Acto3D: an open-source user-friendly volume rendering software for high-resolution 3D fluorescence imaging in biology.

Advances in fluorescence microscopy and tissue-clearing have revolutionised 3D imaging of fluorescently labelled tissues, organs and embryos. However, the complexity and high cost of existing software and computing solutions limit their widespread adoption, especially by researchers with limited resources. Here, we present Acto3D, an open-source software, designed to streamline the generation and analysis of high-resolution 3D images of targets labelled with multiple fluorescent probes. Acto3D provides an intuitive interface for easy 3D data import and visualisation. Although Acto3D offers straightforward 3D viewing, it performs all computations explicitly, giving users detailed control over the displayed images. Leveraging an integrated graphics processing unit, Acto3D deploys all pixel data to system memory, reducing visualisation latency. This approach facilitates accurate image reconstruction and efficient data processing in 3D, eliminating the need for expensive high-performance computers and dedicated graphics processing units. We have also introduced a method for efficiently extracting lumen structures in 3D. We have validated Acto3D by imaging mouse embryonic structures and by performing 3D reconstruction of pharyngeal arch arteries while preserving fluorescence information. Acto3D is a cost-effective and efficient platform for biological research.

Hey2 enhancer activity defines unipotent progenitors for left ventricular cardiomyocytes in juxta-cardiac field of early mouse embryo.

The cardiac crescent is the first structure of the heart and contains progenitor cells of the first heart field, which primarily differentiate into left ventricular cardiomyocytes. The interface between the forming cardiac crescent and extraembryonic tissue is known as the juxta-cardiac field (JCF), and progenitor cells in this heart field contribute to the myocardium of the left ventricle and atrioventricular canal as well as the epicardium. However, it is unclear whether there are progenitor cells that differentiate specifically into left ventricular cardiomyocytes. We have previously demonstrated that an enhancer of the gene encoding the Hey2 bHLH transcriptional repressor is activated in the ventricular myocardium during mouse embryonic development. In this study, we aimed to investigate the characteristics of cardiomyocyte progenitor cells and their cell lineages by analyzing Hey2 enhancer activity at the earliest stages of heart formation. We found that the Hey2 enhancer initiated its activity prior to cardiomyocyte differentiation within the JCF. Hey2 enhancer-active cells were present rostrally to the Tbx5-expressing region at the early phase of cardiac crescent formation and differentiated exclusively into left ventricular cardiomyocytes in a lineage distinct from the Tbx5-positive lineage. By the late phase of cardiac crescent formation, Hey2 enhancer activity became significantly overlapped with Tbx5 expression in cells that contribute to the left ventricular myocardium. Our study reveals that a population of unipotent progenitor cells for left ventricular cardiomyocytes emerge in the JCF, providing further insight into the mode of cell type diversification during early cardiac development.

Raman Spectroscopic Assessment of Myocardial Viability in Langendorff-Perfused Ischemic Rat Hearts.

Spontaneous Raman spectroscopy, which senses changes in cellular contents of reduced cytochrome c, could be a powerful tool for label-free evaluation of ischemic hearts. However, undetermined is whether it is applicable to evaluation of myocardial viability in ischemic hearts. To address this issue, we investigated sequential changes in Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart before and during ligation of the left coronary artery and its subsequent release and re-ligation. Under 532-nm wavelength excitation, the Raman peak intensity of reduced cytochrome c at 747 cm-1 increased quickly after the coronary ligation, and reached a quasi-steady state within 30 min. Subsequent reperfusion of the heart after a short-term (30-min) ligation that simulates reversible conditions resulted in quick recovery of the peak intensity to the baseline. Further re-ligation resulted in resurgence of the peak intensity to nearly the identical value to the first ischemia value. In contrast, reperfusion after prolonged (120-min) ligation that assumes irreversible states resulted in incomplete recovery of the peak intensity, and re-ligation resulted in inadequate resurgence. Electron microscopic observations confirmed the spectral findings. Together, the Raman spectroscopic measurement for cytochrome c could be applicable to evaluation of viability of the ischemic myocardium without labeling.

AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP-170.

AMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated disks.

Endocardium differentiation through Sox17 expression in endocardium precursor cells regulates heart development in mice.

