INO80-Dependent Remodeling of Transcriptional Regulatory Network Underlies the Progression of Heart Failure.

BACKGROUND: Progressive remodeling of cardiac gene expression underlies decline in cardiac function, eventually leading to heart failure. However, the major determinants of transcriptional network switching from normal to failed hearts remain to be determined. METHODS: In this study, we integrated human samples, genetic mouse models, and genomic approaches, including bulk RNA sequencing, single-cell RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, to identify the role of chromatin remodeling complex INO80 in heart homeostasis and dysfunction. RESULTS: The INO80 chromatin remodeling complex was abundantly expressed in mature cardiomyocytes, and its expression further increased in mouse and human heart failure. Cardiomyocyte-specific overexpression of Ino80, its core catalytic subunit, induced heart failure within 4 days. Combining RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, we revealed INO80 overexpression-dependent reshaping of the nucleosomal landscape that remodeled a core set of transcription factors, most notably the MEF2 (Myocyte Enhancer Factor 2) family, whose target genes were closely associated with cardiac function. Conditional cardiomyocyte-specific deletion of Ino80 in an established mouse model of heart failure demonstrated remarkable preservation of cardiac function. CONCLUSIONS: In summary, our findings shed light on the INO80-dependent remodeling of the chromatin landscape and transcriptional networks as a major mechanism underlying cardiac dysfunction in heart failure, and suggest INO80 as a potential preventative or interventional target.

Spatial Multiplexed Protein Profiling of Cardiac Ischemia-Reperfusion Injury.

BACKGROUND: Reperfusion therapy is critical to myocardial salvage in the event of a myocardial infarction but is complicated by ischemia-reperfusion injury (IRI). Limited understanding of the spatial organization of cardiac cells, which governs cellular interaction and function, has hindered the search for targeted interventions minimizing the deleterious effects of IRI. METHODS: We used imaging mass cytometry to characterize the spatial distribution and dynamics of cell phenotypes and communities in the mouse left ventricle following IRI. Heart sections were collected from 12 cardiac segments (basal, mid-cavity, apical, and apex of the anterior, lateral, and inferior wall) and 8 time points (before ischemia [I-0H], and postreperfusion [R-0H, R-2H, R-6H, R-12H, R-1D, R-3D, R-7D]), and stained with 29 metal-isotope-tagged antibodies. Cell community analysis was performed on reconstructed images, and the most disease-relevant cell type and target protein were selected for intervention of IRI. RESULTS: We obtained a total of 251 multiplexed images, and identified 197 063 single cells, which were grouped into 23 distinct cell communities based on the structure of cellular neighborhoods. The cellular architecture was heterogeneous throughout the ventricular wall and exhibited swift changes following IRI. Analysis of proteins with posttranslational modifications in single cells unveiled 13 posttranslational modification intensity clusters and highlighted increased H3K9me3 (tri-methylated lysine 9 of histone H3) as a key regulatory response in endothelial cells during the middle stage of IRI. Erasing H3K9 methylation, by silencing its methyltransferase Suv39h1 or overexpressing its demethylase Kdm4d in isolated endothelial cells, attenuated cardiac dysfunction and pathological remodeling following IRI. in vitro, H3K9me3 binding significantly increased at endothelial cell function-related genes upon hypoxia, suppressing tube formation, which was rescued by inhibiting H3K9me3. CONCLUSIONS: We mapped the spatiotemporal heterogeneity of cellular phenotypes in the adult heart upon IRI, and uncovered H3K9me3 in endothelial cells as a potential therapeutic target for alleviating pathological remodeling of the heart following myocardial IRI.

INO80 governs superenhancer-mediated oncogenic transcription and tumor growth in melanoma.

