RESUMO
RATIONALE: The regenerative potential of the heart is insufficient to fully restore functioning myocardium after injury, motivating the quest for a cell-based replacement strategy. Bone marrow-derived mesenchymal stem cells (MSCs) have the capacity for cardiac repair that appears to exceed their capacity for differentiation into cardiac myocytes. OBJECTIVE: Here, we test the hypothesis that bone marrow derived MSCs stimulate the proliferation and differentiation of endogenous cardiac stem cells (CSCs) as part of their regenerative repertoire. METHODS AND RESULTS: Female Yorkshire pigs (n=31) underwent experimental myocardial infarction (MI), and 3 days later, received transendocardial injections of allogeneic male bone marrow-derived MSCs, MSC concentrated conditioned medium (CCM), or placebo (Plasmalyte). A no-injection control group was also studied. MSCs engrafted and differentiated into cardiomyocytes and vascular structures. In addition, endogenous c-kit(+) CSCs increased 20-fold in MSC-treated animals versus controls (P<0.001), there was a 6-fold increase in GATA-4(+) CSCs in MSC versus control (P<0.001), and mitotic myocytes increased 4-fold (P=0.005). Porcine endomyocardial biopsies were harvested and plated as organotypic cultures in the presence or absence of MSC feeder layers. In vitro, MSCs stimulated c-kit(+) CSCs proliferation into enriched populations of adult cardioblasts that expressed Nkx2-5 and troponin I. CONCLUSIONS: MSCs stimulate host CSCs, a new mechanism of action underlying successful cell-based therapeutics.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Animais , Biópsia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Vasos Coronários/citologia , Vasos Coronários/fisiologia , Meios de Cultivo Condicionados/farmacologia , Feminino , Proteínas de Fluorescência Verde/genética , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Regeneração/fisiologia , Sus scrofaRESUMO
The mechanism(s) underlying cardiac reparative effects of bone marrow-derived mesenchymal stem cells (MSC) remain highly controversial. Here we tested the hypothesis that MSCs regenerate chronically infarcted myocardium through mechanisms comprising long-term engraftment and trilineage differentiation. Twelve weeks after myocardial infarction, female swine received catheter-based transendocardial injections of either placebo (n = 4) or male allogeneic MSCs (200 million; n = 6). Animals underwent serial cardiac magnetic resonance imaging, and in vivo cell fate was determined by co-localization of Y-chromosome (Y(pos)) cells with markers of cardiac, vascular muscle, and endothelial lineages. MSCs engrafted in infarct and border zones and differentiated into cardiomyocytes as ascertained by co-localization with GATA-4, Nkx2.5, and alpha-sarcomeric actin. In addition, Y(pos) MSCs exhibited vascular smooth muscle and endothelial cell differentiation, contributing to large and small vessel formation. Infarct size was reduced from 19.3 +/- 1.7% to 13.9 +/- 2.0% (P < 0.001), and ejection fraction (EF) increased from 35.0 +/- 1.7% to 41.3 +/- 2.7% (P < 0.05) in MSC but not placebo pigs over 12 weeks. This was accompanied by increases in regional contractility and myocardial blood flow (MBF), particularly in the infarct border zone. Importantly, MSC engraftment correlated with functional recovery in contractility (R = 0.85, P < 0.05) and MBF (R = 0.76, P < 0.01). Together these findings demonstrate long-term MSC survival, engraftment, and trilineage differentiation following transplantation into chronically scarred myocardium. MSCs are an adult stem cell with the capacity for cardiomyogenesis and vasculogenesis which contribute, at least in part, to their ability to repair chronically scarred myocardium.