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5+ cardiac progenitor cells (CPCs) that express the Sry-type HMG box gene Sox17 from embryonic day (E) 7.5 to E8.5 specifically differentiate into the endocardium in mouse embryos. Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, likely as a consequence of down-regulation of NOTCH signaling. An unknown signal, regulated by Sox17 and required for nurturing of the myocardium, is responsible for the reduction in NOTCH-related genes in the mutant embryos. Our results thus provide insight into differentiation of the endocardium and its role in heart development.

Loss of Fam60a, a Sin3a subunit, results in embryonic lethality and is associated with aberrant methylation at a subset of gene promoters.

We have examined the role of Fam60a, a gene highly expressed in embryonic stem cells, in mouse development. Fam60a interacts with components of the Sin3a-Hdac transcriptional corepressor complex, and most Fam60a-/- embryos manifest hypoplasia of visceral organs and die in utero. Fam60a is recruited to the promoter regions of a subset of genes, with the expression of these genes being either up- or down-regulated in Fam60a-/- embryos. The DNA methylation level of the Fam60a target gene Adhfe1 is maintained at embryonic day (E) 7.5 but markedly reduced at E9.5 in Fam60a-/- embryos, suggesting that DNA demethylation is enhanced in the mutant. Examination of genome-wide DNA methylation identified several differentially methylated regions, which were preferentially hypomethylated, in Fam60a-/- embryos. Our data suggest that Fam60a is required for proper embryogenesis, at least in part as a result of its regulation of DNA methylation at specific gene promoters.

Fibrin Glue-aided, Instant Epicardial Placement Enhances the Efficacy of Mesenchymal Stromal Cell-Based Therapy for Heart Failure.

Transplantation of mesenchymal stromal cells (MSCs) is a promising new therapy for heart failure. However, the current cell delivery routes result in poor donor cell engraftment. We therefore explored the role of fibrin glue (FG)-aided, instant epicardial placement to enhance the efficacy of MSC-based therapy in a rat ischemic cardiomyopathy model. We identified a feasible and reproducible method to instantly produce a FG-MSC complex directly on the heart surface. This complex exhibited prompt, firm adhesion to the heart, markedly improving initial retention of donor MSCs compared to intramyocardial injection. In addition, maintenance of retained MSCs was enhanced using this method, together contributing the increased donor cell presence. Such increased donor cell quantity using the FG-aided technique led to further improved cardiac function in association with augmented histological myocardial repair, which correlated with upregulation of tissue repair-related genes. We identified that the epicardial layer was eliminated shortly after FG-aided epicardial placement of MSCs, facilitating permeation of the donor MSC's secretome into the myocardium enabling myocardial repair. These data indicate that FG-aided, on-site, instant epicardial placement enhances MSC engraftment, promoting the efficacy of MSC-based therapy for heart failure. Further development of this accessible, advanced MSC-therapy is justified.

Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins.

As core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.

NCK Associated Protein 1 Modulated by miRNA-214 Determines Vascular Smooth Muscle Cell Migration, Proliferation, and Neointima Hyperplasia.

BACKGROUND: MicroRNA miR-214 has been implicated in many biological cellular functions, but the impact of miR-214 and its target genes on vascular smooth muscle cell (VSMC) proliferation, migration, and neointima smooth muscle cell hyperplasia is unknown. METHODS AND RESULTS: Expression of miR-214 was closely regulated by different pathogenic stimuli in VSMCs through a transcriptional mechanism and decreased in response to vascular injury. Overexpression of miR-214 in serum-starved VSMCs significantly decreased VSMC proliferation and migration, whereas knockdown of miR-214 dramatically increased VSMC proliferation and migration. Gene and protein biochemical assays, including proteomic analyses, showed that NCK associated protein 1 (NCKAP1)-a major component of the WAVE complex that regulates lamellipodia formation and cell motility-was negatively regulated by miR-214 in VSMCs. Luciferase assays showed that miR-214 substantially repressed wild-type but not the miR-214 binding site mutated version of NCKAP1 3' untranslated region luciferase activity in VSMCs. This result confirmed that NCKAP1 is the functional target of miR-214 in VSMCs. NCKAP1 knockdown in VSMCs recapitulates the inhibitory effects of miR-214 overexpression on actin polymerization, cell migration, and proliferation. Data from cotransfection experiments also revealed that inhibition of NCKAP1 is required for miR-214-mediated lamellipodia formation, cell motility, and growth. Importantly, locally enforced expression of miR-214 in the injured vessels significantly reduced NCKAP1 expression levels, inhibited VSMC proliferation, and prevented neointima smooth muscle cell hyperplasia after injury. CONCLUSIONS: We uncovered an important role of miR-214 and its target gene NCKAP1 in modulating VSMC functions and neointima hyperplasia. Our findings suggest that miR-214 represents a potential therapeutic target for vascular diseases.