Superenhancers (SEs) are large genomic regions with a high density of enhancer marks. In cancer, SEs are found near oncogenes and dictate cancer gene expression. However, how oncogenic SEs are regulated remains poorly understood. Here, we show that INO80, a chromatin remodeling complex, is required for SE-mediated oncogenic transcription and tumor growth in melanoma. The expression of Ino80, the SWI/SNF ATPase, is elevated in melanoma cells and patient melanomas compared with normal melanocytes and benign nevi. Furthermore, Ino80 silencing selectively inhibits melanoma cell proliferation, anchorage-independent growth, tumorigenesis, and tumor maintenance in mouse xenografts. Mechanistically, Ino80 occupies >90% of SEs, and its occupancy is dependent on transcription factors such as MITF and Sox9. Ino80 binding reduces nucleosome occupancy and facilitates Mediator recruitment, thus promoting oncogenic transcription. Consistently, genes co-occupied by Ino80 and Med1 are selectively expressed in melanomas compared with melanocytes. Together, our results reveal an essential role of INO80-dependent chromatin remodeling in SE function and suggest a novel strategy for disrupting SEs in cancer treatment.

Single-Cell Reconstruction of Progression Trajectory Reveals Intervention Principles in Pathological Cardiac Hypertrophy.

BACKGROUND: Pressure overload-induced pathological cardiac hypertrophy is a common predecessor of heart failure, the latter of which remains a major cardiovascular disease with increasing incidence and mortality worldwide. Current therapeutics typically involve partially relieving the heart's workload after the onset of heart failure. Thus, more pathogenesis-, stage-, and cell type-specific treatment strategies require refined dissection of the entire progression at the cellular and molecular levels. METHODS: By analyzing the transcriptomes of 11,492 single cells and identifying major cell types, including both cardiomyocytes and noncardiomyocytes, on the basis of their molecular signatures, at different stages during the progression of pressure overload-induced cardiac hypertrophy in a mouse model, we characterized the spatiotemporal interplay among cell types, and tested potential pharmacological treatment strategies to retard its progression in vivo. RESULTS: We illustrated the dynamics of all major cardiac cell types, including cardiomyocytes, endothelial cells, fibroblasts, and macrophages, as well as those of their respective subtypes, during the progression of disease. Cellular crosstalk analysis revealed stagewise utilization of specific noncardiomyocytes during the deterioration of heart function. Specifically, macrophage activation and subtype switching, a key event at middle-stage of cardiac hypertrophy, was successfully targeted by Dapagliflozin, a sodium glucose cotransporter 2 inhibitor, in clinical trials for patients with heart failure, as well as TD139 and Arglabin, two anti-inflammatory agents new to cardiac diseases, to preserve cardiac function and attenuate fibrosis. Similar molecular patterns of hypertrophy were also observed in human patient samples of hypertrophic cardiomyopathy and heart failure. CONCLUSIONS: Together, our study not only illustrated dynamically changing cell type crosstalk during pathological cardiac hypertrophy but also shed light on strategies for cell type- and stage-specific intervention in cardiac diseases.

Reading the heart at single-cell resolution.

The burgeoning field of single-cell transcriptomics augments our ability to scrutinize organ systems at unprecedented resolutions. Single-cell RNA sequencing (scRNA-seq) and analytical techniques have shed light on the cellular heterogeneity, developmental trajectories, intercellular communications of the cardiac system, and thus contributed much to the understanding of cardiac development, homeostasis and disorders. Although generalized protocols are well established for scRNA-seq pipelines, customized sample preparation, quality control, and data interpretation are still needed in cardiac research. In this article, we highlight major steps that impact data quality in scRNA-seq experiments, with particular focus on sample and data processing of cardiomyocytes. We also summarize popular applications of scRNA-seq, outlining general tools, caveats and examples in cardiac research.

THO Complex-Dependent Posttranscriptional Control Contributes to Vascular Smooth Muscle Cell Fate Decision.

