functional roles of chaperone molecule (hsp70) for muscle...

Functional roles of chaperone molecule (HSP70) for

muscle atrophy inhibition: future implication

Taesik Gwaga, Eunjung Kima, Takeshi

Nikawab and Inho Choia

aDivision of Biological Science and Technology,

College of Science and Technology, Yonsei University,

Wonju, Gangwon-Do, Republic of Korea

bDepartment of Nutritional Physiology, Institute of

Health Biosciences, University of Tokushima Graduate

School, Tokushima, Japan

The 10th Japan-Korea Joint Seminar on Space Environment Utilization Research

September 12, 2013

Korea University

(1) a significant decrease in mass or volume of skeletal muscle,

with loss of myofibrillar proteins and tone.

(2) caused by various factors including physical unloading and diseases.

(3) Molecular mechanisms underlying the atrophic process are much shared in the causal states..

Cancer

Bed rest Space microgravity

Spinal injury – denervation

Muscle injury

Inactivity by cast

Cancer

AIDS

Sepsis

Space physiology good solutions the quality of life

Introduction: Factors causing muscle atrophy

Muscle atrophy

Muscle force production ↓

Performance and work ↓

Mission capability ↓

Responsiveness to emergency ↓

Muscle atrophy during prolonged spaceflight

Severe muscle damage by reloading (landing)

Skeletal muscle atrophy

due to unloading

Introduction: Space microgravity causes serious muscle atrophy

Space microgravity Muscle atrophy occurs steadily

despite elaborate exercise program in space.

Adams et al., 2003

We may need another means of countermeasure

to effectively prevent muscle atrophy!

Muscle mass = balance between protein synthesis rate and degradation rate

<source from Hoffmand and Nader, Nature Medicine 10(6):584-585 (2004)>

p70/S6K

Activation of Protein synthesis pathway

Activation of protein breakdown pathway

Exercise /overloading

Spaceflight, bedrest /unloading

Upregulation of Proteases via FoxO

HSP↓

dexamethasone

Introduction: Molecular mechanisms underlying muscle mass regulation

CEL: a quinone methide triterpentene obtained from oriental herbs.

DEX: dexamethasone, a synthetic glucocorticoid that is known to induce muscle atrophy.

Inhibition of C2C12 myotube atrophy by a novel HSP70 inducer, celastrol, via

activation of Akt1 and ERK1/2 pathways

A quinine methide triterpene

Heat Shock Protein 72 inducer

Diverse functions: antioxidant activity, neuroprotection, and inhibition of proteasome activity

Previous reports: muscle atrophy prevented by HSP70 overexpression

Celastrol (CEL): general features

Kukreti et al., 2013, Muscle-specific MicroRNA1 (miR1) Targets Heat Shock Protein 70 (HSP70) during

Dexamethasone-mediated Atrophy. Journal of Biological Chemistry. 288:6663-6678

Senf et al., 2008, Hsp70 overexpression inhibits NF-kappaB and Foxo3a transcriptional activities and prevents

skeletal muscle atrophy. FASEB journal : official publication of the Federation of American Societies for

Experimental Biology. 22:3836-3845.

Gehrig et al., 2012, Hsp72 preserves muscle function and slows progression of severe muscular dystrophy.

Nature. 484:394-398.

Cell line: mouse C2C12 myotube

Determination of proper concentration of CEL.

Concentration of CEL used for this study: 1.5 μM for 6 h treatment

Data were obtained from three independent experiments

(each experiment performed in triplicate) and are

presented as mean SEM.

The cells exposed to four doses of CEL for 6 h

Stable dosage



FACS analysis

Data were obtained from three independent experiments

Experimental design

DEX (150µM): time-dependent test

23% loss

in diameter



Myotube diameter affected by CEL and DEX.

White bar = 100 μm

Comparison of the cell diameters among the four groups

Data are presented as the mean SEM. Cell counts: 220 - 250 cells per group.

The symbols a, b, c and d indicate statistical significance among the denoted

groups at P < 0.05 (one-way ANOVA and SNK post hoc multiple comparisons

test).