Cell Size Critically Determines Initial Retention of Bone Marrow Mononuclear Cells in the Heart after Intracoronary Injection: Evidence from a Rat Model.

Intracoronary injection of bone marrow mononuclear cells (BMMNC) is an emerging treatment for heart failure. Initial donor cell retention in the heart is the key to the success of this approach, but this process remains insufficiently characterized. Although it is assumed that cell size of injected cells may influence their initial retention, no scientific evidence has been reported. We developed a unique model utilizing an ex-vivo rat heart perfusion system, enabling quantitative assessment of retention of donor cells after intracoronary injection. The initial (5 minutes after intracoronary injection) retention rate of BMMNC was as low as approximately 20% irrespective of donor cell doses injected (1×106, 8×106, 4×107). Quantitative cell-size assessment revealed a positive relationship between the size of BMMNC and retention ratio; larger subpopulations of BMMNC were more preferentially retained compared to smaller ones. Furthermore, a larger cell type-bone marrow-derived mesenchymal stromal cells (median size = 11.5µm versus 7.0µm for BMMNC)-had a markedly increased retention rate (77.5±1.8%). A positive relationship between the cell size and retention ratio was also seen in mesenchymal stromal cells. Flow-cytometric studies showed expression of cell-surface proteins, including integrins and selectin-ligands, was unchanged between pre-injection BMMNC and those exited from the heart, suggesting that biochemical interaction between donor cells and host coronary endothelium is not critical for BMMNC retention. Histological analyses showed that retained BMMNC and mesenchymal stromal cells were entrapped in the coronary vasculature and did not extravasate by 60 minutes after transplantation. Whilst BMMNC did not change coronary flow after intracoronary injection, mesenchymal stromal cells reduced it, suggesting coronary embolism, which was supported by the histological finding of intravascular cell-clump formation. These data indicate that cell-size dependent, passive (mechanical), intravascular entrapment is responsible for the initial donor cell retention after intracoronary injection of BMMNC in the heart having normal vasculatures (at least).

GFRA2 Identifies Cardiac Progenitors and Mediates Cardiomyocyte Differentiation in a RET-Independent Signaling Pathway.

A surface marker that distinctly identifies cardiac progenitors (CPs) is essential for the robust isolation of these cells, circumventing the necessity of genetic modification. Here, we demonstrate that a Glycosylphosphatidylinositol-anchor containing neurotrophic factor receptor, Glial cell line-derived neurotrophic factor receptor alpha 2 (Gfra2), specifically marks CPs. GFRA2 expression facilitates the isolation of CPs by fluorescence activated cell sorting from differentiating mouse and human pluripotent stem cells. Gfra2 mutants reveal an important role for GFRA2 in cardiomyocyte differentiation and development both in vitro and in vivo. Mechanistically, the cardiac GFRA2 signaling pathway is distinct from the canonical pathway dependent on the RET tyrosine kinase and its established ligands. Collectively, our findings establish a platform for investigating the biology of CPs as a foundation for future development of CP transplantation for treating heart failure.

Alternatively activated macrophages determine repair of the infarcted adult murine heart.

Alternatively activated (also known as M2) macrophages are involved in the repair of various types of organs. However, the contribution of M2 macrophages to cardiac repair after myocardial infarction (MI) remains to be fully characterized. Here, we identified CD206+F4/80+CD11b+ M2-like macrophages in the murine heart and demonstrated that this cell population predominantly increases in the infarct area and exhibits strengthened reparative abilities after MI. We evaluated mice lacking the kinase TRIB1 (Trib1-/-), which exhibit a selective depletion of M2 macrophages after MI. Compared with control animals, Trib1-/- mice had a catastrophic prognosis, with frequent cardiac rupture, as the result of markedly reduced collagen fibril formation in the infarct area due to impaired fibroblast activation. The decreased tissue repair observed in Trib1-/- mice was entirely rescued by an external supply of M2-like macrophages. Furthermore, IL-1α and osteopontin were suggested to be mediators of M2-like macrophage-induced fibroblast activation. In addition, IL-4 administration achieved a targeted increase in the number of M2-like macrophages and enhanced the post-MI prognosis of WT mice, corresponding with amplified fibroblast activation and formation of more supportive fibrous tissues in the infarcts. Together, these data demonstrate that M2-like macrophages critically determine the repair of infarcted adult murine heart by regulating fibroblast activation and suggest that IL-4 is a potential biological drug for treating MI.