RATIONALE: Modulation of vascular smooth muscle cell (VSMC) phenotype plays a fundamental role in vascular development and diseases. Although extensive studies uncovered the roles of transcriptional regulation in VSMC-specific gene expression, how posttranscriptional regulation contributes to VSMC fate decisions remains to be determined. OBJECTIVE: To establish THO complex-dependent VSMC gene expression as a novel regulatory basis controlling VSMC phenotypes. METHODS AND RESULTS: Immunohistochemical staining against THOC2 and THOC5, 2 components of the THO complex, revealed a dramatic reduction in their expression in human arteries undergoing carotid endarterectomy compared with normal internal mammary arteries. Silencing of THOC2 or THOC5 led to dedifferentiation of VSMCs in vitro, characterized by decreased VSMC marker gene expression and increased migration and proliferation. Furthermore, RNA high-throughput sequencing (Seq) revealed that THOC5 silencing closely resembled the gene expression changes induced on PDGF (platelet-derived growth factor)-BB/PDGF-DD treatments in cultured VSMCs. Mechanistically, THOC2 and THOC5 physically interacted with and functionally relied on each other to bind to specific motifs on VSMC marker gene mRNAs. Interestingly, mRNAs that lost THOC2 or THOC5 binding during VSMC dedifferentiation were enriched for genes important for the differentiated VSMC phenotype. Last, THOC5 overexpression in injured rat carotid arteries significantly repressed loss of VSMC marker gene expression and neointima formation. CONCLUSIONS: Our data introduce dynamic binding of THO to VSMC marker gene mRNAs as a novel mechanism contributing to VSMC phenotypic switching and imply THOC5 as a potential intervention node for vascular diseases.

Transcriptional Profiling of Single Cardiomyocytes in Health and Disease.

PURPOSE OF REVIEW: Cardiomyocytes are the chief cell type in the heart, and are central to the pathogenesis of many cardiac diseases. Increasing recognition of its cellular, molecular, and functional heterogeneity prompted us to review the latest advancements in cardiac health and disease at single-cell resolution. RECENT FINDINGS: Single-cell RNA profiling of cardiac lineage commitment events uncovered immense heterogeneity amongst ostensibly homogeneous cell populations. Classic cardiac transcription factors and new regulatory genes exhibit cell subtype-specific and temporally controlled expression patterns that serve the phenotypic changes in development, disease progression, and regeneration. Dissection of dynamically changing cell-cell communications and cardiac cell plasticity offers new opportunities in disease intervention and cardiac repair. Finally, updates in research models, platforms, and pipelines are continuously increasing the efficiency and reliability in data generation and interpretation. Transcriptional profiling of cardiac lineage cells, especially cardiomyocytes, has tremendously enriched our knowledge of the cellular milieu and the transcriptional network in the heart. Implementing technical standards and interrogating underexplored research areas will further our understanding of this organ and increase the likelihood of finding tractable therapeutic targets.

Single-cell reconstruction of differentiation trajectory reveals a critical role of ETS1 in human cardiac lineage commitment.

BACKGROUND: Cardiac differentiation from human pluripotent stem cells provides a unique opportunity to study human heart development in vitro and offers a potential cell source for cardiac regeneration. Compared to the large body of studies investigating cardiac maturation and cardiomyocyte subtype-specific induction, molecular events underlying cardiac lineage commitment from pluripotent stem cells at early stage remain poorly characterized. RESULTS: In order to uncover key molecular events and regulators controlling cardiac lineage commitment from a pluripotent state during differentiation, we performed single-cell RNA-Seq sequencing and obtained high-quality data for 6879 cells collected from 6 stages during cardiac differentiation from human embryonic stem cells and identified multiple cell subpopulations with distinct molecular features. Through constructing developmental trajectory of cardiac differentiation and putative ligand-receptor interactions, we revealed crosstalk between cardiac progenitor cells and endoderm cells, which could potentially provide a cellular microenvironment supporting cardiac lineage commitment at day 5. In addition, computational analyses of single-cell RNA-Seq data unveiled ETS1 (ETS Proto-Oncogene 1) activation as an important downstream event induced by crosstalk between cardiac progenitor cells and endoderm cells. Consistent with the findings from single-cell analysis, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) against ETS1 revealed genomic occupancy of ETS1 at cardiac structural genes at day 9 and day 14, whereas ETS1 depletion dramatically compromised cardiac differentiation. CONCLUSION: Together, our study not only characterized the molecular features of different cell types and identified ETS1 as a crucial factor induced by cell-cell crosstalk contributing to cardiac lineage commitment from a pluripotent state, but may also have important implications for understanding human heart development at early embryonic stage, as well as directed manipulation of cardiac differentiation in regenerative medicine.

Histone Variant H2A.Z Is Required for the Maintenance of Smooth Muscle Cell Identity as Revealed by Single-Cell Transcriptomics.

