Online Appendix for the following January 13 JACC article

TITLE: Sildenafil Stops Progressive Chamber, Cellular, and Molecular Remodeling and

Improves Calcium Handling and Function in Hearts With Pre-Existing Advanced

Hypertrophy Caused by Pressure Overload

AUTHORS: Takahiro Nagayama, PHD, Steven Hsu, BA, Manling Zhang, MD, PHD,

Norimichi Koitabashi, MD, PHD, Djahida Bedja, MS, Kathleen L. Gabrielson, PHD, Eiki

Takimoto, MD, PHD, David A. Kass, MD

APPENDIX

Supplemental Methods

Protein Western blot. Immunoblot analysis used the following primary antibodies:

MAP kinase ERK1/2 and phospho (Thr202/Thr204)-ERK, Akt and phospho (Ser473)-

Akt, glycogen synthase 3 (GSK3), phospho (Ser9)-GSK3source and titers reported

in Takimoto et al. (10)] PDE5a (1:1,000), calmodulin-dependent protein kinase II

(CaMKII; 1:1,000, Cell Signaling Technology, Inc., Danvers, Massachusetts), calcineurin

(Cn; 1:500), protein kinase C- (PKC1,000), and caveolin 3 (1:4,000, BD

Biosciences, Sparks, Maryland), PLB (1:2,000), phospho-Thr286-CaMKII (1:1,000,

Affinity BioReagents, Golden, Colorado), SERCA2a, PKC nPKC(1:750, 1:200,

1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, California), phosphor-Ser16-PLB

(1:2,000, Millipore, Billerica, Massachusetts), phosphor-Ser239-vasodilator-stimulated

protein (VASP; 1:1000, Alexis Corp., Lausen, Switzerland), and protein kinase G-1

(PKG-1; 1:2,000, gift from Dr. Michael Mendelsohn). Secondary antibodies were either

goat anti-rabbit immunoglobulin (Ig) G; goat anti-mouse IgG1, IgG2a, IgG2b; or donkey

anti-goat conjugated with horseradish peroxidase (Santa Cruz Biotechnology). Bands

were detected by chemiluminescence, and band intensity expressed in relative units. The

GAPDH was run as a loading control, and data are presented normalized to GAPDH

density.

Cell fractionation. For membrane/cytosolic fractionation, samples were homogenized in

a sucrose buffer (10 mM imidazole, 300 mM sucrose, 10 mM NaF, 1 mM EDTA) with

protease inhibitors (0.3 mM PMSF, 0.5 mM DTT, 10 µg/ml aprotinin, 10 µg/ml

leupeptin, 2 µg/ml pepstatin A) and centrifuged briefly at 2,000g to pellet the

nuclear/indestructible cell fraction. The supernatant was centrifuged at 20,000g for 1 h,

and supernatant kept as the cytosolic fraction. The pellet was resuspended in high-salt

buffer (25 mM Tris pH 7.4, 500 mM NaCL, 1 mM EDTA, 1 mM EGTA + protease

inhibitors) incubated for 30 min, and centrifuged at 16,000g for 10 min. The pellet was

resuspended in the same high-salt buffer with 0.5% Triton, incubated for 30 min, re-

centrifuged at 16,000g for 10 min, and the resulting supernatant collected as the

membrane fraction. Subfraction protein concentrations were equalized by BCA assay

(Pierce).

RNA analysis. Total RNA was prepared using TRIzol reagent (Invitrogen). Messenger

RNA was analyzed by quantitative real-time polymerase chain reaction (PCR) using

either SYBR green or Taqman probe method. Real-time PCR reactions were performed,

recorded, and analyzed using the ABI PRISM 7900 (Applied Biosystems, Foster City,

California). Samples were run in duplicate and normalized to GAPDH. The ANP, BNP,

MHC, and GAPDH mRNA levels were quantified by using SYBR green PCR primers

as previously described (Tsujita, Sussman PNAS 2006, 103:11946). In addition to ANP,

BNP, and MHC, regulator of calcineurin 1 (RCAN-1), SERCA2a, and PLB mRNA

levels were assessed using commercial primers (Applied Biosystems: RCAN-1,

Mm01213407_m1; SERCA2a, Mm00437634_m1; PLB, Mm00452263_m1; GAPDH,

Mm99999915_g1). The specificity of the SYBR green assays was confirmed by

dissociation curve analysis.

Confocal immunohistochemistry. Immunohistochemistry analysis was performed in

aliquots of adult myocytes isolated for the physiologic studies. Cells were fixed in 50%

methanol/50% acetone and incubated overnight with mouse PKC (1:250 dilution; BD

Biosciences), and secondary incubation with anti-mouse Alexa 488 (Molecular Probes)

for 1 h. Cells were then imaged on a Zeiss inverted epifluorescence microscope with

argon-krypton laser confocal scanning system (UltraVIEW; PerkinElmer Life And

Analytical Sciences, Inc., Waltham, Massachusetts). In additional studies, control

myocytes were isolated and then incubated with either phorbol 12-myristate 13-acetate

(PMA; 1M) alone or PMA plus sildenafil (1 M) for 2 h. After incubation, myocytes

were fixed and probed with PKC antibody as described above.