CCND2 Modified mRNA Activates Cell Cycle of Cardiomyocytes in Hearts With Myocardial Infarction in Mice and Pigs.

BACKGROUND: Experiments in mammalian models of cardiac injury suggest that the cardiomyocyte-specific overexpression of CCND2 (cyclin D2, in humans) improves recovery from myocardial infarction (MI). The primary objective of this investigation was to demonstrate that our specific modified mRNA translation system (SMRTs) can induce CCND2 expression in cardiomyocytes and replicate the benefits observed in other studies of cardiomyocyte-specific CCND2 overexpression for myocardial repair. METHODS: The CCND2-cardiomyocyte-specific modified mRNA translation system (cardiomyocyte SMRTs) consists of 2 modRNA constructs: one codes for CCND2 and contains a binding site for L7Ae, and the other codes for L7Ae and contains recognition elements for the cardiomyocyte-specific microRNAs miR-1 and miR-208. Thus, L7Ae suppresses CCND2 translation in noncardiomyocytes but is itself suppressed by endogenous miR-1 and -208 in cardiomyocytes, thereby facilitating cardiomyocyte-specific CCND2 expression. Experiments were conducted in both mouse and pig models of MI, and control assessments were performed in animals treated with an SMRTs coding for the cardiomyocyte-specific expression of luciferase or green fluorescent protein (GFP), in animals treated with L7Ae modRNA alone or with the delivery vehicle, and in Sham-operated animals. RESULTS: CCND2 was abundantly expressed in cultured, postmitotic cardiomyocytes 2 days after transfection with the CCND2-cardiomyocyte SMRTs, and the increase was accompanied by the upregulation of markers for cell-cycle activation and proliferation (eg, Ki67 and Aurora B kinase). When the GFP-cardiomyocyte SMRTs were intramyocardially injected into infarcted mouse hearts, the GFP signal was observed in cardiomyocytes but no other cell type. In both MI models, cardiomyocyte proliferation (on day 7 and day 3 after treatment administration in mice and pigs, respectively) was significantly greater, left-ventricular ejection fractions (days 7 and 28 in mice, days 10 and 28 in pigs) were significantly higher, and infarcts (day 28 in both species) were significantly smaller in animals treated with the CCND2-cardiomyocyte SMRTs than in any other group that underwent MI induction. CONCLUSIONS: Intramyocardial injections of the CCND2-cardiomyocyte SMRTs promoted cardiomyocyte proliferation, reduced infarct size, and improved cardiac performance in small and large mammalian hearts with MI.

Pichia kurtzmaniana f.a. sp. nov., with the transfer of eight Candida species to Pichia.

Three yeast strains belonging to the ascomycetous yeast genus Pichia were isolated from two soil samples from Yunnan and Guizhou provinces and a marine water sample from Liaoning province, PR China. Phylogenetic analyses based on the sequences of the D1/D2 domains of the large subunit(LSU) rRNA gene and the internal transcribed spacer (ITS) region indicate that these three strains, together with 12 additional strains isolated from various substrates collected in different regions or countries of the world, represent a novel species of the genus Pichia, for which the name Pichia kurtzmaniana sp. nov. (holotype: strain CGMCC 2.7213) is proposed. The novel species differs from its close relatives Candida californica by eight (1.5 %) and 26 (11.1 %) mismatches in the D1/D2 domains and the ITS region, respectively; and from Pichia chibodasensis by 11 (2.1 %) and 20 (8.7 %) mismatches in the D1/D2 domains and the ITS region, respectively. In addition, eight Candida species which belong to the Pichia clade are transferred to the genus Pichia, resulting in the proposal of the following new combinations: Pichia cabralensis comb. nov., Pichia californica comb. nov., Pichia ethanolica comb. nov., Pichia inconspicua comb. nov., Pichia phayaonensis comb. nov., Pichia pseudolambica comb. nov., Pichia rugopelliculosa comb. nov., and Pichia thaimueangensis comb. nov.

Saturnispora sinensis sp. nov., a new ascomycetous yeast species from soil and rotten wood.

