Page 25 - ebook

Tfeb-mediated transcriptional regulation of autophagy induces

autosis during ischemia/reperfusion in the heart

2
2
1,2
2
Jihoon Nah , Eun-Ah Sung , Peiyong Zhai , Daniela Zablocki , and Junichi Sadoshima 2

1 School of Biological Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.

2 Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, Newark, New Jersey, USA

Abstract A B C A B

Sham Is. IR 2h IR 6h IR 24h IR24h
MW kDa 2 10 *
10
70 Tfeb 2.0 .0 ** * 8 8 ** 15
1
Autosis is a unique form of cell death with characteristic morphological 70 TFE3 Tfeb/αTubulin 1.5 .5 TFE3/αTubulin 6 6 AVs
1.0 .0
1
4 4
and biochemical features caused by dysregulated autophagy. Autosis is 70 ATP6V1B1 0.5 .5 22 10 10
0
70 ATP6V1A
0
observed in the heart during the late phase of ischemia/reperfusion (I/R), 55 αTubulin 0 .0 Sham Is. IR2h IR6h IR24h 00 Sham Is. IR2h IR6h IR24h Number of electron densed mitochondria/area (Phase 1a) 15 **

when marked accumulation of autophagosomes is induced. We have 5 5

shown previously that excessive accumulation of autophagosomes D

promotes autosis in cardiomyocytes. Although the inhibition of MW kDa Sham IR 2h IR 6h IR 24h 0 0 Ctrl. shTfeb

autophagic flux via upregulation of Rubicon induces accumulation of 70 Tfeb E F

autophagosomes during I/R, it appears that additional mechanisms 70 LaminB1 6 ** 1.5
1.5

exacerbating autophagosome accumulation are required for the induction 44 ** ** 1.0 * **
1.0
55 αTubulin Nuclear Tfeb/LaminB1 6 C
of autosis. Here we show that Tfeb contributes to the induction of Relative Relative Cytosolic Tfeb/αTubulin 1.5

Nuclear fraction 22 0.5
autosis during the late phase of I/R in the heart. During myocardial 0.0 0 10

reperfusion, Tfeb is activated and translocated into the nucleus, which in 70 Tfeb 0 0 Sham IR2h IR6h IR24h Sham IR2h IR6h IR24h 8
6
turn upregulates genes involved in autophagy and lysosomal function. 70 LaminB1 PNS Cells with PNS (Phase 2; %) 4 **

Overexpression of Tfeb enhanced cardiomyocyte death induced by a 2

55 αTubulin Chromatin
high dose of TAT-Beclin 1, an effect that was inhibited by Condensation 0

Cytosolic fraction Ctrl. shTfeb
downregulation of Atg7. Conversely, knock-down of Tfeb attenuated Ctrl AAV-shTfeb

high dose TAT-Beclin1-induced death in cardiomyocytes. Although Figure 2. Tfeb is translocated into the nucleus in response to I/R.

down-regulation of Tfeb in the heart significantly decreased the number Three-month-old mice were subjected to 30 minutes of ischemia with D E F

of autophagic vacuoles and inhibited autosis during I/R, activation of reperfusion for the times indicated. A - C. Heart lysates were analyzed p < 0.05 n.s.

Tfeb activity by 3,4-dimethoxychalcone, an activator of Tfeb, by Western blot. D - F. The mouse heart samples were subjected to

aggravated myocardial injury during I/R. These findings suggest that nuclear fractionation. Both the nuclear and cytosolic fractions were

Tfeb promotes cardiomyocyte autosis during the late phase of analyzed by Western blot analyses (D). The relative ratio of nuclear Tfeb

reperfusion in the heart. (E) and cytosolic Tfeb (F) was quantified.

Key words : Autophagic cell death, Autosis, Tfeb, Ischemia/reperfusion Control AAV-shTfeb

A
Summary MW kDa Scramble Tat-Beclin1 B C Figure 5. Downregulation of Tfeb attenuates autosis but aggravates
I/R injury. AAV9-shTfeb-IRES-GFP was injected into the jugular vein

70 Tfeb Tfeb/Gapdh pTfeb(Ser142)/Tfeb (1.5 × 1012 vg per 10 g for 4 weeks). Four weeks later, mice were

70 p-Tfeb subject to 30 minutes of ischemia and 24 hours of reperfusion. The heart
(Ser 142)
1) Ischemia / early reperfusion 2) Late reperfusion 37 Gapdh samples were subjected to electron microscopic analyses (A-C).

Representative images of LV myocardial sections after Alcian blue and

Protective Autophagy Autosis triphenyltetrazolium chloride (TTC) staining (D). The ratios of the

D F F lacZ infarction area to area at risk (AAR) (E) and AAR to total LV (F) are

Ad-Tfeb
Isolation Tfeb activation n.s. shown.

membrane MW kDa MW kDa 1.0 ** n.s. A B ** C

Rubicon 70 Tfeb 70 Atg7 * 3,4-DC - 3 24 - 3 24 (hours) 5 1 .5

4

Recycle 37 Gapdh 37 Gapdh Relative cell survival (fold) 0.5 MW(kDa) Tfeb 3 Ratio of cytosolic Tfeb 1 .0
55

Autophagosome 55 αTubulin Ratio of nucleus Tfeb /Histone H3 2 n.s. 0 .5

0.0
Scrambled TB1 TB1/shAtg7 15 Histone H3 1 /αTubulin

Lysosome 0 0 .0

Cytosol Nucleus 3,4-DC (ho- 3 24 3,4-DC (ho- 3 24
urs) urs)
Rubicon Figure 3. Tat-Beclin1-induced autosis is regulated by Tfeb

modulation in NRCMs. A - C. NRCMs were treated with 5 µM D E F

Scrambled or Tat-Beclin 1 for 6 hours and analyzed by Western blotting. 1.5 Tfeb Hexb 30 ns
Tfeb

Results Cell death was quantified by CellTiter-Blue assays (mean values ±S.D., 1.0 20

n=16; *p < 0.05, **p < 0.01, not significant (n.s.)). D - F. NRCMs were

transduced with either Ad-lacZ or Ad-Tfeb with or without Ad-shAtg7 Relative mRNA Relative mRNA 0.5 Relative mRNA FS(%) FS (%) 10

as indicated for 48 hours and then treated with 5 µM Scrambled or Tat-
A B 0.0 Vehicle 3,4-DC Vehicle 3,4-DC 0 Vehicle 3,4-DC

1
Akt1 2.0> 2.0 Tfeb * Tfe3 Beclin 1 for 3 hours. Cell death was quantified by CellTiter-Blue assays. Vehicle 3'4'-DC Vehicle
Ambra1 1.5 1.5 3 8 ** +3'4'-DC
3
App * 6
Atg10 1.0 1.0 2
5
Atg16l1 4 G H I
Atg16l2 0.5 0.5 Relative mRNA Relative mRNA
7
Atg3 0 expression 1 0 expression 2 A B I/R (24 h)
Atg4a 0 80 50
9
Atg4b 0 Ctrl AAV-shTfeb-GFP Vehicle +3,4-DC ** n.s.
Atg4c Ctrl. **
11
Atg4d 40
Atg5 1.5 AAV-shTfeb 60
13
Atg7
Atg9a C Autophagy ** 30
15
Atg9b mRNA (fold) Infarct/AAR (%) 40 AAR/LV (%)
Bcl2
17
Bcl2l1 1.5 Atg12 ** 2.0 Becn1 1.5 Gabarap 3 Vps11 ** 1.0 20
Bnip3 ** * ** 20
19
Cdkn1b 1.5 ** ** 10
Cdkn2a 1.0 1.0 2
21 Relative mRNA 1.0 0.5 0 0
Ctss
Cxcr4 expression 0.5 0.5 0.5 1 Relative Tfeb
23
Dapk1 Veh. 3,4-DC Veh. 3,4-DC
Dram1 0.0 0.0 0.0 0
25
Dram2
Eif2ak3 0.0 Figure 6. Activation of Tfeb by 3,4-Dimethoxychalcone aggravates
27
Sham
IR 24h
Eif4g1 C Ctrl AAV-shTfeb-GFP Sham IR 24h
Esr1
29
Gabarapl1 D Lysosomal hydrolases I/R injury. A - C. NRCMs were treated with 30 µM 3,4-
Gabarapl2
31
Hdac1 Gaa Ctsb ** Ctsd Hexb Dimethoxychalcone (3,4-DC) for indicated time periods and subcellular
Hdac6 1.5 * 2.5 1.5 2.5 **
33 2.0 2.0 ** D
Hgs
Ifng 1.0 * * 1.0 ** fractions were analyzed by Western blotting. D and E. 3,4-DC was
35 Igf1 Relative mRNA 1.5 1.5 Sham
Hsp90aa1 expression 0.5 1.0 0.5 1.0 dissolved in corn oil at a concentration of 230 mg/kg and injected
37
Hspa8 0.5 0.5 40 A V s
Atg12 0.0 0.0 0.0 0.0 4 0 E V s
39
Becn1 Tfeb intraperitoneally and whole heart lysates were analyzed by qPCR
Cln3
41 30 analysis. F. 3,4-DC or vehicle injected mice were examined cardiac
Ctsb
3 0
Ctsd taget
43 E Lysosomal membrane and acidification
Gaa
Gabarap Cln3 Number of vacuoles/area 20 function using echocardiography. G - I. Representative images of LV
45
Hexb genes Atp6v1 2 0
Vps11 2.0 ** ** 4 ** ** ** myocardial sections after Alcian blue TTC staining (G). The ratios of the
47
Atp6v1 1.5 * 3 ** 10 1 0 **
Tfeb
49 Relative mRNA 1.0 2 IR 24h infarction to AAR (H) and AAR to total LV (I) are shown.
Tfe3
Sham IR 2h IR 6h IR 24h expression
A B C D 0.5 1 0 0 Ctrl. shTfeb

0.0
0
Conclusion

Figure 1. Pathway focused qPCR array analysis shows a comparison 2µm

of the autophagy and lysosomal gene profile in Sham and I/R heart

samples. An autophagy-related pathway focused qPCR array was 1) Tfeb is upregulated during the late phase of reperfusion

performed using primers for 75 autophagy-related genes. Three-month- Figure 4. Marked accumulation of autophagic vacuoles is when Rubicon is upregulated and autosis is observed in

old C57BL/6J mice were subjected to 30 minutes of ischemia followed accompanied by upregulation of Tfeb during I/R. the infarction region.

by reperfusion for the indicated times, and whole heart lysates were AAV9-shTfeb-IRES-GFP was injected into the jugular vein (1.5 × 1012 2) Simultaneous upregulation of the factor promoting

analyzed by qPCR analysis. A. Heat map showing changes in gene vg per 10 g for 4 weeks). Four weeks later, mice were subject to 30 autophagy and that inhibiting autophagic flux would

expression levels. B - E. Graphs showing relative gene expression levels minutes of ischemia and 24 hours of reperfusion. The heart samples induce a condition where a marked accumulation of

of Tfeb and Tfe3 (B), autophagy related (Atg) genes (C), lysosomal were subjected to fluorescent microscopic analyses and electron autophagic vacuoles may take place.

hydrolase genes (D) and lysosomal membrane and acidification related microscopic analyzes. 3) Tfeb is involved in the stimulation of cardiomyocyte

genes (E). autosis during the late phase of reperfusion.