N utrient Overload and Divergence in A daptive Redox Responses between Hear t and Skeletal Muscle
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1 N utrient verload and Divergence in A daptive Redox Responses between Hear t and Skeletal Muscle Ethan J. A nderson Department of Pharmacology & Toxicology, and Cardiovascular Sciences, East Carolina University
2 Roles of Mitochondria in the Cell No longer viewed as simply the engine of the cell 2 1 e SD H 2 2 Fe 2+ 1 e H Physiological levels of RS can only be transmitted via REDX pathways 100 um H 2 2 -S S- -S 2-, -S 3 - -Lipid-H - Aldehydes -Stable electrophiles (i.e. proteins)
3 Mitochondrial RS and Lipid Peroxides (LH) Clorgyline MM MATRIX H 2 + R NH 3 INTERMEMBRANE SPACE Complex I 2eˉ 2 Monoamine R CH + NH 4 xidase 2 Ubiquinone/Ubiquinol Complex II (SDH) ETF- QR H 2 2 CuZnSD 2 FAD + Fe 2+ H 2 2 Fe 2+ 2 ˉ eˉ eˉ Complex III H H Cytochrome C 2 H ½ 2 H 2 Complex IV 1 um NE Pyr/Mal Glutam Succinate NADH NAD + FADH 2 FAD 2 ˉ FADH 2 MnSD Fe 2 ˉ H H 2 2 HNE (if n-6 PUFA oxidation) Cardiolipin HHE (if n-3 PUFA oxidation) MDA (n-6 + n-3 PUFA oxidation) H CH 2 4) both possible CH CH 2 CH 2 CH CH 2 2 3) 2) H + n H 2 1) H Anderson, Katunga & Willis (2011) Clin Exp Pharmacol Physiol Mitochondria prepared from Human Heart biopsy
4 Lipid peroxidation and reactive aldehyde (HA E) formation Reactive A ldehydes 2GSH GPx4 GSSG + LH + H 2 Prot ei n, DNA A dduct formation n-3pu FA -derived n-6pu FA -derived
5 Consequences of N utrient verload (i.e. High Fat, High Sucrose Diet).. In Skeletal Muscle.. In Heart
6 Question 1: What are the mechanisms underlying this disparity between Skeletal Muscle and Hear t with nutrient overload?? Question 2: Increased FFA metabolism and oxidative stress occur with exercise (Ex) as well as HFHS diet, yet Ex is beneficial to cardiometabolic health. Why is this? A Hypothesis differences in redox adaptations between Heart and SkM, particularly in mitochondria, explain these disparate responses Hormetic effects of Exercise and Dietary Fats (especially PUFA s) Known to be potent inducers of antioxidant enzymes and mitochondrial biogenesis
7 Experimental Model and Study Design: 8 wk old Sprague-Dawley rats Standard Chow (CN) Fed Standard Chow (CN-Ex) Fed High Fat High Sucrose (HFHS) Fed High Fat High Sucrose (HFHS-Ex) Fed 12 weeks Exercise: 70 min/day, 5 days/wk 12 weeks Exercise: 70 min/day, 5 days/wk 12 weeks Determine effects on Cardiac and Skeletal Muscle Mitochondria, focus is on Redox Adaptations
8 Diet Composition: Ctl Diet HFHS Diet TD TD Protein (% kcal) Carbohydrate (% kcal) Sucrose (g/kg) Corn Starch (g/kg) Fat (% kcal) Anhydrous Milkfat Soybean il Safflower il 6 80 Total kcal/g Fatty Acid Composition SFA (% total fatty acids) MUFA (% total fatty acids) PUFA (% total fatty acids) C18:2 linoleic (% by wt.) C18:3 linolenic (% by wt.) n/6 to n/3 ratio
9 Metabolic Endpoints: Ctl Sed Ctl + Ex HFHS Sed HFHS + Ex Terminal Body Wt. (g) 458 ± ± ± 11.1 ** 456 ± 17.2 Heart Wt. (g) 1.14 ± ± ± ± 0.11 Fat Mass (g) 52.0 ± ± ± ± 8.48 Lean Mass (g) 341 ± ± ± ± 10.3 Body Fat % 13.1 ± ± ± ± 1.52 Glucose (mg/dl) 107 ± ± ± ± 3.7 Insulin (pm) 125 ± ± ± ± 23.4 HMA-IR 4.94 ± ± ± ± 1.09 Cholesterol (mg/dl) 39.0 ± ± ± ± 2.14 * Triglycerides (mg/dl) 58.0 ± ± ± ± 6.1 Citrate Synthase-Heart ± ± ± ± 3.59 Citrate Synthase-Sk M ± ± ± 3.55 ** ± 2.71
10 Mitochondrial experiments using permeabilized myofibers Bundle of muscle fibers ~10 mg wet wt. Trimmed to 2-mm wide x 5-mm long (1-2 mg wet wt) Separate using fine forceps Permeabilized Fiber Bundles Mitochondrial Respiration Mito-H 2 2 emission Mito-Ca2+ uptake RBRS 2 K xygraph -Ex/ Em 570/ 585 Calcium Green 5N -Ex/ Em 503/ 535 Permeabilize with Saponin Spectrofluorometer
11 Mitochondrial 2 capacity in Heart and SkM following HFHS diet and/ or Ex ATP + Glucose Hexokinase Glucose-6-phosphate ΔG ATP < 0 Mitochondria maintained in a permanent phosphorylating state xphos constantly maximal
12 Mitochondrial Ca 2+ overload and PTP Adenine Nucleotide Translocase ATP ADP Cylophilin D ADP + + ATP Ca2+ Ca2+ Ca2+ Ca2+ Ca 2+ Ca 2+ + Ca 2+ Ca 2+ Ca 2+ Ca Ca I e cyto c - e - e - e - QH 2 e - Q III IV + NADH e - e- Membrane potential ( Ψ) e - H + e - e - + NAD + H + + high Ψ low ½ 2 + 2H + When exposed to elevated levels of cytosolic Ca2+, mitochondria take up the ion due to electrical gradient ψ -230mV -130mV e - H 2 low e - flow high + H + Matrix
13 Mitochondrial Ca 2+ overload and PTP
14 Mitochondrial RS following HFHS diet and/ or Ex
15 Redox Signaling in Heart and SkM following HFHS diet and/ or Ex
16 Mitochondrial Redox state and Thioredoxin Reductase-2 (TxnRd2) Auranofin
17 Thioredoxin Reductase-2: A mitochondrial specific antioxidant enzyme Heart RG SkM Ctl Sed Ctl + Ex HFHS Sed HFHS + Ex Ctl Sed Ctl + Ex HFHS Sed HFHS + Ex TxnRd2 α-tubulin
18 Mitochondrial Redox state and TxnRd2 Auranofin
19 What might be happening here?
20 Thank you to. My Lab Justin La Favor Kelsey Fisher-Wellman Kathleen Thayne - Research Specialist Timothy Darden -grad student Funding NIH R21HL Taylor Mattox -grad student Lalage Katunga -grad student
21 Enzymatic and Non-enzymatic removal of Lipid-H and aldehydes in Mitochondria ALDH1, 3A1(FALDH) ALDH6,7,8,9,16,18A1 CYTSL GSH Histidine Taurine Carnosine Vitamin E Coenzyme Q10 Cardiolipin MATRIX NADP + ALDH2 ALDH4A1 ALDH5A1 GPx4 Acrolein MDA 4-hydroxyalkenals RCH + NADH + H + C C C 2 H GSH H + n Histidine Taurine Carnosine H 2 H Anderson, Katunga & Willis (2011) Clin Exp Pharmacol Physiol
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