Yokohama City University School of Medicine Susumu Minamisawa (
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1 Yokohama City University School of Medicine Susumu Minamisawa (
2 skeletal muscle cardiac muscle mitochondria Sarcoplasmic reticulum mitochondria I A T-tubule
3 SR Free Ca 2+ T-tubule SR Opi. The Heart, 3rd edition
4 Phospholamban is an endogenous inhibitor of SERCA activity PLN PLB S16 T17 +K -D -D +K P V Ca 2+ PLB Ca 2+ A Kinase PKA P P P PLN S16 T17 +K -D -D +K P V SERCA2 cytosole SERCA2 V49 V49A lumen Ca 2+
5 SERCA2a and Phospholamban are critical regulators of calcium cycling in the heart
6 PLNKO Mice Exhibit Enhanced Myocardial Contractility and Loss of β-agonist Stimulation dp/dt max (mmhg/sec) PLNKO wildtype Isoproterenol [M] Luo et. al.:circ Res 75: 44-9, 1994
7 Gene Complementary Strategy Copy right : Terrytoons Copy right : Azumi Kosawa PLNKO mouse MLPKO mouse
8 Phospholamban gene ablation prevents the progression of dilated cardiomyopathy MLP KO FS 9% ESD EDD 2mm MLP KO/ PLN KO FS 32% 2mm 2 msec Minamisawa et al. Cell 1999
9 S16E PLN mutant gene transfer by adenoviral vector improved myocyte contractiliy in DCM model mice MLPKO MLPKO S16EPLN wildtype
10 Enhancement of calcium uptake via the sarcoplasmic reticulum is a potent therapeutic strategy for heart failure Strategies to increase SERCA2a protein Strategies to modulate SERCA2a to increase calcium transport Strategies to decrease PLB protein Strategies to disrupt the interaction between SERCA2a and PLN
11 The advantages of phospholamban as a prime target to increase SERCA2a activity (1) PLN is the strongest (probably only) endogenous inhibitor of SERCA2a in the ventricles. PLN is a terminal effector of β-adrenergic signalling pathways. PLN expression is highly cardiac specific and is much higher in ventricles than atria. PLN inhibition does not affect chronotropic responses.
12 The advantages of phospholamban as a prime target to increase SERCA2a activity (2) PLN is a small protein. The genetic modification can be easily manipulated. PLN is remarkably conserved between species. PLN-deficient mice have not displayed any adverse events so far.
13 Seidman JG & Seidman C: Cell 14: , 21 HCM Gene Mutations DCM QuickTime???? - JPEG???????????
14 Arrhythmogenic Right Ventricular Dysplasia Cathecholamine-sensitive Ventricular Tachycardia Ca 2+ Ca 2+ DCM/HCM Ca 2+
15 Calcium cycling defects due to a gene mutation of the SR proteins cause cardiomyopathy
16 PLN PLB Ca 2+ Ca 2+ S16 T17 +K -D -D +K P V SLN? SERCA2 cytosole SERCA2 V49 V49A lumen Sarcalumenin
17 Sarcalumenin(SAR) is a lumenal Ca 2+ binding glycoprotein in the longitudinal SR of striated muscles. Ca 2+ binding Region SP Long isoform (16kD) SAR SP Short isoform (53kD) Putative ATP/GTPase Domain
18 Generation of SAR-KO mice
19 Mild systolic dysfunction was detected in 6-week 6 SAR-KO mice A. LV%FS B. Vcfc C. 4 * 3. * WT KO WT KO. WT KO
20 Survival Rates after TAC were Lower in SAR-KO Mice than in WT Mice A. Survival Rates B. Pressure Gradients 8 WT-TAC (n=11) 6 KO-TAC (n=1) 4 2 Log Rank Test : P<.5 days WT KO
21 SAR Deficiency Caused the Progressive Dysfunction in Response to Pressure Overload A. dp/dt dt max [ratio to control] Sham TAC ** B. tau [ratio to control] Sham ** TAC C. LVEDP [ratio to control] Sham ** TAC * P <.5, vs. WT
22 SERCA2a Protein, but not mrna was Decreased in SAR-KO Mice mrna WT KO SERCA2a GAPDH Protein WT KO SERCA2a n.s * - 18% WT KO WT KO * P <.5, vs. WT
23 Co-localization of SAR and SERCA2a Co- expressed in HEK-293T Cells mserca2 (FITC) DIC/F L SAR (TRITC) DIC/FL DIC/FL Overlay FL FL FL Excitation:488nm Filter:BP55-53 Excitation:543nm Filter:BP * LSM51 (Carl Zeiss) * Scale bar : 1uM
24 Interactions between SAR and SR proteins in cardiac muscles IP Ab : IP (-) SERCA normal mouse IgG IB Ab : SAR SERCA2 Lysate IP-P IP-S IP-P IP-S
25 Rapid Protein Degradation of SERCA2a without SAR 1) SAR(-) 3) Protein Half-life life SERCA2 -Actin CHX(+) 2) SAR(+) CHX(+) days Protein Levels SERCA days.2 -Actin Time(days)
26 Interactions between SAR and SERCA2a were Dissociated by Elevated Ca 2+ Interactions between SAR and SERCA2a Co-expressed in HEK-293 T-Cells (IP) a. IB : SAR pca IB : SERCA SAR SERCA2 Lysate - IP:SERCA 2 + b. IB : SERCA2a IP:mouse IgG + Lysate IP:SAR IP:rabbit IgG [Relative to pca 7] SAR SERCA pca
27 Summary -SAR deficiency induced the progressive impairment of cardiac function, especially in response to pressure overload. -SAR interacts with SERCA2a in a Ca 2+ -dependent manner. -The physical interaction of SAR with SERCA2a increases the protein stability of SERCA2a by its presumable chaperone-like activity.
28 PLN PLB Ca 2+ Ca 2+ S16 T17 +K -D -D +K P V SLN? SERCA2 cytosole SERCA2 V49 V49A lumen Sarcalumenin
29 Sarcolipin (SLN) is a 31-amino acid proteolipid in the SR. SLN is a shorter homologue of PLN.
30 The expression of mouse sarcolipin mrna was most abundant in the atria and was not detected in the ventricles. LA LA LV RA LV RV Mitral valve Minamisawa et al. J Biol Chem 278: 957, 23
31 ed 14.5 skeletal muscle ed 12.5 ventricle ed 1.5 ed 12.5 ed 16.5 ed 18.5 day 1 day 3 day 7 day 21 2 year atria aorta atria SERCA1 sarcolipin GAPDH atrium vent r icle ventr icle s keletal muscle sk eletal muscle mouse human sarcolipin SERCA1 GAPDH
32 Abnormal intracellular Ca 2+ homeostasis may be an important modulator of sustained atrial fibrillation (AF).
33 A SLN ANF Control MVD+NSR MVD+AF #9 #8 #5 #4 #9 #3 Patient s number B Relative abundance of SLN mrna (arbitrary units) GAPDH 1 P<.5 2 p<.1 ns Control MVD+NSR MVD+AF Uemura et al. Eur J Clin Invest 34: 723, 24
34 Pressure overload down-regulates SLN mrna in the loaded atria.
35 Transverse aortic constriction down-regulated SLN mrna in LA A C ** * RA LA RA LA Sham TAC RA LA RA LA Sham TAC SERCA2a/GAPDH mrna (arbitrary unit) BNP/GAPDH mrna (arbitrary unit) SLN/GAPDH mrna (arbitrary unit) PLN/GAPDH mrna (arbitrary unit) B D * * * Shimura et al. BBRC 334: 861, 25 * *
36 Monocrotaline down-regulated SLN mrna in RA * ** SERCA2a/GAPDH mrna (arbitrary unit) SLN/GAPDH mrna (arbitrary unit) ANF/GAPDH mrna (arbitrary unit) PLN/GAPDH mrna (arbitrary unit) ** MCT-RA MCT- LA ** MCT-RA MCT- LA Shimura et al. BBRC 334: 861, 25
37 Heart-specific SLN Transgenic Mice αmhc promoter mouse SLN SV4pA Northern Blot Analysis of Ventricular Tissues SLN ANF GAPDH
38 In vivo hemodynamic assessment on SLN transgenic mice Heart Rate (bpm) ns dp/dtmin (mmhg/sec) p<.1 dp/dtmax (mmhg/sec) NTG ns TG Tau (sec) NTG p<.1 TG
39 ANF and BNP mrnas were increased in SLN TG ventricles ANF BNP SERCA2a ns * 2.5 * PLN ns RyR2 ns NCX1 HRC ns 1.2 Relative expression Relative expression Relative expression Relative expression CSQ2 ns ns C TG C TG C TG C TG Relative expression Relative expression Relative expression Relative expression
40 Summary -SLN is an atrial type of PLN homologue and inhibits diastolic cardiac function by inhibiting the SERCA2a activity. -SLN is down-regulated in the atria with atrial fibrillation and with pressure-overload, which may affect the atrial function through the change in atrial Ca 2+ cycling.
41 University of California, San Diego Masahiko Hoshijima John Ross, Jr. Kenneth R. Chien University of Maryland Yibin Wang University of Cincinnati Evangelia G. Kranias Tohoku University Hiroshi Takeshima Tokyo Women s Medical University Rumiko Matsuoka National Institute of Health Sciences Yoji Sato Yamaguchi University Yasuhiro Ikeda Tomoko Ohkusa Yokohama City University Miei Shimura Yoshihiro Ishikawa
42 The clinical profile of the patient with mutation 77A G Sex: female Age: 58 years old Family history: father (cardiomyopathy) a brother (HCM) Clinical symptom: palpitation at the age of 56 years The 12-lead ECG : LVH and ST-T wave abnormality Echocardiography: The wall thickness of LV septal wall : 3 mm posterior wall: 13 mm The dimension of LV diastolic: 48 mm systolic: 27mm
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