ABSTRACT
BACKGROUND: Remuscularization of the mammalian heart can be achieved after cell transplantation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). However, several hurdles remain before implementation into clinical practice. Poor survival of the implanted cells is related to insufficient vascularization, and the potential for fatal arrhythmogenesis is associated with the fetal cell-like nature of immature CMs. METHODS: We generated 3 lines of hiPSC-derived endothelial cells (ECs) and hiPSC-CMs from 3 independent donors and tested hiPSC-CM sarcomeric length, gap junction protein, and calcium-handling ability in coculture with ECs. Next, we examined the therapeutic effect of the cotransplantation of hiPSC-ECs and hiPSC-CMs in nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice undergoing myocardial infarction (n≥4). Cardiac function was assessed by echocardiography, whereas arrhythmic events were recorded using 3-lead ECGs. We further used healthy non-human primates (n=4) with cell injection to study the cell engraftment, maturation, and integration of transplanted hiPSC-CMs, alone or along with hiPSC-ECs, by histological analysis. Last, we tested the cell therapy in ischemic reperfusion injury in non-human primates (n=4, 3, and 4 for EC+CM, CM, and control, respectively). Cardiac function was evaluated by echocardiography and cardiac MRI, whereas arrhythmic events were monitored by telemetric ECG recorders. Cell engraftment, angiogenesis, and host-graft integration of human grafts were also investigated. RESULTS: We demonstrated that human iPSC-ECs promote the maturity and function of hiPSC-CMs in vitro and in vivo. When cocultured with ECs, CMs showed more mature phenotypes in cellular structure and function. In the mouse model, cotransplantation augmented the EC-accompanied vascularization in the grafts, promoted the maturity of CMs at the infarct area, and improved cardiac function after myocardial infarction. Furthermore, in non-human primates, transplantation of ECs and CMs significantly enhanced graft size and vasculature and improved cardiac function after ischemic reperfusion. CONCLUSIONS: These results demonstrate the synergistic effect of combining iPSC-derived ECs and CMs for therapy in the postmyocardial infarction heart, enabling a promising strategy toward clinical translation.
Subject(s)
Induced Pluripotent Stem Cells , Myocardial Infarction , Humans , Mice , Animals , Myocytes, Cardiac/metabolism , Induced Pluripotent Stem Cells/metabolism , Endothelial Cells/metabolism , Mice, SCID , Mice, Inbred NOD , Myocardial Infarction/pathology , Primates , Cell Differentiation , MammalsABSTRACT
Background: Acute Respiratory Distress syndrome (ARDS) is a clinical syndrome of noncardiac pulmonary edema and inflammation leading to acute respiratory failure. We used the oleic acid infusion pig model of ARDS resembling human disease to explore cytokine changes in white blood cells (WBC) and plasma proteins, comparing baseline to ARDS values. Methods: Nineteen juvenile female swine were included in the study. ARDS defined by a PaO2/FiO2 ratio < 300 was induced by continuous oleic acid infusion. Arterial blood was drawn before and during oleic acid infusion, and when ARDS was established. Cytokine expression in WBC was analyzed by RT-qPCR and plasma protein expression by ELISA. Results: The median concentration of IFN-γ mRNA was estimated to be 59% (p = 0.006) and of IL-6 to be 44.4% (p = 0.003) of the baseline amount. No significant changes were detected for TNF-α, IL-17, and IL-10 mRNA expression. In contrast, the concentrations of plasma IFN-γ and IL-6 were significantly higher (p = 0.004 and p = 0.048 resp.), and TNF-α was significantly lower (p = 0.006) at ARDS compared to baseline. Conclusions: The change of proinflammatory cytokines IFN-γ and IL-6 expression is different comparing mRNA and plasma proteins at oleic acid-induced ARDS compared to baseline. The migration of cells to the lung may be the cause for this discrepancy.
Subject(s)
Acute Lung Injury , Respiratory Distress Syndrome , Humans , Female , Animals , Swine , Oleic Acid , Tumor Necrosis Factor-alpha , Interleukin-6 , Cytokines , Acute Lung Injury/chemically induced , Respiratory Distress Syndrome/chemically inducedABSTRACT
RATIONALE: Flask-shaped invaginations of the cardiomyocyte sarcolemma called caveolae require the structural protein caveolin-3 (Cav-3) and host a variety of ion channels, transporters, and signaling molecules. Reduced Cav-3 expression has been reported in models of heart failure, and variants in CAV3 have been associated with the inherited long-QT arrhythmia syndrome. Yet, it remains unclear whether alterations in Cav-3 levels alone are sufficient to drive aberrant repolarization and increased arrhythmia risk. OBJECTIVE: To determine the impact of cardiac-specific Cav-3 ablation on the electrophysiological properties of the adult mouse heart. METHODS AND RESULTS: Cardiac-specific, inducible Cav3 homozygous knockout (Cav-3KO) mice demonstrated a marked reduction in Cav-3 expression by Western blot and loss of caveolae by electron microscopy. However, there was no change in macroscopic cardiac structure or contractile function. The QTc interval was increased in Cav-3KO mice, and there was an increased propensity for ventricular arrhythmias. Ventricular myocytes isolated from Cav-3KO mice exhibited a prolonged action potential duration (APD) that was due to reductions in outward potassium currents (Ito, Iss) and changes in inward currents including slowed inactivation of ICa,L and increased INa,L. Mathematical modeling demonstrated that the changes in the studied ionic currents were adequate to explain the prolongation of the mouse ventricular action potential. Results from human iPSC-derived cardiomyocytes showed that shRNA knockdown of Cav-3 similarly prolonged APD. CONCLUSION: We demonstrate that Cav-3 and caveolae regulate cardiac repolarization and arrhythmia risk via the integrated modulation of multiple ionic currents.
Subject(s)
Caveolae , Long QT Syndrome , Animals , Humans , Mice , Caveolae/metabolism , Caveolin 3/genetics , Caveolin 3/metabolism , Arrhythmias, Cardiac/metabolism , Action Potentials , Ion Channels/metabolism , Long QT Syndrome/metabolism , Myocytes, Cardiac/metabolism , Caveolin 1/genetics , Caveolin 1/metabolismABSTRACT
Arginine-serine (RS) domain(s) in splicing factors are critical for protein-protein interaction in pre-mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA-binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho-antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle-down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein-RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock-in (KI) mouse model (Rbm20S637A ) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)-like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.
Subject(s)
RNA Splicing , RNA-Binding Proteins , Active Transport, Cell Nucleus , Animals , Mice , Myocytes, Cardiac/metabolism , Phosphorylation , RNA-Binding Motifs , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RatsABSTRACT
Dilated cardiomyopathy (DCM) is a heritable and genetically heterogenous disease often idiopathic and a leading cause of heart failure with high morbidity and mortality. DCM caused by RNA binding motif protein 20 (RBM20) mutations is diverse and needs a more complete mechanistic understanding. RBM20 mutation S637G (S639G in mice) is linked to severe DCM and early death in human patients. In this study, we generated a RBM20 S639G mutation knock-in (KI) mouse model to validate the function of S639G mutation and examine the underlying mechanisms. KI mice exhibited severe DCM and premature death with a ~ 50% mortality in two months old homozygous (HM) mice. KI mice had enlarged atria and increased ANP and BNP biomarkers. The S639G mutation promoted RBM20 trafficking and ribonucleoprotein (RNP) granules in the sarcoplasm. RNA Seq data revealed differentially expressed and spliced genes were associated with arrhythmia, cardiomyopathy, and sudden death. KI mice also showed a reduction of diastolic stiffness and impaired contractility at both the left ventricular (LV) chamber and cardiomyocyte levels. Our results indicate that the RBM20 S639G mutation leads to RNP granules causing severe heart failure and early death and this finding strengthens the novel concept that RBM20 cardiomyopathy is a RNP granule disease.
Subject(s)
Cardiomyopathy, Dilated , Heart Failure , Animals , Cardiomyopathy, Dilated/metabolism , Heart Failure/genetics , Humans , Mice , Mortality, Premature , Mutation , RNA , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Risk FactorsABSTRACT
BACKGROUND: Neonatal mouse cardiomyocytes undergo a metabolic switch from glycolysis to oxidative phosphorylation, which results in a significant increase in reactive oxygen species production that induces DNA damage. These cellular changes contribute to cardiomyocyte cell cycle exit and loss of the capacity for cardiac regeneration. The mechanisms that regulate this metabolic switch and the increase in reactive oxygen species production have been relatively unexplored. Current evidence suggests that elevated reactive oxygen species production in ischemic tissues occurs as a result of accumulation of the mitochondrial metabolite succinate during ischemia via succinate dehydrogenase (SDH), and this succinate is rapidly oxidized at reperfusion. Mutations in SDH in familial cancer syndromes have been demonstrated to promote a metabolic shift into glycolytic metabolism, suggesting a potential role for SDH in regulating cellular metabolism. Whether succinate and SDH regulate cardiomyocyte cell cycle activity and the cardiac metabolic state remains unclear. METHODS: Here, we investigated the role of succinate and SDH inhibition in regulation of postnatal cardiomyocyte cell cycle activity and heart regeneration. RESULTS: Our results demonstrate that injection of succinate into neonatal mice results in inhibition of cardiomyocyte proliferation and regeneration. Our evidence also shows that inhibition of SDH by malonate treatment after birth extends the window of cardiomyocyte proliferation and regeneration in juvenile mice. Remarkably, extending malonate treatment to the adult mouse heart after myocardial infarction injury results in a robust regenerative response within 4 weeks after injury via promoting adult cardiomyocyte proliferation and revascularization. Our metabolite analysis after SDH inhibition by malonate induces dynamic changes in adult cardiac metabolism. CONCLUSIONS: Inhibition of SDH by malonate promotes adult cardiomyocyte proliferation, revascularization, and heart regeneration via metabolic reprogramming. These findings support a potentially important new therapeutic approach for human heart failure.