A yeast strain (CGMCC 2.6937T) belonging to the ascomycetous yeast genus Saturnispora was recently isolated from soil collected in Xinghuacun, Shanxi Province, PR China. The strain produces one or two ellipsoid or spherical ascospores in asci formed by the conjugation between a cell and its bud. Phylogenetic analyses of the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit rRNA gene suggest that this strain is conspecific with strains NYNU 14639 isolated from rotten wood collected in Funiu Mountain, Henan province and ES13S05 from soil collected in Nantou County, Taiwan. The CGMCC 2.6937T group is most closely related to Saturnispora dispora and Saturnispora zaruensis. However, strain CGMCC 2.6937T differs from S. dispora by 17 (3.2 %, 13 substitutions and four gaps) and 77 (18.8 %, 52 substitutions and 25 gaps) mismatches, and from S. zaruensis by 15 (2.9 %, 12 substitutions and three gaps) and 64 (15.6 %, 44 substitutions and 20 gaps) mismatches, in the D1/D2 domain and ITS region, respectively. The results suggest that the CGMCC 2.6937T group represents an undescribed species in the genus Saturnispora, for which the name Saturnispora sinensis sp. nov. is proposed. The holotype strain is CGMCC 2.6937T.

Immediate and delayed effects of environmental temperature on schizophrenia admissions in Liuzhou, China, 2013-2020: a time series analysis.

This study aimed to investigate the associations between environmental temperature and schizophrenia admissions in Liuzhou, China. A Poisson generalized linear model combined with a distributed lag nonlinear model was used to analyze the effects of daily mean temperature on schizophrenia admissions from 2013 to 2020 in Liuzhou. Additionally, subgroup analyses were conducted to investigate possible modifications stratified by gender, marital status, and age. In this study, 10,420 schizophrenia admissions were included. The relative risks of schizophrenia admissions increased as the temperature rose, and the lag effects of high temperature on schizophrenia admissions were observed when the daily mean temperature reached 21.65°C. The largest single effect was observed at lag0, while the largest cumulative effect was observed at lag6. The single effects of high temperatures on schizophrenia admissions were statistically significant in both males and females, but the cumulative effects were statistically significant only in males, with the greatest effect at lag0-7. The single effect of high temperatures on admissions for unmarried schizophrenics was greatest at lag5, while the maximum cumulative effect for unmarried schizophrenia was observed at lag0-7. The single effects of high temperatures on schizophrenia admissions were observed in those aged 0-20, 21-40, and 41-60. The cumulative effects for schizophrenics aged 21-40 were observed from lag0-3 to lag0-7, with the maximum effect at lag0-7. In conclusion, the risk of schizophrenia admissions increased as the environmental temperature increased. The schizophrenics who were unmarried appeared to be more vulnerable to the single and cumulative effects of high temperature.

Vishniacozyma pseudocarnescens sp. nov., a new anamorphic tremellomycetous yeast species.

Three strains belonging to the basidiomycetous yeast genus Vishniacozyma were isolated from marine water samples collected from intertidal zones in Liaoning province, northeast China. Phylogenetic analyses based on the sequences of the small subunit (SSU) ribosomal DNA (rDNA), the D1/D2 domain of the large subunit (LSU) ribosomal DNA (rDNA), the internal transcribed spacer region (ITS), the two subunits of DNA polymerase II (RPB1 and RPB2), the translation elongation factor 1-α (TEF1), and the mitochondrial gene cytochrome b (CYTB) showed that these strains together with 20 strains from various geographic and ecological origins from other regions of the world represent a novel species in the genus Vishniacozyma. We propose the name Vishniacozyma pseudocarnescens sp. nov. (holotype CGMCC 2.6457) for the new species, which differs phenotypically from its close relatives V. carnescens, V. tephrensis, and V. victoriae by its ability to grow at 30 °C and on 50 % (w/v) glucose-yeast extract agar.

Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction.

