RESUMEN
BACKGROUND: Cardiolipin is a mitochondrial-specific phospholipid that maintains integrity of the electron transport chain (ETC) and plays a central role in myocardial ischemia/reperfusion injury. Tafazzin is an enzyme that is required for cardiolipin maturation. Venoarterial extracorporeal membrane oxygenation (VA-ECMO) use to provide hemodynamic support for acute myocardial infarction has grown exponentially, is associated with poor outcomes, and is under active clinical investigation, yet the mechanistic effect of VA-ECMO on myocardial damage in acute myocardial infarction remains poorly understood. We hypothesized that VA-ECMO acutely depletes myocardial cardiolipin and exacerbates myocardial injury in acute myocardial infarction. METHODS: We examined cardiolipin and tafazzin levels in human subjects with heart failure and healthy swine exposed to VA-ECMO and used a swine model of closed-chest myocardial ischemia/reperfusion injury to evaluate the effect of VA-ECMO on cardiolipin expression, myocardial injury, and mitochondrial function. RESULTS: Cardiolipin and tafazzin levels are significantly reduced in the left ventricles of individuals requiring VA-ECMO compared with individuals without VA-ECMO before heart transplantation. Six hours of exposure to VA-ECMO also decreased left ventricular levels of cardiolipin and tafazzin in healthy swine compared with sham controls. To explore whether cardiolipin depletion by VA-ECMO increases infarct size, we performed left anterior descending artery occlusion for a total of 120 minutes followed by 180 minutes of reperfusion in adult swine in the presence and absence of MTP-131, an amphipathic molecule that interacts with cardiolipin to stabilize the inner mitochondrial membrane. Compared with reperfusion alone, VA-ECMO activation beginning after 90 minutes of left anterior descending artery occlusion increased infarct size (36±8% versus 48±7%; P<0.001). VA-ECMO also decreased cardiolipin and tafazzin levels, disrupted mitochondrial integrity, reduced electron transport chain function, and promoted oxidative stress. Compared with reperfusion alone or VA-ECMO before reperfusion, delivery of MTP-131 before VA-ECMO activation reduced infarct size (22±8%; P=0.03 versus reperfusion alone and P<0.001 versus VA-ECMO alone). MTP-131 restored cardiolipin and tafazzin levels, stabilized mitochondrial function, and reduced oxidative stress in the left ventricle. CONCLUSIONS: We identified a novel mechanism by which VA-ECMO promotes myocardial injury and further identify cardiolipin as an important target of therapy to reduce infarct size and to preserve mitochondrial function in the setting of VA-ECMO for acute myocardial infarction.
RESUMEN
Alpha-B-crystallin, a member of the small heat shock family of proteins, has been implicated in a variety of cardiomyopathies and in normal cardiac homeostasis. It is known to function as a molecular chaperone, particularly for desmin, but also interacts with a wide variety of additional proteins. The molecular chaperone function is also enhanced by signal-dependent phosphorylation at specific residues under stress conditions. Naturally occurring mutations in CRYAB, the gene that encodes alpha-B-crystallin, have been suggested to alter ionic intermolecular interactions that affect dimerization and chaperone function. These mutations have been associated with myofibrillar myopathy, restrictive cardiomyopathy, and hypertrophic cardiomyopathy and promote pathological hypertrophy through different mechanisms such as desmin aggregation, increased reductive stress, or activation of calcineurin-NFAT signaling. This review will discuss the known mechanisms by which alpha-B-crystallin functions in cardiac homeostasis and the pathogenesis of cardiomyopathies and provide insight into potential future areas of exploration.
