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1.
Methods Mol Biol ; 2319: 51-60, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331242

RESUMO

Cardiovascular disease is a worldwide health issue that affects millions of lives every year, and thus, researchers are in need of high-throughput model systems with which to investigate mechanisms of disease and to develop and test potential therapies. The use of human-derived induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes aims to address this need. While cardiac differentiation protocols have been established previously in iPSCs, optimization of cardiac differentiation remains crucial to obtaining high quality cardiomyocytes for future experimental analyses. Important factors to consider include cell density and rate of proliferation, temporal regulation of media changes throughout the differentiation process, and the concentration of the chemicals utilized. In this chapter, we present a detailed protocol to outline the process of differentiating cardiomyocytes from human iPSCs via modulation of Wnt signaling, characterization of cardiomyocytes by immunofluorescence, as well as guidelines for troubleshooting and optimizing these techniques.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Células-Tronco Pluripotentes Induzidas/citologia , Desenvolvimento Muscular , Miócitos Cardíacos/citologia , Via de Sinalização Wnt , Imunofluorescência , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo
2.
Hum Mol Genet ; 2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34270708

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by severe, progressive muscle wasting. Cardiomyopathy has emerged as a leading cause of death in patients with DMD. The mechanisms contributing to DMD cardiac disease remain under investigation and specific therapies available are lacking. Our prior work has shown that DMD-iPSC derived cardiomyocytes (DMD-iCMs) are vulnerable to oxidative stress injury and chronic exposure to DMD secreted exosomes impaired the cell's ability to protect against stress. In this study, we sought to examine a mechanism by which DMD cardiac exosomes impair cellular response through altering important stress-responsive genes in the recipient cells. Here, we report that DMD-iCMs secrete exosomes containing altered microRNA (miR) profiles in comparison to healthy controls. In particular, miR-339-5p was upregulated in DMD-iCMs, DMD exosomes, and in mdx mouse cardiac tissue. Restoring dystrophin in DMD-iCMs improved the cellular response to stress and was associated with downregulation of miR-339-5p, suggesting that it is disease-specific. Knockdown of miR-339-5p was associated with increased expression of MDM2, GSK3A and MAP2K3, which are genes involved in important stress-responsive signaling pathways. Finally, knockdown of miR-339-5p led to mitochondrial protection and a reduction in cell death in DMD-iCMs, indicating miR-339-5p is involved in direct modulation of stress-responsiveness. Together, these findings identify a potential mechanism by which exosomal miR-339-5p may be modulating cell signaling pathways which are important for robust stress responses. Additionally, these exosomal miRs may provide important disease specific targets for future therapeutic advancements for the management and diagnosis of DMD cardiomyopathy.

3.
BMC Cardiovasc Disord ; 21(1): 302, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34130633

RESUMO

BACKGROUND: Duchenne muscular dystrophy (DMD) associated cardiomyopathy is a major cause of morbidity and mortality. In an in vitro DMD cardiomyocyte model, nicorandil reversed stress-induced cell injury through multiple pathways implicated in DMD. We aimed to test the efficacy of nicorandil on the progression of cardiomyopathy in mdx mice following a 10-day treatment protocol. METHODS: A subset of mdx mice was subjected to low-dose isoproterenol injections over 5 days to induce a cardiac phenotype and treated with vehicle or nicorandil for 10 days. Baseline and day 10 echocardiograms were obtained to assess cardiac function. At 10 days, cardiac tissue was harvested for further analysis, which included histologic analysis and assessment of oxidative stress. Paired student's t test was used for in group comparison, and ANOVA was used for multiple group comparisons. RESULTS: Compared to vehicle treated mice, isoproterenol decreased ejection fraction and fractional shortening on echocardiogram. Nicorandil prevented isoproterenol induced cardiac dysfunction. Isoproterenol increased cardiac fibrosis, which nicorandil prevented. Isoproterenol increased gene expression of NADPH oxidase, which decreased to baseline with nicorandil treatment. Superoxide dismutase 2 protein expression increased in those treated with nicorandil, and xanthine oxidase activity decreased in mice treated with nicorandil during isoproterenol stress compared to all other groups. CONCLUSIONS: In conclusion, nicorandil is cardioprotective in mdx mice and warrants continued investigation as a therapy for DMD associated cardiomyopathy.


