, high-throughput, cell-free assay to evaluate the capacity of HDL to accept additional cholesterol, which is herein referred to as "cholesterol uptake capacity (CUC)". OBJECTIVE: To clarify the cross-sectional relationship between CUC and coronary plaque properties. METHODS: We enrolled 135 patients to measure CUC and assess the morphological features of angiographic stenosis by optical coherence tomography (OCT). We estimated the extent of the lipid-rich plaque by multiplying the mean lipid arc by lipid length (lipid index). The extent of the OCT-detected macrophage accumulation in the target plaque was semi-quantitatively estimated using a grading system. RESULTS: Lipid-rich plaque lesions were identified in 125 patients (92.6%). CUC was inversely associated with the lipid index (R = -0.348, P < 0.0001). In addition, CUC was also inversely associated with macrophage score (R = -0.327, P < 0.0001). Conversely, neither circulating levels of HDL cholesterol nor apoA1 showed a similar relationship. CONCLUSIONS: We demonstrated that CUC was inversely related to lipid-rich plaque burden and the extent of macrophage accumulation, suggesting that CUC could be useful for cardiovascular risk stratification.

Geometrical Patterning and Constituent Cell Heterogeneity Facilitate Electrical Conduction Disturbances in a Human Induced Pluripotent Stem Cell-Based Platform: An In vitro Disease Model of Atrial Arrhythmias.

Ectopic foci from pulmonary veins (PVs) comprise the main trigger associated with the initiation of atrial fibrillation (AF). An abrupt anatomical narrow-to-wide transition, modeled as in vitro geometrical patterning with similar configuration in the present study, is located at the junction of PVs and the left atrium (LA). Complex cellular composition, i.e., constituent cell heterogeneity, is also observed in PVs and the PVs-LA junction. High frequency triggers accompanied with anatomical irregularity and constituent cell heterogeneity provoke impaired conduction, a prerequisite for AF genesis. However, few experiments investigating the effects of these factors on electrophysiological properties using human-based cardiomyocytes (CMs) with atrial properties have been reported. The aim of the current study was to estimate whether geometrical patterning and constituent cell heterogeneity under high frequency stimuli undergo conduction disturbance utilizing an in vitro two-dimensional (2D) monolayer preparation consisting of atrial-like CMs derived from human induced pluripotent stem cells (hiPSCs) and atrial fibroblasts (Fbs). We induced hiPSCs into atrial-like CMs using a directed cardiac differentiation protocol with the addition of all-trans retinoic acid (ATRA). The atrial-like hiPSC-derived CMs (hiPSC-CMs) and atrial Fbs were transferred in defined ratios (CMs/Fbs: 100%/0% or 70%/30%) on manually fabricated plates with or without geometrical patterning imitating the PVs-LA junction. High frequency field stimulation emulating repetitive ectopic foci originated in PVs were delivered, and the electrical propagation was assessed by optical mapping. We generated high purity CMs with or without the ATRA application. ATRA-treated hiPSC-CMs exhibited significantly higher atrial-specific properties by immunofluorescence staining, gene expression patterns, and optical action potential parameters than those of ATRA-untreated hiPSC-CMs. Electrical stimuli at a higher frequency preferentially induced impaired electrical conduction on atrial-like hiPSC-CMs monolayer preparations with an abrupt geometrical transition than on those with uniform geometry. Additionally, the application of human atrial Fbs to the geometrically patterned atrial-like hiPSC-CMs tended to further deteriorate the integrity of electrical conduction compared with those using the atrial-like hiPSC-CM alone preparations. Thus, geometrical narrow-to-wide patterning under high frequency stimuli preferentially jeopardized electrical conduction within in vitro atrial-like hiPSC-CM monolayers. Constituent cell heterogeneity represented by atrial Fbs also contributed to the further deterioration of conduction stability.

In vitro platform of allogeneic stem cell-derived cardiomyocyte transplantation for cardiac conduction defects.

