AKIP1 Expression Modulates Mitochondrial Function in Rat Neonatal Cardiomyocytes

et al. (2013) AKIP1 Expression Modulates Mitochondrial Function in Rat Neonatal Cardiomyocytes. PLoS ONE 8(11): e80815. doi:10.1371/journal.pone.0080815 AKIP1 Expression Modulates Mitochondrial Function in Rat Neonatal Cardiomyocytes Hongjuan Yu 0 Wardit Tigchelaar 0 Debby P. Y. Koonen 0 Hemal H. Patel 0 Rudolf A. de Boer 0 Wiek H. 0 van Gilst 0 B. Daan Westenbrink 0 Herman H. W. Sillj 0 Xin-Liang Ma, Thomas Jefferson University, United States of America 0 1 Department of Cardiology, University Medical Center Groningen, University of Groningen , Groningen , The Netherlands , 2 Department of Hematology, the First Affiliated Hospital of Harbin Medical University , Harbin, China, 3 Molecular Genetics , University Medical Center Groningen, University of Groningen , Groningen , The Netherlands , 4 VA San Diego Healthcare System, San Diego, California, United States of America, 5 Department of Anesthesiology, University of California San Diego , San Diego, California , United States of America A kinase interacting protein 1 (AKIP1) is a molecular regulator of protein kinase A and nuclear factor kappa B signalling. Recent evidence suggests AKIP1 is increased in response to cardiac stress, modulates acute ischemic stress response, and is localized to mitochondria in cardiomyocytes. The mitochondrial function of AKIP1 is, however, still elusive. Here, we investigated the mitochondrial function of AKIP1 in a neonatal cardiomyocyte model of phenylephrine (PE)-induced hypertrophy. Using a seahorse flux analyzer we show that PE stimulated the mitochondrial oxygen consumption rate (OCR) in cardiomyocytes. This was partially dependent on PE mediated AKIP1 induction, since silencing of AKIP1 attenuated the increase in OCR. Interestingly, AKIP1 overexpression alone was sufficient to stimulate mitochondrial OCR and in particular ATP-linked OCR. This was also true when pyruvate was used as a substrate, indicating that it was independent of glycolytic flux. The increase in OCR was independent of mitochondrial biogenesis, changes in ETC density or altered mitochondrial membrane potential. In fact, the respiratory flux was elevated per amount of ETC, possibly through enhanced ETC coupling. Furthermore, overexpression of AKIP1 reduced and silencing of AKIP1 increased mitochondrial superoxide production, suggesting that AKIP1 modulates the efficiency of electron flux through the ETC. Together, this suggests that AKIP1 overexpression improves mitochondrial function to enhance respiration without excess superoxide generation, thereby implicating a role for AKIP1 in mitochondrial stress adaptation. Upregulation of AKIP1 during different forms of cardiac stress may therefore be an adaptive mechanism to protect the heart. - Funding: HY received a fellowship of the Graduate School for Drug Exploration (GUIDE) from the University of Groningen. This work was partially supported by the Netherlands Heart Foundation (grant 2012T066 to BDW). The Seahorse flux analyzer was obtained via a NWOZonMw Medium Investment Grant (project nr: 91112010). 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. These authors contributed equally to this work. A kinase interacting protein 1 (AKIP1) is a small 23 kDa protein originally identified as a breast cancer associated gene (BCA3) [1]. In humans, there are three splice variants, the fulllength protein (AKIP1a), one that lacks the third exon (AKIP1b), and one that lacks the third and fifth exon (AKIP1c). In contrast, only the full-length protein is present in rodents [2]. It has no homologies to other proteins, is devoid of particular catalytic domains and is therefore believed to have a role as an adaptor or structural intracellular protein. AKIP1 localizes to the cytoplasm, nucleus, and mitochondria and associations with proteins with different sub-cellular localizations have been reported, including PKA [3], NFB [4], apoptin [5], RAC1 [6], TAP73 [7] and AIF [8]. These varied sites of cellular localization suggest that AKIP1 may have multiple functions in the cell. In cancer cell lines a role for AKIP1 in nuclear-cytoplasmic shuttling of PKA and NFB has been proposed [3,4,9,10], but AKIP1 may also be involved in apoptosis [5,7]. AKIP1 has been shown to localize to mitochondria in both cancer cells and cardiomyocytes, but its functional role in mitochondria is still elusive [7,8]. AKIP1 has been mainly studied in cancer cell lines, but is also expressed in many normal, non-tumor, cells in different organs. AKIP1 is abundantly expressed in cardiac tissue predominantly in cardiomyocytes [11]. In a gene array study we identified AKIP1 as a differentially expressed gene that was significantly upregulated in animal models of pathological cardiac hypertrophy and heart failure, including pressure overload and post-myocardial infarction (MI) remodelling [11]; however, exercise mediated physiological hypertrophy also increased AKIP1 expression [12]. Cardiac hypertrophy is initially adaptive in cardiomyocytes to compensate for sustained wall stress, but becomes maladaptive during sustained pathological stress. Interestingly, mitochondrial function is improved during physiological hypertrophy, but diminishes upon sustained pathological hypertrophy [13]. It is possible that AKIP1 may be regulating the compensation phase of pathologic hypertrophy and exercise-induced physiologic hypertrophy through regulation of mitochondrial function. Mitochondria isolated from AKIP1 gene transferred hearts showed amongst others, enhanced calcium tolerance, and decreased mitochondrial cytochrome C release upon ischemic stress. Interestingly, AKIP1 overexpression could protect cardiac function in an ex vivo mouse ischemia/ reperfusion model [8]. Here we test the direct effects of loss or overexpression of AKIP1 on mitochondrial function. Materials and Methods Ethics statement Animal use for these studies was in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The study was submitted to, and approved by, the Committee for Animal Experiments of the University of Groningen (Permit Number: DEC6002). All efforts were made to minimize suffering. Isolation and culturing of primary cardiomyocytes Neonatal rats of 1-3 day old were euthanized by decapitation, hearts excised and atria were removed. Primary neonatal rat ventricular cardiomyocytes (NRVCs) were isolated as previously described [14,15]. Cardiomyocytes were grown in DMEM (Sigma D5671, Missouri, USA) supplemented with 5% fetal calf serum (FCS: Sigma F9665, Missouri, USA) and penicillin-streptomycin (100U/ml-100g/ml; Sigma P0781, Missouri, USA). Adenoviral constructs were generated with the ViraPowerTM adenoviral expression system from Invitrogen as described previously [16]. Primers used for cloning are listed in Table S1. For adenoviral infections, cardiom (...truncated)