Assuntos
Cardiomiopatias/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Nicorandil/farmacologia , Volume Sistólico/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Feminino , Fibrose , Isoproterenol , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/complicações , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Xantina Oxidase/metabolismo
4.
JAMA Cardiol ; 6(7): 841-846, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33439236

RESUMO

Importance: Cardiac fibrosis is exceedingly rare in young adults. Identification of genetic variants that cause early-onset cardiomyopathy may inform novel biological pathways. Experimental models and a single case report have linked genetic deficiency of plasminogen activator inhibitor-1 (PAI-1), a downstream target of cardiac transforming growth factor ß, with cardiac fibrosis. Objective: To perform detailed cardiovascular phenotyping and genotyping in young adults from an Amish family with a frameshift variant (c.699_700dupTA) in SERPINE1, the gene that codes for PAI-1. Design, Setting, and Participants: This observational study included participants from 3 related nuclear families from an Amish community in the primary analysis and participants from the extended family in the secondary analysis. Participants were recruited from May 2015 to December 2016, and analysis took place from June 2015 to June 2020. Main Outcomes and Measures: (1) Multimodality cardiovascular imaging (transthoracic echocardiography and cardiac magnetic resonance imaging), (2) whole-exome sequencing, and (3) induced pluripotent stem cell-derived cardiomyocytes. Results: Among 17 participants included in the primary analysis, the mean (interquartile range) age was 23.7 (20.9-29.9) years and 9 individuals (52.9%) were confirmed to be homozygous for the SERPINE1 c.699_700dupTA variant. Late gadolinium enhancement was present in 6 of 9 homozygous participants (67%) with absolute PAI-1 deficiency vs 0 of 8 in the control group (P = .001). Late gadolinium enhancement patterns tended to be dense and linear, usually subepicardial but also midmyocardial and transmural with noncoronary distributions. Targeted whole-exome sequencing analysis identified that homozygosity for c.699_700dupTA SERPINE1 was the only shared pathogenic variant or variant of uncertain significance after examination of cardiomyopathy genes among those with late gadolinium enhancement. Induced pluripotent stem cell-derived cardiomyocytes from participants homozygous for the SERPINE1 c.699_700dupTA variant exhibited susceptibility to cardiomyocyte injury in response to angiotensin II (increased transforming growth factor ß1 secretion and release of lactate dehydrogenase) compared with control induced pluripotent stem cell-derived cardiomyocytes. In a secondary analysis based on echocardiography in 155 individuals across 3 generations in the extended family, no difference in global longitudinal strain was observed in carriers for the SERPINE1 c.699_700dupTA variant compared with wild-type participants, supporting an autosomal recessive inheritance pattern. Conclusions and Relevance: In this study, a highly penetrant, autosomal recessive, cardiac fibrosis phenotype among young adults with homozygous frameshift variant for SERPINE1 was identified, suggesting an optimal range of PAI-1 levels are needed for cardiac homeostasis.

5.
Front Oncol ; 10: 506739, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33178571

RESUMO

Purpose/Objectives: Node-positive breast cancer patients often receive chemotherapy and regional nodal irradiation. The cardiotoxic effects of these treatments, however, may offset some of the survival benefit. Cardiac magnetic resonance (CMR) is an emerging modality to assess cardiac injury. This is a pilot trial assessing cardiac damage using CMR in patients who received anthracycline-based chemotherapy and three-dimensional conformal radiotherapy (3DCRT) regional nodal irradiation using heart constraints. Materials and Methods: Node-positive breast cancer patients (2000-2008) treated with anthracycline-based chemotherapy and 3DCRT regional nodal irradiation (including the internal mammary chain nodes) with heart ventricular constraints (V25 < 10%) were invited to participate. Cardiac tissues were contoured and analyzed separately for whole heart (pericardium) and for combined ventricles and left atrium (myocardium). CMR obtained ventricular function/dimensions, late gadolinium enhancement (LGE), global longitudinal strain (GLS), and extracellular volume fraction (ECV) as measures of cardiac injury and/or early fibrosis. CMR parameters were correlated with dose-volume constraints using Spearman correlations. Results: Fifteen left-sided and five right-sided patients underwent CMR. Median diagnosis age was 50 (32-77). No patients had baseline cardiac disease before regional nodal irradiation. Median time after 3DCRT was 8.3 years (5.2-14.4). Median left-sided mean heart dose (MHD) was 4.8 Gy (1.1-11.2) and V25 was 5.7% (0-12%). Median left ventricular ejection fraction (LVEF) was 63%. No abnormal LGE was observed. No correlations were seen between whole heart doses and LVEF, LV mass, GLS, or LV dimensions. Increasing ECV did not correlate with increased heart or ventricular doses. However, correlations between higher LV mass and ventricular mean dose, V10, and V25 were seen. Conclusion: At a median follow-up of 8.3 years, this cohort of node-positive breast cancer patients who received anthracycline-based chemotherapy and regional nodal irradiation had no clinically abnormal CMR findings. However, correlations between ventricular mean dose, V10, and V25 and LV mass were seen. Larger corroborating studies that include advanced techniques for measuring regional heart mechanics are warranted.

6.
Dis Model Mech ; 13(11)2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188007

RESUMO

Cardiomyopathy is a leading cause of early mortality in Duchenne muscular dystrophy (DMD). There is a need to gain a better understanding of the molecular pathogenesis for the development effective therapies. Exosomes (exo) are secreted vesicles and exert effects via their RNA, lipid and protein cargo. The role of exosomes in disease pathology is unknown. Exosomes derived from stem cells have demonstrated cardioprotection in the murine DMD heart. However, it is unknown how the disease status of the donor cell type influences exosome function. Here, we sought to determine the phenotypic responses of DMD cardiomyocytes (DMD-iCMs) after long-term exposure to DMD cardiac exosomes (DMD-exo). DMD-iCMs were vulnerable to stress, evidenced by production of reactive oxygen species, the mitochondrial membrane potential and cell death levels. Long-term exposure to non-affected exosomes (N-exo) was protective. By contrast, long-term exposure to DMD-exo was not protective, and the response to stress improved with inhibition of DMD-exo secretion in vitro and in vivo The microRNA (miR) cargo, but not exosome surface peptides, was implicated in the pathological effects of DMD-exo. Exosomal surface profiling revealed N-exo peptides associated with PI3K-Akt signaling. Transcriptomic profiling identified unique changes with exposure to either N- or DMD-exo. Furthermore, DMD-exo miR cargo regulated injurious pathways, including p53 and TGF-beta. The findings reveal changes in exosomal cargo between healthy and diseased states, resulting in adverse outcomes. Here, DMD-exo contained miR changes, which promoted the vulnerability of DMD-iCMs to stress. Identification of these molecular changes in exosome cargo and effectual phenotypes might shed new light on processes underlying DMD cardiomyopathy.This article has an associated First Person interview with the first author of the paper.

7.
BMC Cardiovasc Disord ; 20(1): 367, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32795252

RESUMO

BACKGROUND: Subclinical diastolic dysfunction is a precursor for developing heart failure with preserved ejection fraction (HFpEF); yet not all patients progress to HFpEF. Our objective was to evaluate clinical and echocardiographic variables to identify patients who develop HFpEF. METHODS: Clinical, laboratory, and echocardiographic data were retrospectively collected for 81 patients without HF and 81 matched patients with HFpEF at the time of first documentation of subclinical diastolic dysfunction. Density-based clustering or hierarchical clustering to group patients was based on 65 total variables including 19 categorical and 46 numerical variables. Logistic regression analysis was conducted on the entire study population as well as each individual cluster to identify independent predictors of HFpEF. RESULTS: Unsupervised clustering identified 3 subgroups which differed in gender composition, severity of cardiac hypertrophy and aortic stenosis, NT-proBNP, percentage of patients who progressed to HFpEF, and timing of disease progression from diastolic dysfunction to HFpEF to death. Clusters that had higher percentages of women had progressively milder cardiac hypertrophy, less severe aortic stenosis, lower NT-proBNP, were diagnosed at an older age with HFpEF, and survived to an older age. Independent predictors of HFpEF for the entire cohort included diabetes, chronic kidney disease, atrial fibrillation, and diuretic use, with additional predictive variables found for each cluster. CONCLUSIONS: Cluster analysis can identify phenotypically distinct subgroups of patients with diastolic dysfunction. Clusters differ in HFpEF and mortality outcome. In addition, the variables that correlate with and predict HFpEF outcome differ among clusters.