Assuntos
Cardiomiopatias/patologia , Isquemia/patologia , Células-Tronco Mesenquimais/citologia , Cromossomo Y/metabolismo , Actinas/metabolismo , Animais , Células da Medula Óssea , Diferenciação Celular , Sobrevivência Celular , Feminino , Fator de Transcrição GATA4/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Masculino , Placebos , Suínos , Porco Miniatura , Fatores de Transcrição/metabolismoRESUMO
The mechanism(s) regulating nitric oxide synthase-1 (NOS1) localization within the cardiac myocyte in health and disease remains unknown. Here we tested the hypothesis that the PDZ-binding domain interaction between CAPON (carboxy-terminal PDZ ligand of NOS1), a NOS1 adaptor protein and NOS1, contribute to NOS1 localization in specific organelles within cardiomyocytes. Ventricular cardiomyocytes and whole heart homogenates were isolated from sham and post-myocardial infarction (MI) wild-type (C57BL/6) and NOS1(-/-) female mice for quantification of CAPON protein expression levels. NOS1, CAPON, xanthine oxidoreductase and Dexras1, a CAPON binding partner, were all present and enriched in isolated cardiac sarcoplasmic reticulum (SR) fractions. CAPON co-immunoprecipitated with the mu and alpha isoforms of NOS1 in whole heart lysates, and co-localization of CAPON and NOS1 was demonstrated in the SR and mitochondria with dual immuno-gold electron microscopy. Following MI, CAPON and NOS1 both redistributed to caveolae and colocalized with caveolin-3. In addition, following MI, expression level of CAPON remained unchanged and Dexras1 was reduced, CAPON binding to xanthine oxidoreductase was augmented and the plasma membrane calcium ATPase (PMCA) increased. In NOS1 deficient myocytes, CAPON abundance in the SR was reduced, and redistribution to caveolae and PMCA binding after MI was absent. Together these findings support the hypothesis that NOS1 redistribution in injured myocardium requires the formation of a complex with the PDZ adaptor protein CAPON.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Membrana Celular/metabolismo , Feminino , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo I/genética , Retículo Sarcoplasmático/metabolismoAssuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Cardiopatias/genética , Cardiopatias/terapia , Animais , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/fisiologia , Glicogênio Sintase Quinase 3 beta , Cardiopatias/fisiopatologia , Transplante de Células-Tronco Mesenquimais , Camundongos , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Whereas cardiac-derived c-kit(+) stem cells (CSCs) and bone marrow-derived mesenchymal stem cells (MSCs) are undergoing clinical trials testing safety and efficacy as a cell-based therapy, the relative therapeutic and biologic efficacy of these two cell types is unknown. We hypothesized that human CSCs have greater ability than MSCs to engraft, differentiate, and improve cardiac function. We compared intramyocardial injection of human fetal CSCs (36,000) with two doses of adult MSCs (36,000 and 1,000,000) or control (phosphate buffered saline) in nonobese diabetic/severe combined immune deficiency mice after coronary artery ligation. The myocardial infarction-induced enlargement in left ventricular chamber dimensions was ameliorated by CSCs (p < .05 for diastolic and systolic volumes), as was the decline in ejection fraction (EF; p < .05). Whereas 1 × 10(6) MSCs partially ameliorated ventricular remodeling and improved EF to a similar degree as CSCs, 36,000 MSCs did not influence chamber architecture or function. All cell therapies improved myocardial contractility, but CSCs preferentially reduced scar size and reduced vascular afterload. Engraftment and trilineage differentiation was substantially greater with CSCs than with MSCs. Adult-cultured c-kit(+)CSCs were less effective than fetal, but were still more potent than high-dose MSCs. These data demonstrate enhanced CSC engraftment, differentiation, and improved cardiac remodeling and function in ischemic heart failure. MSCs required a 30-fold greater dose than CSCs to improve cardiac function and anatomy. Together, these findings demonstrate a greater potency of CSCs than bone marrow MSCs in cardiac repair.
Assuntos
Células-Tronco Fetais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Infarto do Miocárdio/terapia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transplante de Células-Tronco , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Células Cultivadas , Cicatriz/metabolismo , Cicatriz/patologia , Cicatriz/terapia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Células-Tronco Fetais/fisiologia , Testes de Função Cardíaca , Hemodinâmica , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Contração Miocárdica , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Remodelação VentricularRESUMO
Cardiac injury induces myocardial expression of the thyroid hormone inactivating type 3 deiodinase (D3), which in turn dampens local thyroid hormone signaling. Here, we show that the D3 gene (Dio3) is a tissue-specific imprinted gene in the heart, and thus, heterozygous D3 knockout (HtzD3KO) mice constitute a model of cardiac D3 inactivation in an otherwise systemically euthyroid animal. HtzD3KO newborns have normal hearts but later develop restrictive cardiomyopathy due to cardiac-specific increase in thyroid hormone signaling, including myocardial fibrosis, impaired myocardial contractility, and diastolic dysfunction. In wild-type littermates, treatment with isoproterenol-induced myocardial D3 activity and an increase in the left ventricular volumes, typical of cardiac remodeling and dilatation. Remarkably, isoproterenol-treated HtzD3KO mice experienced a further decrease in left ventricular volumes with worsening of the diastolic dysfunction and the restrictive cardiomyopathy, resulting in congestive heart failure and increased mortality. These findings reveal crucial roles for Dio3 in heart function and remodeling, which may have pathophysiologic implications for human restrictive cardiomyopathy.
Assuntos
Cardiomiopatia Restritiva/metabolismo , Iodeto Peroxidase/metabolismo , Miocárdio/enzimologia , Animais , Animais Recém-Nascidos , Cardiomiopatia Restritiva/patologia , Cardiomiopatia Restritiva/fisiopatologia , Cardiotônicos/administração & dosagem , Cardiotônicos/farmacologia , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/crescimento & desenvolvimento , Coração/fisiopatologia , Insuficiência Cardíaca/etiologia , Infusões Intravenosas , Iodeto Peroxidase/genética , Isoproterenol/administração & dosagem , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , RNA Mensageiro/metabolismo , Remodelação VentricularRESUMO
Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.
Assuntos
Células-Tronco Embrionárias/transplante , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Ecocardiografia , Feminino , Estimativa de Kaplan-Meier , Camundongos , Infarto do Miocárdio/mortalidade , Miócitos Cardíacos/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Células-Tronco/métodosRESUMO
The underlying mechanism(s) of improved left ventricular function (LV) due to mesenchymal stem cell (MSC) administration after myocardial infarction (MI) remains highly controversial. Myocardial regeneration and neovascularization, which leads to increased tissue perfusion, are proposed mechanisms. Here we demonstrate that delivery of MSCs 3 days after MI increased tissue perfusion in a manner that preceded improved LV function in a porcine model. MI was induced in pigs by 60-min occlusion of the left anterior descending coronary artery, followed by reperfusion. Pigs were assigned to receive intramyocardial injection of allogeneic MSCs (200 million, approximately 15 injections) (n = 10), placebo (n = 6), or no intervention (n = 8). Resting myocardial blood flow (MBF) was serially assessed by first-pass perfusion magnetic resonance imaging (MRI) over an 8-wk period. Over the first week, resting MBF in the infarct area of MSC-treated pigs increased compared with placebo-injected and untreated animals [0.17 +/- 0.03, 0.09 +/- 0.01, and 0.08 +/- 0.01, respectively, signal intensity ratio of MI to left ventricular blood pool (LVBP); P < 0.01 vs. placebo, P < 0.01 vs. nontreated]. In contrast, the signal intensity ratios of the three groups were indistinguishable at weeks 4 and 8. However, MSC-treated animals showed larger, more mature vessels and less apoptosis in the infarct zones and improved regional and global LV function at week 8. Together these findings suggest that an early increase in tissue perfusion precedes improvements in LV function and a reduction in apoptosis in MSC-treated hearts. Cardiac MRI-based measures of blood flow may be a useful tool to predict a successful myocardial regenerative process after MSC treatment.