Autophagy Pathway

1. NF-κB is Required for the Upregulation of Beclin in High-Fat Fed Mice Trisha Tee, Lauren Haar, Michael Tranter, W. Keith Jones Department of Pharmacology & Cell Biophysics, University of Cincinnati, Cincinnati, OH OBJECTIVE INTRODUCTION PRELIMINARY DATA CONCLUSIONS HYPOTHESIS RESULTS BIBLIOGRAPHY NEXT STEPS • Previous research has indicated that heart failure paired with chronic high fat diets can improve cardiac function as well as influence cardioprotective genes • Beclin-1 (BECN1) is an essential autophagy protein that has been linked to protection against some cardiac degenerative diseases. • Autophagy is a process involving the degradation of a cell’s intracellular components via the lysosome. Beclin-1 interacts with the Vps34 protein complex by increasing its activity, and is thus a promoter of autophagy • Studies have shown that nuclear factor NF-κB is involved in the regulation of BECN1 expression in autophagy • NF-κB is a transcriptional factor that plays a key role in regulating immune response to infection. It is activated when the I-κB protein subunits p50 and p65 are phosphorylated • p65 is known to directly bind to the BECN1 promoter and modulate transcription and autophagy • Autophagy Pathway • Acute HFD causes cardioprotection against I/R injury in an in vivo murine model • Blockade of NF- κB nullifies cardioprotection at 24 hrs. Western Blots for 2M and WT cardiac tissue samples Quantitative Data for Western Blots • In 2M mice, NF-kB cannot be activated. The lack of change in beclin-1 signals between the control fed vs. the high fat fed implicates that BECN1 is dependent on NF-kB. • Compared to the WT control fed mice, the high fat fed WT mice had significantly higher BECN1 signals, indicating that a high fat diet leads to an increase in upregulation of beclin-1; which in turn implies an increase in autophagy. TTC Staining Area at Risk A) Area of Infarct A) Control 24 Hr HFD 6 wk HFD Infarct Size Measured by TTC Stain after High Fat Feeding Infarct Size Measured by TTC Stain after High Fat Feeding B) 24hr HFD Wt c57 Infarct Size (as a % of area at risk) Infarct Size (as a % of area at risk) B. A. 24hr HFD 2M c57 • Obtain ischemic and non-ischemic cardiac tissue samples from control and high fat fed WT and 2M mice in order to further explore the cardioprotective effects of acute high fat feedings via western blots • During autophagy, LC3-I is converted to LC3-II through lipidationthat allows it to become associated with autophagic vesicles. Thus LC3 markers can be used to further indicate the occurrence of autophagy • Further exploration can be done with a caspase marker; caspases are essential in cells for apoptosis. Cardioprotective effects can be further examined by using caspase markers to quantify potential apoptotic differences between control and high fat fed WT and 2M mice Fig. 4. A. WT control diet (first 3 lanes) vs. WT high fat diet (latter 4 lanes) on actin antibody. B. WT control diet vs. WT high fat diet on beclin-1 antibody. C. 2M control diet (first 4 lanes) vs. 2M high fat diet (latter 4 lanes) on actin antibody. D. 2M control diet vs. 2M high fat diet on beclin-1 antibody. Fig. 1. Schematic of the autophagy process. C. D. To determine the role of NF-κB in upregulation of Beclin in high fat fed mice. To determine the cardioprotective effect of acute high fat feedings paired with induced myocardial infarction A high fat diet leads to an increase in upregulation of Beclin, and consequently an increase in autophagy Beclinupregulation is NF-κB dependent Fig. 2. A. Histology of LV after 30 m LAD occlusion and 2- hr. reperfusion for mice fed control diet (15% kcal/fat, 29% protein, 56% carbohydrate), 24-hr or 6-wk on high fat diet (60% kcal, 20% protein, 20% carbohydrate). B. Mice fed ad libitum on (p<0.05, n>4) HFD (60%kcal/fat) or control (11%kcal/fat) ad libitum for time indicated then subject to 30 minute LAD occlusion and 24-hr reperfusion prior to sacrifice. R Kang, HJ Zeh, MT Lotze, D Tang, The Beclin 1 network regulates autophagy and Apoptosis, Cell Death and Differentiation, 2011, 571-580 L Haar, X Ren, N Bertaux-Skeirik, M Tranter, J Rubinstein, WK Jones, High fat mediated cardioprotection is NF-κB dependent and alters ischemic zone apoptosis and autophagy, 2012 http://www.phosphosite.org/ MATERIALS & METHODS • Samples collected from WT and 2M murine models 24-hr after high fat and control feedings • Samples analyzed using standard western blotting protocol in order to determine the quantitative amount of beclin-1 compared to actin antibody control. Fig. 3. A. Dominant negative (2M) model of NF- κBtranscription. Phosphorylation of serine inhibited and p50/p65 subunits confined to cytosolic compartment. B. Histology of infarct for 30 minute ischemia/24h reperfusion in WT relative to 2M on HFD. C. Quantified infarct size of 24-hr fed high fat control and 2M mice (p<0.05, n>4) Fig. 5. WT mice fed HFD for 24-hr resulted in higher BECN1 signals than mice fed control diet (top). 2M mice fed HFD for 24-hr resulted in relatively equal BECN1 signals as control fed 2M mice (bottom).