Subject(s)
Cardiovascular Diseases/drug therapy , Malonates/therapeutic use , Myocardium/pathology , Myocytes, Cardiac/drug effects , Regeneration/drug effects , Animals , Cell Proliferation , Humans , Male , Malonates/pharmacology , Mice , Signal TransductionABSTRACT
Sickle cell disease (SCD) is a hereditary blood disorder affecting millions of people in which red blood cells (RBCs) become sickled and lyse easily driven by polymerization of hemoglobin. Chronically, SCD causes anemia and biventricular dysfunction. GBT440 is an experimental treatment for SCD that prevents hemoglobin polymerization. We hypothesized that 17-month-old Berkeley SCD mice treated with GBT440 would have increased hematocrit (Hct) and better biventricular function compared to vehicle treated SCD mice. Our results demonstrate that 3 weeks of GBT440 treatment eliminated chronic anemia, increased left ventricular ejection fraction (LVEF) and stroke volume index, and improved right ventricular function. Overall, our findings support a therapeutic effect of GBT440 in vivo in a small animal model of SCD. Next steps in investigating mechanisms of improved cardiac function are warranted.
Subject(s)
Anemia, Sickle CellABSTRACT
BACKGROUND: The impact of gut microbiota on the regulation of host physiology has recently garnered considerable attention, particularly in key areas such as the immune system and metabolism. These areas are also crucial for the pathophysiology of and repair after myocardial infarction (MI). However, the role of the gut microbiota in the context of MI remains to be fully elucidated. METHODS: To investigate the effects of gut microbiota on cardiac repair after MI, C57BL/6J mice were treated with antibiotics 7 days before MI to deplete mouse gut microbiota. Flow cytometry was applied to examine the changes in immune cell composition in the heart. 16S rDNA sequencing was conducted as a readout for changes in gut microbial composition. Short-chain fatty acid (SCFA) species altered after antibiotic treatment were identified by high-performance liquid chromatography. Fecal reconstitution, transplantation of monocytes, or dietary SCFA or Lactobacillus probiotic supplementation was conducted to evaluate the cardioprotective effects of microbiota on the mice after MI. RESULTS: Antibiotic-treated mice displayed drastic, dose-dependent mortality after MI. We observed an association between the gut microbiota depletion and significant reductions in the proportion of myeloid cells and SCFAs, more specifically acetate, butyrate, and propionate. Infiltration of CX3CR1+ monocytes to the peri-infarct zone after MI was also reduced, suggesting impairment of repair after MI. Accordingly, the physiological status and survival of mice were significantly improved after fecal reconstitution, transplantation of monocytes, or dietary SCFA supplementation. MI was associated with a reorganization of the gut microbial community such as a reduction in Lactobacillus. Supplementing antibiotic-treated mice with a Lactobacillus probiotic before MI restored myeloid cell proportions, yielded cardioprotective effects, and shifted the balance of SCFAs toward propionate. CONCLUSIONS: Gut microbiota-derived SCFAs play an important role in maintaining host immune composition and repair capacity after MI. This suggests that manipulation of these elements may provide opportunities to modulate pathological outcome after MI and indeed human health and disease as a whole.
Subject(s)
Anti-Bacterial Agents/toxicity , Bacteria/drug effects , Gastrointestinal Microbiome/drug effects , Monocytes/immunology , Myocardial Infarction/microbiology , Myocardium/immunology , Animals , Bacteria/immunology , Bacteria/metabolism , Disease Models, Animal , Dysbiosis , Fatty Acids/administration & dosage , Fatty Acids/metabolism , Fecal Microbiota Transplantation , Female , Host-Pathogen Interactions , Lactobacillus/immunology , Lactobacillus/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Monocytes/metabolism , Monocytes/transplantation , Myocardial Infarction/immunology , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardium/metabolism , Myocardium/pathology , Probiotics/administration & dosage , RAW 264.7 CellsABSTRACT
Although women are more susceptible to pulmonary arterial hypertension (PAH) than men, their right ventricular (RV) function is better preserved. Estrogen receptor-α (ERα) has been identified as a likely mediator for estrogen protection in the RV. However, the role of ERα in preserving RV function and remodeling during pressure overload remains poorly understood. We hypothesized that loss of functional ERα removes female protection from adverse remodeling and is permissive for the development of a maladapted RV phenotype. Male and female rats with a loss-of-function mutation in ERα (ERαMut) and wild-type (WT) littermates underwent RV pressure overload by pulmonary artery banding (PAB). At 10 wk post-PAB, WT and ERαMut demonstrated RV hypertrophy. Analysis of RV pressure waveforms demonstrated RV-pulmonary vascular uncoupling and diastolic dysfunction in female, but not male, ERαMut PAB rats. Similarly, female, but not male, ERαMut exhibited increased RV fibrosis, comprised primarily of thick collagen fibers. There was an increased protein expression ratio of TIMP metallopeptidase inhibitor 1 (Timp1) to matrix metalloproteinase 9 (Mmp9) in female ERαMut compared with WT PAB rats, suggesting less collagen degradation. RNA-sequencing in female WT and ERαMut RV revealed kallikrein-related peptidase 10 (Klk10) and Jun Proto-Oncogene (Jun) as possible mediators of female RV protection during PAB. In summary, ERα in females is protective against RV-pulmonary vascular uncoupling, diastolic dysfunction, and fibrosis in response to pressure overload. ERα appears to be dispensable for RV adaptation in males. ERα may be a mediator of superior RV adaptation in female patients with PAH.NEW & NOTEWORTHY Using a novel loss-of-function mutation in estrogen receptor-α (ERα), we demonstrate that female, but not male, ERα mutant rats display right ventricular (RV)-vascular uncoupling, diastolic dysfunction, and fibrosis following pressure overload, indicating a sex-dependent role of ERα in protecting against adverse RV remodeling. TIMP metallopeptidase inhibitor 1 (Timp1), matrix metalloproteinase 9 (Mmp9), kallikrein-related peptidase 10 (Klk10), and Jun Proto-Oncogene (Jun) were identified as potential mediators in ERα-regulated pathways in RV pressure overload.
Subject(s)
Estrogen Receptor alpha/metabolism , Hypertrophy, Right Ventricular/prevention & control , Myocardium/metabolism , Ventricular Dysfunction, Right/prevention & control , Ventricular Function, Right , Ventricular Remodeling , Animals , Disease Models, Animal , Estrogen Receptor alpha/genetics , Female , Fibrillar Collagens/metabolism , Fibrosis , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/pathology , Hypertrophy, Right Ventricular/physiopathology , Kallikreins/genetics , Kallikreins/metabolism , Male , Mitochondria, Heart/metabolism , Mitochondria, Heart/pathology , Mutation , Myocardium/pathology , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Rats, Mutant Strains , Rats, Sprague-Dawley , Sex Factors , Signal Transduction , Ventricular Dysfunction, Right/metabolism , Ventricular Dysfunction, Right/pathology , Ventricular Dysfunction, Right/physiopathologyABSTRACT
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) exhibit a fetal phenotype that limits in vitro and therapeutic applications. Strategies to promote cardiomyocyte maturation have focused interventions on differentiated hPSC-CMs, but this study tests priming of early cardiac progenitor cells (CPCs) with polyinosinic-polycytidylic acid (pIC) to accelerate cardiomyocyte maturation. CPCs were differentiated from hPSCs using a monolayer differentiation protocol with defined small molecule Wnt temporal modulation, and pIC was added during the formation of early CPCs. pIC priming did not alter the expression of cell surface markers for CPCs (>80% KDR+/PDGFRα+), expression of common cardiac transcription factors, or final purity of differentiated hPSC-CMs (â¼90%). However, CPC differentiation in basal medium revealed that pIC priming resulted in hPSC-CMs with enhanced maturity manifested by increased cell size, greater contractility, faster electrical upstrokes, increased oxidative metabolism, and more mature sarcomeric structure and composition. To investigate the mechanisms of CPC priming, RNAseq revealed that cardiac progenitor-stage pIC modulated early Notch signaling and cardiomyogenic transcriptional programs. Chromatin immunoprecipitation of CPCs showed that pIC treatment increased deposition of the H3K9ac activating epigenetic mark at core promoters of cardiac myofilament genes and the Notch ligand, JAG1. Inhibition of Notch signaling blocked the effects of pIC on differentiation and cardiomyocyte maturation. Furthermore, primed CPCs showed more robust formation of hPSC-CMs grafts when transplanted to the NSGW mouse kidney capsule. Overall, epigenetic modulation of CPCs with pIC accelerates cardiomyocyte maturation enabling basic research applications and potential therapeutic uses. Stem Cells 2019;37:910-923.
Subject(s)
Cell Differentiation/drug effects , Epigenesis, Genetic , Induced Pluripotent Stem Cells/drug effects , Myocytes, Cardiac/drug effects , Poly I-C/pharmacology , Receptors, Notch/genetics , Animals , Cell Size , Histones/genetics , Histones/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , Jagged-1 Protein/genetics , Jagged-1 Protein/metabolism , Kidney , Mice , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Oxidative Phosphorylation , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Receptors, Notch/metabolism , Sarcomeres/metabolism , Sequence Analysis, RNA , Signal Transduction , Stem Cell Transplantation/methods , Transplantation, Heterotopic , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.
