ABSTRACT
BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is an emerging major unmet need and one of the most significant clinic challenges in cardiology. The pathogenesis of HFpEF is associated with multiple risk factors. Hypertension and metabolic disorders associated with obesity are the 2 most prominent comorbidities observed in patients with HFpEF. Although hypertension-induced mechanical overload has long been recognized as a potent contributor to heart failure with reduced ejection fraction, the synergistic interaction between mechanical overload and metabolic disorders in the pathogenesis of HFpEF remains poorly characterized. METHOD: We investigated the functional outcome and the underlying mechanisms from concurrent mechanic and metabolic stresses in the heart by applying transverse aortic constriction in lean C57Bl/6J or obese/diabetic B6.Cg-Lepob/J (ob/ob) mice, followed by single-nuclei RNA-seq and targeted manipulation of a top-ranked signaling pathway differentially affected in the 2 experimental cohorts. RESULTS: In contrast to the post-transverse aortic constriction C57Bl/6J lean mice, which developed pathological features of heart failure with reduced ejection fraction over time, the post-transverse aortic constriction ob/ob mice showed no significant changes in ejection fraction but developed characteristic pathological features of HFpEF, including diastolic dysfunction, worsened cardiac hypertrophy, and pathological remodeling, along with further deterioration of exercise intolerance. Single-nuclei RNA-seq analysis revealed significant transcriptome reprogramming in the cardiomyocytes stressed by both pressure overload and obesity/diabetes, markedly distinct from the cardiomyocytes singularly stressed by pressure overload or obesity/diabetes. Furthermore, glucagon signaling was identified as the top-ranked signaling pathway affected in the cardiomyocytes associated with HFpEF. Treatment with a glucagon receptor antagonist significantly ameliorated the progression of HFpEF-related pathological features in 2 independent preclinical models. Importantly, cardiomyocyte-specific genetic deletion of the glucagon receptor also significantly improved cardiac function in response to pressure overload and metabolic stress. CONCLUSIONS: These findings identify glucagon receptor signaling in cardiomyocytes as a critical determinant of HFpEF progression and provide proof-of-concept support for glucagon receptor antagonism as a potential therapy for the disease.
Subject(s)
Heart Failure , Mice, Inbred C57BL , Stroke Volume , Animals , Heart Failure/physiopathology , Heart Failure/metabolism , Heart Failure/drug therapy , Heart Failure/etiology , Stroke Volume/drug effects , Mice , Male , Receptors, Glucagon/antagonists & inhibitors , Receptors, Glucagon/metabolism , Receptors, Glucagon/genetics , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Mice, Obese , Ventricular Function, Left/drug effects , Obesity/metabolism , Obesity/physiopathology , Obesity/complications , Disease Models, Animal , Signal TransductionABSTRACT
Exercise promotes pulsatile shear stress in the arterial circulation and ameliorates cardiometabolic diseases. However, exercise-mediated metabolic transducers for vascular protection remain under-investigated. Untargeted metabolomic analysis demonstrated that wild-type mice undergoing voluntary wheel running exercise expressed increased endothelial stearoyl-CoA desaturase 1 (SCD1) that catalyzes anti-inflammatory lipid metabolites, namely, oleic (OA) and palmitoleic acids (PA), to mitigate NF-κB-mediated inflammatory responses. In silico analysis revealed that exercise augmented time-averaged wall shear stress but mitigated flow recirculation and oscillatory shear index in the lesser curvature of the mouse aortic arch. Following exercise, endothelial Scd1-deleted mice (Ldlr-/- Scd1EC-/-) on high-fat diet developed persistent VCAM1-positive endothelium in the lesser curvature and the descending aorta, whereas SCD1 overexpression via adenovirus transfection mitigated endoplasmic reticulum stress and inflammatory biomarkers. Single-cell transcriptomics of the aorta identified Scd1-positive and Vcam1-negative endothelial subclusters interacting with other candidate genes. Thus, exercise mitigates flow recirculation and activates endothelial SCD1 to catalyze OA and PA for vascular endothelial protection.