BACKGROUND: Human induced pluripotent stem cells with normal (wild-type) or upregulated (overexpressed) levels of CCND2 (cyclin D2) expression were differentiated into cardiomyocytes (CCND2WTCMs or CCND2OECMs, respectively) and injected into infarcted pig hearts. METHODS: Acute myocardial infarction was induced by a 60-minute occlusion of the left anterior descending coronary artery. Immediately after reperfusion, CCND2WTCMs or CCND2OECMs (3×107 cells each) or an equivalent volume of the delivery vehicle was injected around the infarct border zone area. RESULTS: The number of the engrafted CCND2OECMs exceeded that of the engrafted CCND2WTCMs from 6- to 8-fold, rising from 1 week to 4 weeks after implantation. In contrast to the treatment with the CCND2WTCMs or the delivery vehicle, the administration of CCND2OECM was associated with significantly improved left ventricular function, as revealed by magnetic resonance imaging. This correlated with reduction of infarct size, fibrosis, ventricular hypertrophy, and cardiomyocyte apoptosis, and increase of vascular density and arterial density, as per histologic analysis of the treated hearts. Expression of cell proliferation markers (eg, Ki67, phosphorylated histone 3, and Aurora B kinase) was also significantly upregulated in the recipient cardiomyocytes from the CCND2OECM-treated than from the CCND2WTCM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured human induced pluripotent stem cell cardiomyocytes were significantly greater after treatment with exosomes isolated from the CCND2OECMs (CCND2OEExos) than from the CCND2WTCMs (CCND2WTExos). As demonstrated by our study, CCND2OEExos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2OEExos versus CCND2WTExos identified 206 and 91 miRNAs that were significantly upregulated and downregulated, respectively. Gene ontology enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis. CONCLUSIONS: We demonstrated that enhanced potency of CCND2OECMs promoted myocyte proliferation in both grafts and recipient tissue in a large mammal acute myocardial infarction model. These results suggest that CCND2OECMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.

Relationship Between the Efficacy of Cardiac Cell Therapy and the Inhibition of Differentiation of Human iPSC-Derived Nonmyocyte Cardiac Cells Into Myofibroblast-Like Cells.

RATIONALE: Myofibroblasts are believed to evolve from precursor cells; however, whether noncardiomyocyte cardiac cells (NMCCs; ie, endothelial cells, smooth muscle cells, pericytes, and fibroblasts) that have been derived from human-induced pluripotent stem cells (hiPSCs) can transdifferentiate into myofibroblast-like cells, and if so, whether this process reduces the efficacy of hiPSC-NMCC therapy, is unknown. OBJECTIVE: To determine whether hiPSC-NMCCs can differentiate to myofibroblast-like cells and whether limiting the transdifferentiation of hiPSC-NMCCs can improve their effectiveness for myocardial repair. METHODS AND RESULTS: When endothelial cells, smooth muscle cells, pericytes, and fibroblasts that had been generated from hiPSCs were cultured with TGF-ß (transforming growth factor-ß), the expression of myofibroblast markers increased, whereas endothelial cell, smooth muscle cell, pericyte, and fibroblast marker expression declined. TGF-ß-associated myofibroblast differentiation was accompanied by increases in the signaling activity of Smad, Snail, and mTOR (mammalian target of rapamycin). However, measures of pathway activation, proliferation, apoptosis, migration, and protein expression in hiPSC-endothelial cell-derived, smooth muscle cell-derived, pericyte-derived, and fibroblast-derived myofibroblast-like cells differed. Furthermore, when hiPSC-NMCCs were transplanted into the hearts of mice after myocardial infarction, ≈21% to 35% of the transplanted hiPSC-NMCCs expressed myofibroblast markers 1 week later, compared with <7% of transplanted cells ( P<0.01, each cell type) in animals that were treated with both hiPSC-NMCCs and the TGF-ß inhibitor galunisertib. Galunisertib coadministration was also associated with significant improvements in fibrotic area, left ventricular dilatation, vascular density, and cardiac function. CONCLUSIONS: hiPSC-NMCCs differentiate into myofibroblast-like cells when cultured with TGF-ß or when transplanted into infarcted mouse hearts, and the phenotypes of the myofibroblast-like cells can differ depending on the lineage of origin. TGF-ß inhibition significantly improved the efficacy of transplanted hiPSC-NMCCs for cardiac repair, perhaps by limiting the differentiation of hiPSC-NMCCs into myofibroblast-like cells.

Comparison of Transfemoral Cerebral Angiography and Transradial Cerebral Angiography Following a Shift in Practice During Four Years at a Single Center in China.

BACKGROUND Cerebral angiography, or intra-arterial digital subtraction angiography (DSA), is a fluoroscopic imaging technique. In China, until recently, transfemoral access (TFA) has been used, rather than transradial access (TRA). This retrospective study aimed to compare transfemoral cerebral angiography (TFCA) with transradial cerebral angiography (TRCA) consecutively performed by the same operator, at a single center in China, to determine whether there were benefits from the shift from TFA to TRA in terms of efficiency, safety, and feasibility. MATERIAL AND METHODS A review of 1,048 cerebral angiograms in 980 patients was performed by a single operator from June 2014 to May 2018, including the TFA group (n=513) and the transradial access (TRA) group (n=535), and 39 patients underwent both TFA and TRA. The total procedure time, duration of fluoroscopy, catheterization success rate, image quality, length of stay in hospital, complications of the procedure, and patient preference were compared between the groups. RESULTS Compared with TFCA, TRCA resulted in significantly shorter total procedure time, a higher catheterization success rate, better image quality, and shorter duration of hospital stay (P<0.05). There was no significant difference between the TFA and TRA groups for cardiovascular, cerebral, and access site complications. Patients in the TRA group showed a significantly reduced fluoroscopy time at the early stages of operator training (P<0.05). Patient preference included TRA (76.74%), TFA (16.28%), and no preference (6.89%). CONCLUSIONS During four years at a single center, and with a single operator, TRCA was safe, feasible, and more rapid when compared with TFCA.

Identification of a conserved linear B-cell epitope in the Staphylococcus aureus GapC protein.

The GapC protein of Staphylococcus aureus (S. aureus) is a surface protein that is highly conserved among Staphylococcus strains, and it can induce protective humoral immune responses. However, B-cell epitopes in S. aureus GapC have not been reported. In this study, we generated a monoclonal antibody (mAb2A9) targeting S. aureus GapC. Through a passive immunity test, mAb2A9 was shown to partially protect mice against S. aureus infection. We screened the motif 236PVATGSLTE243 that is recognized by mAb2A9 using a phage-display system. The motif sequence exactly matched amino acids 236-243 of the S. aureus GapC protein. Then, we identified the key amino acids in the motif using site-directed mutagenesis. Site-directed mutagenesis revealed that residues P236, G240, L242, and T243 formed the core of the 236PVATGSLT243 motif. In addition, this epitope was proven to be located on the surface of S. aureus, and it induced a protective humoral immune response against S. aureus infection in immunized mice. Overall, our results characterized a conserved B-cell epitope, which will be an attractive target for designing effective epitope-based vaccines against S. aureus infection.

Identification and characterization of CD4⁺ T-cell epitopes on GapC protein of Streptococcus dysgalactiae.

The GapC protein is highly conserved surface dehydrogenase among Streptococcus dysgalactiae (S. dysgalactiae) and is shown to be involved in bacterial virulence. Immunization of GapC protein can induce specific CD4(+) T-cell immune responses and protect against S. dysgalactiae infection. However, there are no studies to identify immunodominant CD4(+) T-cell epitopes on GapC protein. In this study, in silico MHC affinity measurement method was firstly used to predict potential CD4(+) T-cell epitopes on GapC protein. Six predictive 15-mer peptides were synthesized and two novel GapC CD4(+) T-cell epitopes, GapC63-77 and GapC96-110, were for the first time identified using CD4(+) T-cells obtained from GapC-immunized BALB/c (H-2(d)) and C57BL/6 (H-2(b)) mice spleen based on cell proliferation and cytokines response. The results showed that peptides containing 63-77 and 96-110 induced significant antigen-specific CD4(+) T-cells proliferation response in vivo. At the same time, high levels of IFN-γ and IL-17A, as well as moderate levels of IL-10 and IL-4 were detected in CD4(+) T-cells isolated from both GapC and peptide-immunized mice in vivo, suggesting that GapC63-77 and GapC96-110 preferentially elicited polarized Th1/Th17-type responses. The characterization of GapC CD4(+) T-cell epitopes not only helps us understand its protective immunity, but also contributes to design effective T-cell epitope-based vaccine against S. dysgalactiae infection.