Assuntos
Aprendizado Profundo , Diagnóstico por Computador , Insuficiência Cardíaca/diagnóstico , Volume Sistólico , Aprendizado de Máquina não Supervisionado , Disfunção Ventricular Esquerda/diagnóstico , Função Ventricular Esquerda , Idoso , Idoso de 80 Anos ou mais , Doenças Assintomáticas , Biomarcadores/sangue , Análise por Conglomerados , Diástole , Progressão da Doença , Ecocardiografia , Feminino , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Valor Preditivo dos Testes , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Disfunção Ventricular Esquerda/fisiopatologia
8.
J Muscle Res Cell Motil ; 41(4): 269-284, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-31564031

RESUMO

microRNAs are short, (18-22 nt) non-coding RNAs involved in important cellular processes due to their ability to regulate gene expression at the post-transcriptional level. Exosomes are small (50-200 nm) extracellular vesicles, naturally secreted from a variety of living cells and are believed to mediate cell-cell communication through multiple mechanisms, including uptake in destination cells. Circulating microRNAs and exosome-derived microRNAs can have key roles in regulating muscle cell development and differentiation. Several microRNAs are highly expressed in muscle and their regulation is important for myocyte homeostasis. Changes in muscle associated microRNA expression are associated with muscular diseases including muscular dystrophies, inflammatory myopathies, and congenital myopathies. In this review, we aim to highlight the biology of microRNAs and exosomes as well as their roles in muscle health and diseases. We also discuss the potential crosstalk between skeletal and cardiac muscle through exosomes and their contents.

9.
J Am Heart Assoc ; 8(22): e012792, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31718444

RESUMO

Background The SNRK (sucrose-nonfermenting-related kinase) enzyme is critical for cardiac function. However, the underlying cause for heart failure observed in Snrk cardiac conditional knockout mouse is unknown. Methods and Results Previously, 6-month adult mice knocked out for Snrk in cardiomyocytes (CMs) displayed left ventricular dysfunction. Here, 4-month adult mice, on angiotensin II (Ang II) infusion, show rapid decline in cardiac systolic function, which leads to heart failure and death in 2 weeks. These mice showed increased expression of nuclear factor κ light chain enhancer of activated B cells (NF-κB), inflammatory signaling proteins, proinflammatory proteins in the heart, and fibrosis. Interestingly, under Ang II infusion, mice knocked out for Snrk in endothelial cells did not show significant systolic or diastolic dysfunction. Although an NF-κB inflammation signaling pathway was increased in Snrk knockout endothelial cells, this did not lead to fibrosis or mortality. In hearts of adult mice knocked out for Snrk in CMs, we also observed NF-κB pathway activation in CMs, and an increased presence of Mac2+ macrophages was observed in basal and Ang II-infused states. In vitro analysis of Snrk knockdown HL-1 CMs revealed similar upregulation of the NF-κB signaling proteins and proinflammatory proteins that was exacerbated on Ang II treatment. The Ang II-induced NF-κB pathway-mediated proinflammatory effects were mediated in part through protein kinase B or AKT, wherein AKT inhibition restored the proinflammatory signaling protein levels to baseline in Snrk knockdown HL-1 CMs. Conclusions During heart failure, SNRK acts as a cardiomyocyte-specific repressor of cardiac inflammation and fibrosis.


Assuntos
Células Endoteliais/metabolismo , Insuficiência Cardíaca/genética , Inflamação/genética , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/genética , Angiotensina II/farmacologia , Animais , Linhagem Celular , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Coração/efeitos dos fármacos , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Técnicas In Vitro , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , Miocárdio/patologia , Vasoconstritores/farmacologia , Disfunção Ventricular Esquerda
10.
Am J Physiol Heart Circ Physiol ; 316(6): H1267-H1280, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30848680