BACKGROUND: Histone variants endow chromatin with specific structures, and play essential roles in development and diseases. However, little is known about their roles in controlling cell identity in vascular diseases. METHODS: Given the cell heterogeneity in atherosclerotic lesions, we applied single-cell RNA-Sequencing to analyze diseased human arteries, and identified histone variant H2A.Z as a key histone signature to maintain vascular smooth muscle cell (VSMC) identity. RESULTS: We show that H2A.Z occupies genomic regions near VSMC marker genes, and its occupancy is decreased in VSMCs undergoing dedifferentiation. Mechanistically, H2A.Z occupancy preferentially promotes nucleosome turnover, and facilitates the recruitment of SMAD3 and MED1, thereby activating VSMC marker gene expression. In addition, H2A.Z expression is dramatically reduced at both mRNA and protein levels in diseased human vascular tissues compared to those in normal arteries. Notably, in vivo overexpression of H2A.Z rescues injury-induced loss of VSMC identity and neointima formation. CONCLUSIONS: Together, our data introduce dynamic occupancy of a histone variant as a novel regulatory basis contributing to cell fate decisions, and imply H2A.Z as a potential intervention node for vascular diseases.

The nuclear localization signal is required for the function of squamosa promoter binding protein-like gene 9 to promote vegetative phase change in Arabidopsis.

KEY MESSAGE: A mutation in the nuclear localization signal of squamosa promoter binding like-protein 9 (SPL9) delays vegetative phase change by disrupting its nuclear localization. The juvenile-to-adult phase transition is a critical developmental process in plant development, and it is regulated by a decrease in miR156/157 and a corresponding increase in their targets, squamosa promoter binding protein-like (SPL) genes. SPL proteins contain a conserved SBP domain with putative nuclear localization signals (NLSs) at their C-terminals. Some SPLs promote vegetative phase change by promoting miR172 expression, but the function of nuclear localization signals in those SPLs remains unknown. Here, we identified a loss-of-function mutant, which we named del6, with delayed vegetative phase change phenotypes in a forward genetic screen. Map-based cloning, the whole genome resequencing, and allelic complementation test demonstrate that a G-to-A substitution in the SPL9 gene is responsible for the delayed vegetative phase change phenotypes. In del6, the mutation causes a substitution of the glutamine (Gln) for the conserved basic amino acid arginine (Arg) in the NLS of the SBP domain, and disrupts the normal nuclear localization and function of SPL9. Therefore, our work demonstrates that the NLSs in the SBP domain of SPL9 are indispensable for its nuclear localization and normal function in Arabidopsis.

Age-dependent heteroblastic development of leaf hairs in Arabidopsis.

Higher plants progress through a juvenile and an adult phase of development before they enter the reproductive phase. The transition from the juvenile to the adult phase is referred to as vegetative phase change, and this is signified by the production of trichomes on the abaxial side of leaf blades in Arabidopsis; however, the molecular mechanism underlying this process is poorly understood. We identified a dominant mutation (gl1-D) in a forward genetic screen that accelerates abaxial trichome production during shoot development. This phenotype is the result of a G-to-A substitution in the 3' noncoding region in the GLABRA1(GL1) gene. We show that TOE1, an AP2-like transcription factor that acts downstream of the miR156-SPL pathway, represses GL1 expression by directly binding to this site, and that gl1-D prevents TOE1 binding. Our work reveals a molecular link between vegetative phase change and abaxial trichome production in Arabidopsis, and answers a long-standing question of how the vegetative phase change pathway regulates vegetative traits.

The role of wild type RAS isoforms in cancer.

