Supplementary Methods. Animals and TAC surgery. Transgenic C57BL/6J mice expressing p115-rgs were

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1 Supplementary Methods Animals and TAC surgery. Transgenic C57BL/6J mice expressing p5-rgs were tried to generate three times. We obtained only one line that was used in the present study. Low success rate may be explained by the report that α-mhc promoter is active even during embryogenesis, and knockout mice of Gα are embryonic lethal due to defective organization of the vascular system. Therefore, p5-rgs may not completely inhibit functions of Gα / in this transgenic line. Transgenic mice after birth are normal for weight, blood pressure, and other characteristics except dp/dt max and dp/dt min. p5-tg mice showed slight reduction of dp/dt max and dp/dt min. We think that slight reduction of dp/dt max and dp/dt min reflects the effects of inhibition of Gα /, as real time PCR showed small decreases in expression of SERCA and phospholamban in p5-tg hearts. Two lines of transgenic mice expressing CA-Gα were generated (line and line 5). These two lines of transgenic mice showed similar expression levels of CA-Gα, and similar degree of fibrosis. Heterozygote of line 5 was used in this study. Age-matched male C57BL/6J mice were used as control. Histological analyses. Perfused and embedded hearts were sectioned and stained with hematoxylin and eosin (H&E) or picrosirius red. Digital photographs were taken at x magnification using AxioCam microscope camera (Carl Zeiss) or Biozero microscope (BZ-8, Keyence), and 6 regions selected at random for each specimen of the heart and then collagen volume fraction (CVF) was analyzed using Adobe Photoshop Software and Scion Image Software. The number of cardiomyocytes and the cross-sectional area (CSA) of each cardiomyocyte were measured within a sampling

2 region of x 4 μm. Six regions were selected at random for each specimen, and the average values of CSA were calculated using BZ-II Analyzer (Keyence) for all regions. Isolation of cardiomyocytes and transfection. Myocytes ( x 5 /cm ) plated onto laminin-coated silicone rubber culture dishes ( cm, STREX) were stretched by % with stretch system (STB-4-, STREX) (Naruse et al, 998). Cells were transfected with μg p5-rgs or an interaction-deficient mutant of p5-rgs (p5-mut) and.5 μg GFP using electroporation ( V, msec x 4, LMS Co. Ltd.). We confirmed that more than 7 % of cells are GFP-positive. For reporter assays, cells (.5 x 6 ) were transfected with.5 μg luciferase reporter constructs and internal control plasmid (prl-sv4) using electroporation. Luciferase activities were measured as described previously (Onohara et al, 6). Knockdown of PY receptors, PY 6 receptors or pannexin- in cardiomyocytes was performed using Stealth sirna oligonucleotides as described previously (Nishida et al, 7). Pulldown assay. Measurement of Rho, Gα and Gα activities in cardiomyocytes were performed as described previously (Nishida et al, 5). Cells were mechanically stretched for the indicated time, and lysed in buffer containing 5 mm Tris (ph 7.5),.% Triton X-, % glycerol, 5 mm NaCl, mm MgCl, mm dithiothreitol, μg/ml leupeptin, μ/ml aprotinin, mm phenylmethylsulfonyl fluoride. The supernatant of cell lysates (.5 mg proteins for Rho and. mg proteins for Gα / ) was incubated with μg of GST-Rho-binding domain (RBD) for Rho activation or μg of GST-protein phosphatase 5-tetratricopeptide repeat domain (GST-TPR) for Gα / activation in the presence of μl bed volume of

3 glutathione-sepharose beads rocking for min at 4 C. The bead was washed times, and finally suspended in SDS sample buffer. Pulled down Rho was detected with anti-rhoa antibody. For measurement of Rho and Rac activity in the heart, hearts were removed week after sham or TAC surgery. Supernatants of heart homogenates obtained after centrifugation ( mg proteins for Rho and mg proteins for Rac) were incubated with 5 μg of GST-RBD or μg of GST-protein p-activated kinase Cdc4/Rac interactive binding protein (PAK-CRIB) in the presence of μl bed volume of glutathione-sepharose beads rocking overnight at 4 ºC. The pulled down complexes were washed twice with ml of lysis buffer, and resuspended in 6 μl of SDS sample buffer. The protein samples ( μl) were fractionated by 5% SDS-PAGE. Aliquots of cell lysates from each sample were subjected to Western blot analysis to confirm that equal amounts of RhoA, Rac, and Gα / were present under each condition. Western blot analysis. The proteins (5 μg) were loaded and detected by antibodies: mouse OSF-/periostin (dilution rate: /, R&D Systems) and ACE (/, R&D Systems), HSP9 (/5, Santa Cruz), GAPDH (/5, Santa Cruz), RhoA (/, Santa Cruz) and JNK (/5, Santa Cruz), phospho-erk (/5, Promega), phospho-jnk (/, Promega), and TGF-β (/5, Cell Signaling), phospho-p8 (/, Cell Signaling), p8 (/, Cell Signaling), ERK (/5, Cell Signaling), phospho-akt (/, Cell Signaling) and Akt (/, Cell Signaling). We visualized the reactive bands by the enhanced chemiluminescence method (Perkin Elmer). The optical density of the film was scanned and measured with Scion Image Software.

4 Measurement of extracellular nucleotides concentration. The determination of extracellular ATP concentration ( x 5 cells/well) was performed using ATP Bioluminescence Assay Kit CLSII (Roche). The concentration of extracellular UDP in the supernatant of culture medium was analyzed with an HPLC system (Jasco) as described (Koizumi et al, 7). Twenty min before mechanical stretch, the culture medium was removed and substituted for Hank s balanced salt (HBS) solution containing 5 μm of ARL6756 (Sigma) with or without inhibitor. After mechanical stretch was performed, μl of supernatants was collected and quickly frozen with liquid nitrogen. For measurement of changes in [Ca + ] i, the cells on coverslips were loaded with μm fura-/am for min and then fluorescence images of the cells were recorded and analyzed as described previously (Onohara et al, 6). PY 6 receptors were stimulated by a perfusion with the supernatants or UDP-containing HBS solutions. Measurement of mrna expressions. Real time RT-PCR was performed as described (Nagamatsu et al, 6; Nishida et al, 7). Sequences for PCR primers and Taqman probes were described in Supplementary information (Supplementary Table 5). Primers and probes for 8S rrna, glyceraldehydes--phosphate dehydrogenase, pannexin (Assay ID: pannexin-: Rn447979_m (rat) and Mm459_m (mouse), pannexin-: Rn47567_g and Mm854_m, pannexin-: Rn647_m and Mm55586_m), and mouse TGF-β (β: Mm4474_m, β: Mm4695_m, β: Mm4696_m) were used according to the manufacturer s instructions (Applied Biosystems). For RT-PCR analysis, PCR amplification was performed in 5 μl with Pfx polymerase (Invitrogen) using a Takara PCR thermal cycler. The program of PCR amplification consisted of min denaturation at 94 ºC followed by 5 sec denaturation 4

5 at 94 ºC, sec annealing at 57 ºC, min extension at 68 ºC for 5 cycles, and min extension at 68 ºC. The PCR primers used for expression analysis of PY receptors are described in Supplementary Table 6. Statistical Analysis. Data were shown as means ± s.e.m. Statistical comparisons were made with two-tailed Student s t-test or analysis of variance followed by Student-Newman-Keuls procedure, with significance imparted at P values <.5. Supplementary Data. Supplementary data include 8 figures and 6 tables and can be found with this article online. References Koizumi S, Shigemoto-Mogami Y, Nasu-Tada K, Shinozaki Y, Ohsawa K, Tsuda M, Joshi BV, Jacobson KA, Kohsaka S, Inoue K (7) UDP acting at PY 6 receptors is a mediator of microglial phagocytosis. Nature 446: 9-95 Nagamatsu Y, Nishida M, Onohara N, Fukutomi M, Maruyama Y, Kobayashi H, Sato Y, Kurose H (6) Heterotrimeric G protein Gα -induced induction of cytokine mrnas through two distinct pathways in cardiac fibroblasts. J Pharmacol Sci : 44-5 Naruse K, Yamada T, Sokabe M (998) Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch. Am J Physiol 74: H5-H58 Nishida M, Tanabe S, Maruyama Y, Mangmool S, Urayama K, Nagamatsu Y, 5