α-Lipoic Acid Treatment Improves Vision-Related Quality of Life in Patients with Dry Age-Related Macular Degeneration.

Dry form of age-related macular degeneration (AMD) constitutes 90% of AMD cases, and it is characterized by the formation of drusen under the retina and the slow breakdown of the light-sensing cells in the macula, which causes a gradual loss of central vision. Since oxidative stress is involved in the pathogenesis of dry AMD, α-lipoic acid (LA) with antioxidant properties was selected, and its effect on anti-oxidative markers and visual quality in patients with dry AMD was assessed. A total of 100 dry AMD patients (60-83 years old) were randomly assigned to LA treatment group (n = 50) and placebo control group (n = 50). We measured the serum superoxide dismutase (SOD) activity, an important marker of antioxidant defense, best-corrected visual acuity (BCVA), contrast sensitivity, and Chinese-Version Low Vision Quality of Life (CLVQOL) before and after LA or placebo intervention. Pearson correlation coefficients were calculated to explore the relationship between contrast sensitivity values and CLVQOL scores. There was a statistically significant increase in serum SOD activity after LA intervention. The CLVQOL score was improved significantly after LA treatment. The contrast sensitivity measured at middle and low spatial frequency was significantly higher after LA treatment. CLVQOL scores were positively correlated with contrast sensitivity at low spatial frequency (3 cyc/degree) in LA-treated group. These results indicate that LA treatment improves vision-related quality of life in patients with dry AMD probably by increasing antioxidant activity. Thus, LA can be regarded as a promising agent for the treatment of AMD.

Identification of CD4+ T-cell epitopes on iron-regulated surface determinant B of Staphylococcus aureus.

Iron-regulated surface determinant B (IsdB) of Staphylococcus aureus (S. aureus) is a highly conserved surface protein that can induce protective CD4(+) T-cell immune response. A pivotal role of CD4(+) T-cells in effective immunity against S. aureus infection has been proved, but CD4(+) T-cell epitopes on the S. aureus IsdB have not been well identified. In this study, MHC binding assay was firstly used to predict CD4(+) T-cell epitopes on S. aureus IsdB protein, and six peptides were synthesized to validate the probable epitopes. Two novel IsdB CD4(+) T-cell epitopes, P1 (residues 159-178) and P4 (residues 287-306), were for the first time identified using CD4(+) T-cells obtained from IsdB-immunized C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice spleen based on cell proliferation and cytokines response. The results showed that P1 and P4 emulsified in Freund's adjuvant (FA) induced much higher cell proliferation compared with PBS emulsified in FA. CD4(+) T-cells stimulated with peptides P1 and P4 secreted significantly higher levels of IFN-γ and IL-17A. However, the level of the cytokine IL-4 almost remained unchanged, suggesting that P1 and P4 preferentially elicited polarized Th1-type responses. In addition, BALB/c mice just respond to P4 not P1, while C57BL/6 mice respond to P1 not P4, implying that epitope P1 and P4 were determined as H-2(b) and H-2(d) restricted epitope, respectively. Taken together, our data may provide an explanation of the IsdB-induced protection against S. aureus and highlight the possibility of developing the epitope-based vaccine against the S. aureus.

Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines.

The J bs-5YP peptide can alleviate dementia in senile mice by restoring the transcription of Slc40a1 to secrete the excessive iron from brain.