Mutationally activated RAS proteins are critical oncogenic drivers in nearly 30% of all human cancers. As with mutant RAS, the role of wild type RAS proteins in oncogenesis, tumour maintenance and metastasis is context-dependent. Complexity is introduced by the existence of multiple RAS genes (HRAS, KRAS, NRAS) and protein "isoforms" (KRAS4A, KRAS4B), by the ever more complicated network of RAS signaling, and by the increasing identification of numerous genetic aberrations in cancers that do and do not harbour mutant RAS. Numerous mouse model carcinogenesis studies and examination of patient tumours reveal that, in RAS-mutant cancers, wild type RAS proteins are likely to serve as tumour suppressors when the mutant RAS is of the same isoform. This evidence is particularly robust in KRAS mutant cancers, which often display suppression or loss of wild type KRAS, but is not as strong for NRAS. In contrast, although not yet fully elucidated, the preponderance of evidence indicates that wild type RAS proteins play a tumour promoting role when the mutant RAS is of a different isoform. In non-RAS mutant cancers, wild type RAS is recognized as a mediator of oncogenic signaling due to chronic activation of upstream receptor tyrosine kinases that feed through RAS. Additionally, in the absence of mutant RAS, activation of wild type RAS may drive cancer upon the loss of negative RAS regulators such as NF1 GAP or SPRY proteins. Here we explore the current state of knowledge with respect to the roles of wild type RAS proteins in human cancers.

Protein Kinase CK2α Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2α Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma.

The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies.

Regulation of Vegetative Phase Change by SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA.

Plants progress from a juvenile vegetative phase of development to an adult vegetative phase of development before they enter the reproductive phase. miR156 has been shown to be the master regulator of the juvenile-to-adult transition in plants. However, the mechanism of how miR156 is transcriptionally regulated still remains elusive. In a forward genetic screen, we identified that a mutation in the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) exhibited an accelerated vegetative phase change phenotype by reducing the expression of miR156, which in turn caused a corresponding increase in the levels of SQUAMOSA PROMOTER BINDING PROTEIN LIKE genes. BRM regulates miR156 expression by directly binding to the MIR156A promoter. Mutations in BRM not only increased occupancy of the -2 and +1 nucleosomes proximal to the transcription start site at the MIR156A locus but also the levels of trimethylated histone H3 at Lys 27. The precocious phenotype of brm mutant was partially suppressed by a second mutation in SWINGER (SWN), but not by a mutation in CURLEY LEAF, both of which are key components of the Polycomb Group Repressive Complex 2 in plants. Our results indicate that BRM and SWN act antagonistically at the nucleosome level to fine-tune the temporal expression of miR156 to regulate vegetative phase change in Arabidopsis.

Modulation of miR156 to identify traits associated with vegetative phase change in tobacco (Nicotiana tabacum).

After germination, plants progress through juvenile and adult phases of vegetative development before entering the reproductive phase. The character and timing of these phases vary significantly between different plant species, which makes it difficult to know whether temporal variations in various vegetative traits represent the same, or different, developmental processes. miR156 has been shown to be the master regulator of vegetative development in plants. Overexpression of miR156 prolongs the juvenile phase of development, whereas knocking-down the level of miR156 promotes the adult phase of development. Therefore, artificial modulation of miR156 expression is expected to cause corresponding changes in vegetative-specific traits in different plant species, particularly in those showing no substantial difference in morphology during vegetative development. To identify specific traits associated with the juvenile-to-adult transition in tobacco, we examined the phenotype of transgenic tobacco plants with elevated or reduced levels of miR156. We found that leaf shape, the density of abaxial trichomes, the number of leaf veins, the number of stomata, the size and density of epidermal cells, patterns of epidermal cell staining, the content of chlorophyll and the rate of photosynthesis, are all affected by miR156. These newly identified miR156-regulated traits therefore can be used to distinguish between juvenile and adult phases of development in tobacco, and provide a starting point for future studies of vegetative phase change in the family Solanaceae.

Methylcellulose improves dissociation quality of adult human primary cardiomyocytes.

Obtaining high-quality adult human primary cardiomyocytes (hPCM) have been technically challenging due to isolation-induced biochemical and mechanical stress. Building upon a previous tissue slicing-assisted digestion method, we introduced polymers into the digestion solution to reduce mechanical damage to cells. We found that low-viscosity methylcellulose (MC) significantly improved hPCM viability and yield. Mechanistically, it protected cells from membrane damage, which led to decreased apoptosis and mitochondrial reactive oxygen species production. MC also improved the electrophysiological properties of hPCMs by maintaining the density of sodium channels. The effects on cell viability and cell yield effects were not recapitulated by MC of larger viscosities, other cellulose derivatives, nor shear protectants polyethylene glycol and polyvinyl alcohol. Finally, MC also enhanced the isolation efficiency and the culture quality of hPCMs from diseased ventricular myocardium, expanding its potential applications. Our findings showed that the isolation quality of hPCMs can be further improved through the addition of a polymer, rendering hPCMs a more reliable cellular model for cardiac research.