Asunto(s)
Cardiomiopatías , Cardiomiopatía Restrictiva , Humanos , Desmina/genética , Cardiomiopatías/patología , Mutación , Cardiomiopatía Restrictiva/complicaciones , Chaperonas Moleculares/genéticaRESUMEN
Cardiomyopathy is the predominant defect in Barth syndrome (BTHS) and is caused by a mutation of the X-linked Tafazzin (TAZ) gene, which encodes an enzyme responsible for remodeling mitochondrial cardiolipin. Despite the known importance of mitochondrial dysfunction in BTHS, how specific TAZ mutations cause diverse BTHS heart phenotypes remains poorly understood. We generated a patient-tailored CRISPR/Cas9 knock-in mouse allele (TazPM) that phenocopies BTHS clinical traits. As TazPM males express a stable mutant protein, we assessed cardiac metabolic dysfunction and mitochondrial changes and identified temporally altered cardioprotective signaling effectors. Specifically, juvenile TazPM males exhibit mild left ventricular dilation in systole but have unaltered fatty acid/amino acid metabolism and normal adenosine triphosphate (ATP). This occurs in concert with a hyperactive p53 pathway, elevation of cardioprotective antioxidant pathways, and induced autophagy-mediated early senescence in juvenile TazPM hearts. However, adult TazPM males exhibit chronic heart failure with reduced growth and ejection fraction, cardiac fibrosis, reduced ATP, and suppressed fatty acid/amino acid metabolism. This biphasic changeover from a mild-to-severe heart phenotype coincides with p53 suppression, downregulation of cardioprotective antioxidant pathways, and the onset of terminal senescence in adult TazPM hearts. Herein, we report a BTHS genotype/phenotype correlation and reveal that absent Taz acyltransferase function is sufficient to drive progressive cardiomyopathy.
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Aciltransferasas , Síndrome de Barth , Cardiomiopatías , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Síndrome de Barth/patología , Animales , Ratones , Aciltransferasas/genética , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Masculino , Humanos , Mutación Puntual , Modelos Animales de Enfermedad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , FenotipoRESUMEN
Hypertrophic Cardiomyopathy (HCM) is a common inherited disorder that can lead to heart failure and sudden cardiac death, characterized at the histological level by focal areas of myocyte disarray, hypertrophy and fibrosis, and only a few disease-targeted therapies exist. To identify the focal and spatially restricted alterations in the transcriptional pathways and reveal novel therapeutic targets, we performed a spatial transcriptomic analysis of the areas of focal myocyte disarray compared to areas of normal tissue using a commercially available platform (GeoMx, nanoString). We analyzed surgical myectomy tissue from four patients with HCM and the control interventricular septum tissue from two unused organ donor hearts that were free of cardiovascular disease. Histological sections were reviewed by an expert pathologist, and 72 focal areas with varying degrees of myocyte disarray (normal, mild, moderate, severe) were chosen for analysis. Areas of interest were interrogated with the Human Cancer Transcriptome Atlas designed to profile 1800 transcripts. Differential expression analysis revealed significant changes in gene expression between HCM and the control tissue, and functional enrichment analysis indicated that these genes were primarily involved in interferon production and mitochondrial energetics. Within the HCM tissue, differentially expressed genes between areas of normal and severe disarray were enriched for genes related to mitochondrial energetics and the extracellular matrix in severe disarray. An analysis of the gene expression of the ligand-receptor pair revealed that the HCM tissue exhibited downregulation of platelet-derived growth factor (PDGF), NOTCH, junctional adhesion molecule, and CD46 signaling while showing upregulation of fibronectin, CD99, cadherin, and amyloid precursor protein signaling. A deconvolution analysis utilizing the matched single nuclei RNA-sequencing (snRNA-seq) data to determine cell type composition in areas of interest revealed significant differences in fibroblast and vascular cell composition in areas of severe disarray when compared to normal areas in HCM samples. Cell composition in the normal areas of the control tissue was also divergent from the normal areas in HCM samples, which was consistent with the differential expression results. Overall, our data identify novel and potential disease-modifying targets for therapy in HCM.
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Cardiomiopatía Hipertrófica , Trasplante de Corazón , Humanos , Transcriptoma , Donantes de Tejidos , Cardiomiopatía Hipertrófica/genética , Células MuscularesRESUMEN
Barth Syndrome, a rare X-linked disorder affecting 1:300,000 live births, results from defects in Tafazzin, an acyltransferase that remodels cardiolipin and is essential for mitochondrial respiration. Barth Syndrome patients develop cardiomyopathy, muscular hypotonia and cyclic neutropenia during childhood, rarely surviving to middle age. At present, no effective therapy exists, and downstream transcriptional effects of Tafazzin dysfunction are incompletely understood. To identify novel, cell-specific, pathological pathways that mediate heart dysfunction, we performed single-nucleus RNA-sequencing (snRNA-seq) on wild-type (WT) and Tafazzin-knockout (Taz-KO) mouse hearts. We determined differentially expressed genes (DEGs) and inferred predicted cell-cell communication networks from these data. Surprisingly, DEGs were distributed heterogeneously across the cell types, with fibroblasts, cardiomyocytes, endothelial cells, macrophages, adipocytes and pericytes exhibiting the greatest number of DEGs between genotypes. One differentially expressed gene was detected for the lymphatic endothelial and mesothelial cell types, while no significant DEGs were found in the lymphocytes. A Gene Ontology (GO) analysis of these DEGs showed cell-specific effects on biological processes such as fatty acid metabolism in adipocytes and cardiomyocytes, increased translation in cardiomyocytes, endothelial cells and fibroblasts, in addition to other cell-specific processes. Analysis of ligand-receptor pair expression, to infer intercellular communication patterns, revealed the strongest dysregulated communication involved adipocytes and cardiomyocytes. For the knockout hearts, there was a strong loss of ligand-receptor pair expression involving adipocytes, and cardiomyocyte expression of ligand-receptor pairs underwent reorganization. These findings suggest that adipocyte and cardiomyocyte mitochondria may be most sensitive to mitochondrial Tafazzin deficiency and that rescuing adipocyte mitochondrial dysfunction, in addition to cardiomyocyte mitochondrial dysfunction, may provide therapeutic benefit in Barth Syndrome patients.
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Síndrome de Barth , Cardiomiopatías , Ratones , Animales , Síndrome de Barth/metabolismo , Células Endoteliales/metabolismo , Ligandos , Transcriptoma , Modelos Animales de Enfermedad , Aciltransferasas/genética , Cardiolipinas/metabolismo , Ratones Noqueados , Comunicación CelularRESUMEN
Hypertrophic cardiomyopathy is one of the most common inherited cardiomyopathies and a leading cause of sudden cardiac death in young adults. Despite profound insights into the genetics, there is imperfect correlation between mutation and clinical prognosis, suggesting complex molecular cascades driving pathogenesis. To investigate this, we performed an integrated quantitative multi-omics (proteomic, phosphoproteomic, and metabolomic) analysis to illuminate the early and direct consequences of mutations in myosin heavy chain in engineered human induced pluripotent stem-cell-derived cardiomyocytes relative to late-stage disease using patient myectomies. We captured hundreds of differential features, which map to distinct molecular mechanisms modulating mitochondrial homeostasis at the earliest stages of pathobiology, as well as stage-specific metabolic and excitation-coupling maladaptation. Collectively, this study fills in gaps from previous studies by expanding knowledge of the initial responses to mutations that protect cells against the early stress prior to contractile dysfunction and overt disease.
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Cardiomiopatía Hipertrófica , Células Madre Pluripotentes Inducidas , Adulto Joven , Humanos , Dinámicas Mitocondriales , Multiómica , Proteómica , Cardiomiopatía Hipertrófica/genética , Miocitos Cardíacos/metabolismo , Mutación , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
Barth Syndrome is an X-linked disorder of mitochondrial cardiolipin metabolism caused by pathogenic variants in TAFAZZIN with pleiotropic effects including cardiomyopathy, neutropenia, growth delay, and skeletal myopathy. Management requires a multidisciplinary approach to the organ-specific manifestations including specialists from cardiology, hematology, nutrition, physical therapy, genetics, and metabolism. Currently, treatment is centered on management of specific clinical features, and is not targeted toward remediating the underlying biochemical defect. However, two clinical trials have been recently undertaken which target the mitochondrial pathology of this disease: a study to examine the effects of elamipretide, a cardiolipin targeted agent, and a study to examine the effects of bezafibrate, a peroxisome proliferator-activated receptor (PPAR) agonist. Treatments to directly target the defective TAFAZZIN pathway are under development, including enzyme and gene therapies.
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Síndrome de Barth/terapia , Bezafibrato/uso terapéutico , Oligopéptidos/uso terapéutico , Aciltransferasas/genética , Animales , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Cardiolipinas/metabolismo , Cardiomiopatías/metabolismo , Cardiomiopatías/terapia , Ensayos Clínicos como Asunto , Terapia Enzimática , Terapia Genética , Humanos , Ratones , Enfermedades Musculares/metabolismo , Enfermedades Musculares/terapia , Neutropenia/metabolismo , Neutropenia/terapia , Receptores Activados del Proliferador del Peroxisoma/agonistasRESUMEN
Hypertrophic Cardiomyopathy (HCM) is a common inherited disorder characterized by unexplained left ventricular hypertrophy with or without left ventricular outflow tract (LVOT) obstruction. Single-nuclei RNA-sequencing (snRNA-seq) of both obstructive and nonobstructive HCM patient samples has revealed alterations in communication between various cell types, but no direct and integrated comparison between the two HCM phenotypes has been reported. We performed a bioinformatic analysis of HCM snRNA-seq datasets from obstructive and nonobstructive patient samples to identify differentially expressed genes and distinctive patterns of intercellular communication. Differential gene expression analysis revealed 37 differentially expressed genes, predominantly in cardiomyocytes but also in other cell types, relevant to aging, muscle contraction, cell motility, and the extracellular matrix. Intercellular communication was generally reduced in HCM, affecting the extracellular matrix, growth factor binding, integrin binding, PDGF binding, and SMAD binding, but with increases in adenylate cyclase binding, calcium channel inhibitor activity, and serine-threonine kinase activity in nonobstructive HCM. Increases in neuron to leukocyte and dendritic cell communication, in fibroblast to leukocyte and dendritic cell communication, and in endothelial cell communication to other cell types, largely through changes in the expression of integrin-ß1 and its cognate ligands, were also noted. These findings indicate both common and distinct physiological mechanisms affecting the pathogenesis of obstructive and nonobstructive HCM and provide opportunities for the personalized management of different HCM phenotypes.
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Cardiomiopatía Hipertrófica/genética , Redes Reguladoras de Genes , Hipertrofia Ventricular Izquierda/genética , Análisis de Secuencia de ARN/métodos , Obstrucción del Flujo Ventricular Externo/genética , Comunicación Celular , Biología Computacional , Bases de Datos Genéticas , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Humanos , Masculino , Análisis de la Célula IndividualRESUMEN
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a common inherited heart disorder complicated by left ventricle outflow tract (LVOT) obstruction, which can be treated with surgical myectomy. To date, no reliable biomarkers for LVOT obstruction exist. We hypothesized that metabolomic biomarkers for LVOT obstruction may be detectable in plasma from HCM patients. METHODS: We conducted metabolomic profiling on plasma samples of 18 HCM patients before and after surgical myectomy, using a commercially available metabolomics platform. RESULTS: We found that 215 metabolites were altered in the postoperative state (p-value < 0.05). 12 of these metabolites were notably significant after adjusting for multiple comparisons (q-value < 0.05), including bilirubin, PFOS, PFOA, 3,5-dichloro-2,6-dihydroxybenzoic acid, 2-hydroxylaurate, trigonelline and 6 unidentified compounds, which support improved organ metabolic function and increased lean soft tissue mass. CONCLUSIONS: These findings suggest improved organ metabolic function after surgical relief of LVOT obstruction in HCM and further underscore the beneficial systemic effects of surgical myectomy.