Allogeneic Mesenchymal Stromal Cells Transplanted Onto the Heart Surface Achieve Therapeutic Myocardial Repair Despite Immunologic Responses in Rats.

BACKGROUND: Transplantation of allogeneic mesenchymal stromal cells (MSCs) is a promising treatment for heart failure. We have shown that epicardial placement of cell sheets markedly increases donor cell survival and augments therapeutic effects compared with the current methods. Although immune rejection of intramyocardially injected allogeneic MSCs have been suggested, allogeneic MSCs transplanted on the heart surface (virtual space) may undergo different courses. This study aimed to elucidate immunologic response against epicardially placed allogeneic MSCs, rejection or acceptance of these cells, and their therapeutic effects for heart failure. METHODS AND RESULTS: At 4 weeks after coronary artery ligation, Lewis rats underwent epicardial placement of MSC sheets from syngeneic Lewis or allogeneic Fischer 344 rats or sham treatment. At days 3 and 10 after treatment, similar ratios (≈50% and 30%, respectively) of grafted MSCs survived on the heart surface in both MSC sheet groups. By day 28, survival of syngeneic MSCs was substantially reduced (8.9%); survival of allogeneic MSCs was more extensively reduced (0.2%), suggesting allorejection. Correspondingly, allogeneic MSCs were found to have evoked an immunologic response, albeit low level, as characterized by accumulation of CD4(+) T cells and upregulation of interleukin 6. Despite this alloimmune response, the allogeneic MSC sheet achieved myocardial upregulation of reparative factors, enhanced repair of the failing myocardium, and improved cardiac function to the equivalent degree observed for the syngeneic MSC sheet. CONCLUSIONS: Allogeneic MSCs placed on the heart surface evoked an immunologic response; however, this allowed sufficient early phase donor cell survival to induce equivalent therapeutic benefits to syngeneic MSCs. Further development of this approach toward clinical application is warranted.

Cardiomyocyte differentiation from mouse embryonic stem cells using a simple and defined protocol.

BACKGROUND: Embryonic stem (ES) cells are pluripotent cells with the ability to differentiate to any cell type of the resident organism. In recent years, significant advances have been made in using these cells to obtain large numbers of cardiomyocyte (CM)-like cells for scientific research and clinical application. A vast number of protocols have emerged describing differentiation methods without the use of animal serum or extracts restrictive for use in a human clinical setting. These techniques follow a complicated procedure, which although successful, show a relatively varied yield among cell batches. RESULTS: We have designed a three-step differentiation protocol using defined reagents and a monolayer culture without feeder cells, avoiding embryoid body formation and multiple trypsin treatment, in which beating foci appeared as early as day 6 in in vitro differentiating conditions. Our results show a high yield of CM reaching approximately 60% of the differentiated cells after 13 days in vitro. CONCLUSIONS: We provide a fast, simple, reliable and reproducible protocol for inducing murine ES cells toward a CM-like phenotype comparable to available high-yield protocols, without the use of intermediate trypsinization/passage steps.

Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo.

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study.

Collagenous gastroduodenitis with recurrent gastric ulcer in 12-year-old girl.

This report describes a rare case of collagenous gastroduodenitis found in a 12-year-old Japanese girl who had recurrent hematemesis. Gastrointestinal endoscopy showed many lotus leaf-like lesions on the gastric mucosa surrounded by atrophic gastric mucosa in the antrum, with a cobblestone appearance and a scarred duodenal ulcer in the duodenal bulb. A biopsy of the gastric mucosa indicated subepithelial collagen band. The patient was treated with H2-blockers for her symptoms for 4 years following the endoscopic findings. Follow-up endoscopy showed the same appearance as before. The pathology, however, showed a more prominent subepithelial collagen deposition. To make the correct diagnosis, it is critical to know from which part the pathological biopsy specimens were taken because there were numerous collagen bands in the atrophic membrane. It is important to monitor the patient regularly for evaluation of the etiology, pathogenesis and prognosis of this rare disease.