2/12/2014 *** + Chloro + Chloro. Bruce Ito, Nandini Ravindran, Robert Mentzer, unpublished data

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1 LC3-II/Actin Ratio 1 Young *** Aged B. 0 + Chloro + Chloro Bruce Ito, Nandini Ravindran, Robert Mentzer, unpublished data 1

Chow HFD p62 LC3 Normal Chow High Fat Diet (16

Mentzer, unpublished data 100 90 80 Infarct Size

7 20 10 0 Chow (6) DIO (4) Chengqun Huang, Bruce

minipigs maintained for 4mo on regular diet or

experimental heart attack with or without

2 Chow HFD p62 LC3 Normal Chow High Fat Diet (16 wk) Bruce Ito, Nandini Ravindran, Robert Mentzer, unpublished data Infarct Size (% ) Deaths: Chow: 1 / 8 DIO: 4 / Chow (6) DIO (4) Chengqun Huang, Bruce Ito, Robert Mentzer, unpublished data Yucatan minipigs maintained for 4mo on regular diet or high-fat, high-fructose diet Subjected to experimental heart attack with or without cardioprotective intervention Measured autophagy markers and infarct size 2

3 Bruce Ito, Robert Mentzer, unpublished data Bruce Ito, Robert Mentzer, unpublished data Bruce Ito, Robert Mentzer, unpublished data 3

4 Atrial biopsies obtained before aortic cross-clamp and after its removal Patients undergoing CABG or valve repair Tissue snap-frozen and assessed for autophagy proteins by western blot Jahania, colleagues, and Mentzer. J Am Coll Surg 2013 Pt Sample 6a 6b 7a 7b 8a 8b 9a 9b Beclin-1 Atg5-12 p p Atg p = p = Before After 0.0 Before After Actin 0.4 N.S Before After Pt Sample 6a 6b 7a 7b 8a 8b 9a 9b 10a 10b 11a 11b 12a 12b Actin 4

5 Age Ejection fraction Risk Predictors Metabolic syndrome features Obesity Hypercholesterolemia, dyslipidemia Hyperglycemia, diabetes 5

Mice were deprived of food for 24hr on Monday and Thursday Free access to food all other days Compared to mice with free access to food all seven days of

6 Mice were deprived of food for 24hr on Monday and Thursday Free access to food all other days Compared to mice with free access to food all seven days of the week Enrolled at age 10mo (middle age) and maintained for 1 year (old age, 22mo) Evaluated T memory cells and severity of injury after heart attack 6

7 No difference in weight More active in cage Less hair loss Higher levels of autophagy proteins More naive T-cells (immunologic youth) Better tolerance to myocardial infarction Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet. Hatori M, Vollmers C, Zarrinpar A, DiTacchio L, Bushong EA, Gill S, Leblanc M, Chaix A, Joens M, Fitzpatrick JA, Ellisman MH, Panda S. Cell Metab. 2012, 15: After 100 days, the mice who ate fatty food throughout the day gained weight and developed high cholesterol, high blood glucose, liver damage and diminished motor control, while the mice who had access to the same fatty food for only 8 hours/day weighed 28% less and showed no adverse health effects despite consuming the same amount of calories from the same fatty food. The timerestricted mice also did better on an exercise test than the mice who ate either fatty food or normal chow throughout the day. Autophagy is probably the key factor in preventing age-related diseases Overnutrition suppresses autophagy Intermittent fasting shows promise for preventing age-related diseases But we don t know how much is enough, AND, We need better tools to monitor autophagy in humans 7

Mridula Ramesh Moutusee Islam Diana Lui Xinghao Zhang Jon Sin Allen Andres Chengqun Huang Jen Ramil

Bruce Ito Nandini Ravindran Salik Jahania David Sengstock Peter Vaitkevicius Phyllis-Jean Linton

R01-HL92136 Allen Andres Genaro Hernandez Yoshimi Hiraumi Chengqun Huang Moutusee Islam Pamela Lee

Xiong Xinghao Zhang Tamotsu Yoshimori Richard Youle William Claycomb Support from NIH P01-HL112730

8 Mridula Ramesh Moutusee Islam Diana Lui Xinghao Zhang Jon Sin Allen Andres Chengqun Huang Jen Ramil Rick Sayen Robert Mentzer, Jr. Bruce Ito Nandini Ravindran Salik Jahania David Sengstock Peter Vaitkevicius Phyllis-Jean Linton Michael Gurney Reagents from Support from NIH P01-HL112730, R01-HL60590, R01-AG33283, R01-HL34579, R01-HL92136 Allen Andres Genaro Hernandez Yoshimi Hiraumi Chengqun Huang Moutusee Islam Pamela Lee Giovanni Quarato Bruno Queliconi Mridula Ramesh Jen Ramil Rick Sayen Jon Sin David Taylor Ying Xiong Xinghao Zhang Tamotsu Yoshimori Richard Youle William Claycomb Support from NIH P01-HL NIH R01-HL NIH R01-HL Robert Mentzer, Jr. Bruce Ito Salik Jahania Nandini Ravindran David Sengstock Peter Vaitkevicius Phyllis Phyllis--Jean Linton Michael Gurney 8

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