David A. Schipper, BS, Ryan Palsma, BS, Katherine M

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Chronic Myocardial Ischemia Leads to Loss of Maximal Oxygen Consumption and Complex I Dysfunction David A. Schipper, BS, Ryan Palsma, BS, Katherine M. Marsh, BS, Connor O’Hare, BS, Destiny S. Dicken, BA, Scott Lick, MD, Toshinobu Kazui, MD, PhD, Kitsie Johnson, BA, Ryszard T. Smolenski, PhD, Dirk J. Duncker, MD, PhD, Zain Khalpey, MD, PhD The Annals of Thoracic Surgery Volume 104, Issue 4, Pages 1298-1304 (October 2017) DOI: 10.1016/j.athoracsur.2017.03.004 Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Representative Seahorse XF assay on isolated mitochondria. (A) Illustrative example of mitochondrial coupling assay performed on mitochondria isolated from chronic ischemic and control left atrial appendage (LAA), normalized per μG of mitochondrial protein. (B) Electron transport chain assay performed on the same isolates of chronic ischemic and control LAA. Each value shown has a minimum of 6 replicates, with error bars indicating SEM. (OCR = oxygen consumption rate.) The Annals of Thoracic Surgery 2017 104, 1298-1304DOI: (10.1016/j.athoracsur.2017.03.004) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Mitochondrial respiration states in ischemic and control patients. Oxygen consumption rates of isolated mitochondria from chronic ischemic left atrial appendage (LAA) compared with isolated mitochondria of control LAA, normalized per μG of mitochondrial protein. Mitochondrial assay media was enriched with rotenone and succinate to induce state 2. State 3ADP, state 4O, and state 3U were achieved by the sequential addition of ADP, oligomycin, and FCCP, respectively. (OCR = oxygen consumption rate.) The Annals of Thoracic Surgery 2017 104, 1298-1304DOI: (10.1016/j.athoracsur.2017.03.004) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Respiratory control ratios in ischemic and control patients. (A) Respiratory control ratio (RCR) was determined as the ratio of state 3ADP and state 4O. (B) RCR was determined as state 3ADP and state 4O. (C) Both ratios were then compared. All values displayed are mean ± SEM, * indicates p < 0.05. The Annals of Thoracic Surgery 2017 104, 1298-1304DOI: (10.1016/j.athoracsur.2017.03.004) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Mitochondrial complex driven respiration in ischemic and control patients. Oxygen consumption rates (OCR) of isolated mitochondrial from ischemic left atrial appendage (LAA) compared with isolated mitochondria of control LAA, normalized per μG of mitochondrial protein. Mitochondrial assay media was enriched with malate, FCCP, and sodium pyruvate to induce complex I respiration. (A) Complex I respiration, measured as the resulting OCR after subtraction of rotenone driven OCR. (B) Complex II respiration, measured as the succinate driven respiration after subtraction of the antimycin A OCR. (C) Complex IV respiration, measured as the TMPD and ascorbate driven respiration after subtraction of antimycin A respiration. All values displayed are mean ± SEM, ** indicates p < 0.01, * indicates p < 0.05. The Annals of Thoracic Surgery 2017 104, 1298-1304DOI: (10.1016/j.athoracsur.2017.03.004) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Mitochondrial complex respiration as ratios. Respiratory complex ratios (%) of isolated mitochondrial from ischemic left atrial appendage (LAA) compared with isolated mitochondria of control LAA. (A) Complex I respiration as a ratio of complex II respiration. (B) Complex I respiration as a ratio of complex IV respiration. (C) Complex II respiration as a ratio of complex IV respiration. All values displayed are mean ± SEM, * indicates p < 0.05. The Annals of Thoracic Surgery 2017 104, 1298-1304DOI: (10.1016/j.athoracsur.2017.03.004) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions