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Continuous outcomes were compared using MannCWhitney U test or linear regression and K

Human clinical tissue samples were obtained from NNTC. biology approaches to investigate the effect of Nef on cardiomyocyte-homeostasis by concentrating on protein quality control (PQC) pathway and autophagy. We found that HIV-1 Nef-mediated inhibition of autophagy flux leads to cytotoxicity and death of cardiomyocytes. Nef compromises autophagy at the maturation stage of autophagosomes by interacting SR9238 with Beclin 1/Rab7 and dysregulating TFEB localization and cellular lysosome content. These effects were reversed by rapamycin treatment. Our results indicate that HIV-1 Nef-mediated inhibition of cellular PQC is one possible mechanism involved in the development of HIV-associated cardiomyopathy. Introduction A large number of HIV-1 infected individuals develop cardiovascular diseases (CVD) with abnormal heart function causing significant morbidity and mortality1, 2. Antiretroviral therapy (ART) has dramatically improved the life span of HIV-1 infected patients, yet heart failure affects 7.4% of HIV-1 positive patients and is now a leading cause of death in HIV-1 infected persons3. HIV-1 patients show cardiomyopathy along with cardiac fibrosis4C8. HIV-1 can compromise cardiac function in the background of opportunistic infection, alcohol abuse, micronutrient deficiency and tobacco use9, 10, or native HIV-1 infected patients with or without SR9238 ART. HIV-1 has been detected in the heart, yet the mechanisms of any such direct HIV-mediated cardiomyopathy, and progression towards heart failure, are unknown11C13. Actively infected cells such as CD4+ T lymphocytes often release viral and cellular proteins with toxic activities that can be taken up by uninfected bystander cells leading to cell injury and death14C16. These observations have led to speculation that an indirect pathway potentially involving viral proteins may contribute to disease in some cell and tissue types including heart17C21. Nef is a 27?kDa HIV-1 auxiliary protein that localizes to the perinuclear space and distributes diffusely within the host cell membrane and cytosol22C24. This protein is relevant due to its important roles in HIV-1 replication, pathogenesis, reactivation and sexual transmission25C27. Even after ART, Nef is expressed by the proviral DNA and secreted to the extracellular environment28 and is present in high concentrations in the serum of HIV-1 positive patients, providing likely access to heart and other organs14, 17, 29C32. Nef may also be produced locally by HIV-1 infected immune cells that persist within the hearts of HIV-1 patients33. Additionally, Nef is sequestered within exosomes released from HIV-1 infected cells, and causes cell toxicity and apoptosis14, 17, 31, 34. The molecular and cellular events associated with cardiomyocyte injury and heart failure in the context of HIV-1 remains elusive. Autophagy is a dynamic, self-digestive ATF3 process important to cellular homeostasis that is responsible for regulating energy stores, clearing damaged organelles, and assuring protein quality control (PQC)35, 36. Various biological and physiological stimuli can dysregulate autophagy in myocardial cells causing adverse effects in heart muscle37, 38. HIV-1 manipulates autophagy in host cells to foster efficient viral production during an early stage of the viral life cycle, but at later stages may inhibit this pathway to promote host cell survival39C43. It is unknown whether such a mechanism contributes to cardiomyopathy in HIV-1 infected patients. In the present study we tested the hypothesis that Nef directly impairs cardiomyocyte autophagy, viability and cell death. We found that Nef dysregulates autophagy in cardiomyocytes and abolishes autophagic flux. Also, we found that rapamycin, an mTOR pathway inhibitor, improves autophagy flux in Nef-expressing cardiomyocytes and promotes autophagosome-lysosome fusion by enhancing lysosomal biogenesis44, 45. Together, these findings suggest that rapamycin treatment can induce autophagy and improve function in Nef-bearing cardiomyocytes, raising the novel possibility of its use to ameliorate cardiomyopathy in HIV-1 infected patients. Results Detection of Nef in cardiomyocytes and circulating blood from HIV-1 positive patients and SIV infected animals As a first step, we investigated the presence of Nef in cardiac tissue from HIV-1 patients and SIV-infected macaques. Confocal microscopy images of formalin-fixed heart tissue sections from HIV-1 positive individuals and control HIV-1 SR9238 negative subjects detected the presence of Nef within the HIV-1 positive individuals even in patients undergoing ART (Fig.?1A). This was further confirmed by Western blot analysis of protein extracts obtained from heart tissue of HIV-1 positive individuals (Fig.?1B). Similarly, hearts of SIV-infected and ART treated macaques showed the presence of SIV Nef protein in the cardiomyocytes (Fig.?1C and D). We also detected HIV-1 Nef protein by ELISA in the sera of HIV-1.