RESUMEN
Chaperone-mediated autophagy (CMA) is a chaperone-dependent process of selective cytosolic protein turnover that targets specific proteins to lysosomes for degradation. Enhancing protein degradation mechanisms has been shown to be beneficial in multiple models of cardiac disease, including myocardial infarction (MI) and ischemia-reperfusion (I/R) injury. However, the causal role of CMA in cardiomyocyte injury and death is largely unknown. Hypoxia is an important contributor to both MI and I/R damage, which are major, precedent causes of heart failure. Upregulating CMA was hypothesized to protect against hypoxia-induced cardiomyocyte death. Lysosome-associated membrane protein 2a (Lamp2a) overexpression and knockdown were used to causally study CMA's role in hypoxically stressed cardiomyocytes. LAMP2a protein levels were used as both a primary indicator and driver of CMA function. Hypoxic stress was stimulated by CoCl2 treatment, which increased LAMP2a protein levels (+1.4-fold) and induced cardiomyocyte apoptosis (+3.2-4.0-fold). Lamp2a siRNA knockdown (-3.2-fold) of control cardiomyocytes increased apoptosis (+1.8-fold) suggesting that loss of CMA is detrimental for cardiomyocyte survival. However, there was neither an additive nor a synergistic effect on cell death when Lamp2a-silenced cells were treated with CoCl2. Conversely, Lamp2a overexpression (+3.0-fold) successfully reduced hypoxia-induced apoptosis by â¼50%. LAMP2a was also significantly increased (+1.7-fold) in ischemic heart failure patient samples, similar to hypoxically stressed cardiomyocytes. The failing ischemic hearts may have had insufficient CMA activation. To our knowledge, this study for the first time establishes a protective role for CMA (via Lamp2a overexpression) against hypoxia-induced cardiomyocyte loss and reveals the intriguing possibility that CMA activation may offer a cardioprotective treatment for ischemic heart disease.
Asunto(s)
Autofagia Mediada por Chaperones , Insuficiencia Cardíaca , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Miocitos Cardíacos/metabolismo , Autofagia/genética , Lisosomas/metabolismo , Hipoxia/metabolismo , Apoptosis , Insuficiencia Cardíaca/metabolismoRESUMEN
There has been considerable interest in pursuing phospholamban as a putative therapeutic target for overcoming depressed calcium handling in human heart failure. Studies predominantly done in mice have shown that phospholamban is a key regulator of sarcoplasmic reticulum calcium cycling and cardiac function. However, mice differ significantly from humans in how they regulate calcium, whereas rabbits better recapitulate human cardiac function and calcium handling. To investigate phospholamban's role in the rabbit heart, transgenic rabbits that overexpressed wild-type phospholamban in the ventricular cardiomyocytes and slow-twitch skeletal muscles were generated. Rabbits expressing high levels of phospholamban were not viable due to severe skeletal muscle wasting, the onset of cardiac pathology and early death. A viable transgenic line exhibited a 30% increase in PLN protein levels in the heart. These animals showed isolated foci of cardiac pathology, but cardiac function as well as the response to beta-adrenergic stimulation were normal. SR-calcium uptake measurements showed that the transgenic hearts had the expected reduced affinity for calcium. The data show that phospholamban-overexpressing transgenic rabbits differ markedly in phenotype from analogous transgenic mice in that rabbits are quite sensitive to alterations in phospholamban levels. Exceeding a relatively narrow window of phospholamban expression results in significant morbidity and early death.