Subject(s)
Aorta , Motor Activity , Animals , Mice , Aorta/metabolism , Diet, High-Fat , Endothelium, Vascular/metabolism , Stearoyl-CoA Desaturase/genetics , Stearoyl-CoA Desaturase/metabolismSubject(s)
Myocytes, Cardiac , RNA Splicing , Humans , RNA Splicing Factors/genetics , Alternative SplicingABSTRACT
Exercise modulates vascular plasticity in multiple organ systems; however, the metabolomic transducers underlying exercise and vascular protection in the disturbed flow-prone vasculature remain under-investigated. We simulated exercise-augmented pulsatile shear stress (PSS) to mitigate flow recirculation in the lesser curvature of the aortic arch. When human aortic endothelial cells (HAECs) were subjected to PSS ( τ ave = 50 dyne·cm -2 , ∂τ/∂t = 71 dyne·cm -2 ·s -1 , 1 Hz), untargeted metabolomic analysis revealed that Stearoyl-CoA Desaturase (SCD1) in the endoplasmic reticulum (ER) catalyzed the fatty acid metabolite, oleic acid (OA), to mitigate inflammatory mediators. Following 24 hours of exercise, wild-type C57BL/6J mice developed elevated SCD1-catalyzed lipid metabolites in the plasma, including OA and palmitoleic acid (PA). Exercise over a 2-week period increased endothelial SCD1 in the ER. Exercise further modulated the time-averaged wall shear stress (TAWSS or τ ave) and oscillatory shear index (OSI ave ), upregulated Scd1 and attenuated VCAM1 expression in the disturbed flow-prone aortic arch in Ldlr -/- mice on high-fat diet but not in Ldlr -/- Scd1 EC-/- mice. Scd1 overexpression via recombinant adenovirus also mitigated ER stress. Single cell transcriptomic analysis of the mouse aorta revealed interconnection of Scd1 with mechanosensitive genes, namely Irs2 , Acox1 and Adipor2 that modulate lipid metabolism pathways. Taken together, exercise modulates PSS ( τ ave and OSI ave ) to activate SCD1 as a metabolomic transducer to ameliorate inflammation in the disturbed flow-prone vasculature.
ABSTRACT
Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.
Subject(s)
Myocardium/metabolism , Myocytes, Cardiac/metabolism , Phosphoric Diester Hydrolases/metabolism , Pyrimidines/biosynthesis , Pyrophosphatases/metabolism , Regeneration , Signal Transduction , Adenosine Monophosphate/genetics , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Animals , Heart Injuries/genetics , Heart Injuries/metabolism , Mice , Phosphoric Diester Hydrolases/genetics , Pyrophosphatases/geneticsABSTRACT
Calcium carbonate is used as bone-filling material due to its good biocompatibility, bioactivity, and bioabsorbability, but the prevalence of infectious complications associated with calcium carbonate has created a persisting challenge in the treatment of bone defect. Therefore, this greatly necessitate the need to endow calcium carbonate with antibacterial properties. In this study, calcium carbonate powders loaded with silver nanoparticles (Ag-CaCO3) were prepared in attempt to serve as a novel antibacterial inorganic filler material. This objective was achieved using ultrasonic spray-pyrolysis (USSP) route to produce Ag-CaCO3 with 1, 5 and 10 mol% silver. The size of silver nanoparticles on CaCO3 microspheres could be regulated by adjusting silver concentration to facilitate effective release of Ag+ ions. This was demonstrated in Ag-CaCO3 (1), where the lowest silver content at 1 mol% achieved the highest Ag+ ions release over 28 days. This in turn gave rise to effective antibacterial efficiency against Staphylococcus aureus and Escherichia coli. Furthermore, CaCO3 (1) could also support osteoblast-like cells (MG-63) at a cell viability of 80%. Overall, this work extends the capabilities in employing USSP to produce inorganic filler materials with sustained antibacterial properties, bringing one step closer to the development of antibacterial products.