Modeling drug-induced mitochondrial toxicity with human primary cardiomyocytes.

Mitochondrial toxicity induced by therapeutic drugs is a major contributor for cardiotoxicity, posing a serious threat to pharmaceutical industries and patients' lives. However, mitochondrial toxicity testing is not incorporated into routine cardiac safety screening procedures. To accurately model native human cardiomyocytes, we comprehensively evaluated mitochondrial responses of adult human primary cardiomyocytes (hPCMs) to a nucleoside analog, remdesivir (RDV). Comparison of their response to human pluripotent stem cell-derived cardiomyocytes revealed that the latter utilized a mitophagy-based mitochondrial recovery response that was absent in hPCMs. Accordingly, action potential duration was elongated in hPCMs, reflecting clinical incidences of RDV-induced QT prolongation. In a screen for mitochondrial protectants, we identified mitochondrial ROS as a primary mediator of RDV-induced cardiotoxicity. Our study demonstrates the utility of hPCMs in the detection of clinically relevant cardiac toxicities, and offers a framework for hPCM-based high-throughput screening of cardioprotective agents.

Mitochondrial Dysfunction in Cardiac Diseases and Therapeutic Strategies.

Mitochondria are the main site of intracellular synthesis of ATP, which provides energy for various physiological activities of the cell. Cardiomyocytes have a high density of mitochondria and mitochondrial damage is present in a variety of cardiovascular diseases. In this paper, we describe mitochondrial damage in mitochondrial cardiomyopathy, congenital heart disease, coronary heart disease, myocardial ischemia-reperfusion injury, heart failure, and drug-induced cardiotoxicity, in the context of the key roles of mitochondria in cardiac development and homeostasis. Finally, we discuss the main current therapeutic strategies aimed at alleviating mitochondrial impairment-related cardiac dysfunction, including pharmacological strategies, gene therapy, mitochondrial replacement therapy, and mitochondrial transplantation. It is hoped that this will provide new ideas for the treatment of cardiovascular diseases.

Isolation of porcine adult cardiomyocytes: Comparison between Langendorff perfusion and tissue slicing-assisted enzyme digestion.

Tissue slicing-assisted digestion (TSAD) of adult cardiomyocytes has shown significant improvements over conventional chunk methods. However, it remains unclear how this method compares to Langendorff perfusion, the current standard of adult cardiomyocyte isolation. Using adult Bama minipigs, we performed cardiomyocyte isolation via these two distinct methods, and compared the resulting cellular quality, including viability, cellular structure, gene expression, and electrophysiological properties, of cardiomyocytes from 3 distinct anatomical regions, namely the left ventricle, right ventricle, and left atrial appendage. Our results revealed largely indistinguishable cell quality in all of the measured parameters. These findings suggest that that TSAD can be reliably used to isolate adult mammalian cardiomyocytes as a reliable alternative to perfusion in cardiomyocyte isolation from larger mammals, particularly when Langendorff perfusion is not feasible.

Coordinated regulation of vegetative phase change by brassinosteroids and the age pathway in Arabidopsis.

Vegetative phase change in plants is regulated by a gradual decline in the level of miR156 and a corresponding increase in the expression of its targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. Gibberellin (GA), jasmonic acid (JA), and cytokinin (CK) regulate vegetative phase change by affecting genes in the miR156-SPL pathway. However, whether other phytohormones play a role in vegetative phase change remains unknown. Here, we show that a loss-of-function mutation in the brassinosteroid (BR) biosynthetic gene, DWARF5 (DWF5), delays vegetative phase change, and the defective phenotype is primarily attributable to reduced levels of SPL9 and miR172, and a corresponding increase in TARGET OF EAT1 (TOE1). We further show that GLYCOGEN SYNTHASE KINASE3 (GSK3)-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2) directly interacts with and phosphorylates SPL9 and TOE1 to cause subsequent proteolytic degradation. Therefore, BRs function to stabilize SPL9 and TOE1 simultaneously to regulate vegetative phase change in plants.