INTRODUCTION: With age and ATP decrease in the body, the transcription factors hypophosphorylation weakens the transcription of Slc40a1 and hinders the expression of the iron discharger ferroportin. This may lead to iron accumulation in the brain and the catalysis of free radicals that damage cerebral neurons and eventually lead to Alzheimer's disease (AD). OBJECTIVES: To prevent AD caused by brain iron excretion disorders and reveal the mechanism of J bs-5YP peptide restoring ferroportin. METHODS: We prepared J bs-YP peptide and administered it to the senile mice with dementia. Then, the intelligence of the mice was tested using a Morris Water Maze. The ATP content in the body was detected using the ATP hydrophysis and Phosphate precipitation method. The activation of Slc40a1 transcription was assayed with ATAC seq and the ferroportin, as well as the phosphorylation levels of Ets1 in brain were detected by Western Blot. RESULTS: The phosphorylation level of Ets1in brain was enhanced, and subsequently, the transcription of Slc40a1 was activated and ferroportin was increased in the brain, the levels of iron and free radicals were reduced, with the neurons protection, and the dementia was ultimately alleviated in the senile mice. CONCLUSION: J bs-5YP can recover the expression of ferroportin to excrete excessive iron in the brain of senile mice with dementia by enhancing the transcription of Slc40a1 via phosphorylating Ets1, revealing the potential of J bs-5YP as a drug to alleviate senile dementia.

Analysis of cardiac single-cell RNA-sequencing data can be improved by the use of artificial-intelligence-based tools.

Single-cell RNA sequencing (scRNAseq) enables researchers to identify and characterize populations and subpopulations of different cell types in hearts recovering from myocardial infarction (MI) by characterizing the transcriptomes in thousands of individual cells. However, the effectiveness of the currently available tools for processing and interpreting these immense datasets is limited. We incorporated three Artificial Intelligence (AI) techniques into a toolkit for evaluating scRNAseq data: AI Autoencoding separates data from different cell types and subpopulations of cell types (cluster analysis); AI Sparse Modeling identifies genes and signaling mechanisms that are differentially activated between subpopulations (pathway/gene set enrichment analysis), and AI Semisupervised Learning tracks the transformation of cells from one subpopulation into another (trajectory analysis). Autoencoding was often used in data denoising; yet, in our pipeline, Autoencoding was exclusively used for cell embedding and clustering. The performance of our AI scRNAseq toolkit and other highly cited non-AI tools was evaluated with three scRNAseq datasets obtained from the Gene Expression Omnibus database. Autoencoder was the only tool to identify differences between the cardiomyocyte subpopulations found in mice that underwent MI or sham-MI surgery on postnatal day (P) 1. Statistically significant differences between cardiomyocytes from P1-MI mice and mice that underwent MI on P8 were identified for six cell-cycle phases and five signaling pathways when the data were analyzed via Sparse Modeling, compared to just one cell-cycle phase and one pathway when the data were analyzed with non-AI techniques. Only Semisupervised Learning detected trajectories between the predominant cardiomyocyte clusters in hearts collected on P28 from pigs that underwent apical resection (AR) on P1, and on P30 from pigs that underwent AR on P1 and MI on P28. In another dataset, the pig scRNAseq data were collected after the injection of CCND2-overexpression Human-induced Pluripotent Stem Cell-derived cardiomyocytes (CCND2hiPSC) into injured P28 pig heart; only the AI-based technique could demonstrate that the host cardiomyocytes increase proliferating by through the HIPPO/YAP and MAPK signaling pathways. For the cluster, pathway/gene set enrichment, and trajectory analysis of scRNAseq datasets generated from studies of myocardial regeneration in mice and pigs, our AI-based toolkit identified results that non-AI techniques did not discover. These different results were validated and were important in explaining myocardial regeneration.

Single-cell RNA sequencing analysis identifies one subpopulation of endothelial cells that proliferates and another that undergoes the endothelial-mesenchymal transition in regenerating pig hearts.

Background: In our previous work, we demonstrated that when newborn pigs undergo apical resection (AR) on postnatal day 1 (P1), the animals' hearts were completely recover from a myocardial infarction (MI) that occurs on postnatal day 28 (P28); single-nucleus RNA sequencing (snRNAseq) data suggested that this recovery was achieved by regeneration of pig cardiomyocyte subpopulations in response to MI. However, coronary vasculature also has a key role in promoting cardiac repair. Method: Thus, in this report, we used autoencoder algorithms to analyze snRNAseq data from endothelial cells (ECs) in the hearts of the same animals. Main results: Our results identified five EC clusters, three composed of vascular ECs (VEC1-3) and two containing lymphatic ECs (LEC1-2). Cells from VEC1 expressed elevated levels of each of five cell-cyclespecific markers (Aurora Kinase B [AURKB], Marker of Proliferation Ki-67 [MKI67], Inner Centromere Protein [INCENP], Survivin [BIRC5], and Borealin [CDCA8]), as well as a number of transcription factors that promote EC proliferation, while (VEC3 was enriched for genes that regulate intercellular junctions, participate in transforming growth factor ß (TGFß), bone morphogenic protein (BMP) signaling, and promote the endothelial mesenchymal transition (EndMT). The remaining VEC2 did not appear to participate directly in the angiogenic response to MI, but trajectory analyses indicated that it may serve as a reservoir for the generation of VEC1 and VEC3 ECs in response to MI. Notably, only the VEC3 cluster was more populous in regenerating (i.e., ARP1MIP28) than non-regenerating (i.e., MIP28) hearts during the 1-week period after MI induction, which suggests that further investigation of the VEC3 cluster could identify new targets for improving myocardial recovery after MI. Histological analysis of KI67 and EndMT marker PDGFRA demonstrated that while the expression of proliferation of endothelial cells was not significantly different, expression of EndMT markers was significantly higher among endothelial cells of ARP1MIP28 hearts compared to MIP28 hearts, which were consistent with snRNAseq analysis of clusters VEC1 and VEC3. Furthermore, upregulated secrete genes by VEC3 may promote cardiomyocyte proliferation via the Pi3k-Akt and ERBB signaling pathways, which directly contribute to cardiac muscle regeneration. Conclusion: In regenerative heart, endothelial cells may express EndMT markers, and this process could contribute to regeneration via a endothelial-cardiomyocyte crosstalk that supports cardiomyocyte proliferation.

Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes.

Introduction: The potentially unlimited number of cardiomyocyte (CMs) derived from human induced pluripotent stem cells (hiPSCs) in vitro facilitates high throughput applications like cell transplantation for myocardial repair, disease modelling, and cardiotoxicity testing during drug development. Despite promising progress in these areas, a major disadvantage that limits the use of hiPSC derived CMs (hiPSC-CMs) is their immaturity. Methods: Three hiPSC lines (PCBC-hiPSC, DP3-hiPSCs, and MLC2v-mEGFP hiPSC) were differentiated into CMs (PCBC-CMs, DP3-CMs, and MLC2v-CMs, respectively) with or without retinoic acid (RA). hiPSC-CMs were either maintained up to day 30 of contraction (D30C), or D60C, or purified using lactate acid and used for experiments. Purified hiPSC-CMs were cultured in basal maturation medium (BMM) or BMM supplemented with ascorbic acid (AA) for 14 days. The AA treated and non-treated hiPSC-CMs were characterized for sarcomeric proteins (MLC2v, TNNI3, and MYH7), ion channel proteins (Kir2.1, Nav1.5, Cav1.2, SERCA2a, and RyR), mitochondrial membrane potential, metabolomics, and action potential. Bobcat339, a selective and potent inhibitor of DNA demethylation, was used to determine whether AA promoted hiPSC-CM maturation through modulating DNA demethylation. Results: AA significantly increased MLC2v expression in PCBC-CMs, DP3-CMs, MLC2v-CMs, and RA induced atrial-like PCBC-CMs. AA treatment significantly increased mitochondrial mass, membrane potential, and amino acid and fatty acid metabolism in PCBC-CMs. Patch clamp studies showed that AA treatment induced PCBC-CMs and DP3-CMs adaptation to a ventricular-like phenotype. Bobcat339 inhibited MLC2v protein expression in AA treated PCBC-CMs and DP3-CMs. DNA demethylation inhibition was also associated with reduced TET1 and TET2 protein expressions and reduced accumulation of the oxidative product, 5 hmC, in both PCBC-CMs and DP3-CMs, in the presence of AA. Conclusions: Ascorbic acid induced MLC2v protein expression and promoted ventricular-like CM subtype in hiPSC-CMs. The effect of AA on hiPSC-CM was attenuated with inhibition of TET1/TET2 mediated DNA demethylation.