Subject(s)
Metal Nanoparticles , Silver , Anti-Bacterial Agents/pharmacology , Calcium Carbonate/pharmacology , Delayed-Action Preparations , Microbial Sensitivity Tests , Silver/pharmacology , UltrasonicsABSTRACT
Extrapulmonary manifestations of COVID-19 are associated with a much higher mortality rate than pulmonary manifestations. However, little is known about the pathogenesis of systemic complications of COVID-19. Here, we create a murine model of SARS-CoV-2-induced severe systemic toxicity and multiorgan involvement by expressing the human ACE2 transgene in multiple tissues via viral delivery, followed by systemic administration of SARS-CoV-2. The animals develop a profound phenotype within 7 days with severe weight loss, morbidity, and failure to thrive. We demonstrate that there is metabolic suppression of oxidative phosphorylation and the tricarboxylic acid (TCA) cycle in multiple organs with neutrophilia, lymphopenia, and splenic atrophy, mirroring human COVID-19 phenotypes. Animals had a significantly lower heart rate, and electron microscopy demonstrated myofibrillar disarray and myocardial edema, a common pathogenic cardiac phenotype in human COVID-19. We performed metabolomic profiling of peripheral blood and identified a panel of TCA cycle metabolites that served as biomarkers of depressed oxidative phosphorylation. Finally, we observed that SARS-CoV-2 induces epigenetic changes of DNA methylation, which affects expression of immune response genes and could, in part, contribute to COVID-19 pathogenesis. Our model suggests that SARS-CoV-2-induced metabolic reprogramming and epigenetic changes in internal organs could contribute to systemic toxicity and lethality in COVID-19.
Subject(s)
COVID-19/complications , Epigenesis, Genetic/immunology , Failure to Thrive/etiology , SARS-CoV-2/pathogenicity , Wasting Syndrome/etiology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Animals, Genetically Modified , COVID-19/metabolism , COVID-19/physiopathology , COVID-19/virology , Citric Acid Cycle/physiology , DNA Methylation/physiology , Disease Models, Animal , Failure to Thrive/physiopathology , Humans , Immunity/genetics , Male , Mice , Oxidative Phosphorylation , Renin-Angiotensin System/physiology , SARS-CoV-2/metabolism , Wasting Syndrome/physiopathologyABSTRACT
Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.
Subject(s)
Cicatrix/metabolism , Collagen Type V/deficiency , Collagen Type V/metabolism , Heart Injuries/metabolism , Myocardial Contraction/genetics , Myofibroblasts/metabolism , Animals , Cicatrix/genetics , Cicatrix/physiopathology , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Collagen Type III/genetics , Collagen Type III/metabolism , Collagen Type V/genetics , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Female , Fibrosis/genetics , Fibrosis/metabolism , Gene Expression Regulation/genetics , Integrins/antagonists & inhibitors , Integrins/genetics , Integrins/metabolism , Isoproterenol/pharmacology , Male , Mechanotransduction, Cellular/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Atomic Force/instrumentation , Microscopy, Electron, Transmission , Myocardial Contraction/drug effects , Myofibroblasts/cytology , Myofibroblasts/pathology , Myofibroblasts/ultrastructure , Principal Component Analysis , Proteomics , RNA-Seq , Single-Cell AnalysisABSTRACT
In the mammalian heart, the left ventricle (LV) rapidly becomes more dominant in size and function over the right ventricle (RV) after birth. The molecular regulators responsible for this chamber-specific differential growth are largely unknown. We found that cardiomyocytes in the neonatal mouse RV had lower proliferation, more apoptosis, and a smaller average size compared with the LV. This chamber-specific growth pattern was associated with a selective activation of p38 mitogen-activated protein kinase (MAPK) activity in the RV and simultaneous inactivation in the LV. Cardiomyocyte-specific deletion of both the Mapk14 and Mapk11 genes in mice resulted in loss of p38 MAPK expression and activity in the neonatal heart. Inactivation of p38 activity led to a marked increase in cardiomyocyte proliferation and hypertrophy but diminished cardiomyocyte apoptosis, specifically in the RV. Consequently, the p38-inactivated hearts showed RV-specific enlargement postnatally, progressing to pulmonary hypertension and right heart failure at the adult stage. Chamber-specific p38 activity was associated with differential expression of dual-specific phosphatases (DUSPs) in neonatal hearts, including DUSP26. Unbiased transcriptome analysis revealed that IRE1α/XBP1-mediated gene regulation contributed to p38 MAPK-dependent regulation of neonatal cardiomyocyte proliferation and binucleation. These findings establish an obligatory role of DUSP/p38/IRE1α signaling in cardiomyocytes for chamber-specific growth in the postnatal heart.
