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1 DECLARATION OF CONFLICT OF No disclosure INTEREST
2 ESC Congress Impaired vacuolar H + -ATPase function causes cardiomyocyte death with extensive vacuolation and impaired autophagic degradation Department of cardiology Keio University School of Medicine Motoaki Sano M.D., Ph.D.
3 Background Recent studies have demonstrated that the existence of autophagic cell death in hypertrophied failing and hibernating myocardium. Autophagic cell death is a morphological term derived from electron microscopic observations and denotes a form of cell death in which abundant autophagic vaculoles are present in the cytoplasm.
4 The (pro)renin receptor, the gene product of ATP6AP2, plays a key role in the activation of local RAS Plasma angiotensinogen prorenin ANG I ANG II Handle region prorenin (P)RR ACE AT1R Intracellular signaling (ERK, p38mapk) Tissue angiotensinogen Internalization
5 A truncated form of (P)RR was also found to be associated with the vacuolar H + -ATPase (V-ATPase) V-ATPase Cytosol Lumen ATP6AP2 ATP6AP2 Ludwig, J. et al. J Biol Chem 1998
6 V-ATPase acidifies the contents of intracellular compartments Forgac, M. Nat Rev Mol Cell Biol. 2007
7 Methods (P)RR lox neo /0 neo cassette deleter (P)RR lox /Y X loxneo Y XX αmhc-cre +/0 XY XX lox NPHS-Cre +/0 Atp6ap2 lox /Y αmhc-cre +/0 X lox Y Atp6ap2 lox /Y NPHS-Cre +/0 X lox Y Cardiomyocyte specific (P)RRKO mice (CKO) Podocyte specific (P)RRKO mice (PKO) XY
8 Cardiomyocyte-specific Atp6ap2 KO resulted in lethal heart failure (%) 100 Survival curve WT (n=12) CKO (n=15) Days
9 Echocardiography (M-mode) Postnatal day 18 WT CKO
10 Echocardiography (M-mode) WT Postnatal day 18 CKO
11 Histologic examination of the Atp6ap2 KO mice WT CKO 2 mm 50 μm
12 Electron microscopic examination of the Atp6ap2 KO cardiomyocytes WT 5 μm 2 μm 1 μm CKO A A V A 5 μm 2 μm 2 μm A Multi-vesicular vacuoles autophagic vacuoles
13 Electron microscopic examination of the Atp6ap2 KO cardiomyocytes
14 The cardiomyocytes contained RAB7-, LAMP2-positive multivesicular vacuoles in the perinuclear regions actinin RAB7 TOPRO3 actinin LAMP2 TOPRO3 WT CKO 10 μm
15 Defective autophagic protein degradation in the Atp6ap2 KO mice P62 SQSTM1 LC3-I LC3-II GAPDH WT CKO (kda)
16 Defective autophagic protein degradation in the Atp6ap2 KO mice WT CKO Asns Atf4 Gadd153 (fold) (fold) (fold) * * * * * 4 * 10 * 100 * 2 5 * 0 P10 P14 P18 0 P10 P14 P18 0 P10 P14 P18 *P < 0.05 vs WT
17 A truncated form of (P)RR was also found to be associated with the vacuolar H + -ATPase (V-ATPase) V-ATPase Cytosol Lumen ATP6AP2 ATP6AP2 Ludwig, J. et al. J Biol Chem 1998
18 (P)RR lox neo /0 (P)RR lox /Y X loxneo Y XX lox Ade-Cre MEF MEF (P)RR lox /Y (P)RR lox /Y Ade-Cre +/0 neo cassette deleter XX B6 XY X lox Y X lox Y
19 Deletion of ATP6AP2 resulted in the reduction of VO subunits Days after Cre virus infection a1 a2 a3 c E2 WT MEF Flox (kda) (P)RR β-actin 40 Kinouchi K, et al. Circ Res
20 Deletion of ATP6AP2 caused impaired endoluminal acidification LysoTracker staining a1 a2 a3 LT WT a1 a2 a3 LT Flox 50 μm Genetic ablation of ATP6AP2 selectively affects the stability and assembly of the V O subunits, thereby compromising vesicular acidification
21 Vehicle Bafilomycin 100 nm Sequential time-lapse analysis Chloroquine 10 μm Immunocytochemistry RAB7 LT
22
23 Chloroquine Bafilomycin Arbitrary unit Control The inhibition of intracellular acidification by bafilomycin A1 or chloroquine reproduced the phenotype observed for the (P)RR/ATP6AP2-deficient cardiomyocytes. Phase contrast RAB7 DAPI LT (fold) 4 (P)RR mrna * * μm 20 μm 20 μm 0 control Baf CQ *P < 0.05 vs control
24 Results Genetic ablation of Atp6ap2 created a loss-of-function model for V-ATPase. The gene product of ATP6AP2 is considered to act as in 2 ways: (1) as (P)RR, exerting a RAS-related function; and (2) as the V-ATPase-associated protein, exerting a non-ras-related function that is essential for cell survival.
25 Conclusions Heart failure (energy starvation) V-ATPase dysfunction Activation of local RAS Impaired vesicular acidification Up-regulation of ATP6AP2 Accumulation of autophagic vacuoles (autophagic cell death)
26 Keio University School of Medicine Jin Endo Takaharu Katayama Satori Tokudome Tomohiro Matsuhashi Shintaro Morizane Hideyuki Ito Yan Zhang Heping Zhou Yan Xiaoxiang Yoshinori Katsumata Ken-ichiro Kinouchi Shugo Toyama Keiichi Fukuda Kayoko Tamaki Ken Shinmura Takako Hishiki Makoto Suematsu Acknowledgements Atsushi Ichihara Hiroshi Ito
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