RESUMO
BACKGROUND: Matrix metalloproteinases (MMPs) contribute to left ventricular remodeling after myocardial infarction (MI). Specific causative roles of particular MMPs, however, remain unclear. MMP-7 is abundant in cardiomyocytes and macrophages, but MMP-7 function after MI has not been defined. METHODS AND RESULTS: Wild-type (WT; n=55) and MMP-7-null (MMP-7-/-; n=32) mice underwent permanent coronary artery ligation for 7 days. MI sizes were similar, but survival was greatly improved in MMP-7-/- mice. The survival difference could not be attributed to differences in left ventricular dilation because end-diastolic volumes increased similarly. ECG analysis revealed a prolonged PR interval in WT but not in MMP-7-/- post-MI mice. Post-MI conduction velocity, determined by optically mapping electrical wavefront propagation, decreased to 78+/-6% of control for WT and was normalized in MMP-7-/- mice. In WT mice, slower conduction velocity correlated with a 53% reduction in the gap junction protein connexin-43. Direct binding of MMP-7 to connexin-43, determined by surface plasmon resonance technology, occurred in a dose-dependent manner. Connexin-43 processing by MMP-7 was confirmed by in silico and in vitro substrate analyses and MMP-7 infusion induced arrhythmias in vivo. CONCLUSIONS: MMP-7 deletion results in improved survival and myocardial conduction patterns after MI. This is the first report to implicate MMP-7 in post-MI remodeling and to demonstrate that connexin-43 is a novel MMP-7 substrate.
Assuntos
Conexina 43/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Metaloproteinase 7 da Matriz/fisiologia , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/fisiopatologia , Sequência de Aminoácidos , Animais , Comunicação Celular , Relação Dose-Resposta a Droga , Condutividade Elétrica , Eletrocardiografia , Junções Comunicantes/química , Junções Comunicantes/patologia , Deleção de Genes , Bloqueio Cardíaco/etiologia , Bloqueio Cardíaco/fisiopatologia , Imuno-Histoquímica , Macrófagos/química , Macrófagos/citologia , Macrófagos/patologia , Metaloproteinase 7 da Matriz/análise , Metaloproteinase 7 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Infarto do Miocárdio/patologia , Miocárdio/química , Miocárdio/patologia , Miócitos Cardíacos/química , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Análise de Sobrevida , Remodelação Ventricular/fisiologiaRESUMO
The matrix metalloproteinases (MMPs), in particular, membrane type 1 MMP (MT1-MMP), are increased in the context of myocardial ischemia and reperfusion (I/R) and likely contribute to myocardial dysfunction. One potential upstream induction mechanism for MT1-MMP is endothelin (ET) release and subsequent protein kinase C (PKC) activation. Modulation of ET and PKC signaling with respect to MT1-MMP activity with I/R has yet to be explored. Accordingly, this study examined in vivo MT1-MMP activation during I/R following modification of ET signaling and PKC activation. With the use of a novel fluorogenic microdialysis system, myocardial interstitial MT1-MMP activity was measured in pigs (30 kg; n = 9) during I/R (90 min I/120 min R). Local ET(A) receptor antagonism (BQ-123, 1 microM) and PKC inhibition (chelerythrine, 1 microM) were performed in parallel microdialysis probes. MT1-MMP activity was increased during I/R by 122 +/- 10% (P < 0.05) and was unchanged from baseline with ET antagonism and/or PKC inhibition. Selective PKC isoform induction occurred such that PKC-betaII increased by 198 +/- 31% (P < 0.05). MT1-MMP phosphothreonine, a putative PKC phosphorylation site, was increased by 121 +/- 8% (P < 0.05) in the I/R region. These studies demonstrate for the first time that increased interstitial MT1-MMP activity during I/R is a result of the ET/PKC pathway and may be due to enhanced phosphorylation of MT1-MMP. These findings identify multiple potential targets for modulating a local proteolytic pathway operative during I/R.
Assuntos
Endotelinas/fisiologia , Metaloproteinase 14 da Matriz/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Transdução de Sinais/fisiologia , Animais , Antagonistas do Receptor de Endotelina B , Corantes Fluorescentes , Imunoprecipitação , Isoenzimas/metabolismo , Microdiálise , Traumatismo por Reperfusão Miocárdica/enzimologia , Peptídeos Cíclicos/farmacologia , Fosforilação , Proteína Quinase C/metabolismo , Receptor de Endotelina B/metabolismo , Volume Sistólico/fisiologia , Suínos , Treonina/metabolismoRESUMO
Adequate wound healing and scar formation is an essential response to myocardial infarction (MI), and fibroblasts are primary cellular components regulating the process. How fibroblast functions are altered post-MI and to what extent these abnormalities persist in vitro is not well understood. Accordingly, we isolated myocardial fibroblasts from MI and non-MI (remote) regions at 7 days post-MI (n=35) and from the free wall and septum of unoperated control C57BL/6 mice (n=14). Proliferation was increased 182+/-28% in MI, but not in remote, fibroblasts compared with unoperated controls (P=0.01). Migration decreased 61+/-8%, adhesion to laminin decreased 79+/-8%, adhesion to collagen IV increased 196+/-27%, and collagen synthesis increased 169+/-24% in fibroblasts isolated from the MI region (all P<0.05). Migration, adhesion, and collagen synthesis changes were similar in remote fibroblasts, and the phenotypic differences were maintained through passage four. Transforming growth factor beta1 (TGFbeta1) is a bioactive molecule that has been shown to affect fibroblast function. Stimulation of unoperated control fibroblasts with 10 ng/ml TGFbeta(1) increased proliferation 137+/-7% (P=0.03 vs. unstimulated), increased adhesion to collagen IV 149+/-6% (P<0.01), and increased collagen I levels 187+/-10% (P=0.01). TGFbeta1 may, therefore, explain some of the changes in post-MI fibroblast phenotype. These data demonstrate for the first time region specific alterations in post-MI fibroblast biology that are maintained in vitro. Additionally, our model provides a novel in vitro template for examining the cellular mechanisms of wound healing and scar formation post-MI.