Subject(s)
Arteries/cytology , Endothelial Cells/transplantation , Neovascularization, Physiologic , Pluripotent Stem Cells/physiology , Tissue Engineering/methods , Animals , CRISPR-Cas Systems , Cell Line , Endothelial Cells/cytology , Humans , Mice , Myocardial Infarction/therapy , Sequence Analysis, RNAABSTRACT
ABSTARCT: To investigate the use of two-site regional oxygen saturations (rSO2) and end tidal carbon dioxide (EtCO2) to assess the effectiveness of resuscitation and return of spontaneous circulation (ROSC). Eight mechanically ventilated juvenile swine underwent 28 ventricular fibrillatory arrests with open cardiac massage. Cardiac massage was administered to achieve target pulmonary blood flow (PBF) as a percentage of pre-cardiac arrest baseline. Non-invasive data, including, EtCO2, cerebral rSO2 (C-rSO2) and renal rSO2 (R-rSO2) were collected continuously. Our data demonstrate the ability to measure both rSO2 and EtCO2 during CPR and after ROSC. During resuscitation EtCO2 had a strong correlation with goal CO with r = 0.83 (p < 0.001) 95% CI [0.67-0.92]. Both C-rSO2 and R-rSO2 had moderate and statistically significant correlation with CO with r = 0.52 (p = 0.003) 95% CI (0.19-0.74) and 0.50 (p = 0.004) 95% CI [0.16-0.73]. The AUCs for sudden increase of EtCO2, C-rSO2, and R-rSO2 at ROSC were 0.86 [95% CI, 0.77-0.94], 0.87 [95% CI, 0.8-0.94], and 0.98 [95% CI, 0.96-1.00] respectively. Measurement of continuous EtCO2 and rSO2 may be used during CPR to ensure effective chest compressions. Moreover, both rSO2 and EtCO2 may be used to detect ROSC in a swine pediatric ventricular fibrillatory arrest model.
Subject(s)
Capnography/methods , Heart Arrest/therapy , Oxygen/metabolism , Resuscitation/methods , Ventricular Fibrillation , Animals , Carbon Dioxide , Cardiopulmonary Resuscitation , Cerebrovascular Circulation/physiology , Disease Models, Animal , Heart Arrest/physiopathology , Hemodynamics , Monitoring, Physiologic , Oximetry , ROC Curve , Spectroscopy, Near-Infrared , Stress, Mechanical , SwineABSTRACT
Right ventricular failure (RVF) is a common cause of death in patients suffering from pulmonary arterial hypertension (PAH). The current treatment for PAH only moderately improves symptoms, and RVF ultimately occurs. Therefore, it is necessary to develop new treatment strategies to protect against right ventricle (RV) maladaptation despite PAH progression. In this study, we hypothesize that local mesenchymal stem cell (MSC) delivery via a novel bioscaffold can improve RV function despite persistent PAH. To test our hypothesis, we induced PAH in adult rats with SU5416 and chronic hypoxia exposure; treated with rat MSCs delivered by intravenous injection, intramyocardial injection, or epicardial placement of a bioscaffold; and then examined treatment effectiveness by in vivo pressure-volume measurement, echocardiography, histology, and immunohistochemistry. Our results showed that compared with other treatment groups, only the MSC-seeded bioscaffold group resulted in RV functional improvement, including restored stroke volume, cardiac output, and improved stroke work. Diastolic function indicated by end-diastolic pressure-volume relationship was improved by the local MSC treatments or bioscaffold alone. Cardiomyocyte hypertrophy and RV fibrosis were both reduced, and von Willebrand factor expression was restored by the MSC-seeded bioscaffold treatment. Overall, our study suggests a potential new regenerative therapy to rescue the pressure-overload failing RV with persistent pulmonary vascular disease, which may improve quality of life and/or survival of PAH patients. NEW & NOTEWORTHY We explored the effects of mesenchymal stem cell-seeded bioscaffold on right ventricles (RVs) of rats with established pulmonary arterial hypertension (PAH). Some beneficial effects were observed despite persistent PAH, suggesting that this may be a new therapy for RV to improve quality of life and/or survival of PAH patients.
Subject(s)
Arterial Pressure , Hypertrophy, Right Ventricular/surgery , Mesenchymal Stem Cell Transplantation/methods , Pulmonary Arterial Hypertension/surgery , Pulmonary Artery/physiopathology , Tissue Scaffolds , Ventricular Dysfunction, Right/surgery , Ventricular Function, Right , Animals , Cells, Cultured , Disease Models, Animal , Fibrosis , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/physiopathology , Hypoxia/complications , Indoles , Male , Myocardial Contraction , Myocardium/metabolism , Myocardium/pathology , Pulmonary Arterial Hypertension/etiology , Pulmonary Arterial Hypertension/metabolism , Pulmonary Arterial Hypertension/physiopathology , Pyrroles , Rats, Sprague-Dawley , Recovery of Function , Regeneration , Ventricular Dysfunction, Right/etiology , Ventricular Dysfunction, Right/metabolism , Ventricular Dysfunction, Right/physiopathology , Ventricular Remodeling , von Willebrand Factor/metabolismABSTRACT
Rats exposed to postnatal hyperoxia develop right ventricular (RV) dysfunction, mild pulmonary hypertension, and dysregulated cardiac mitochondrial biogenesis when aged to one year, with the degree of cardiac dysfunction and pulmonary hypertension similar to that previously described in young adults born preterm. Here, we sought to understand the impact of postnatal hyperoxia exposure on RV hemodynamic and mitochondrial function across the life span. In Methods, pups from timed-pregnant Sprague-Dawley rats were randomized to normoxia or hyperoxia [fraction of inspired oxygen (FIO2), 0.85] exposure for the first 14 days of life, a commonly used model of chronic lung disease of prematurity. RV hemodynamic and mitochondrial function were assessed by invasive measurement of RV pressure-volume loops and by high-resolution respirometry at postnatal day 21 (P21), P90, and P365. In Results, at P21, hyperoxia-exposed rats demonstrated severe pulmonary hypertension and RV dysfunction, accompanied by depressed mitochondrial oxidative capacity. However, significant upregulation of mitochondrial biogenesis at P21 as well as improved afterload led to complete RV hemodynamic and mitochondrial recovery at P90. Mitochondrial DNA mutations were significantly higher by P90 and associated with significant late RV mitochondrial and hemodynamic dysfunction at P365. In conclusion, there appears to be a "honeymoon period" where cardiac hemodynamic and mitochondrial function normalizes following postnatal hyperoxia exposure, only to decline again with ongoing aging. This finding may have significant implications if a long-term pulmonary vascular screening program were to be developed for children or adults with a history of severe prematurity. Further investigation into the mechanisms of recovery are warranted.NEW & NOTEWORTHY Premature birth is associated with increased risk for cardiac dysfunction and failure throughout life. Here, we identify bimodal right ventricular dysfunction after postnatal hyperoxia exposure. Mitochondrial biogenesis serves as an early adaptive feature promoting recovery of cardiac hemodynamic and mitochondrial function. However, the accumulation of mitochondrial DNA mutations results in late mitochondrial and right ventricular dysfunction. This bimodal right ventricular dysfunction may have important implications for the development of screening programs in the preterm population.
Subject(s)
Hyperoxia/complications , Ventricular Dysfunction, Right/physiopathology , Animals , DNA, Mitochondrial/genetics , Female , Heart/growth & development , Heart/physiopathology , Male , Mitochondria/metabolism , Mutation , Organelle Biogenesis , Rats , Rats, Sprague-Dawley , Ventricular Dysfunction, Right/etiology , Ventricular Dysfunction, Right/genetics , Ventricular Dysfunction, Right/metabolismABSTRACT
Left heart failure (LHF) is the most common cause of pulmonary hypertension, which confers an increase in morbidity and mortality in this context. Pulmonary vascular resistance has prognostic value in LHF, but otherwise the mechanical consequences of LHF for the pulmonary vasculature and right ventricle (RV) remain unknown. We sought to investigate mechanical mechanisms of pulmonary vascular and RV dysfunction in a rodent model of LHF to address the knowledge gaps in understanding disease pathophysiology. LHF was created using a left anterior descending artery ligation to cause myocardial infarction (MI) in mice. Sham animals underwent thoracotomy alone. Echocardiography demonstrated increased left ventricle (LV) volumes and decreased ejection fraction at 4 wk post-MI that did not normalize by 12 wk post-MI. Elevation of LV diastolic pressure and RV systolic pressure at 12 wk post-MI demonstrated pulmonary hypertension (PH) due to LHF. There was increased pulmonary arterial elastance and pulmonary vascular resistance associated with perivascular fibrosis without other remodeling. There was also RV contractile dysfunction with a 35% decrease in RV end-systolic elastance and 66% decrease in ventricular-vascular coupling. In this model of PH due to LHF with reduced ejection fraction, pulmonary fibrosis contributes to increased RV afterload, and loss of RV contractility contributes to RV dysfunction. These are key pathologic features of human PH secondary to LHF. In the future, novel therapeutic strategies aimed at preventing pulmonary vascular mechanical changes and RV dysfunction in the context of LHF can be tested using this model. NEW & NOTEWORTHY In this study, we investigate the mechanical consequences of left heart failure with reduced ejection fraction for the pulmonary vasculature and right ventricle. Using comprehensive functional analyses of the cardiopulmonary system in vivo and ex vivo, we demonstrate that pulmonary fibrosis contributes to increased RV afterload and loss of RV contractility contributes to RV dysfunction. Thus this model recapitulates key pathologic features of human pulmonary hypertension-left heart failure and offers a robust platform for future investigations.