RESUMO

Radiation therapy is used in ~50% of cancer patients to reduce the risk of recurrence and in some cases improve survival. Despite these benefits, doses can be limited by toxicity in multiple organs, including the heart. The underlying causes and biomarkers of radiation-induced cardiotoxicity are currently unknown, prompting the need for experimental models with inherent differences in sensitivity and resistance to the development of radiation-induced cardiotoxicity. We have identified the parental SS (Dahl salt-sensitive/Mcwi) rat strain to be a highly-sensitized model of radiation-induced cardiotoxicity. In comparison, substitution of rat chromosome 3 from the resistant BN (Brown Norway) rat strain onto the SS background (SS-3BN consomic) significantly attenuated radiation-induced cardiotoxicity. SS-3BN rats had less radiation-induced cardiotoxicity than SS rats, as measured by survival, pleural and pericardial effusions, echocardiogram parameters, and histological damage. Mast cells, previously shown to have predominantly protective roles in radiation-induced cardiotoxicity, were increased in the more resistant SS-3BN hearts postradiation. RNA sequencing from SS and SS-3BN hearts at 1 wk postradiation revealed 5,098 differentially expressed candidate genes across the transcriptome and 350 differentially expressed genes on rat chromosome 3, which coincided with enrichment of multiple pathways, including mitochondrial dysfunction, sirtuin signaling, and ubiquitination. Upstream regulators of enriched pathways included the oxidative stress modulating transcription factor, Nrf2, which is located on rat chromosome 3. Nrf2 target genes were also differentially expressed in the SS vs. SS-3BN consomic hearts postradiation. Collectively, these data confirm the existence of heritable modifiers in radiation-induced cardiotoxicity and provide multiple biomarkers, pathways, and candidate genes for future analyses. NEW & NOTEWORTHY This novel study reveals that heritable genetic factors have the potential to modify normal tissue sensitivity to radiation. Gene variant(s) on rat chromosome 3 can contribute to enhanced cardiotoxicity displayed in the SS rats vs. the BN and SS-3BN consomic rats. Identifying genes that lead to understanding the mechanisms of radiation-induced cardiotoxicity represents a novel method to personalize radiation treatment, as well as predict the development of radiation-induced cardiotoxicity.


Assuntos
Mapeamento Cromossômico , Cromossomos de Mamíferos , Genes Modificadores , Variação Genética , Cardiopatias/genética , Lesões por Radiação/genética , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Cardiopatias/metabolismo , Cardiopatias/patologia , Masculino , Mastócitos/metabolismo , Mastócitos/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Lesões por Radiação/metabolismo , Lesões por Radiação/patologia , Ratos Endogâmicos BN , Ratos Endogâmicos Dahl , Transdução de Sinais
11.
Health Phys ; 116(4): 558-565, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30624347

RESUMO

Total-body irradiation causes acute and delayed toxicity to hematopoietic, pulmonary, cardiac, gastrointestinal, renal, and other organ systems. Angiotensin-converting enzyme inhibitors mitigate many of the delayed injuries to these systems. The purpose of this study was to define echocardiographic features in rats at two times after irradiation, the first before lethal radiation pneumonitis (50 d) and the second after recovery from pneumonitis but before lethal radiation nephropathy (100 d), and to determine the actions of the angiotensin-converting enzyme inhibitor lisinopril. Four groups of female WAG/RijCmcr rats at 11-12 wk of age were studied: nonirradiated, nonirradiated plus lisinopril, 13-Gy partial-body irradiation sparing one hind leg (leg-out partial-body irradiation), and 13-Gy leg-out partial-body irradiation plus lisinopril. Lisinopril was started 7 d after radiation. Echocardiograms were obtained at 50 and 100 d, and cardiac histology was assessed after 100 d. Irradiation without lisinopril demonstrated echocardiographic transient pulmonary hypertension by 50 d which was largely resolved by 100 d in survivors. Irradiated rats given lisinopril showed no increase in pulmonary artery pressures at 50 d but exhibited left ventricular remodeling. By 100 d these rats showed some signs of pulmonary hypertension. Lisinopril alone had no impact on echocardiographic end points at either time point in nonirradiated rats. Mild increases in mast cells and fibrosis in the heart were observed after 100 d following 13-Gy leg-out partial-body irradiation. These data demonstrate irradiation-induced pulmonary hypertension which was reversed in survivors of pneumonitis. Lisinopril modified cardiovascular remodeling to enhance survival in this model from 41% to 86% (p = 0.0013).


Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Hipertensão Pulmonar/etiologia , Lisinopril/uso terapêutico , Lesões Experimentais por Radiação/tratamento farmacológico , Pneumonite por Radiação/tratamento farmacológico , Remodelação Ventricular/efeitos da radiação , Animais , Ecocardiografia , Feminino , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/prevenção & controle , Miocárdio/patologia , Lesões Experimentais por Radiação/patologia , Lesões Experimentais por Radiação/prevenção & controle , Pneumonite por Radiação/complicações , Pneumonite por Radiação/prevenção & controle , Ratos
12.
JACC Case Rep ; 1(2): 213-217, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34316787

RESUMO

A 56-year-old man with multiple cardiac manifestations of type 1 myotonic dystrophy, including severe, nonischemic cardiomyopathy, presented in refractory cardiogenic shock requiring inotropic therapy. Given his wishes to die without having any intravenous medications, he was started on oral probenecid therapy, which allowed for successful elimination of his intravenous therapies. (Level of Difficulty: Intermediate.).

13.
Sci Rep ; 8(1): 16519, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30410044

RESUMO

As mediators of intercellular communication, exosomes containing molecular cargo are secreted by cells and taken up by recipient cells to influence cellular phenotype and function. Here we have investigated the effects of exosomes in dystrophin-deficient (Dys) induced pluripotent stem cell derived cardiomyocytes (iCMs). Our data demonstrate that exosomes secreted from either wild type (WT) or Dys-iCMs protect the Dys-iCM from stress-induced injury by decreasing reactive oxygen species and delaying mitochondrial permeability transition pore opening to maintain the mitochondrial membrane potential and decrease cell death. The protective effects of exosomes were dependent on the presence of exosomal surface proteins and activation of ERK1/2 and p38 MAPK signaling. Based on our findings, the acute effects of exosomes on recipient cells can be initiated from exosome membrane proteins and not necessarily their internal cargo.


Assuntos
Distrofina/deficiência , Exossomos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Sistema de Sinalização das MAP Quinases , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Distrofina/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Potencial da Membrana Mitocondrial , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/metabolismo , Deleção de Sequência , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Surg Obes Relat Dis ; 14(1): 117-122, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29108893

RESUMO

Heart failure due to severe obesity is a complex disease due to multiple mechanisms, including increased body mass, inflammation, and impaired cardiac metabolism that is complicated by obesity-associated co-morbidities, such as type 2 diabetes and obstructive sleep apnea. Bariatric surgery significantly improves cardiac geometry, function, and symptoms related to obesity cardiomyopathy. There is a consistently positive impact of bariatric surgery on diastolic function with the potential to significantly improve systolic function as measured by ejection fraction in patients with advanced heart failure. For end-stage heart failure patients, including those requiring mechanical circulatory support who are ineligible for organ transplant due to morbid obesity, bariatric surgery has been successfully used for weight loss as a bridge to cardiac transplantation.


Assuntos
Cirurgia Bariátrica , Insuficiência Cardíaca/cirurgia , Obesidade Mórbida/complicações , Cardiomiopatias/etiologia , Cardiomiopatias/cirurgia , Diabetes Mellitus Tipo 2/complicações , Insuficiência Cardíaca/etiologia , Transplante de Coração , Humanos , Obesidade Mórbida/cirurgia , Síndromes da Apneia do Sono/complicações , Tempo para o Tratamento , Resultado do Tratamento
16.
J Immunol ; 200(2): 523-537, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29229678

RESUMO

Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971-990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971-990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk By creating IAk/and IEk/SERCA2a 971-990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971-990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971-990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971-990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971-990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans.


Assuntos
Mapeamento de Epitopos , Epitopos/imunologia , Miocardite/imunologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/imunologia , Alelos , Animais , Proteínas de Bactérias , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , Imunofluorescência , Expressão Gênica , Átrios do Coração/imunologia , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Ventrículos do Coração/imunologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Epitopos Imunodominantes/imunologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos , Miocardite/diagnóstico por imagem , Miocardite/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Peptídeos/imunologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
17.
Front Immunol ; 8: 1567, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29209317

