Cardio-Metabolic Effects of HIV Protease Inhibitors (Lopinavir/Ritonavir) Kathleen M.S.E . Reyskens , Tarryn-Lee Fisher , Jonathan C. Schisler , Wendi G. O’Connor , Arlin B. Rogers , 1 1 2 2 2 Monte S. Willis 2 , Cynthia Planesse 3 , Florence Boyer 3 , Philippe Rondeau 3 , Emmanuel Bourdon 3 , M. Faadiel Essop 1 * 1 Cardio-Metabolic Research Group (CMRG), Department of Physiological Sciences, Stellenbosch University, Stellenbosch 7600, South Africa, 2 McAllister Heart Institute, Department of Pathology & Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina, USA, 3 Groupe d’Etude sur l’Inflammation Chronique et l’Obe ´ site ´ ´ de La Re ´ union, Saint Denis de La Re ´ union, France Abstract Although antiretroviral treatment decreases HIV-AIDS morbidity/mortality, long-term side effects may include the onset of insulin resistance and cardiovascular diseases. However, the underlying molecular mechanisms responsible for highly active antiretroviral therapy (HAART)-induced cardio-metabolic effects are poorly understood. In light of this, we hypothesized that HIV protease inhibitor (PI) treatment (Lopinavir/Ritonavir) elevates myocardial oxidative stress and concomitantly inhibits the ubiquitin proteasome system (UPS), thereby attenuating cardiac function. Lopinavir/Ritonavir was dissolved in 1% ethanol (vehicle) and injected into mini-osmotic pumps that were surgically implanted into Wistar rats for 8 weeks vs. vehicle and sham controls. We subsequently evaluated metabolic parameters, gene/protein markers and heart function (ex vivo Langendorff perfusions). PI-treated rats exhibited increased serum LDL-cholesterol, higher tissue triglycerides (heart, liver), but no evidence of insulin resistance. In parallel, there was upregulation of hepatic gene expression, i.e. acetyl-CoA carboxylase b and 3-hydroxy-3-methylglutaryl-CoA-reductase, key regulators of fatty acid oxidation and cholesterol synthesis, respectively. PI-treated hearts displayed impaired cardiac contractile function together with attenuated UPS activity. However, there was no significant remodeling of hearts exposed to PIs, i.e. lack of ultrastructural changes, fibrosis, cardiac hypertrophic response, and oxidative stress. Western blot analysis of PI-treated hearts revealed that perturbed calcium handling may contribute to the PI-mediated contractile dysfunction. Here chronic PI administration led to elevated myocardial calcineurin, nuclear factor of activated T-cells 3 (NFAT3), connexin 43, and phosphorylated phospholamban, together with decreased calmodulin expression levels. This study demonstrates that early changes triggered by PI treatment include increased serum LDL-cholesterol levels together with attenuated cardiac function. Furthermore, PI exposure inhibits the myocardial UPS and leads to elevated calcineurin and connexin 43 expression that may be associated with the future onset of cardiac contractile dysfunction. Citation: Reyskens KMSE, Fisher T-L, Schisler JC, O’Connor WG, Rogers AB, et al. (2013) Cardio-Metabolic Effects of HIV Protease Inhibitors (Lopinavir/ Ritonavir). PLoS ONE 8(9): e73347. doi:10.1371/journal.pone.0073347 Editor: Sudhiranjan Gupta, Texas A & M, Division of Cardiology, United States of America Received May 13, 2013; Accepted July 18, 2013; Published September 30, 2013 Copyright: ß 2013 Reyskens et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the South African National Research Foundation and Stellenbosch University (MFE), the Conseil Re ´gional de La Re ´ union and l’Europethe Jefferson (EB), and a Pilot Fellowship in Academic Medicine (MSW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mfessop@sun.ac.za Introduction The human immunodeficiency virus (HIV) has infected over 40 million individuals over the last decade, with more than 5 million residing in sub-Saharan Africa [1,2]. Although highly active antiretroviral therapy (HAART) enhances life expectancy and quality of infected individuals [3,4], there is increased emphasis on HAART-mediated metabolic derangements [5] and its potential risk for cardiovascular diseases (CVD) in the long- term. Protease inhibitors (PIs) form an integral part of HAART and side-effects include development of dyslipidemia, i.e. greater production of plasma triglycerides and lipids together with an adverse cholesterol profile [6–8]. Together such derangements elicit inflammation, stress the myocardium (9), and may potentially predict the onset of insulin resistance (IR) [10,11] and cardiac dysfunction (11). PIs are also linked to increased risk for myocardial infarction [13] and cardiovascular abnormalities [14,15], with many changes resembling coronary artery disease [16]. It is unclear whether metabolic side effects of PIs are independently and/or causally linked with cardiovascular pertur- bations. Moreover, the effects of PIs per se on the heart in this context are also poorly understood. Therefore, an emerging focus is to identify key metabolic and transcriptional pathways that may mediate PI-induced cardio-metabolic pathophysiology. For exam- ple, we recently found that rats exposed to 8 weeks of PI treatment displayed cardiac dysfunction [17]. Moreover, PI-treated HIV- infected individuals exhibit elevated reactive oxygen species (ROS) production [18–20] that may trigger the activation of detrimental signaling and cell death pathways [21]. HIV-PIs may also exert unfavorable effects at the gene transcriptional level, e.g. activating sterol regulatory element binding protein (SREBP) [22], a key lipid transcriptional PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e73347 (GEICO), Plateforme CYROI, Universite