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Procedimientos Quirúrgicos Cardíacos , Cardiomiopatía Hipertrófica/sangre , Cardiomiopatía Hipertrófica/cirugía , Metaboloma , Metabolómica , Obstrucción del Flujo Ventricular Externo/sangre , Obstrucción del Flujo Ventricular Externo/cirugía , Adulto , Anciano , Biomarcadores/sangre , Cardiomiopatía Hipertrófica/diagnóstico , Cardiomiopatía Hipertrófica/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Recuperación de la Función , Resultado del Tratamiento , Función Ventricular Izquierda , Obstrucción del Flujo Ventricular Externo/diagnóstico , Obstrucción del Flujo Ventricular Externo/fisiopatologíaRESUMEN
Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder, affecting 1 in 500 people in the general population. Although characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray, and cardiac fibrosis, HCM is in fact a highly complex disease with heterogenous clinical presentation, onset, and complications. While HCM is generally accepted as a disease of the sarcomere, variable penetrance in families with identical genetic mutations challenges the monogenic origin of HCM and instead implies a multifactorial cause. Furthermore, large-scale genome sequencing studies revealed that many genes previously reported as causative of HCM in fact have little or no evidence of disease association. These findings thus call for a re-evaluation of the sarcomere-centered view of HCM pathogenesis. Here, we summarize our current understanding of sarcomere-independent mechanisms of cardiomyocyte hypertrophy, highlight the role of extracellular signals in cardiac fibrosis, and propose an alternative but integrated model of HCM pathogenesis.
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Cardiomiopatía Hipertrófica/patología , Predisposición Genética a la Enfermedad , Fenotipo , Sarcómeros/patología , Cardiomiopatía Hipertrófica/etiología , HumanosRESUMEN
Left Ventricular Outflow Tract (LVOT) obstruction occurs in approximately 70% of Hypertrophic Cardiomyopathy (HCM) patients and currently requires imaging or invasive testing for diagnosis, sometimes in conjunction with provocative physiological or pharmaceutical stimuli. To identify potential biomarkers of LVOT obstruction, we performed proteomics profiling of 1305 plasma proteins in 12 HCM patients with documented LVOT obstruction, referred for surgical myectomy. Plasma was collected at the surgical preoperative visit, approximately one month prior to surgery and then at the post-surgical visit, approximately 3 months later. Proteomic profiles were generated using the aptamer-based SOMAscan assay. Principal Component Analysis using the highest statistically significant proteins separated all preoperative samples from all postoperative samples. Further analysis revealed a set of 25 proteins that distinguished the preoperative and postoperative states with a paired t-test p-value of <0.01. Ingenuity Pathway analysis facilitated the generation of protein interaction networks and the elucidation of key upstream regulators of differentially expressed proteins, such as interferon-γ, TGF-ß1, and TNF. Biological pathways affected by surgery included organ inflammation, migration, and motility of leukocytes, fibrosis, vasculogenesis, angiogenesis, acute coronary events, endothelial proliferation, eicosanoid metabolism, calcium flux, apoptosis, and morphology of the cardiovascular system. Our results indicate that surgical relief of dynamic outflow tract obstruction in HCM patients is associated with unique alterations in plasma proteomic profiles that likely reflect improvement in organ inflammation and physiological function.
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Biomarcadores/sangre , Procedimientos Quirúrgicos Cardíacos/métodos , Cardiomiopatía Hipertrófica/cirugía , Inflamación/prevención & control , Proteoma/análisis , Adulto , Anciano , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Femenino , Humanos , Inflamación/sangre , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Developmental exposure to particulate matter air pollution is harmful to cardiovascular health, but the mechanisms by which this exposure mediates susceptibility to heart disease is poorly understood. We have previously shown, in a mouse model, that gestational exposure to diesel exhaust (DE) results in increased cardiac hypertrophy, fibrosis and susceptibility to heart failure in the adult offspring following transverse aortic constriction. RESULTS: In this study, we have analyzed gene expression in neonatal cardiomyocytes after gestational exposure by RNA-sequencing and have identified 300 genes that are dysregulated, including many involved in cardiac metabolism. We subsequently determined that these cardiomyocytes exhibit reduced metabolic activity as measured by Seahorse extracellular flux analysis. We also surveyed for modifications in DNA methylation at global regulatory regions using reduced representation bisulfite sequencing and found hypomethylation of DNA in neonatal cardiomyocytes isolated from in utero DE exposed neonates. CONCLUSION: We have demonstrated that in utero exposure to diesel exhaust alters the neonatal cardiomyocyte transcriptional and epigenetic landscapes, as well as the metabolic capability of these cells. Understanding how exposure alters the developing heart through dysregulation of gene expression, metabolism and DNA methylation is vital for identifying therapeutic interventions for air pollution-related heart failure.