Aims: The aim of the present study is to develop in vitro experimental analytical method for the electrophysiological properties of allogeneic induced pluripotent stem cell-derived cardiomyocytes (CMs) in cardiac conduction defect model. Methods and results: Cardiomyocytes were derived from rat induced pluripotent stem cells CMs (riPSC-CMs) using an embryoid body-based differentiation method with the serial application of growth factors including activin-A, bone morphogenetic protein 4 (BMP-4), and inhibitor of wnt production 2 (IWP-2). Flow cytometry analysis showed that 74.0 ± 2.7% of riPSC-CMs expressed cardiac troponin-T (n = 3). Immunostaining analysis revealed organized sarcomeric structure in riPSC-CMs and the expression of connexin 43 between riPSC-CMs and neonatal rat ventricular CMs (NRVMs). Ca2+ transient recordings revealed the simultaneous excitement of riPSC-CMs and NRVMs, and prolonged Ca2+ transient duration of riPSC-CMs as compared with NRVMs (731 ± 15.9 vs. 610 ± 7.72 ms, P < 0.01, n = 3). Isolated NRVMs were cultured in two discrete regions to mimic cardiac conduction defects on multi-electrode array dish, and riPSC-CMs were seeded in the channel between the two discrete regions. Membrane potential imaging with di-8-ANEPPS discerned the propagation of the electrical impulse from one NRVM region to the other through a riPSC-CM pathway. This pathway had significantly longer action potential duration as compared with NRVMs. Electrophysiological studies using a multi-electrode array platform demonstrated the longer conduction time and functional refractory period of the riPSC-CM pathway compared with the NRVM pathway. Conclusion: Using an in vitro experimental system to mimic cardiac conduction defect, transplanted allogeneic riPSC-CMs showed electrical coupling between two discrete regions of NRVMs. Electrophysiological testing using our platform will enable electrophysiological screening prior to transplantation of stem cell-derived CMs.

Quantitative distinction of the morphological characteristic of erythrocyte precursor cells with texture analysis using gray level co-occurrence matrix.

BACKGROUND: Morphological characteristics of blood cells are still qualitatively defined. So a texture analysis (Tx) method using gray level co-occurrence matrices (GLCMs; CM-Tx method) was applied to images of erythrocyte precursor cells (EPCs) for quantitatively distinguishing four types of EPC stages: proerythroblast, basophilic erythroblast, polychromatic erythroblast, and orthochromatic erythroblast. METHODS: Fifty-five images of four types of EPCs were downloaded from an atlas uploaded by the Blood Cell Morphology Standardization Subcommittee (BCMSS) of the Japanese Society of Laboratory Hematology (JSLH). Using in-house programs, two types of GLCMs-(R: d=1, θ=0°) and (U: d=1, θ=270°)-and nine types of texture distinction index (TDI) were calculated with images removed outer part of cell. RESULTS: Three binary decision trees were sequentially divided among four types of EPC with the sum average of GLCM (U), the contrast of GLCM (R), and the sum average of GLCM (U). The average concordance rate (sensitivity) of CM-Tx method with the judgments of eleven experts in the BCMSS of the JSLH was 95.8% (87.5-100.0), and the average specificity was 97.6% (92.5-100.0). CONCLUSIONS: The CM-Tx method is an effective tool for quantitative distinction of EPC with their morphological features.

Quantitative Distinction of Morphological Characteristics of White Blood Cells in Peripheral Blood Through Texture Analysis Using Gray Level Co-Occurrence Matrix.

BACKGROUND: Texture features are valuable clues for skilled technicians to differentiate peripheral blood (PB) white blood cells (WBCs). Some studies have tried to distinguish WBCs automatically by using texture analysis. However, no study so far has applied a gray level co-occurrence matrix (GLCM) to images of PB WBCs. Here, we developed a new GLCM method, called the CM-Tx method, for automatically distinguishing PB WBCs. METHODS: We used a total of 199 images of six different types of PB WBCs, taken from PB smears of 12 healthy volunteers, as objective standard images for the analysis. The six types were band form neutrophil, segmented form neutrophil, eosinophil, basophil, lymphocyte, and monocyte. Using in-house FORTRAN programs, three types of GLCM (R: distance (d) = 1, direction (θ) = 0°), (U: d = 1, θ = 270°) and (AE: d = 1, θ = 15° x q: q = 0, ..., 23), the mean intensity (MI) of each image and nine different texture distinction indexes (TDIs) for each GLCM were calculated. Then, a threshold value (TV) for distinguishing the type of PB WBC was selected from the dot plots of all TDIs and the MI. RESULTS: In total, we made 1,194 GLCMs. Using the selected TVs of the TDI, four sequential binary divisions could distinguish five types of PB WBCs. First, monocytes were distinguished (sensitivity 100%, specificity 100%, p < 0.0001) with the TV of the inverse difference moment of the GLCM (U). Then, segmented and band form neutrophils were distinguished from the remaining (100%, 99%, p < 0.0001) with the TV of the contrast of the GLCM (AE). Next, lymphocytes were distinguished (100%, 98%, p < 0.0001) with the TV of the entropy of the GLCM (AE). Finally, basophils were distinguished (82.4%, 100%, p < 0.0001) from eosinophils with the TV of the summed entropy of the GLCM (R). Band form neutrophils could not be distinguished from segmented form neutrophils. The average sensitivity of the CM-Tx method for the five types was 95.6%, and its average specificity was 99.3%. CONCLUSIONS: The CM-Tx method can distinguish five types of PB WBCs by using numerical differences only in texture futures quantified with GLCM. However, some other method was needed to distinguish the band and segmented form neutrophils from each other.

Quantification of sympathetic hyperinnervation and denervation after myocardial infarction by three-dimensional assessment of the cardiac sympathetic network in cleared transparent murine hearts.

BACKGROUND: The sympathetic nervous system is critical in maintaining the normal physiological function of the heart. Its dysfunction in pathological states may exacerbate the substrate for arrhythmias. Obviously, knowledge of its three-dimensional (3D) structure is important, however, it has been revealed by conventional methods only to a limited extent. In this study, a new method of tissue clearance in combination with immunostaining unravels the 3D structure of the sympathetic cardiac network as well as its changes after myocardial infarction. METHODS AND RESULTS: Hearts isolated from adult male mice were optically cleared using the CUBIC-perfusion protocol. After making the hearts transparent, sympathetic nerves and coronary vessels were immunofluorescently labeled, and then images were acquired. The spatial distribution of sympathetic nerves was visualized not only along the epicardial surface, but also transmurally. They were distributed over the epicardial surface and penetrated into the myocardium to twist around coronary vessels, but also independent from the coronary vasculature. At 2 weeks after myocardial infarction, we were able to quantify both denervation distal from the site of infarction and nerve sprouting (hyperinnervation) at the ischemic border zone of the hearts in a 3D manner. The nerve density at the ischemic border zone was more than doubled in hearts with myocardial infarction compared to intact mice hearts (3D analyses; n = 5, p<0.05). CONCLUSIONS: There is both sympathetic hyperinnervation and denervation after myocardial infarction. Both can be visualized and quantified by a new imaging technique in transparent hearts and thereby become a useful tool in elucidating the role of the sympathetic nervous system in arrhythmias associated with myocardial infarction.

Semi-Automatic Rating Method for Neutrophil Alkaline Phosphatase Activity.

BACKGROUND: The neutrophil alkaline phosphatase (NAP) score is a valuable test for the diagnosis of myeloproliferative neoplasms, but it has still manually rated. Therefore, we developed a semi-automatic rating method using Photoshop® and Image-J, called NAP-PS-IJ. METHODS: Neutrophil alkaline phosphatase staining was conducted with Tomonaga's method to films of peripheral blood taken from three healthy volunteers. At least 30 neutrophils with NAP scores from 0 to 5+ were observed and taken their images. From which the outer part of neutrophil was removed away with Image-J. These were binarized with two different procedures (P1 and P2) using Photoshop® . NAP-positive area (NAP-PA) and granule (NAP-PGC) were measured and counted with Image-J. RESULTS: The NAP-PA in images binarized with P1 significantly (P < 0.05) differed between images with NAP scores from 0 to 3+ (group 1) and those from 4+ to 5+ (group 2). The original images in group 1 were binarized with P2. NAP-PGC of them significantly (P < 0.05) differed among all four NAP score groups. The mean NAP-PGC with NAP-PS-IJ indicated a good correlation (r = 0.92, P < 0.001) to results by human examiners. CONCLUSIONS: The sensitivity and specificity of NAP-PS-IJ were 60% and 92%, which might be considered as a prototypic method for the full-automatic rating NAP score.

Cholesterol Uptake Capacity: A New Measure of HDL Functionality for Coronary Risk Assessment.

BACKGROUND: Recent studies have shown that the cholesterol efflux capacity of HDL is a better predictor of cardiovascular disease (CVD) than HDL cholesterol. However, the standard procedures used for measuring cholesterol efflux capacity involve radioisotope-labeled cholesterol and cultured macrophages. Thus, a simpler method to measure HDL functionality is needed for clinical application. METHODS: We established a cell-free assay system to evaluate the capacity of HDL to accept additional cholesterol, which we named cholesterol "uptake capacity," using fluorescently labeled cholesterol and an anti-apolipoprotein A1 antibody. We quantified cholesterol uptake capacity of apolipoprotein B (apoB)-depleted serum samples from patients with coronary artery disease who had previously undergone revascularization. RESULTS: This assay system exhibited high reproducibility (CV <10%) and a short processing time (<6 h). The myeloperoxidase-mediated oxidation of apoB-depleted serum impaired cholesterol uptake capacity. Cholesterol uptake capacity correlated significantly with cholesterol efflux capacity (r2 = 0.47, n = 30). Furthermore, cholesterol uptake capacity correlated inversely with the requirement for revascularization because of recurrence of coronary lesions in patients with optimal control of LDL cholesterol (P < 0.01, n = 156). A multivariate analysis adjusted for traditional coronary risk factors showed that only cholesterol uptake capacity remained significant (odds ratio, 0.48; 95% CI, 0.29-0.80; P = 0.0048). CONCLUSIONS: Cholesterol uptake capacity assay evaluates the functionality of HDL in a sensitive and high-throughput manner without using radioisotope label and cells. This assay system could be used for the assessment of CVD risk in the clinical settings.

Semiautomated Segmentation and Measurement of Cytoplasmic Vacuoles in a Neutrophil With General-Purpose Image Analysis Software.

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.

A New In Vitro Co-Culture Model Using Magnetic Force-Based Nanotechnology.

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.

Pirfenidone exhibits cardioprotective effects by regulating myocardial fibrosis and vascular permeability in pressure-overloaded hearts.

Although cardiac fibrosis causes heart failure, its molecular mechanisms remain elusive. In this study, we investigated the mechanisms of cardiac fibrosis and examined the effects of the antifibrotic drug pirfenidone (PFD) on chronic heart failure. To understand the responsible mechanisms, we generated an in vivo pressure-overloaded heart failure model via transverse aortic constriction (TAC) and examined the effects of PFD on chronic-phase cardiac fibrosis and function. In the vehicle group, contractile dysfunction and left ventricle fibrosis progressed further from 4 to 8 wk after TAC but were prevented by PFD treatment beginning 4 wk after TAC. We isolated cardiac fibroblasts and vascular endothelial cells from the left ventricles of adult male mice and investigated the cell-type-specific effects of PFD. Transforming growth factor-ß induced upregulated collagen 1 expression via p38 phosphorylation and downregulated claudin 5 (Cldn5) expression in cardiac fibroblasts and endothelial cells, respectively; both processes were inhibited by PFD. Moreover, PFD inhibited changes in the collagen 1 and Cldn5 expression levels, resulting in reduced fibrosis and serum albumin leakage into the interstitial space during the chronic phase in TAC hearts. In conclusion, PFD inhibited cardiac fibrosis by suppressing both collagen expression and the increased vascular permeability induced by pressure overload.

Excitation propagation in three-dimensional engineered hearts using decellularized extracellular matrix.

Engineering of three-dimensional (3D) cardiac tissues using decellularized extracellular matrix could be a new technique to create an "organ-like" structure of the heart. To engineer artificial hearts functionally comparable to native hearts, however, much remain to be solved including stable excitation-propagation. To elucidate the points, we examined conduction properties of engineered tissues. We repopulated the decellularized hearts with neonatal rat cardiac cells and then, we observed excitation-propagation of spontaneous beatings using high resolution cameras. We also conducted immunofluorescence staining to examine morphological aspects. Live tissue imaging revealed that GFP-labeled-isolated cardiac cells were migrated into interstitial spaces through extravasation from coronary arteries. Engineered hearts repopulated with Ca(2+)-indicating protein (GCaMP2)-expressing cardiac cells were subjected to optical imaging experiments. Although the engineered hearts generally showed well-organized stable excitation-propagation, the hearts also demonstrated arrhythmogenic propensity such as disorganized propagation. Immunofluorescence study revealed randomly-mixed alignment of cardiomyocytes, endothelial cells and smooth muscle cells. The recellularized hearts also showed disarray of cardiomyocytes and markedly decreased expression of connexin43. In conclusion, we successfully demonstrated that the recellularized hearts showed dynamic excitation-propagation as a "whole organ". Our strategy could provide prerequisite information to construct a 3D-engineered heart, functionally comparable to the native heart.

Sympathetic innervation induced in engrafted engineered cardiomyocyte sheets by glial cell line derived neurotrophic factor in vivo.

The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue. However, this strategy has been hampered by lack of functional integration of grafts with native myocardium. Autonomic innervation may be crucial for grafts to function properly with host myocardium. In this study, we explored the feasibility of in vivo induction of autonomic innervation to engineered myocardial tissue using genetic modulation by adenovirus encoding glial cell line derived neurotrophic factor (GDNF). GFP-transgene (control group) or GDNF overexpressing (GDNF group) engineered cardiomyocyte sheets were transplanted on cryoinjured hearts in rats. Nerve fibers in the grafts were examined by immunohistochemistry at 1, 2, and 4 weeks postoperatively. Growth associated protein-43 positive growing nerves and tyrosine hydroxylase positive sympathetic nerves were first detected in the grafts at 2 weeks postoperatively in control group and 1 week in GDNF group. The densities of growing nerve and sympathetic nerve in grafts were significantly increased in GDNF group. No choline acetyltransferase immunopositive parasympathetic nerves were observed in grafts. In conclusion, sympathetic innervation could be effectively induced into engrafted engineered cardiomyocyte sheets using GDNF.

Cuprizone short-term exposure: astrocytic IL-6 activation and behavioral changes relevant to psychosis.

A growing body of evidence suggests the involvement of inflammatory processes in the pathophysiology of schizophrenia. Four- to 8-week exposure to cuprizone, a copper chelator, causes robust demyelination and has been used to build a model for multiple sclerosis. In contrast, we report here the effects of 1-week cuprizone exposure in mice. This short-term cuprizone exposure elicits behavioral changes that include augmented responsiveness to methamphetamine and phencyclidine, as well as impaired working memory. The cellular effects of 1-week cuprizone exposure differ substantially from the longer-term exposure; perturbation of astrocytes and microglia is induced without any sign of demyelination. Furthermore, the proinflammatory cytokine interleukin-6 was significantly up-regulated in glial fibrillary acidic protein (GFAP)-positive cells. We propose that this cuprizone short-term exposure may offer a model to study some aspects of biology relevant to schizophrenia and related conditions.