B In vivo QD. Ca pacgfp-actinin. Lipofectamine LTX 1 2,3

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2 X -CT MRI Ca 2+ 1 BIn vivo QD Transfection FuGENE HD Wistar ~300 g 50 mg/kg, i.p. FuGENE HD -actinin -QD 30 1 Tyrode Ca 2+ AB532 nm QD in vivo 2AcGFP 1 Wistar 2pAcGFP-actinin Invitrogen Lipofectamine LTX A Invitrogen Qdot655 3Ex vivo Ca 2+ Antibody Conjugation Kit 2.5% Z -actinin 8 Ca 2+ QDQD Cal-520 Tyrode 655 nm Wistar Cal-520 ~300 g0.3% Tyrode w/v Triton X-100 2,3 QD Ca 2+ Tyrode ATPase 2,3 2,3-butanedione monoxime BDM 30 fps

In vivo -Actinin-AcGFP ADVin vivo Z AcGFP ADV 2 AcGFP 488 nm AcGFP 40 N/A, 0.

00 100 Hz 1in vivo Z α-actinin -QD SPOCSL SPOC 2 10-6 MCa 2+ Ca-SPOC ADP ADP-SPOC

3 In vivo -Actinin-AcGFP ADVin vivo Z AcGFP ADV 2 AcGFP 488 nm AcGFP 40 N/A, N/A, Hz 1in vivo Z α-actinin -QD SPOCSL SPOC MCa 2+ Ca-SPOC ADP ADP-SPOC Ishiwata et al., Prog Biophys Mol Biol 2011 SPOC Sasaki et al., BBRC 2006QD SL 1 SL m SPOC 1 min 30 nm 5Alexa nm 1 min ~200 nm QD () α-actinin - QD Z 10 m SPOC ADP-SPOC 0.6 QD in vivo FuGENE-HD α-actinin -QD 3 2 m QD T Z Z QD 25 A) 2Aα-actinin -QD 1 Z QD BA QD T Z TT ZZ 2 Frank-Starling 1 Z α-actininacgfp 3 S.D.3 nm

Ca 2+ Fluo-4 8 nm SPOC Ca 2+ Ca-SPOC pca 6.0; 10 mm EGTA Ca 2+ Fluo-4 SPOC SPOC 2 A) C) 2.2 B) Z-disc position ( m m) 12 9 1 2 3 45 6 6 Phase 1 Phase 2 3 No.

4 Ca 2+ Fluo-4 8 nm SPOC Ca 2+ Ca-SPOC pca 6.0; 10 mm EGTA Ca 2+ Fluo-4 SPOC SPOC 2 A) C) 2.2 B) Z-disc position ( m m) Phase 1 Phase 2 3 No Time (s) 10 m m Phase Phase 2 3Ca 2+ Ca 2+ in vivo Ca 2+ BDM Ca 2+ Ca 2+ Ca 2+ Ca 2+ 4 SL ( m m) DSL=0.370±0.019 mm in vivo Ca 2+ A Time (s) Time (s) 3 AZ -actininacgfp B BCell-SPOC Z C BPhase 1,2 SLZ Ca 2+ C Cell-SPOC Ca 2+ SL SL 4 1 Hz SPOC / SPOC SL ( m m ) DSL=0.384 ±0.030 mm in vivo α-actinin-acgfp 100 fps BDM 2.0 μm 20 nm actinin-acgfp