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Metilación de ADN/efectos de los fármacos , Metaboloma/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Material Particulado/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Transcriptoma/efectos de los fármacos , Emisiones de Vehículos/toxicidad , Animales , Animales Recién Nacidos , Femenino , Exposición por Inhalación/efectos adversos , Exposición Materna/efectos adversos , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/metabolismoRESUMEN
Mutations in the gene tafazzin (TAZ) result in Barth syndrome (BTHS). Patients present with hypotonia, cyclic neutropenia, 3-methyglutaconic aciduria, and cardiomyopathy, which is the major cause of mortality. The recessive, X-linked TAZ gene encodes a mitochondrial membrane-associated phospholipid modifying enzyme, which adds unsaturated fatty acid species to monolysocardiolipin to generate mature cardiolipin in the mitochondrial membrane that is essential for mitochondrial morphology and function. To identify intrinsic mitochondrial localization sequences in the human TAZ protein, we made sequential TAZ peptide-eGFP fusion protein expression constructs and analyzed the localization of eGFP fluorescence by confocal microscopy. We assessed these fusion proteins for mitochondrial localization through cotransfection of H9c2 cells with plasmids encoding organellar markers linked to TdTomato. We have identified two peptides of TAZ that are independently responsible for mitochondrial localization. Using CRISPR-generated TAZ knock out cell lines, we found that these peptides are able to direct proteins to mitochondria in the absence of endogenous TAZ. These peptides are not located within the predicted enzymatic clefts of TAZ, implying that some BTHS disease causing mutations may affect mitochondrial localization without affecting transacylase activity. These novel peptides improve our understanding of TAZ intracellular trafficking, provide insight into the molecular basis of BTHS and provide molecular reagents for developing targeted mitochondrial therapies.
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Síndrome de Barth/metabolismo , Cardiomiopatías/metabolismo , Mitocondrias/metabolismo , Señales de Clasificación de Proteína , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Aciltransferasas , Animales , Secuencia de Bases , Línea Celular , Femenino , Técnicas de Inactivación de Genes , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mutación Missense/genética , Miocitos Cardíacos/metabolismo , Péptidos/metabolismo , Isoformas de Proteínas/metabolismo , Ratas , Factores de Transcripción/genéticaRESUMEN
In utero exposure to diesel exhaust air pollution has been associated with increased adult susceptibility to heart failure in mice, but the mechanisms by which this exposure promotes susceptibility to heart failure are poorly understood. To identify the potential transcriptional effects that mediate this susceptibility, we have performed RNA sequencing analysis on adult hearts from mice that were exposed to diesel exhaust in utero and that have subsequently undergone transverse aortic constriction. We identified 3 target genes, Mir133a-2, Ptprf, and Pamr1, which demonstrate dysregulation after exposure and aortic constriction. Examination of expression patterns in human heart tissues indicates a correlation between expression and heart failure. We subsequently assessed DNA methylation modifications at these candidate loci in neonatal cultured cardiac myocytes after in utero exposure to diesel exhaust and found that the promoter for Mir133a-2 is differentially methylated. These target genes in the heart are the first genes to be identified that likely play an important role in mediating adult sensitivity to heart failure. We have also shown a change in DNA methylation within cardiomyocytes as a result of in utero exposure to diesel exhaust.-Goodson, J. M., Weldy, C. S., MacDonald, J. W., Liu, Y., Bammler, T. K., Chien, W.-M., Chin, M. T. In utero exposure to diesel exhaust particulates is associated with an altered cardiac transcriptional response to transverse aortic constriction and altered DNA methylation.
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Enfermedades de la Aorta , Metilación de ADN/efectos de los fármacos , Exposición Materna/efectos adversos , Miocardio/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Transcripción Genética/efectos de los fármacos , Emisiones de Vehículos/toxicidad , Animales , Enfermedades de la Aorta/inducido químicamente , Enfermedades de la Aorta/congénito , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , MicroARNs/biosíntesis , Miocardio/patología , Embarazo , Efectos Tardíos de la Exposición Prenatal/patología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/biosíntesis , Serina Endopeptidasas/biosíntesis , Serina ProteasasRESUMEN
Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.
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Péptidos de Penetración Celular/administración & dosificación , Portadores de Fármacos/administración & dosificación , Proteínas Recombinantes/uso terapéutico , Animales , Membrana Celular/metabolismo , Péptidos de Penetración Celular/química , Ensayos Clínicos como Asunto , Portadores de Fármacos/química , Humanos , Transporte de Proteínas , Proteínas Recombinantes/químicaRESUMEN
CHF1/Hey2 is a Notch-responsive basic helix-loop-helix transcription factor involved in cardiac development. Common variants in Hey2 are associated with Brugada syndrome. We hypothesized that absence of CHF1/Hey2 would result in abnormal cellular electrical activity, altered cardiac conduction system (CCS) development, and increased arrhythmogenesis. We isolated neonatal CHF/Hey2-knockout (KO) cardiac myocytes and measured action potentials and ion channel subunit gene expression. We also crossed myocardial-specific CHF1/Hey2-KO mice with cardiac conduction system LacZ reporter mice and stained for conduction system tissue. We also performed ambulatory ECG monitoring for arrhythmias and heart rate variability. Neonatal cardiomyocytes from CHF1/Hey2-KO mice demonstrate a 50% reduction in action potential dV/dT, a 50-75% reduction in SCN5A, KCNJ2, and CACNA1C ion channel subunit gene expression, and an increase in delayed afterdepolarizations from 0/min to 12/min. CHF1/Hey2 cKO CCS-lacZ mice have a â¼3-fold increase in amount of CCS tissue. Ambulatory ECG monitoring showed no difference in cardiac conduction, arrhythmias, or heart rate variability. Wild-type cells or animals were used in all experiments. CHF1/Hey2 may contribute to Brugada syndrome by influencing the expression of SCN5A and formation of the cardiac conduction system, but its absence does not cause baseline conduction defects or arrhythmias in the adult mouse.-Hartman, M. E., Liu, Y., Zhu, W.-Z., Chien, W.-M., Weldy, C. S., Fishman, G. I., Laflamme, M. A., Chin, M. T. Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias.
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Potenciales de Acción/genética , Arritmias Cardíacas/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sistema de Conducción Cardíaco/anomalías , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Arritmias Cardíacas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Síndrome de Brugada , Trastorno del Sistema de Conducción Cardíaco , Sistema de Conducción Cardíaco/metabolismo , Frecuencia Cardíaca/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The Cre-loxP recombination system has been used to promote DNA recombination both in vitro and in vivo. For in vivo delivery, Cre expression is commonly achieved through the use of tissue/cell type-specific promoters, viral infection, or drug inducible transcription and protein translocation to promote targeted DNA excision. The development of cell permeable (or penetrating) peptide tagged proteins has facilitated the delivery of Cre recombinase protein into cells in culture, organotypic slide culture, or in living animals. In this report, we generated bacterially expressed, his-tagged Cre protein with either a cardiac targeting peptide or an antennapedia peptide at the C-terminus and demonstrated efficient uptake and recombination in both cell culture and mice. To facilitate delivery to cardiac and skeletal muscle, we mixed proteins with pluronic F-127 hydrogel and delivered Cre protein into reporter Rosa26mTmG mouse skeletal muscle or Rosa26LacZ cardiac muscle via ultrasound guided injection. Activation of reporter gene expression indicated that these Cre proteins were enzymatically active. Recombination events were detected only in the vicinity of injection areas. In conclusion, we have developed a method to deliver enzymatically active Cre protein locally to skeletal muscle and cardiac muscle that may be adapted for use with other proteins.
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Péptidos de Penetración Celular/metabolismo , Técnicas de Transferencia de Gen , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Animales , Técnicas de Cultivo de Célula , Integrasas/genética , Integrasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Recombinación GenéticaRESUMEN
OBJECTIVE: Elevated serum phosphate has emerged as a major risk factor for vascular calcification. The sodium-dependent phosphate cotransporter, PiT-1, was previously shown to be required for phosphate-induced osteogenic differentiation and calcification of cultured human vascular smooth muscle cells (VSMCs), but its importance in vascular calcification in vivo and the potential role of its homologue, PiT-2, have not been determined. We investigated the in vivo requirement for PiT-1 in vascular calcification using a mouse model of chronic kidney disease and the potential compensatory role of PiT-2 using in vitro knockdown and overexpression strategies. APPROACH AND RESULTS: Mice with targeted deletion of PiT-1 in VSMCs were generated (PiT-1(Δsm)). PiT-1 mRNA levels were undetectable, whereas PiT-2 mRNA levels were increased 2-fold in the vascular aortic media of PiT-1(Δsm) compared with PiT-1(flox/flox) control. When arterial medial calcification was induced in PiT-1(Δsm) and PiT-1(flox/flox) by chronic kidney disease followed by dietary phosphate loading, the degree of aortic calcification was not different between genotypes, suggesting compensation by PiT-2. Consistent with this possibility, VSMCs isolated from PiT-1(Δsm) mice had no PiT-1 mRNA expression, increased PiT-2 mRNA levels, and no difference in sodium-dependent phosphate uptake or phosphate-induced matrix calcification compared with PiT-1(flox/flox) VSMCs. Knockdown of PiT-2 decreased phosphate uptake and phosphate-induced calcification of PiT-1(Δsm) VSMCs. Furthermore, overexpression of PiT-2 restored these parameters in human PiT-1-deficient VSMCs. CONCLUSIONS: PiT-2 can mediate phosphate uptake and calcification of VSMCs in the absence of PiT-1. Mechanistically, PiT-1 and PiT-2 seem to serve redundant roles in phosphate-induced calcification of VSMCs.
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Músculo Liso Vascular/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Calcificación Vascular/fisiopatología , Animales , Aorta/citología , Aorta/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Fosfatos/metabolismo , ARN Mensajero/metabolismo , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Uremia/genética , Uremia/metabolismo , Uremia/fisiopatología , Calcificación Vascular/genética , Calcificación Vascular/metabolismoRESUMEN
[This corrects the article DOI: 10.3389/fcvm.2023.1223244.].
RESUMEN
BACKGROUND: The genetic basis of hypertrophic cardiomyopathy (HCM) is complex, and the relationship between genotype status and clinical outcome is incompletely resolved. METHODS AND RESULTS: We assessed a large international HCM cohort to define in contemporary terms natural history and clinical consequences of genotype. Consecutive patients (n=1468) with established HCM diagnosis underwent genetic testing. Patients with pathogenic (or likely pathogenic) variants were considered genotype positive (G+; n=312; 21%); those without definite disease-causing mutations (n=651; 44%) or variants of uncertain significance (n=505; 35%) were considered genotype negative (G-). Patients were followed up for a median of 7.8 years (interquartile range, 3.5-13.4 years); HCM end points were examined by cumulative event incidence. Over follow-up, 135 (9%) patients died, 33 from a variety of HCM-related causes. After adjusting for age, all-cause and HCM-related mortality did not differ between G- versus G+ patients (hazard ratio [HR], 0.78 [95% CI, 0.46-1.31]; P=0.37; HR, 0.93 [95% CI, 0.38-2.30]; P=0.87, respectively). Adverse event rates, including heart failure progression to class III/IV, heart transplant, or heart failure death, did not differ (G- versus G+) when adjusted for age (HR, 1.20 [95% CI, 0.63-2.26]; P=0.58), nor was genotype independently associated with sudden death event risk (HR, 1.39 [95% CI, 0.88-2.21]; P=0.16). In multivariable analysis, age was the only independent predictor of all-cause and HCM-related mortality, heart failure progression, and sudden death events. CONCLUSIONS: In this large consecutive cohort of patients with HCM, genotype (G+ or G-) was not a predictor of clinical course, including all-cause and HCM-related mortality and risk for heart failure progression or sudden death. G+ status should not be used to dictate clinical management or predict outcome in HCM.