Autophagy Pathway

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1. Autophagy Pathway 2. Acute HFD causes cardioprotection against I/R injury in an in vivo murine model 3. Blockade of NF- κB nullifies cardioprotection at 24 hrs. 4. Western Blots for 2M and WT cardiac tissue samples 5. Quantitative Data for Western Blots 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 • 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. 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 INTRODUCTION PRELIMINARY DATA CONCLUSIONS HYPOTHESIS 1. A high fat diet leads to an increase in upregulation of Beclin, and consequently an increase in autophagy 2. Beclin upregulation is NF-κB dependent NEXT STEPS 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 lipidation that 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 OBJECTIVE 1. To determine the role of NF-κB in upregulation of Beclin in high fat fed mice. 2. To determine the cardioprotective effect of acute high fat feedings paired with induced myocardial infarction Fig. 1. Schematic of the autophagy process. 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. 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). BIBLIOGRAPHY 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 Infarct Size Measured by TTC Stain after High Fat Feeding Infarct Size (as a % of area at risk) Contr ol 24 Hr HFD 6 wk HFD A) Area at Risk Area of Infarct TTC Staining 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. Infarct Size (as a % of area at risk) 24hr HFD 2M c57 24hr HFD Wt c57 A) B) Infarct Size Measured by TTC Stain after High Fat Feeding Fig. 3. A. Dominant negative (2M) model of NF- κB transcription. 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) RESULTS A. B. C. D. Control Diet High Fat Diet 0 0.5 1 1.5 2 Beclin Density for WT HFD vs. Control Control Diet High Fat Diet 0.75 0.85 0.95 1.05 Beclin Density for 2M HFD vs. Control 1. R Kang, HJ Zeh, MT Lotze, D Tang, The Beclin 1 network regulates autophagy and Apoptosis, Cell Death and Differentiation, 2011, 571-580 2. 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 3. http://www.phosphosite.org/

description

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. - PowerPoint PPT Presentation

Transcript of Autophagy Pathway

Page 1: Autophagy Pathway

1. Autophagy Pathway

2. Acute HFD causes cardioprotection against I/R injury in an in vivo murine model

3. Blockade of NF- κB nullifies cardioprotection at 24 hrs.

4. Western Blots for 2M and WT cardiac tissue samples

5. Quantitative Data for Western Blots

NF-κB is Required for the Upregulation of Beclin in High-Fat Fed MiceTrisha Tee, Lauren Haar, Michael Tranter, W. Keith Jones

Department of Pharmacology & Cell Biophysics, University of Cincinnati, Cincinnati, OH

• 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.

• 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

INTRODUCTION PRELIMINARY DATA CONCLUSIONS

HYPOTHESIS

1. A high fat diet leads to an increase in upregulation of Beclin, and consequently an increase in autophagy

2. Beclin upregulation is NF-κB dependent

NEXT STEPS

• 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 lipidation that 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

OBJECTIVE

1. To determine the role of NF-κB in upregulation of Beclin in high fat fed mice.

2. To determine the cardioprotective effect of acute high fat feedings paired with induced myocardial infarction

Fig. 1. Schematic of the autophagy process. 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. 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).

BIBLIOGRAPHY

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.

Infarct Size Measured by TTC Stain after High Fat Feeding

Infa

rct

Siz

e (a

s a

% o

f ar

ea a

t ris

k)

Control 24 Hr HFD 6 wk HFD

A)Area at Risk

Area of Infarct

TTC Staining

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.

Infa

rct

Siz

e (a

s a

%

of a

rea

at r

isk)

24hr HFD

2M c57

24hr HFD

Wt c57

A)

B)

Infarct Size Measured by TTC Stain after High Fat Feeding

Fig. 3. A. Dominant negative (2M) model of NF- κB transcription. 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)

RESULTS

A. B.

C. D.

Control Diet High Fat Diet0

0.4

0.8

1.2

1.6

Beclin Density for WT HFD vs. Control

Control Diet High Fat Diet0.75

0.8

0.85

0.9

0.95

1

1.05

Beclin Density for 2M HFD vs. Control1. R Kang, HJ Zeh, MT Lotze, D Tang, The Beclin 1

network regulates autophagy and Apoptosis, Cell Death and Differentiation, 2011, 571-580

2. 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

3. http://www.phosphosite.org/