CEL abolished DEX-induced myotube atrophy

The cell diameter was significantly decreased in myotubes

exposed to 150 μM DEX for ≥12 h

atrophy

hypertrophy

recovery

Data: mean SEM (n = 6). a versus b, P < 0.05 .

Expression of HSP72 upregulated by CEL.

Elevation of HSP72 protein expression by CEL

(A and B) Whole cell lysates subjected to

immunoblot analyses with antibodies rea

cting with total or Ser230-phosphorylate

d HSF1. Mean SEM (n = 6), a versus b

, P < 0.05. (C) Confocal immunofluoresc

ent analysis to visualize the expression a

nd subcellular localization of p-HSF1 in

C2C12 cells. The cells were cultured on

chamber slides and treated as indicated i

n the Methods, and stained with p-HSF1

antibody followed by a secondary antibo

dy conjugated to Rhodamine. The nucleu

s was stained with DAPI (blue). Scale bars = 10 μm.

Upregulation of HSP72 via heat shock factor 1 (HSF1)

transcriptional activity



Anabolic signalling activities upregulated; catabolic signaling activities abolished by celastrol

Anabolic activities compared among the four groups

Data: Mean SEM (n = 6). a versus b, P < 0.05.

Catabolic activities compared among the four groups

Data: Mean SEM (n = 6). *P < 0.05.



HSP72 expression was affected by HSP72 siRNA and CEL in the L6 myotubes

Elevation of HSP72 expression by CEL

Activation of HSF1 by CEL

(A and B) Whole cell lysates

subjected to immunoblot analyses

with Ser230-phosphorylated HSF1.

Mean ± SEM (n = 5). The

symbols a, b and c indicate

statistical significance among the

denoted groups. (C) Confocal

immunofluorescent analysis to

visualize subcellular localization

of HSF1. The cells were stained

with HSF1 antibody followed by a

secondary antibody conjugated to

Alexa488 (green) and DAPI (blue).

White bars = 10 μm.

HSF1 was transactivated and accumulated in the

nucleus by CEL treatment.

HSP72 mRNA and protein expression was upregulated

by CEL treatment despite Hsp72 gene knock down.

248~ 306 fold

Data : mean ± SEM (immunoblot

analyis; n = 6, real-time PCR

analysis; n=4) The symbols a, b, c

and d; significant differences among

the designated groups at P < 0.05.

HSP70 overexpression induced by celastrol abolished the atrophic effect of

HSP siRNA in L6 myotubes

Myotube diameters compared among the four groups.

Data: mean ± SEM (n = 178 -183 cells per group with 3 independent experiments).

The symbols a, b and c indicate significant differences among the denoted groups at P < 0.05.

White bars = 100 µm

The cell diameter was significantly decreased by HSP72 siRNA

atrophy

hypertrophy

recovery

(A) NC siRNA, (B) HSP72 siRNA,

(C) NC siRNA+CEL and (D) HSP72 siRNA+CEL

CEL abolished the atrophic effect of HSP72 siRNA on muscle cell

HSP70 overexpression induced by celastrol abolished the atrophic effect of

HSP siRNA in L6 myotubes

Conclusions

1. The anti-atrophic effect of CEL is mediated by activation of HSF1 and

consequent overexpression of HSP72.

2. HSP overexpression by CEL treatment not only activated the Akt1, S6K and

ERK1/2 signaling pathways but also suppressed FoxO activation, E3 ligase

expression and proteasome activity by DEX in C2C12 myotube.

3. The lack of HSP70 was directly responsible for the L6 myotube atrophy, and

CEL abolished such atrophy of the myotube.

4. An in vivo study on the anti-atrophic potency of celastrol is currently under

way with a direct application of CEL in the presence or absence of siRNA in

the skeletal muscle of a mouse model.

Thank you for your attention!

The 10th Japan-Korea Joint Seminar on Space Environment Utilization Research

Functional roles of chaperone molecule (HSP70) for muscle atrophy inhibition: future implication

Taesik Gwag, Eunjung Kim, Takeshi Nikawa and Inho Choi

functional roles of chaperone molecule (hsp70) for muscle...

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