RESUMO

Myocarditis/dilated cardiomyopathy (DCM) patients can develop autoantibodies to various cardiac antigens and one major antigen is ß1-adrenergic receptor (ß1AR). Previous reports indicate that animals immunized with a ß1AR fragment encompassing, 197-222 amino acids for a prolonged period can develop DCM by producing autoantibodies, but existence of T cell epitopes, if any, were unknown. Using A/J mice that are highly susceptible to lymphocytic myocarditis, we have identified ß1AR 171-190, ß1AR 181-200, and ß1AR 211-230 as the major T cell epitopes that bind major histocompatibility complex class II/IAk or IEk alleles, and by creating IAk and IEk dextramers, we demonstrate that the CD4 T cell responses to be antigen-specific. Of note, all the three epitopes were found also to stimulate CD8 T cells suggesting that they can act as common epitopes for both CD4 and CD8 T cells. While, all epitopes induced only mild myocarditis, the disease-incidence was enhanced in animals immunized with all the three peptides together as a cocktail. Although, antigen-sensitized T cells produced mainly interleukin-17A, their transfer into naive animals yielded no disease. But, steering for T helper 1 response led the T cells reacting to one epitope, ß1AR 181-200 to induce severe myocarditis in naive mice. Finally, we demonstrate that all three ß1AR epitopes to be unique for T cells as none of them induced antibody responses. Conversely, animals immunized with a non-T cell activator, ß1AR 201-220, an equivalent of ß1AR 197-222, had antibodies comprising of all IgG isotypes and IgM except, IgA and IgE. Thus, identification of T cell and B cell epitopes of ß1AR may be helpful to determine ß1AR-reactive autoimmune responses in various experimental settings in A/J mice.

18.
Immun Inflamm Dis ; 5(4): 421-434, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28597552

RESUMO

INTRODUCTION: Organ-specific autoimmune diseases are believed to result from immune responses generated against self-antigens specific to each organ. However, when such responses target antigens expressed promiscuously in multiple tissues, then the immune-mediated damage may be wide spread. METHODS: In this report, we describe a mitochondrial protein, branched chain α-ketoacid dehydrogenase kinase (BCKDk ) that can act as a target autoantigen in the development of autoimmune inflammatory reactions in both heart and liver. RESULTS: We demonstrate that BCKDk protein contains at least nine immunodominant epitopes, three of which, BCKDk 71-90, BCKDk 111-130 and BCKDk 141-160, were found to induce varying degrees of myocarditis in immunized mice. One of these, BCKDk 111-130, could also induce hepatitis without affecting lungs, kidneys, skeletal muscles, and brain. In immunogenicity testing, all three peptides induced antigen-specific T cell responses, as verified by proliferation assay and/or major histocompatibility complex class II/IAk dextramer staining. Finally, the disease-inducing abilities of BCKDk peptides were correlated with the production of interferon-γ, and the activated T cells could transfer disease to naive recipients. CONCLUSIONS: The disease induced by BCKDk peptides could serve as a useful model to study the autoimmune events of inflammatory heart and liver diseases.


Assuntos
Doenças Autoimunes/imunologia , Epitopos de Linfócito T/imunologia , Hepatite Autoimune/imunologia , Miocardite/imunologia , Proteínas Quinases/imunologia , Sequência de Aminoácidos , Animais , Autoantígenos/química , Autoantígenos/imunologia , Doenças Autoimunes/patologia , Biópsia , Citocinas/metabolismo , Modelos Animais de Doenças , Epitopos de Linfócito T/química , Hepatite Autoimune/patologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Imunização , Camundongos , Miocardite/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Proteínas Quinases/química , Multimerização Proteica , Linfócitos T/imunologia , Linfócitos T/metabolismo
19.
Stem Cell Res ; 18: 33-36, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28395799

RESUMO

We have generated a human induced pluripotent stem cell (iPSC) line under feeder-free culture conditions using the urine derived cells (UCs) collected from non-affected control subjects to use as a comparison group for the iPSC lines containing a Plasminogen Activator Inhibitor-1 (PAI-1 homozygous/heterozygous) mutation. The Sendai Virus (SeV) vector encoding pluripotent Yamanaka transcription factors was used at a low multiplicity of infection to reprogram the UCs.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Urina/citologia , Sequência de Bases , Técnicas de Cultura de Células/métodos , Linhagem Celular , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Genótipo , Heterozigoto , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Microscopia de Fluorescência , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Vírus Sendai/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Stem Cell Res ; 18: 41-44, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28395801

RESUMO

We have generated a human induced pluripotent stem cell (iPSC) line under feeder-free culture conditions using the urine derived cells (UCs) collected from subjects heterozygous for a novel Plasminogen Activator Inhibitor-1 (PAI-1) mutation. The Sendai Virus (SeV) vector encoding pluripotent Yamanaka transcription factors was used at a low multiplicity of infection to reprogram the PAI-1 UCs.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Inibidor 1 de Ativador de Plasminogênio/genética , Urina/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Microscopia de Fluorescência , Polimorfismo Genético , Vírus Sendai/genética
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