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1 Supporting Information Muraski et al /pnas SI Text Generation of Transgenic Animals. Pim-WT and Pim-DN cdnas were subcloned NheI/SmaI from pegfp-c1 Pim-1 and pegfp-c1 Pim-DN plasmids described previously (16), into the -MHC plasmid. Following linearization by NotI restriction digest, the fragment was gel purified and injected into 0.5 dpc zygotes of FVB/NJ mice using standard microinjection techniques. For our initial characterization of the transgenic lines, we established that multiple lines resulted in the same cardiac phenotype. After confirmation, we focused our studies on one transgenic line of each. Immunoblotting. The following primary antibodies were used: Pim-1 (Cell Signaling Technology and Zymed), GFP (Molecular Probes), GAPDH (Research Diagnostics), diluted in blocking solution overnight at 4 C. Blots were washed in TBS-0.5% Tween three times and probed with fluorescent or alkaline phosphatase-conjugated secondary antibodies diluted 1:5000 in blocking solution for 1hatroom temperature followed by three washes in TBS-0.5% Tween. Blots were scanned using a Typhoon 9410 (GE Healthcare). In Vitro Kinase and Luciferase Assay. In vitro kinase assay was performed using immunoprecipitated GFP-Pim-1 proteins (WT and KD) from whole heart lysates incubated in the presence of [ - 32 P]ATP with GST-p21 as substrates. For luciferase assays, C2C12 myoblast cells were transfected with the indicated plasmids together with the pgata-luc reporter construct (pgl3epor from Dr. Boyes), and the GATA-dependent luciferase activities were measured 2 days later. Amounts of plasmids used were pgata-luc, 0.2 g; pcmv-gata-1, 0.1 g; pcmv-pim-1, 0.2 g; pcmv-pim-1(kinase dead, KD), 0.2, 0.4, and 0.6 g, respectively. Where no plasmid is indicated, equal amounts of empty control plasmid were included. Cardiac Hemodynamics. Cardiac hemodynamics were performed under ketamine-acepromazine anesthesia. At time of death, mice were anesthetized with chloral hydrate (400 mg/kg b.w., i.p.) and the right carotid artery was cannulated with a microtip pressure transducer (FT111B Scisense Ontario, Canada) connected to an A/D converter to record data on a computer. Subsequently, the catheter was advanced into the LV cavity for the evaluation of LV pressures and and dp/dt in the closed-chest preparation (22). Animals were killed at the time points described in the text and following hemodynamic measurements hearts were arrested in diastole and perfused with phosphate-buffered formalin (22). Neonatal Rat Cardiomyocyte Cultures, Infections, and Treatments. Cardiomyocytes were infected with adenovirus for 2 h, washed in PBS, and then refed M199 with 2% FBS and 50 g/ml pen/strep, and 100 M glutamine. Cells were given 10 7 M ET-1 for 16 h in media M199, 1% BSA, and harvested or fixed for staining. For analysis of hypertrophy cells were treated as above however pretreated for 1hwiththePim-1 inhibitor Quercetagetin (10 nm, Calbiochem) and then incubated for 48 h with ET-1, replacing the media (inhibitor and ET-1) every 16 h. Myocyte Isolation. Following chloral hydrate anesthesia (400 mg/kg body weight, i.p.), the heart was excised and left ventricular (LV) myocytes were enzymatically dissociated (22). Briefly, the myocardium was perfused retrogradely through the aorta at 37 C with a Ca 2 -free solution gassed with 85% O 2 and 15% N 2. After 5 min, 0.1 mm CaCl 2, 274 units/ml collagenase (type 2, Worthington Biochemical Corp, Lakewood, NJ) and 0.57 units/ml protease (type XIV, Sigma, St. Louis, MO) were added to the solution which contained (mm): NaCl 126, KCl 4.4, MgCl 2 5, Hepes 5, Glucose 22, Taurine 20, Creatine 5, Na Pyruvate 5, and NaH 2 PO 4 5 (ph 7.4, adjusted with NaOH). At completion of digestion, the LV was mechanically dissociated to obtain myocytes for physiological measurements and paraformaldehyde fixation or resuspended in Ca mm solution. Volume measurements and total cardiomyocyte number calculations were performed as described previously (25). Myocyte Volume. Isolated myocytes were fixed in 4% paraformaldehyde for determination of myocyte volume (2). Cell length and width were measured with ImagePro software (Media Cybernetics, Silver Spring, MD) image analysis system and the ratio of the minor to the major axis of the ellipse was obtained by confocal microscopy. Cell volume was then computed assuming an elliptical cross-section with the major axis equivalent to cell width; the minor axis was computed from the measured ratios. Cell Shortening and Ca 2 Transients. Isolated LV myocytes were placed in a bath on the stage of an inverted microscope for contractility and Ca 2 transient measurements. Cells were perfused continuously at 37 C with a Tyrode solution containing 1 mm CaCl 2 and stimulated at 1 Hz pacing rate using platinum electrodes. Measurements were performed using epifuorescence (IonOptix) and video edge detection systems (Crescent Electronics). Myocytes were loaded with 10 M Fluo-3 a.m. (Invitrogen, Carlsbad, CA). Excitation length was 480 nm with emission collected at 535 nm using a 40 objective. Fluo-3 signals were expressed as normalized fluorescence (F/F 0 ). 1of10