6 Takagahara S, Turner JH, Kozasa T, Kobayashi H, Sato Y, Kawanishi T, Inoue R, Nagao T, Kurose H (5) Gα / - and reactive oxygen species-dependent activation of c-jun NH -terminal kinase and p8 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes. J Biol Chem 8: Nishida M, Onohara N, Sato Y, Suda R, Ogushi M, Tanabe S, Inoue R, Mori Y, Kurose H (7) Gα / -mediated up-regulation of TRPC6 negatively regulates endothelin--induced cardiac myofibroblast formation and collagen synthesis through nuclear factor of activated T cells activation. J Biol Chem 8: 7-8 Onohara N, Nishida M, Inoue R, Kobayashi H, Sumimoto H, Sato Y, Mori Y, Nagao T, Kurose H (6) TRPC and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy. EMBO J 5:

7 Supplementary Figure Legends Supplementary Figure. Generation of cardiac-specific p5-rgs-expressing transgenic mice. (A) Diagram of p5-rgs transgene construct. The α-myosin heavy chain (MHC) promoter was used to drive p5-rgs cdna expression. F, forward primer; R, reverse primer. α- and β (gray box) represent the exons encoding 5 -UTR of α-mhc and -UTR of β-mhc. (B) Genotype of wild type () and p5-rgs transgenic (p5-tg) mice. The bands represent the -bp PCR product with primers shown in (A) (F and R). (C) Cardiac-specific expression of p5-rgs proteins. The p5-rgs protein detected as about kda band was shown only in p5-tg heart. (D) Ang II-induced activation of Rho in and p5-tg heart. Thirty-min after intraperitoneal injection of Ang II (6 μg/kg), hearts were removed and washed in ice-cold phosphate-buffered saline containing nm Ang II. (E) TAC-induced activation of Rac in and p5-tg heart. Error bars indicate s.e.m.; n=. Supplementary Figure. Generation and functional analyses of cardiac-specific CA-Gα -expressing transgenic mice. (A) Genotype of wild type () and heterozygous CA-Gα (CA-Gα ) mice. (B) Cardiac-specific expression of CA-Gα proteins. The CA-Gα protein was detected as about 4 kda band, and was overexpressed only in CA-Gα heart. (C) Gα protein expression levels and activities in and CA-Gα heart (line and line 5). Results of Western blotting analyses of Gα were shown. Active Gα was detected by pulldown method 7

8 (Nishida et al., 5). GAPDH protein levels were used as an internal control. Error bars indicate s.e.m.; n=. *** indicate P <. versus. ## indicates P <. versus line. Supplementary Figure. Activation of Gα in cardiomyocytes induces interstitial fibrosis. (A) H&E-stained (scale bar, μm) and picrosirius red-stained sections of the heart isolated from -week-old and heterozygous CA-Gα mice. (B) Average CSA of cardiomyocytes. (C) Average collagen volume fraction (CVF) of heart sections. (D) Expression of ANP, β-mhc, α-ska, TGFβ-β to -β, CTGF, periostin, and ACE mrnas. (E) Expression of periostin, mature TGF-βs, ACE, and GAPDH proteins. The expression was plotted by the ratio to GAPDH. (F) Echocardiographic images of -week-old and homozygous CA-Gα mice. (G) Results of echocardiogram in and CA-Gα mice. Error bars indicate s.e.m.; n= (B, C), n=4 (D), n=-6 (E), and n= (G). Representative result of hearts from (n=) and CA-Gα -Tg mice (n=) was shown (A). Supplementary Figure 4. Requirement of extracellular nucleotides in mechanical stretch-induced Rho activation. (A) Effects of G protein-coupled receptor antagonists on mechanical stretch (MS)-induced Rho activation. Cardiomyocytes were treated with CV974 ( μm), PD9 ( μm), CGP7A ( nm), prazosin ( μm), and BQ ( μm) min before mechanical stretch stimulation. (B) Analyses of peak Rho activation induced by mechanical stretch. (C) Effects of apyrase on mechanical stretch-induced Rho activation. Cells were treated with apyrase ( U/ml) min prior to mechanical stretch. (D) Time courses of Rho activation 8

9 induced by various nucleotides. Cells were stimulated with μm of ATP, adenosine 5 -diphosphate (ADP), adenosine (ADO), uridine 5 -triphosphate (UTP), and uridine 5 -diphosphate (UDP) for the indicated time. Results of Western blotting analyses of Rho were shown (E and F). (G) Effects of p5-rgs on nucleotide-induced Rho activation. Cells were stimulated with μm of ATP, ADP and UTP for min. Rho activation by ATP in LacZ-expressing myocytes is set as %. (H) Concentration-dependent increase in Rho activity induced by -phenacyl-udp. Cells were stimulated by -phenacyl-udp for 5 min. Error bars indicate s.e.m.; n= (B, C, G, H) and n=6 (E, F). * indicates P <.5. Supplementary Figure 5. Expression of pannexin mrnas in mouse hearts and rat cardiomyocytes. (A) Expression of mrnas of pannexin- to - in control (sham) and TAC mouse hearts. N.D., not detected. (B) Expression of pannexin- and pannexin- mrnas in rat cardiomyocytes treated with respective pannexin- sirnas (si-, si-97, si-84) for 7 h. Error bars indicate s.e.m.; n=-4 (A) and n=5-6 (B). Supplementary Figure 6. Expression of PY receptors in mouse hearts. (A) RT-PCR analysis of RNA expression of PY receptor subtypes in mouse hearts. (B and C) Real time RT-PCR analysis of mrna expression of PY, PY, PY 6, and PY receptors in sham and TAC hearts (B), and wild type- and CA-Gα transgenic mouse hearts (C). The expression was plotted by the ratio to 8S rrna. Error bars indicate s.e.m.; n=-4 (B, C). Supplementary Figure 7. Involvement of P receptors in pressure 9

10 overload-induced cardiac fibrosis. (A-D) Effects of suramin ( mg/kg/day, started from days after TAC surgery) on TAC-induced fibrosis and hypertrophy (A and B), and cardiac function (C and D). Histological (A and B) and echocardiographic (C and D) analyses. (A) Picrosirius red staining. (B) CVF and HW/BW ratios. (C and D) M-mode echocardiograms. LVIDs, LV internal dimension at end systole; FS, fractional shortening. (E) Expression of hypertrophic and fibrogenic genes. Error bars indicate s.e.m.; n=5- (B, D), n=4-6 (E), and n= (F). Representative result from vehicle-(-)tac (n=5), vehicle-(+)tac (n=), suramin-(-)tac (n=6), and suramin-(+)tac mice (n=7) was shown (A). Representative result from vehicle-(-)tac (n=), vehicle-(+)tac (n=), suramin-(-)tac (n=), and suramin-(+)tac mice (n=) was shown (C). * indicates P<.5 and ** indicate P<.. Supplementary Figure 8. p5-rgs attenuates mechanical stress-induced activation of JNK and p8 MAPK and hypertrophic gene expression. (A and B) Effects of p5-rgs on mechanical stretch-induced increase in NFAT-luciferase activity (A) and BNP-luciferase activity (B). Cells were infected with LacZ or p5-rgs at MOI for 6 h, and then stimulated by mechanical stretch for 6 h (A) or 6 h (B). N.S. indicates not significant. (C and D) Effects of p5-rgs on mechanical stretch-induced activation of ERK, JNK, and p8 MAPK. Cells were stimulated with mechanical stretch for min (ERK and p8 MAPK) and for min (JNK). Error bars indicate s.e.m.; n=-4 (A-D).

11 A Not I α-mhc promoter p5-rgs β α : poly A Not I ~7kbp F R B C p5-tg p5 -Tg brain heart ileum skeltal muscle lung liver testis brain heart ileum skeltal muscle lung liver testis 4 bp 58 bp D Ang II active-rho p5-tg + + E TAC active-rac p5-tg + + total-rho total-rac Rho activity (fold) Ang II P<. P<. + + p5-tg Rac activity (fold) 6 P<. P<. 5 4 TAC + + p5-tg Supplementary Figure Nishida M et al.

12 A B 585 bp 4 bp Gα Gα CA -Gα CA-Gα brain thymus heart lung liver spleen intestine kidney testis skeltal muscle C active Gα total Gα GAPDH total / GAPDH (fold) active / GAPDH (fold) ## CA-Gα (line ) CA-Gα (line 5) Supplementary Figure Nishida M et al.

13 mrna / 8S rrna (fold) CVF (%) CSA (μm ) Supplementary Figure Nishida M et al. A B C 4 CA-Gα P<. CA -Gα D.. CA-Gα P<.5 CA-Gα P<.5. ANP βmhcα-ska TGF -β TGF -β TGF -β CTGF perio -stin ACE E periostin CA-Gα F TGF-β protein expression (fold) ACE GAPDH P<. 4 CA-Gα P<.5 P<.5 periostin TGF-β ACE G fractional shortening (%) CA-Gα 6 4 P<. CA -Gα ejection fraction (%) P<. CA -Gα

14 A DMSO (control) CV974 MS (min) active-rho total-rho PD9 CGP7A MS (min) active-rho total-rho prazosin BQ MS (min) active-rho total-rho B Rho activtiy (fold) 5 4 No-stretch control CV974 PD9 CGP7A prazosin BQ stretch C MS (min) active-rho total-rho Rho activtiy (fold) 4 apyrase Control apyrase stretch (min) D active-rho total-rho active-rho total-rho active-rho total-rho G active-rho total-rho Rho activtiy (%) ATP (min) 5 5 ADO (min) 5 5 UTP (min) ATP LacZ p5 ADP UTP LacZ p5 LacZ p5 ADP (min) 5 5 UDP (min) 5 5 E Rho activtiy (fold) H 5 4 ATP ADP ADO stimulation (min) F Rho activtiy (fold) 5 4 -Phenacyl UDP (M) active-rho total-rho Rho activity (fold) Phenacyl-UDP (M) UTP UDP stimulation (min) Supplementary Figure 4 Nishida M et al.

15 A pannexin mrna / 8S rrna (fold) P <. pannexin - pannexin - sham TAC N.D. pannexin - B pannexin mrna / 8S rrna (fold) control sirna si- si-97 pannexin- pannexin- si-84 pannexin- sirna Supplementary figure 5 Nishida M et al.

16 A (bp) 75 5 M PYR PYR PY4R PY6R PYR PYR PY4R B.5 sham TAC P<.5 C CA-Gα P<.5. P<.5 mrna (fold).5. mrna (fold).5. PYR PYR PY6R PYR PYR PY6R Supplementary Figure 6 Nishida M et al.

17 A vehicle (-) TAC (+) TAC B CVF (%) suramin TAC ( ) (+) ( ) (+) TAC ( ) (+) ( ) (+) vehicle suramin vehicle suramin C E F vehicle suramin mrna / 8S rrna (fold) active-rho total-rho (-) TAC (+) TAC ANP (-)TAC Rho activity (fold) collagen typei (+)TAC ACE D LVIDs (mm) suramin (-)TAC TAC ( ) (+) ( ) (+) vehicle suramin 9 6 HW / BW (mg/g) 6 FS (%) TAC ( ) (+) ( ) (+) TAC ( ) (+) ( ) (+) vehicle suramin vehicle suramin vehicle-sham vehicle-tac suramin-sham suramin-tac CTGF periostin TGF-β TGF-β suramin (+)TAC Rho activity (fold) CA-Gα (-) CA-Gα + suramin ( ) suramin CA-Gα Supplementary Figure 7 Nishida M et al.

18 A NFAT-Luciferase activtiy (fold) LacZ p5 -RGS LacZ N.S. p5 -RGS B BNP-Luciferase activtiy (fold) LacZ p5 -RGS LacZ N.S. p5 -RGS (-) stretch (+) stretch (-) stretch (+) stretch C (-) stretch (+) stretch D phospho-erk total-erk phospho-jnk total-jnk phospho-p8 total-p8 LacZ p5 -RGS LacZ p5 -RGS (kda) MAP kinase activtiy (fold) P<. P<. ERK JNK p8 Supplementary Figure 8 Nishida M et al.

19 Table Cardiac parameters measured by Millar Catheter -sham (n=) -TAC (n=6) p5-tg-sham (n=) p5-tg-tac (n=9) Heart Rate (bpm) 475 ± 474 ± 478 ± 48 ± 4 LVESP (mmhg) 5 ± 86 ± ± 85 ± 9 LVEDP (mmhg).9 ±.4 6. ±.5. ±.6 4. ±.4 # dp/dt max (mmhg/sec) 875 ± ± ± 4# 857 ± 4 dp/dt min (mmhg/sec) 664 ± ± 558 ± 8 # 7767 ± 8 tau (msec).9 ±..5 ±..7 ±.5#.5 ±.6 HR, heart rate; LVESP, left ventricular end systolic pressure; LVEDP, left ventricular end diastolic pressure; dp/dt max, maximal rate of pressure development; dp/dt min, maximal rate of decay of pressure; tau, monoexponential time constant of relaxation. P <.5, P <. vs Sham, and # P <.5 vs. Supplementary Table Nishida M et al.

20 Table Results of cardiac functions and organ weights CA-Gα Millar Catheter n 7 8 Heart Rate (bpm) 499 ± 6 49 ± 8 LVESP (mmhg) LVEDP (mmhg) dp/dt max (mmhg/sec) dp/dt min (mmhg/sec) tau (msec) 5 ± 4 ± 4.9 ±. 9. ±.7 9 ± ± 9 79 ± ± 98. ±.. ±. Organ weights n BW (g) 4.5 ± ±.7 HW (mg) 8 ±.8 7 ±.8 HW/BW (mg/g) 4.4 ± ±.9 LungW/BW (mg/g) 5.7 ±. 6. ±. LiverW/BW (mg/g) 44 ± 4 ± HR, heart rate; LVESP, left ventricular end systolic pressure; LVEDP, left ventricular end diastolic pressure; dp/dt max, maximal rate of pressure development; dp/dt min, maximal rate of decay of pressure; tau, monoexponential time constant of relaxation. P <.5, P <. vs. Supplementary Table Nishida M et al.

21 Table Results of cardiac functions and oragan weights vehicle-sham vehicle-tac MRS578-sham MRS578-TAC Millar Catheter n HR (bpm) ± ± 8 54 ± 4 5 ± 4 ESP(mmHg) 7 ± ± 7 9 ± 5 ± 7 EDP(mmHg).9 ±. 7.7 ±.7. ±. 4.4 ±.4 ## dp/dt max (mmhg/sec) 888 ± ± ± ± 55# dp/dt min (mmhg/sec) 6848 ± ± ± 899 ± ## tau (msec). ±. 6.9 ±..6 ±.4.6 ±.# Echocardiography n HR (bpm) 468 ± ± ± 448 ± 9 IVSd (mm).76 ±..6 ±..77 ±..7 ±. LVPWd (mm).77 ±..5 ±..77 ±..6 ±. LVIDd (mm).88 ±..47 ±.7.86 ±.8. ±.7 # LVIDs (mm).9 ±.8.75 ±.6.9 ±.4. ±.7 ## EF(%) 88. ± ± ±.6 7. ±. ## FS(%) 5.9 ±.. ±.8 5 ±.8 4. ±.9 ## Organ weights n BW(g) 5. ± ±. 4.6 ± ±. HW(mg) ± 97 ± 8 ± 89 ± 6 HW/BW(mg/g) 4.45 ± ± ± ±.8 LuW/BW (mg/g) 6. ±.. ±. 6. ±. 8.8 ±. LivW/BW (mg/g) 48. ±. 4. ± ± ±.8 HR, heart rate; IVSd, interventricular septum diastolic diameter; LVPWd, left ventricular posterior wall diastolic diameter; LVIDd, left ventricular internal diameters at end-diastole; LVIDs, left ventricular internal diameters at end-systole; EF,ejection fraction; FS, fractional shortening; ESP, end systolic pressure; EDP, end diastolic pressure; dp/dt max, maximal rate of pressure development; dp/dt min, maximal rate of decay of pressure; tau, monoexponential time constant of relaxation; BW, body weight; HW, heart weight; LuW, lung weight; LivW, liver weight. P<.5, P<. vs sham, and # P<.5, ##P<. vs vehicle-tac. Supplementary Table Nishida M et al.

22 Table 4 Results of cardiac functions Vehicle-Sham Vehicle-TAC Suramin-Sham Suramin-TAC Millar Catheter n HR (bpm) ± ± ± 7 5 ± 4 dp/dt max (mmhg/sec) 9864 ± ± ± ± 45## dp/dt min (mmhg/sec) 7689 ± 47 ± ± ± 49## tau (msec) 9.6 ±.. ±.5.9 ±.6. ±.4 Echocardiography n HR (bpm) 48 ± 5 47 ± 449 ± 447 ± 7 IVSd (mm).75 ±..5 ±..7 ±.. ±. LVPWd (mm).75 ±.. ±.4.7 ±.4. ±. LVIDd (mm).87 ±..4 ±.8.75 ±.7.8 ±.## LVIDs (mm).4 ±..5 ±.7.44 ±.8.79 ±.9## HR, heart rate; IVSd, interventricular septum diastolic diameter; LVPWd, left ventricular posterior wall diastolic diameter; LVIDd, left ventricular internal diameters at end-diastole; LVIDs, left ventricular internal diameters at end-systole; dp/dt max, maximal rate of pressure development; dp/dt min, maximal rate of decay of pressure; tau, monoexponential time constant of relaxation. P <.5, P <. vs Sham, and #P <.5, ##P <. vs Vehicle-TAC. Supplementary Table 4 Nishida M et al.

23 Table 5 Primers used for real time RT-PCR analysis Gene Name mouse -skeltal actin CTATGTGGCCCTGGACTTCGAGAATGAGAT forward GTGCGCGACATCAAAGAGAAG ( -SKA) reverse GCAACGGAAACGCTCATTG -myosin heavy chain ACCCCTACGATTATGCGTTCATCTCCCAA forward TTTCTACCAAATCCTGTCTAATAAAAAGC ( MHC) reverse GTCATCAATGGAGGCCACAGT atrial natriuretic peptide ATTTCAAGAACCTGCTAGACCACCTGGA forward CATCACCCTGGGCTTCTTCCT (ANP) reverse TGGGCTCCAATCCTGTCAATC connective tissue TCAAATGCCCCGATGGCGAGAT forward CTGCACCAGTGTGAAGACATACAG growth factor (CTGF) reverse TCCCCAGGACAGTTGTAATGG procollagen type CTATCCAGCTGACCTTCCTGCGCCTAAT forward GAGAGAGCATGACCGATGGATT (collagen type I) reverse GCTACGCTGTTCTTGCAGTGAT procollagen type CCACCTTGGTCAGTCCTATGAGTCT forward CAGCAGTCCAACGTAGATGAATTG (collagen type III) reverse CATGGTTCTGGCTTCCAGACA periostin TAACCAAGGACCTGAAACACGGCATGG forward GAATGCCTTACACAGCCACATG reverse GCCCCAGATTGTTGTACATTGA PY receptor CCTGCCGGCTGTCTACATTTTAGTGTTCAT forward CCAAGACCGGTTTCCAGTTC reverse TCCAGATAGCCACGCTGTTG PY receptor CCTGGAACGCCTCCACCACCTACAT forward TCTTCCTATGCCGCCTCAAA reverse CCGAAACTGCCAGGTGAAA PY6 receptor TGGCCCTCACGGTCATCGGC forward AACCGCACTGTGTGCTACGA reverse GCGACAATAACAAGCCAGTAAGG PY receptor AAACGTCCAGCCCCAGCAATCTCTTG forward TGAAGACCACCAGGCCATTT reverse AGGCCCAGATGACAACAGAAA rat CTGF AGGCCCTGTGAAGCTGACCTAGAGGAAAAC forward TACCTTCTGCAGGCTGGAGAA reverse GCGTCCGGATGCACTTTTT TGF TGGTGGACCGCAACAACGCAATC forward AGAAGTCACCCGCGTGCTA reverse TGTGTGATGTCTTTGGTTTTGTCA TGF ATGCCATCCCGCCCACTTTCTACAG forward CCAGGTGCTCTGTGGGTACCT reverse CGAAGCGGACGATTCTGAA PY receptor CTCATGGCCCGACGGCTGC forward TCCATCATCCTGGTCTGTTACGT reverse GGTCCCATAAGCCGGTTTG PY6 receptor TGGCCCTCACGGTCATCGGC forward CACCCGCTACCTGCCGTAT reverse CGACAGTAGCAGGCCAGTAAGG Supplementary Table 5 Nishida M et al.

24 Table 6 Primers used for RT-PCR analysis Gene Name PCR Primer Sequence mouse PY receptor forward TTTGGCTCTGGCTGACTTTT reverse AAGTGGCATAAACCCTGTCG PY receptor forward TTCAAGTACGTGCTGTTGCC reverse TGGTGACGAAGTAGAGCACG PY4 receptor forward ACTAGGTCCCAGCCCAAGTT reverse GCACCATGATTGTGGAACTG PY6 receptor forward CATTAGCTTCCAGCGCTACC reverse CCGGAACTTCTGTTGTGTGA PY receptor forward CACCTCAGCCAATACCACCT reverse AACATGAAGGCCCAGATGAC PY receptor forward CTTCTTTGTCTGCTTTGCCC reverse TGTTGCATGTGCCTCTCTTC PY4 receptor forward GGCAGGTGAATGTGTTTGTG reverse AATACTTGGAAGGGGATGGG Supplementary Table 6 Nishida M et al.