Supple-Zhang Page 1 Wild-type locus Targeting construct Targeted allele Exon Exon3 Exon Probe P1 P P3 FRT FRT loxp loxp neo vector amh I Homologous recombination neo P1 P P3 FLPe recombination Q,!?R'!! /!!!!1"?R'! SOS! E TOS! $R7! $&R7! SOS! TOS! TOT! Floxed allele Knock out FRT P1 Exon Exon3 P3 Exon Exon re recombination - mtor - GPH F G <,=! /! Supplemental Figure 1. Generation and assessment of an inducible, cardiac-specific mtor knockout mice model. () Strategy used to knock out mtor in mouse cardiomyocytes and generate the mtor-c model. Exon and 3 of the mtor gene was flanked by loxp sites, and a neomycin (Neo) resistance cassette, flanked by FRT sites, inserted upstream. Maps of the wild-type mtor locus, the targeting vector, the floxed allele, and the excised allele are shown. Exons are shown as boxes. The position of the 3 probes used for Southern blot analysis in are indicated. rrows indicate primer positions for PR. rrowheads indicate the loxp sites. () Southern blot of N obtained from wild-type (+/+) and 3 heterozygous (F/+) embryonic stem cell clones using probe shown in after digestion with HindIII. The 1.5 kb and 8 kb bands represent respectively the WT and the mutant allele. () Successful recombination of mtor confirmed by PR on genomic N isolated from amh-mm/mtor F/F (F/F) mouse, wild-type mouse (+/+), and heterozygous mouse (F/+) characterized by PR with primers P1 and P allowing the amplification of the loxp-containing deletion region shown in ; a 389-bp fragment for the floxed mtor and 319-bp fragments for the wild-type allele. () Experimental schedule of tamoxifen administration and echocardiographic (EHO) analyses. (E) Western blot demonstrating significantly reduced mtor protein in heart homogenate from an mtor-c () mouse at weeks post-tmx., WT-re control; GPH, glyceraldehyde 3-phosphate dehydrogenase. (F) Gravitometric data analyses (mean±s, n=6 each group). White bars, control, lack bars, mtor-c. HW, heart weight. W, body weight. LV, left ventricle. RV, right ventricle. L, left atrium. R, right atrium. (G) Representative -echocardiograms of and mice at wks post-tmx. FS, fractional shortening; HR, heart rate. mtor regulates heart survival and function through E-P1
Supple-Zhang Page (x) E-P1- P<.5 P<.5 P<.5 d P<.5 P<.5 Supplemental Figure. () Representative fluorescence micrographs of and mtor-c myocardium sections stained with wheat-germ agglutinin to visualize the cell membrane (red). () Representative photo-micrographs of isolated cardiomyocytes from the various mice lines (original magnification, X). () Measurement (mean±sem) of length and width (left) and calculation of cell area (right) of isolated cardiomyocytes. () Immunofluorescence for KI67 (left) and phosphorylated histone H3 (right) did not reveal a difference in cellular proliferation in the myocardium of control and mtor-c mice. mtor regulates heart survival and function through E-P1
Supple-Zhang Page 3 (x) -1wk -wks (x) Tunel postive nuclei (x) -1wk -wks E-P1 mrn (GPH-normalized fold change) P=.18 1 x 1x Supplemental Figure 3. () Representative immunofluorescence images of heart sections processed with TUNEL to reveal apoptotic nuclei (arrows). ardiomyocytes were decorated for!-sarcomeric actin (red) and nuclei were stained with PI (blue). () Representative heart sections stained with Malloy's trichrome to reveal collagen deposition. Increased interstitial fibrosis (turquoise) is evident in mtor-c myocardium at weeks (wks) post-tmx. () E-P1 mrn expression, by qrtpr, in mtor-c and control myocardium at weeks post-tmx (mean±s, n=3- per group). () Immunohistochemical staining for E-P1 in WT myocardium. mtor regulates heart survival and function through E-P1
Supple-Zhang Page T!" ay 1 6 7 19 7 Western blot qpr EHO (wk ) Tamoxifen 3mg/Kg/day EHO (wk 1) EHO (wk 3) Gradient check and SRIFIE (wk ) Supplemental Figure. () Experimental schedule of tamoxifen administration and transverse aortic constriction (T). EHO, echocardiographic analyses. The pressure gradients were checked at week post-t, before sacrifice. () Weights of the left ventricle (LV) and of hearts (HW) given as a ratio with tibial length (TL) after weeks of T (3 weeks post-tmx) (n=5 per group). () Kaplan-Meier survival curves of mtor-c subjected or not to transverse aortic banding (-T n=1 or -sham n=5, respectively) and of sham-operated WT-re (-sham n=5) mice. Mortality of mtor-c mice is significantly increased by pressure overload, with a % death rate after the first week of T. Time shown as weeks post- TMX. mtor regulates heart survival and function through E-P1
Supple-Zhang Page 5 E-P1- dp/dtmax (X1) P<.5 1. 1. 15 1 5 &1"! O (U).8.6.. dp/dtmin (X1) [3H]Leucine Incorporation (fold change) E-P1-.75 6 5 1 15 E E-P1- d F poptosis (positive nuclei/mm) Fibrotic area (%) 3 1 E-P1- d 1 1 8 6 E-P1- d E-P1- d Supplemental Figure 5. nalysis of E-P1 knockout and mtor/e-p1 double knockout mice. () [3H]Leucine incorporation is enhanced in mice harboring deletion of E-P1. () E-P1 knockout mice have improved inotropy and lusitropy at baseline and improved reactivity to dobutamine (O) (means±s, n=6 for each group). () Representative photomicrographs of heart sections stained with Malloy's trichrome to evidence collagen deposition (turquoise) (original magnification, X). () nalysis of fibrosis at weeks post-tmx (means±s). (E) Representative photomicrographs of heart sections stained with TUNEL to evidence apoptotic nuclei (arrows) and decorated for!-sarcomeric actin (Red) (original magnification, X). (F) nalysis of apoptosis at weeks post-tmx (means±s). mtor regulates heart survival and function through E-P1