2 Fig. S1. Generation and confirmation of Pim-WT and Pim-DN constructs for the creation of transgenic mice. (a) Schematic of fusion constructs whereby both the WT and DN Pim-1 genes are driven off the cardiac specific -MHC promoter. (b and -c) PCR of genomic DNA and Western blot of Pim-WT and Pim-DN transgenic animals. (d) Luciferase assay showing dominant negative action of Pim-1-DN construct using C2C12 myoblast cells transfected with the indicated plasmids together with the pgata-luc reporter construct. Error bars represent mean SD (n 6) from two independent experiments, each performed in triplicate. (e) In vitro kinase assay using immunoprecipitated GFP-Pim-1 proteins (WT and KD) from whole heart lysates incubated in the presence of [ - 32 P]ATP with GST-p21 as substrates. Samples were resolved on SDS/PAGE, and 32 P-labeled proteins were detected by autoradiography. Half of the reaction was subjected to Western blot analysis using the indicated antibodies. 2of10

3 Fig. S2. Increased necrosis in Pim-DN hearts. (a) Quantitation of necrosis in Pim-DN transgenic hearts at baseline levels compared to NTG and Pim-WT hearts weeks of age. 3of10

4 Fig. S3. Pim-1 is anti-hypertrophic and is regulated in response to pressure overload. (a) Cardiomyocyte size from NRCMs treated with or without Pim-1 adenovirus, Pim-1 inhibitor, and endothelin-1. (b) NTG mouse Pim-1 protein expression post-tac banding in NTG hearts with quantitation. (c) NTG mouse Pim-1 nuclear expression by immunohistochemistry 9 weeks post-tac banding as compared to nontreated controls. 4of10

5 Fig. S4. Pim-1 increases the number of new myocytes after banding. (a) Number of BrdU positive myocytes per unit area in the left ventricle 4 weeks after TAC banding (*, P 0.05). (b) Assessment of chromosome number in Pim-WT cells after TAC banding. 5of10

6 Fig. S5. Decreased ratio of phospho-plb to total PLB in Pim-DN hearts. Western blot and quantitation of basal protein expression of phospho-plb and total PLB in NTG, Pim-KO, Pim-DN, and Pim-WT hearts. GAPDH is used as a loading control (*, P 0.05). 6of10

7 Fig. S6. Pim-1 increases the total number of myocytes in the heart while decreasing myocyte size. (a) Histogram representing the volume distribution of myocytes in the left ventricle of NTG (top) and Pim-WT (bottom) hearts. Percentage of total myocytes represented (n 483 myocytes from n 5 animals). (b and c) Average volume and number of myocytes in NTG and Pim-WT hearts (**, P 0.01). Represented as number of myocytes 106. (d) Micrograph of myocytes in Pim-WT and NTG hearts. Sections are stained for laminin in green, tropomyosin in red, and topro (nuclei) in blue. 7 of 10

8 Fig. S7. Isolated Pim-DN myocytes are larger compared to NTG controls. Histogram representing the volume distribution of myocytes in the left ventricle of NTG (Top) and Pim-DN (Bottom) hearts. Percentage of total myocytes represented (n 700 myocytes from n 4 animals). 8of10

9 Fig. S8. Assessment of Pim-1 overexpression on MAPK pathway regulation. (a) In vivo analysis of ANP, pjnk, and perk protein expression in NTG and Pim-WT hearts after TAC. (b) In vitro protein analysis of ANP, pjnk, and perk in uninfected, EGFP, or Pim-WT expressing NRCMs after ET-1 treatment. 9of10

10 Table S1. Morphometric analysis confirms Pim-DN hearts are dilated. Morphometric analysis in NTG and Pim-DN hearts weeks of age Parameter NTG Pim-DN P-value Longitudinal axis, mm LV diameter, mm LV volume, mm Free wall thickness, mm Septal thickness, mm Wall thick/chamber radius LV mass/chamber vol of 10