RESUMO
Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.
Assuntos
Miofibrilas , Sarcômeros , Animais , Conectina/química , Conectina/genética , Conectina/metabolismo , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Humanos , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Ratos , Sarcômeros/metabolismoRESUMO
Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy-based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Doxorrubicina/administração & dosagem , Células-Tronco Embrionárias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Animais , Apoptose/efeitos dos fármacos , Cardiotoxicidade/etiologia , Cardiotoxicidade/prevenção & controle , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Células-Tronco Embrionárias/transplante , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Camundongos SCID , Espécies Reativas de Oxigênio/metabolismo , Teratoma/prevenção & controleRESUMO
PURPOSE: Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets polyubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. EXPERIMENTAL DESIGN: Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. RESULTS: We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. CONCLUSION AND CLINICAL RELEVANCE: This is the most comprehensive characterization of cardiac proteasome ubiquitination to date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes.