Supplementaary Material

SUPPLEMENTAARY MATERIAL

Title : Targeted ablation of the histidine-rich Ca2+-binding protein (HRC) gene is associated with abnormal SR Ca2+-cycling and severe pathology under pressure-overload stress

Chang Sik Park1,*; Shan Chen2,*; Hoyong Lee1,*; Hyeseon Cha1; Sunghee Hong1; Peidong Han2; Kenneth S. Ginsburg3; Sora Jin1; Inju Park1; Jae Kyun Oh1; Yeong Seop Yun1; Hong-Sheng Wang2; Vivek P. Singh2; Clara Franzini-Armstrong4; Woo Jin Park1; Donald M. Bers3; Evangelia G. Kranias2; Chunghee Cho1; Do Han Kim1

1School of Life Sciences and Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea. 2Department of Pharmacology and Cell Biophysics, University of CincinnatiCollege of Medicine, Cincinnati, OH. 3Department of Pharmacology, University of California Davis, Davis, CA. 4Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA. 5Molecular Biology Division, Center for Basic Research, Foundation for Biomedical Research of the Academy of Athens, Greece

*Authors contributed equally to this work

Address of correspondence: Do Han Kim, College of Life Sciences, GIST, 123 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Korea, Tel: 82-62-715-2485; E-mail: , Chunghee Cho, College of Life Sciences, GIST, Korea, Tel: 82-62-715-2490; E-mail ; Evangelia G. Kranias, Department of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267, U.S.A., Tel: 1-513-558-2377; E-mail

Supplemental Figure legend

Supplemental Figure1.Measurementsof NCX orDHPR Ca2+current in WT and HRC-KO animal hearts.

a: Average current density-voltage relationship of the NCX current recorded from HRC-KO (n=9 cells from 3 hearts) and WT cells (n=7 cells from 3 hearts). Note that there wasno differenceof density-voltage relationship between isolated WT and HRC-KO ventricular myocytes.b: Average peak current density-voltage relationship of the L-type Ca2+ currents in WT (n=10 cell from 3 hearts) and HRC-KO (n=10 cells from 3 hearts) ventricular cells. Note that there was no difference in the average current-voltage relationship of the L-type Ca2+ currents between WT and KO cells. Data are means ± SEM.

Supplemental Figure 2.Expression levels of Ca2+ handling proteins from WT and HRC-KO hearts under sham and TAC condition

Cardiac protein samples were separated by SDS-PAGE and immunoblotting were performed with antibodies against SERCA2a, RyR2, DHPR, CSQ, PLB and TRN protein. a: Representative Western blot results of the Ca2+handling proteins in WT and HRC-KO hearts under sham and TAC condition. b: Quantified Ca2+handling protein levels. n=6 hearts from each group.Data are means ± SEM. * P < 0.05.

Supplemental Figure 3. TRN and HRC expressions in WT and HRC-KO hearts.

Cardiac protein samples were separated by SDS-PAGE and immunoblotting was performed with antibodies against TRN and HRC proteins. a: Representative Western blot result of HRC, cardiac TRN 1 and alpha tubulin in 16- and 24-weeks old WT and HRC-KO hearts under basal condition. b: Relative expressions of TRN and HRC. n=6 hearts from each group.Data are means ± SEM. ** P < 0.01.

Supplemental Figure 4. Ca2+ sparks in HRC-KO cardiomyocytes under basal conditions.

Properties of Ca2+ sparks in WT and HRC-KO cardiomyocytes in the presence of 1 μmol/L isoproterenol.n=30 cells from 3 WT hearts; n=40 from 3 HRC-KO hearts.Data are presented as mean ± SEM. *P<0.05.

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