5 μm 5 1. Kobirumaki-Shimozawa F, Inoue T, Shintani SA, Oyama K, Terui T, Minamisawa S, Ishiwata S, Fukuda N. Cardiac thin filament regulation and the Frank-Starling mechanism. J Physiol Sci May 1. [Epub ahead of print] 2. Shintani S, Oyama K, Kobirumaki-Shimozawa F, Ohki T, Ishiwata S, Fukuda N. Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with -actinin-acgfp in Z-discs. J Gen Physiol. 2014;143: Inoue T, Kobirumaki-Shimozawa F, Kagemoto T, Fujii T, Terui T, Kusakari Y, Hongo K, Morimoto S, Ohtsuki I, Hashimoto K, Fukuda N. Depressed Frank-Starling mechanism in the left ventricular muscle of the knock-in mouse model of dilated cardiomyopathy with troponin T deletion mutation ΔK210. J Mol Cell Cardiol. 2013;63: Kobirumaki-Shimozawa F, Oyama K, Serizawa T, Mizuno A, Kagemoto T, Shimozawa T, Ishiwata S, Kurihara S, Fukuda N. Sarcomere imaging by quantum dots for the study of cardiac muscle physiology. J Biomed Biotechnol. 2012;2012: Serizawa T, Terui T, Kagemoto T, Mizuno A, Shimozawa T, Kobirumaki F, Ishiwata S, Kurihara S, Fukuda N. Real-time measurement of the length of a single sarcomere in rat ventricular myocytes: a novel analysis with quantum dots.am J Physiol Cell Physiol 2011;301:C1116-C in vivo Yellow Cameleon-Nano α-actinin-acgfp Z Kotaro Oyama, Shuya Ishii, Tomomi Arai, Seine A. Shintani, Hideki Itoh, Norio Fukuda, Madoka Suzuki, Shin ichi Ishiwata THERMAL ACTIVATION OF CARDIAC THIN FILAMENTS INDUCES CONTRACTION WITHOUT INTRACELLULAR CA 2+ CHANGES: STUDIES WITH CARDIOMYOCYTES AND AN IN VITRO MOTILITY ASSAY San Francisco Seiichi Tsukamoto, Kotaro Oyama, Seine A. Shintani, Norio Fukuda, Shin ichi Ishiwata SIMULTANEOUS IMAGING OF LOCAL CALCIUM AND SINGLE SARCOMERE LENGTH IN RAT NEONATAL CARDIOMYOCYTES VIA EXPRESSION OF CAMELEON-NANO IN Z-DISCS San Francisco Seine A. Shintani, Kotaro Oyama, Takashi Ohki, Shin ichi Ishiwata, Norio Fukuda SARCOMERE LENGTH NANOMETRY IN CARDIOMYOCYTES EXPRESSEDWITHα-ACTININ-ACGFP IN Z-DISCS San Francisco Fuyu Kobirumaki-Shimozawa, Kotaro Oyama, Akari Mizuno, Takako Terui, Togo Shimozawa, Takashi Ohki, Shin ichi Ishiwata, Norio Fukuda REAL-TIME IMAGING OF SARCOMERE DYNAMICS IN THE MOUSE HEART IN VIVO San Francisco Norio Fukuda Real-time imaging of single sarcomeres in the mouse heart in vivo International Symposium on Nanomedicine Molecular Science ) In vivo Real-time imaging of single sarcomeres in the mouse heart in vivo Seine A. Shintani, Kotaro Oyama, Shin ichi Ishiwata, Norio Fukuda High-resolution analysis of sarcomeric auto-oscillations in rat neonatal cardiomyocytes Seiichi Tsukamoto, Kotaro Oyama, Seine Shintani, Fuyu Kobirumaki, Shin ichi Ishiwata, Norio Fukuda Simultaneous imaging of intracellular Ca 2+ and sarcomere length in neonatal cardiomyocytes via expression of cameleon-nano in Z-discs Fuyu Kobirumaki-Shimozawa, Kotaro Oyama, Seine A. Shintani, Erisa Hirokawa, Togo Shimozawa, Takako Terui, Shin'ichi Ishiwata, Norio Fukuda Real-time high-resolution cardiac imaging in vivo

6 nm in vivo 21 JSPPPEC Nano-imaging of sarcomeres in the heart in vivo Basis for Cardiac Nanophysiology and Nanomedicine Akari Mizuno, Fuyu Kobirumaki-Shimozawa, Kotaro Oyama, Takako Terui, Erisa Hirokawa, Togo Shimozawa, Shin ichi Ishiwata, Norio Fukuda Real-time measurement of sarcomere length in the mouse heart in vivo by using-actinin-gfp Tatsuya Kagemoto, Mitsunori Yamane, Cristobal G. Dos Remedios, Norio Fukuda, Shin ichi Ishiwata The effects of disease and aging on human myocardial SPOC Kotaro Oyama, Akari Mizuno, Seine Shintani, Hideki Itoh, Takahiro Serizawa, Norio Fukuda, Madoka Suzuki, Shin ichi Ishiwawta Ca 2+ -independent on-off regulation of a cardiomyocyte by microscopic heat pulses Seine Shintani, Kotaro Oyama, Norio Fukuda, Shin ichi Ishiwata SPOC Actinin-GFP (1) FUKUDA NORIO TAKAKO TERUI FUYU KOBIRUMAKI-SHIMOZAWA SATOSHI KURIHARA IWAO OHTSUKI SHIN ICHI ISHIWATA HIDEO HIGUCHI

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