Subject(s)
Heart Failure/physiopathology , Myocardial Infarction/physiopathology , Pulmonary Arterial Hypertension/physiopathology , Pulmonary Artery/physiopathology , Pulmonary Circulation , Ventricular Dysfunction, Left/physiopathology , Ventricular Dysfunction, Right/physiopathology , Ventricular Function, Left , Ventricular Function, Right , Animals , Disease Models, Animal , Fibrosis , Heart Failure/diagnostic imaging , Heart Failure/etiology , Male , Mice, Inbred C57BL , Myocardial Infarction/diagnostic imaging , Myocardial Infarction/etiology , Pulmonary Arterial Hypertension/diagnostic imaging , Pulmonary Arterial Hypertension/etiology , Pulmonary Artery/diagnostic imaging , Pulmonary Artery/pathology , Stroke Volume , Vascular Remodeling , Ventricular Dysfunction, Left/diagnostic imaging , Ventricular Dysfunction, Left/etiology , Ventricular Dysfunction, Right/diagnostic imaging , Ventricular Dysfunction, Right/etiology , Ventricular PressureABSTRACT
Right ventricular (RV) failure (RVF) is the major cause of death in pulmonary hypertension. Recent studies have characterized changes in RV structure in RVF, including hypertrophy, fibrosis, and abnormalities in mitochondria. Few, if any, studies have explored the relationships between these multiscale structural changes and functional changes in RVF. Pulmonary artery banding (PAB) was used to induce RVF due to pressure overload in male rats. Eight weeks postsurgery, terminal invasive measurements demonstrated RVF with decreased ejection fraction (70 ± 10 vs. 45 ± 15%, sham vs. PAB, P < 0.005) and cardiac output (126 ± 40 vs. 67 ± 32 ml/min, sham vs. PAB, P < 0.05). At the organ level, RV hypertrophy was directly correlated with increased contractility, which was insufficient to maintain ventricular-vascular coupling. At the tissue level, there was a 90% increase in fibrosis that had a direct correlation with diastolic dysfunction measured by reduced chamber compliance ( r2 = 0.43, P = 0.008). At the organelle level, transmission electron microscopy demonstrated an abundance of small-sized mitochondria. Increased mitochondria was associated with increased ventricular oxygen consumption and reduced mechanical efficiency ( P < 0.05). These results demonstrate an association between alterations in mitochondria and RV oxygen consumption and mechanical inefficiency in RVF and a link between fibrosis and in vivo diastolic dysfunction. Overall, this work provides key insights into multiscale RV remodeling in RVF due to pressure overload. NEW & NOTEWORTHY This study explores the functional impact of multiscale ventricular remodeling in right ventricular failure (RVF). It demonstrates correlations between hypertrophy and increased contractility as well as fibrosis and diastolic function. This work quantifies mitochondrial ultrastructural remodeling in RVF and demonstrates increased oxygen consumption and mechanical inefficiency as features of RVF. Direct correlation between mitochondrial changes and ventricular energetics provides insight into the impact of organelle remodeling on organ level function.
Subject(s)
Hypertrophy, Right Ventricular/physiopathology , Ventricular Dysfunction, Right/physiopathology , Ventricular Remodeling , Animals , Hypertension, Pulmonary/complications , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/pathology , Male , Mitochondria, Heart/ultrastructure , Myocardial Contraction , Rats , Rats, Wistar , Stroke Volume , Ventricular Dysfunction, Right/etiology , Ventricular Dysfunction, Right/pathologyABSTRACT
OBJECTIVE: Nucleotide P2Y2 receptor (P2Y2R) contributes to vascular inflammation by increasing vascular cell adhesion molecule-1 expression in endothelial cells (EC), and global P2Y2R deficiency prevents fatty streak formation in apolipoprotein E null (ApoE-/-) mice. Because P2Y2R is ubiquitously expressed in vascular cells, we investigated the contribution of endothelial P2Y2R in the pathogenesis of atherosclerosis. APPROACH AND RESULTS: EC-specific P2Y2R-deficient mice were generated by breeding VEcadherin5-Cre mice with the P2Y2R floxed mice. Endothelial P2Y2R deficiency reduced endothelial nitric oxide synthase activity and significantly altered ATP- and UTP (uridine 5'-triphosphate)-induced vasorelaxation without affecting vasodilatory responses to acetylcholine. Telemetric blood pressure and echocardiography measurements indicated that EC-specific P2Y2R-deficient mice did not develop hypertension. We investigated the role of endothelial P2Y2R in the development of atherosclerotic lesions by crossing the EC-specific P2Y2R knockout mice onto an ApoE-/- background and evaluated lesion development after feeding a standard chow diet for 25 weeks. Histopathologic examination demonstrated reduced atherosclerotic lesions in the aortic sinus and entire aorta, decreased macrophage infiltration, and increased smooth muscle cell and collagen content, leading to the formation of a subendothelial fibrous cap in EC-specific P2Y2R-deficient ApoE-/- mice. Expression and proteolytic activity of matrix metalloproteinase-2 was significantly reduced in atherosclerotic lesions from EC-specific P2Y2R-deficient ApoE-/- mice. Furthermore, EC-specific P2Y2R deficiency inhibited nitric oxide production, leading to significant increase in smooth muscle cell migration out of aortic explants. CONCLUSIONS: EC-specific P2Y2R deficiency reduces atherosclerotic burden and promotes plaque stability in ApoE-/- mice through impaired macrophage infiltration acting together with reduced matrix metalloproteinase-2 activity and increased smooth muscle cell migration.
