ORIGINAL RESEARCH ARTICLE 883 Rong Tian, permeability hypertrophy hypertrophy ◼ ◼ cardiac metabolism cardiac September 20, 2016 September 20, DVM heart failure heart failure mitochondria Correspondence to: Correspondence MD, PhD, Mitochondria and University of Metabolism Center, Room N130, 850 Washington, Seattle, WA Republican Street, 98109-8057. E-mail rongtian@u. washington.edu of Funding, see page 893 Sources Key Words: ◼ ◼ transition pore © 2016 American Heart Association, Inc. Chi Fung Lee, PhD Juan D. Chavez, PhD Garcia-Menendez, Lorena Choi, MD Yongseon Nathan D. Roe, PhD Ann Chiao, PhD Ying PhD John S. Edgar, Ah Goo, PhD Young David R. Goodlett, PhD James E. Bruce, PhD Rong Tian, MD, PhD salvage + edox Balance ailure redox balance either redox ratio. Hyperacetylation + + R + D eart F RTICLE NA H imbalance in the failing hearts. These A ratio) and protein acetylation in the ratio) and protein and protein hyperacetylation, previously hyperacetylation, previously and protein + + + levels by stimulating the NAD + precursors are feasible in humans. are precursors ESEARCH + We show that mitochondrial protein hyperacetylation due show that mitochondrial protein We Impairments of mitochondrial function in the heart are Impairments of mitochondrial function in the heart are R herapy for We assessed the reduced/oxidized ratio of nicotinamide assessed the reduced/oxidized We Increased NADH/NAD Increased T redox imbalance contributes to the pathologic remodeling of the imbalance contributes to the pathologic remodeling redox 2016;134:883–894. DOI: 10.1161/CIRCULATIONAHA.116.022495 DOI: 2016;134:883–894. + pool also were tested. pool also were + ormalization of pathway suppressed mitochondrial protein hyperacetylation and cardiac hyperacetylation and cardiac mitochondrial protein pathway suppressed to stresses. function in responses cardiac and improved hypertrophy, Acetylome analysis identified a subpopulation of mitochondrial proteins that was sensitive to changes in the NADH/NAD Circulation. METHODS: adenine dinucleotide (NADH/NAD BACKGROUND: is no therapy but there linked intricately to the development of heart failure, for mitochondrial dysfunction. N ORIGINAL RESULTS: heart via 2 distinct mechanisms. Our preclinical data demonstrate a clear heart via 2 distinct mechanisms. Our preclinical benefit of normalizing NADH/NAD observed in genetic models of defective mitochondrial function, also are observed in genetic models of defective mitochondrial function, also are in human failing hearts as well as in mouse heartspresent with pathologic Elevation of NAD hypertrophy. failing heart. Proteome and acetylome analyses were followed by docking and acetylome analyses were failing heart. Proteome calculation, mutagenesis, and mitochondrial calcium uptake assays to of specific acetylation sites. The therapeutic determine the functional role acetylation by expanding the effects of normalizing mitochondrial protein NAD findings have a high translational potential as the pharmacologic strategy NAD of increasing of mitochondrial malate-aspartate shuttle proteins impaired the transport impaired of mitochondrial malate-aspartate shuttle proteins in altered and oxidation of cytosolic NADH in the mitochondria, resulting acetylation Furthermore, deficiency. state and energy cytosolic redox at lysine-70 in adenosine of oligomycin-sensitive conferring protein its interaction with cyclophilin D, triphosphate synthase complex promoted and sensitized the opening of mitochondrial permeability transition pore. Both could be alleviated by normalizing the NAD CONCLUSIONS: to NAD genetically or pharmacologically. as a Downloaded from http://circ.ahajournals.org/ by guest on October 6, 2016 Lee et al transferase, the later is determined by the deacetylase Clinical Perspective activity, and primarily sirtuins in the mitochondria. The sirtuin deacetylases consume NAD+ as a cosubstrate9; What Is New? mitochondrial function is critical for setting the NADH/ + + • We show that mitochondrial dysfunction increases NAD balance thus the NAD available for sirtuin activity. NADH/NAD+ ratio and protein hyperacetylation result- Using a mouse model with primary mitochondrial dys- ing in a greater sensitivity of the heart to chronic stress. function (cardiac-specific deletion of a Complex-I protein, • Mechanistically, we have identified that hyperacety- Ndufs4: cKO), we recently found that elevation in NADH/ lation of the regulators of mitochondrial permeability NAD+ ratio induce mitochondrial protein hyperacetylation transition pore and malate aspartate shuttle medi- and renders the heart highly susceptible to stresses.10 In ates the increased susceptibility to stresses. this study we defined the molecular intermediaries link- • Increased acetylation levels of cyclophilin D and ing specific NAD+-sensitive hyperacetylation targets to oligomycin sensitive conferring protein on the ade- the development of heart failure and demonstrated the nosine triphosphate synthase complex promote the interaction between oligomycin sensitive conferring relevance of these mechanisms in human heart failure. + protein and cyclophilin D and increase mitochondrial Furthermore, we showed that restoring the NADH/NAD permeability transition pore sensitivity. Moreover, ratio by genetic and pharmacologic approaches is an ef- hyperacetylation and inhibition of malate aspartate fective and potentially translatable strategy for the treat- Downloaded from shuttle limits the mitochondrial import and oxidation ment of heart failure in clinical practice. of NADH generated in the cytosol resulting in cyto- solic redox imbalance. METHODS What are the Clinical Implications? Animal Care, Surgical Procedures, and http://circ.ahajournals.org/ • The identified mechanisms described above are Echocardiography observed in animal models and human failing hearts. All procedures involving animal use were performed with the • Our preclinical results show that expanding the cardiac approval of Institutional Animal Care and Use Committee of NAD+ pool via pharmacologic or genetic approaches the University of Washington. Procedures for animal care, normalizes the NADH/NAD+ ratio and protein acetyla- surgeries, and echocardiography were in the online-only Data tion in hypertrophied and failing hearts. Supplement. • Importantly, these measures improve cardiac func- tion and reduce pathologic hypertrophy in mice. + by guest on October 6, 2016 Thus, the study identifies NADH/NAD ratio a viable Ex Vivo Measurements of Cardiac Function and therapeutic target for mitochondrial dysfunction and Energetics heart failure. Langendorff perfused mouse hearts were isolated as described in the online-only Data Supplement.11 ardiovascular disease is a leading cause of death Mitochondrial Isolation, Proteome, and worldwide.1 As the life expectancy increases and the mortality of acute ischemic events decreases, Acetylome Analyses C 12 the incidence of heart failure is mounting at a pace of Mitochondria were isolated as described. Peptide generation and mass spectrometric protocols were in the online-only Data 900 000 per year.2 However, medical therapy for heart Supplement. failure has been stalled for almost 2 decades. Novel con- Acquired tandem mass spectra were searched for sequence cepts and strategies in the treatment of heart failure are matches against the International Protein Index mouse database needed urgently. using SEQUEST. The following modifications were set as search The heart is a high energy-consuming organ. Mi- parameters: peptide mass tolerance at 500 ppm, trypsin diges- tochondrion is the powerhouse of the cell and mito- tion cleavage after K or R (except when followed by P), one chondrial dysfunction is a well-recognized maladaptive allowed missed cleavage site, carboxymethylated cysteines mechanism during the development of heart failure.3,4 (static modification), and oxidized methionines or acetylation on Targeting mitochondria for heart failure therapy has long K (variable modification). PeptideProphet13 and ProteinProphet14 been sought; however, previous work focusing on im- were used to assign confidence in the identified spectra result- proving mitochondrial energy production and reducing ing from the SEQUEST search. It relies on probability models reactive oxygen species yielded few successful clinical and an empirical Bayesian approach to model fitting. First a score is produced to reflect the quality of each spectrum. Then applications.5 In recent years, protein lysine acetylation a probability-based model is produced for the distribution of emerged as an important mechanism linking mitochon- correctly and incorrectly identified spectra and fit to the scores 6–8 drial metabolism to cellular pathologies. The level of of all identified spectra. The confidence in individual spectra protein acetylation reflects the balance of acetylation are evaluated using the posterior probability. A cutoff is applied and deacetylation. Although the former is dependent on on the scores for the set of correctly identified spectra to the abundance of acetyl-CoA and the activity of acetyl- control the false discovery rate, defined as the percentage of 884 September 20, 2016 Circulation. 2016;134:883–894. DOI: 10.1161/CIRCULATIONAHA.116.022495 Normalizing NADH/NAD+ as Heart Failure Therapy false positives which pass the cutoff. This method produces a caused by mitochondrial Complex-I deficiency (cKO) in- similar estimation of the false discovery rate as the commonly creased cardiac susceptibility to stress.10 We sought used