Subject(s)
Heart/growth & development , Mitogen-Activated Protein Kinase 14/metabolism , Mitogen-Activated Protein Kinases/metabolism , Myocardium/enzymology , Animals , Animals, Newborn , Apoptosis , Cell Proliferation , Cell Size , Enzyme Activation , Female , Gene Expression Profiling , Heart Ventricles/cytology , Heart Ventricles/enzymology , Heart Ventricles/growth & development , Male , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 14/deficiency , Mitogen-Activated Protein Kinase 14/genetics , Mitogen-Activated Protein Kinases/deficiency , Mitogen-Activated Protein Kinases/genetics , Myocardium/cytology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/enzymology , Organ Specificity , Vascular Remodeling/genetics , Vascular Remodeling/physiologyABSTRACT
INTRODUCTION: Secondary bladder, colon, and rectal cancers are relatively common after prostate radiotherapy. However, secondary squamous cell carcinoma of the prostate is rare. CASE PRESENTATION: An 85-year-old man presented with dysuria and low-serum prostate-specific antigen levels. His medical history included localized prostate adenocarcinoma (Gleason score of 4 + 5) treated with combined three-dimensional conformal radiotherapy and androgen deprivation therapy, 11 years ago. Urethroscopy and magnetic resonance imaging showed a bulging mass around the prostatic urethra. Transurethral resection of the prostate performed for histopathological diagnosis revealed squamous cell carcinoma. CONCLUSION: Hereby, a rare case of secondary squamous cell carcinoma of the prostate after radiotherapy for adenocarcinoma was reported, which was found after 11 years of radiotherapy with symptom of dysuria including urinary hesitancy, difficulty, pain during urination, and low-serum prostate-specific antigen levels.
ABSTRACT
Heart failure is associated with induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The serine/threonine protein kinase/endoribonuclease IRE1α is a key protein in ER stress signal transduction. IRE1α activity can induce both protective UPR and apoptotic downstream signaling events, but the specific role for IRE1α activity in the heart is unknown. A major aim of this study was to characterize the specific contribution of IRE1α in cardiac physiology and pathogenesis. We used both cultured myocytes and a transgenic mouse line with inducible and cardiomyocyte-specific IRE1α overexpression as experimental models to achieve targeted IRE1α activation. IRE1α expression induced a potent but transient ER stress response in cardiomyocytes and did not cause significant effects in the intact heart under normal physiological conditions. Furthermore, the IRE1α-activated transgenic heart responding to pressure overload exhibited preserved function and reduced fibrotic area, associated with increased adaptive UPR signaling and with blunted inflammatory and pathological gene expression. Therefore, we conclude that IRE1α induces transient ER stress signaling and confers a protective effect against pressure overload-induced pathological remodeling in the heart. To our knowledge, this report provides first direct evidence of a specific and protective role for IRE1α in the heart and reveals an interaction between ER stress signaling and inflammatory regulation in the pathologically stressed heart.
Subject(s)
Endoplasmic Reticulum Stress , Endoribonucleases/physiology , Heart Failure/prevention & control , Insulinoma/prevention & control , Pressure/adverse effects , Protective Agents/pharmacology , Protein Serine-Threonine Kinases/physiology , Animals , Apoptosis , Cells, Cultured , Female , Heart Failure/etiology , Heart Failure/pathology , Insulinoma/metabolism , Insulinoma/pathology , Male , Mice, Inbred C57BL , Mice, Transgenic , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Signal Transduction , Unfolded Protein ResponseABSTRACT
Stress-induced p38 mitogen-activated protein kinase (MAPK) activity is implicated in pathological remodeling in the heart. For example, constitutive p38 MAPK activation in cardiomyocytes induces pathological features, including myocyte hypertrophy, apoptosis, contractile dysfunction, and fetal gene expression. However, the physiological function of cardiomyocyte p38 MAPK activity in beneficial compensatory vascular remodeling is unclear. This report investigated the functional role and the underlying mechanisms of cardiomyocyte p38 MAPK activity in cardiac remodeling induced by chronic stress. Using both in vitro and in vivo model systems, we found that p38 MAPK activity is required for hypoxia-induced pro-angiogenic activity from cardiomyocytes and that p38 MAPK activation in cardiomyocyte is sufficient to promote paracrine signaling-mediated, pro-angiogenic activity. We further demonstrate that VEGF is a paracrine factor responsible for the p38 MAPK-mediated pro-angiogenic activity from cardiomyocytes and that p38 MAPK pathway activation is sufficient for inducing VEGF secretion from cardiomyocytes in an Sp1-dependent manner. More significantly, cardiomyocyte-specific inactivation of p38α in mouse heart impaired compensatory angiogenesis after pressure overload and promoted early onset of heart failure. In summary, p38αMAPK has a critical role in the cross-talk between cardiomyocytes and vasculature by regulating stress-induced VEGF expression and secretion in cardiomyocytes. We conclude that as part of a stress-induced signaling pathway, p38 MAPK activity significantly contributes to both pathological and compensatory remodeling in the heart.
Subject(s)
Endothelium, Vascular/metabolism , Mitogen-Activated Protein Kinase 14/metabolism , Myocardial Ischemia/metabolism , Myocardial Revascularization , Myocytes, Cardiac/metabolism , Animals , Animals, Newborn , Cell Hypoxia , Cells, Cultured , Crosses, Genetic , Endothelium, Vascular/cytology , Endothelium, Vascular/pathology , Enzyme Activation , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Male , Mice, Knockout , Mice, Transgenic , Mitogen-Activated Protein Kinase 14/chemistry , Mitogen-Activated Protein Kinase 14/genetics , Myocardial Ischemia/pathology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/pathology , RNA Interference , Rats, Sprague-Dawley , Recombinant Proteins/metabolism , Sp1 Transcription Factor/antagonists & inhibitors , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Sus scrofa , Vascular Endothelial Growth Factor A/agonists , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/agonists , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Cardiovascular dysautonomia as well as the deterioration of circadian rhythms are among the earliest detectable pathophysiological changes in individuals with Huntington's disease (HD). Preclinical research requires mouse models that recapitulate disease symptoms and the Q175 knock-in model offers a number of advantages but potential autonomic dysfunction has not been explored. In this study, we sought to test the dual hypotheses that cardiovascular dysautonomia can be detected early in disease progression in the Q175 model and that this dysfunction varies with the daily cycle. Using radiotelemetry implants, we observed a significant reduction in the diurnal and circadian activity rhythms in the Q175 mutants at the youngest ages. By middle age, the autonomically driven rhythms in core body temperature were highly compromised, and the Q175 mutants exhibited striking episodes of hypothermia that increased in frequency with mutant huntingtin gene dosage. In addition, Q175 mutants showed higher resting heart rate (HR) during sleep and greatly reduced correlation between activity and HR HR variability was reduced in the mutants in both time and frequency domains, providing more evidence of autonomic dysfunction. Measurement of the baroreceptor reflex revealed that the Q175 mutant could not appropriately increase HR in response to a pharmacologically induced decrease in blood pressure. Echocardiograms showed reduced ventricular mass and ejection fraction in mutant hearts. Finally, cardiac histopathology revealed localized points of fibrosis resembling those caused by myocardial infarction. Thus, the Q175 mouse model of HD exhibits cardiovascular dysautonomia similar to that seen in HD patients with prominent sympathetic dysfunction during the resting phase of the activity rhythm.
Subject(s)
Autonomic Nervous System/physiopathology , Heart/physiopathology , Huntingtin Protein/genetics , Huntington Disease/physiopathology , Animals , Baroreflex , Blood Pressure , Body Temperature , Circadian Rhythm , Heart/innervation , Heart Rate , Huntington Disease/genetics , Male , Mice , Mice, Inbred C57BL , Mutation , Stroke VolumeABSTRACT
BACKGROUND: There is a strong impetus for the development of alternative treatments for bone disease that avoid the complications associated with autografts and allografts. To address this, we previously developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional interconnected pores, and constructed tissue-engineered bone by culturing rat bone marrow cells (RBMCs) using AFSs in a radial-flow bioreactor (RFB). OBJECTIVE: To generate additional baseline data for the development of tissue-engineered bone constructed for clinical application using a RFB, we cultured RBMCs using AFSs under static conditions (hereafter, RBMC AFS culture), and monitored RBMC growth and differentiation characteristics in vitro, and two weeks after subcutaneous inoculation into recipient rats. METHODS: RBMCs were seeded to AFSs and growth, differentiation and calcification were monitored in vitro and in vivo by histological evaluation using hematoxylin eosin, alkaline phosphatase and alizarin red S stains. RESULTS: RBMCs in/on AFSs proliferated and differentiated normally in vitro and in vivo, and calcification was evident two weeks after subcutaneous AFS culture implantation. Histological assays revealed that AFSs and RBMC AFS cultures were biocompatible, and did not induce inflammation or immunological rejection in vivo. CONCLUSIONS: These findings suggest that AFSs are a conducive microenvironment for bone regeneration and are well tolerated in vivo. The results provide valuable baseline data for the design of implant studies using tissue-engineered bone constructed by RFB.
Subject(s)
Apatites/chemistry , Bone and Bones/cytology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Subcutaneous Tissue/ultrastructure , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Biocompatible Materials/chemistry , Cells, Cultured , Male , Mesenchymal Stem Cell Transplantation/methods , Osteogenesis , Rats , Rats, Wistar , Subcutaneous Tissue/surgeryABSTRACT
Light-sheet fluorescence microscopy (LSFM) serves to advance developmental research and regenerative medicine. Coupled with the paralleled advances in fluorescence-friendly tissue clearing technique, our cardiac LSFM enables dual-sided illumination to rapidly uncover the architecture of murine hearts over 10 by 10 by 10 mm3 in volume; thereby allowing for localizing progenitor differentiation to the cardiomyocyte lineage and AAV9-mediated expression of exogenous transmembrane potassium channels with high contrast and resolution. Without the steps of stitching image columns, pivoting the light-sheet and sectioning the heart mechanically, we establish a holistic strategy for 3-dimentional reconstruction of the "digital murine heart" to assess aberrant cardiac structures as well as the spatial distribution of the cardiac lineages in neonates and ion-channels in adults.
Subject(s)
Imaging, Three-Dimensional , Myocardium/cytology , Proteins/metabolism , Animals , Animals, Newborn , Calibration , Cell Lineage , Fluorescence , Green Fluorescent Proteins/metabolism , Heart Ventricles/cytology , Mice , Microscopy, Fluorescence , Potassium Channels/metabolismABSTRACT
Epigenetic reprogramming is a critical process of pathological gene induction during cardiac hypertrophy and remodeling, but the underlying regulatory mechanisms remain to be elucidated. Here we identified a heart-enriched long noncoding (lnc)RNA, named cardiac-hypertrophy-associated epigenetic regulator (Chaer), which is necessary for the development of cardiac hypertrophy. Mechanistically, Chaer directly interacts with the catalytic subunit of polycomb repressor complex 2 (PRC2). This interaction, which is mediated by a 66-mer motif in Chaer, interferes with PRC2 targeting to genomic loci, thereby inhibiting histone H3 lysine 27 methylation at the promoter regions of genes involved in cardiac hypertrophy. The interaction between Chaer and PRC2 is transiently induced after hormone or stress stimulation in a process involving mammalian target of rapamycin complex 1, and this interaction is a prerequisite for epigenetic reprogramming and induction of genes involved in hypertrophy. Inhibition of Chaer expression in the heart before, but not after, the onset of pressure overload substantially attenuates cardiac hypertrophy and dysfunction. Our study reveals that stress-induced pathological gene activation in the heart requires a previously uncharacterized lncRNA-dependent epigenetic checkpoint.
Subject(s)
Cardiomegaly/genetics , Epigenesis, Genetic/genetics , Heart/diagnostic imaging , Histone Code/genetics , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Polycomb Repressive Complex 2/metabolism , RNA, Long Noncoding/genetics , Animals , Blotting, Northern , Cardiomegaly/metabolism , Chromatin Immunoprecipitation , Computer Simulation , Echocardiography , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Immunoblotting , In Situ Hybridization, Fluorescence , In Vitro Techniques , Induced Pluripotent Stem Cells , Mechanistic Target of Rapamycin Complex 1 , Methylation , Mice , Mice, Knockout , Multiprotein Complexes/metabolism , Rats , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , TOR Serine-Threonine Kinases/metabolismABSTRACT
p38 kinases are members of the mitogen-activated protein kinases (MAPK) with established contribution to a wide range of signaling pathways and different biological processes. The prototypic p38 MAPK, p38α was originally identified as an essential signaling kinase for inflammatory cytokine production Extensive studies have now revealed that p38s have critical roles in many different tissues far beyond immune regulation and inflammatory responses. In this review, we will focus on the structure and molecular biology of p38s, and their specific roles in heart, especially regarding myocyte proliferation, apoptosis, and hypertrophic responses.
Subject(s)
Cardiomegaly/enzymology , Myocardium/enzymology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Apoptosis , Cell Proliferation , Humans , MAP Kinase Signaling System , Myocardium/cytology , Myocytes, Cardiac/physiology , p38 Mitogen-Activated Protein Kinases/chemistryABSTRACT
Sarcolipin (SLN) is a small proteolipid and a regulator of sarco(endo)plasmic reticulum Ca(2+)-ATPase. In heart tissue, SLN is exclusively expressed in the atrium. Previously, we inserted Cre recombinase into the endogenous SLN locus by homologous recombination and succeeded in generating SLN-Cre knockin (Sln(Cre/+)) mice. This Sln(Cre/+) mouse can be used to generate an atrium-specific gene-targeting mutant, and it is based on the Cre-loxP system. In the present study, we used adult Sln(Cre/+) mice atria and analyzed the effects of heterozygous SLN deletion by Cre knockin before use as the gene targeting mouse. Both SLN mRNA and protein levels were decreased in Sln(Cre/+) mouse atria, but there were no morphological, physiological, or molecular biological abnormalities. The properties of contractility and Ca(2+) handling were similar to wild-type (WT) mice, and expression levels of several stress markers and sarcoplasmic reticulum-related protein levels were not different between Sln(Cre/+) and WT mice. Moreover, there was no significant difference in sarco(endo)plasmic reticulum Ca(2+)-ATPase activity between the two groups. We showed that Sln(Cre/+) mice were not significantly different from WT mice in all aspects that were examined. The present study provides basic characteristics of Sln(Cre/+) mice and possibly information on the usefulness of Sln(Cre/+) mice as an atrium-specific gene-targeting model.
Subject(s)
Gene Deletion , Heterozygote , Muscle Proteins/genetics , Myocardial Contraction/genetics , Myocytes, Cardiac/metabolism , Proteolipids/genetics , Ventricular Function, Left/genetics , Adrenergic beta-Agonists/pharmacology , Animals , Calcium Signaling/genetics , Female , Fibrosis , Genotype , Isoproterenol/pharmacology , Male , Mice, Inbred C57BL , Mice, Knockout , Muscle Proteins/deficiency , Myocardial Contraction/drug effects , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Phenotype , Proteolipids/deficiency , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Ventricular Function, Left/drug effectsABSTRACT
Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ~9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.