Subject(s)
Aorta, Thoracic/metabolism , Aortic Diseases/metabolism , Apolipoproteins E/deficiency , Atherosclerosis/metabolism , Endothelial Cells/metabolism , Plaque, Atherosclerotic , Receptors, Purinergic P2Y2/deficiency , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/pathology , Aorta, Thoracic/physiopathology , Aortic Diseases/genetics , Aortic Diseases/pathology , Aortic Diseases/prevention & control , Apolipoproteins E/genetics , Atherosclerosis/genetics , Atherosclerosis/pathology , Atherosclerosis/prevention & control , Cell Movement , Cells, Cultured , Collagen/metabolism , Disease Models, Animal , Disease Progression , Endothelial Cells/drug effects , Endothelial Cells/pathology , Fibrosis , Macrophages/metabolism , Macrophages/pathology , Male , Matrix Metalloproteinase 2/metabolism , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiopathology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , Purinergic P2Y Receptor Agonists/pharmacology , Receptors, Purinergic P2Y2/genetics , Rupture, Spontaneous , Signal Transduction , Vasodilation/drug effects , Vasodilator Agents/pharmacologyABSTRACT
Cardiovascular disease has been the leading cause of death worldwide for the last 15 years, accounting for 15 million deaths per year. While interventions are saving more lives, more than 20% of survivors will end up in heart failure. Cell-based and other types of therapy for advanced heart and vascular disease may offer new hope for those afflicted. Although a variety of cell types are under investigation, common issues include cell survival, retention, engraftment, and proliferation. Cardiac extracellular matrix (C-ECM) has compelling features that offer advantages to not only aid cell survival, retention, engraftment, and proliferation but likely has independent therapeutic (paracrine) and mechanical effects. Animal studies and clinical trials are underway to characterize the role of C-ECM and demonstrate efficacy for acute and chronic heart disease. This chapter reviews animal models used to enhance our knowledge of C-ECMs in heart disease and its use in the treatment of heart disease.
Subject(s)
Extracellular Matrix/physiology , Heart/physiology , Models, Animal , Myocardium/metabolism , Animals , Animals, Genetically Modified , Cells, Cultured , Disease Models, Animal , Heart Diseases/pathology , Heart Diseases/physiopathology , Research DesignABSTRACT
Prematurity complicates 12% of births, and young adults with a history of prematurity are at risk to develop right ventricular (RV) hypertrophy and impairment. The long-term risk for pulmonary vascular disease, as well as mechanisms of RV dysfunction and ventricular-vascular uncoupling after prematurity, remain poorly defined. Using an established model of prematurity-related lung disease, pups from timed-pregnant Sprague Dawley rats were randomized to normoxia or hyperoxia (fraction of inspired oxygen, 0.85) exposure for the first 14 days of life. After aging to 1 year in standard conditions, rats underwent hemodynamic assessment followed by tissue harvest for biochemical and histological evaluation. Aged hyperoxia-exposed rats developed significantly greater RV hypertrophy, associated with a 40% increase in RV systolic pressures. Although cardiac index was similar, hyperoxia-exposed rats demonstrated a reduced RV ejection fraction and significant RV-pulmonary vascular uncoupling. Hyperoxia-exposed RV cardiomyocytes demonstrated evidence of mitochondrial dysregulation and mitochondrial DNA damage, suggesting potential mitochondrial dysfunction as a cause of RV dysfunction. Aged rats exposed to postnatal hyperoxia recapitulate many features of young adults born prematurely, including increased RV hypertrophy and decreased RV ejection fraction. Our data suggest that postnatal hyperoxia exposure results in mitochondrial dysregulation that persists into adulthood with eventual RV dysfunction. Further evaluation of long-term mitochondrial function is warranted in both animal models of premature lung disease and in human adults who were born preterm.
Subject(s)
Hyperoxia/metabolism , Hyperoxia/physiopathology , Organelle Biogenesis , Ventricular Function, Right , Aging/pathology , Animals , Animals, Newborn , Autophagy , Body Weight , DNA Damage , DNA, Mitochondrial/metabolism , Female , Fibrosis , Gene Expression Profiling , Hemodynamics , Hyperoxia/complications , Hyperoxia/diagnostic imaging , Hypertrophy, Right Ventricular/diagnostic imaging , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/genetics , Hypertrophy, Right Ventricular/physiopathology , Male , Myocardium/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Organ Size , Rats, Sprague-DawleyABSTRACT
Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca(2+) cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca(2+) signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca(2+) current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy.