(A) Left panel: representative Western blots of SirT1, histone H1, acetyl-H1 (Lys26), detected in nuclear extracts, and of p53 and acetyl-p53 (Lys382), detected in whole tissue lysates, from wild type and mIGF-1 Tg mice. Four animals of a total of 10 are shown; right panel: densitometric quantification of SirT1, acetyl-H1(Lys26)/H1 and acetyl-p53(Lys382)/p53 levels in cardiomyocytes from mIGF-1 mice, expressed as % of those in wild type cardiomyocytes. (B) representative Western Blot of mIGF-1 detected in the extracellular medium of HL-1 cardiomyocytes, transfected with a plasmid carrying mouse mIGF-1 cDNA. (C) Left panel: representative Western Blot of SirT1, histone H1, acetyl-H1 (Lys26) detected in nuclear extracts, and of p53 and acetyl-p53 (Lys382) detected in whole cell lysates, from HL-1 cardiomyocytes transfected with the indicated constructs (SirT1 or SirT1 H363Y) or treated with 20 ng/ml IGF-1 for 24 hours; right panel: densitometric quantification of SirT1, acetyl-H1(Lys26)/H1 and acetyl-p53(Lys382)/p53 levels in transfected or treated cells, expressed as % of control (CTL). (D) Representative Western blots of IGF-1 receptor (IGF-1R) or phospho-IGF-1R (on Thr 1135/1136) in HL-1 cardiomyocytes lysates. Results in (A) and (B) are means ± SE of 3 independent experiments (^**,***p versus unstimulated control cells or untreated WT cardiomyocytes).

(A) HL-1 cardiomyocytes were transfected or treated as in Legend of Figure 2A. Sarcomeric myosin was stained with MF-20 antibody and images were acquired using a Leica confocal microscope. (B) Cell size and cell hypertrophy quantified according to MF-20 staining in HL-1 cardiomyocytes in the different experimental conditions as in as in Legend of Figure 2A. Results are means ± SE of 3 independent experiments (^*,**p versus unstimulated control cells). Bar: 25 μM.

(A) Neonatal primary cardiomyocytes from wild type or mIGF-1 Tg mice were treated as in Legend of Figure 2B. (B) Cell size and hypertrophy were quantified according to MF-20 staining in the different experimental conditions as in as in Legend of Figure 2B. Results are means ± SE of 3 independent experiments (^*,**p versus unstimulated control cells). Bar: 25 μM.

PQ was injected intraperitoneally at a concentration of 30 mg/kg, while control animals were injected with a saline solution. All mice were sacrificed 24 hours after injections. The figure shows representative Western blots of 4-hydroxy-2-nonenal (4-HNE) adduct products (left panel) and of malondialdehyde (MDA) adduct products (right panel) from wild type, mIGF-1 Tg, wild type plus PQ and mIGF-1 mice plus PQ. Three animals of a total of 10 are shown in both panels A and B.

(A) Neonatal primary cardiomyocytes from wild type or mIGF-1 Tg mice were treated with sirtinol (100 μM) or EX-527 (1 μM), or treated with 20 ng/ml IGF-1 for 24 h. (B) HL-1 cardiomyocytes were transfected with the indicated plasmids, or treated with 20 ng/ml IGF-1 for 24 h. Untransfected cells were used as control (CTL). (A, B) The expression levels of adiponectin, UCP-1 and MT-2 mRNAs were examined by Real Time-PCR. (C) Neonatal primary cardiomyocytes from wild type or mIGF-1 Tg mice, and HL-1 cardiomyocytes, were transfected with 1 μg of plasmids carrying Firefly luciferase under the control of promoters of adiponectin (Adipo-Luc), UCP1 (UCP1-Luc) and MT-2 (MT-2-Luc) genes, respectively, together with 1 μg of Renilla Luciferase plasmid. Neonatal primary cardiomyocytes were also treated with different inhibitors or IGF-1 as described in (A). Dual luciferase assays were performed in duplicate for each condition. (A-C) Results are means ± SE of 3 independent experiments (^*,**,***p versus untreated cardiomyocytes).

Question point and dashed line indicate unanswered issues and hypothetical signaling cross-talk, respectively.

(A) HL-1 cardiomyocytes were transfected with the indicated plasmids, or treated with 20 ng/ml IGF-1 for 24 h, before exposure to Ang II (1 μM for 24 h). Untransfected cells were used as control (CTL). (B) Neonatal mouse cardiomyocytes from wild type (WT) or heart overexpressing mIGF-1 mice (mIGF-1 Tg) were pre-incubated with sirtinol (100 μM) or EX-527 (1 μM), or treated with 20 ng/ml IGF-1 for 24 h, prior to exposure to Ang II (1 μM for 24 h). Untreated WT cardiomyocytes were used as control. (A, B) The expression levels of MYH6, MYH7, BNP, ACTA-1, ANP and SERCA2 mRNAs were examined by qRT-PCR. Results are means ± SE of 3 independent experiments (^*,**,***p versus unstimulated control cells).

(A) HL-1 cardiomyocytes were transfected with the indicated plasmids, or treated with 20 ng/ml IGF-1 for 24 h, or exposed to Ang II (1 μM for 24 h). Untransfected cells were used as control (CTL). Together with Ang II, HL-1 cells were also incubated with 1μCi/ml of [3H]-labeled leucine (24 h). (B) Neonatal primary cardiomyocytes from wild type or mIGF-1 Tg mice were treated with SirT1 inhibitors (sirtinol, 100 μM; EX-527, 1 μM), or treated with 20 ng/ml IGF-1 for 24 h, or exposed to Ang II (1 μM for 24 h); concomitantly to Ang II addition, cells were incubated with 1mCi/ml of [3H]-labeled leucine (24 h). (A, B) [3H]-leucine incorporation values were normalized to total protein content and expressed as % of control. Results are means ± SE of 3 independent experiments (^**,***p versus unstimulated control cells or untreated WT cardiomyocytes).

(A, B) HL-1 cardiomyocytes were transfected or treated as in Legend of Figure 2A, except that Ang II (1 μM) or PQ (100 μM) were added for only 60 min. Untransfected cells were used as control (CTL). (C, D) Neonatal primary cardiomyocytes from wild type or mIGF-1 Tg mice were treated as in Legend of Figure 2A, except that Ang II (1 μM) or PQ (100 μM) were added for only 60 min. (A-D) ROS production was monitored with the fluorescent probe dichlorofluorescein diacetate (CM-DCFDA) and fluorescence values were normalized to protein content. Results are means ± SE of 3 independent experiments (^*,**,***p versus unstimulated control cells or untreated WT cardiomyocytes).

(A, B) HL-1 cardiomyocytes were transfected or treated as in Legend of Figure 2A. (C, D) Neonatal primary cardiomyocytes from wild type or mIGF-1 Tg mice were treated as in Legend of Figure 2B. (A-D) Cell viability was monitored with propidium iodide (PI) by flow cytometry and values were normalized to protein content. Results are means ± SE of 3 independent experiments (^*,**,***p versus unstimulated control cells or untreated wild type cardiomyocytes).

HL-1 cardiomyocytes were transfected with the indicated plasmids, and/or treated with 20 ng/ml IGF-1 for 24 h. HL-1 cardiomyocytes were also co-transfected with 1 μg of plasmids carrying Firefly luciferase under the control of promoters of adiponectin (Adipo-Luc), UCP1 (UCP1-Luc) and MT-2 (MT-2-Luc) genes, respectively, together with 1 μg of Renilla Luciferase plasmid. Untransfected cells were used as control. Dual luciferase assays were performed in duplicate for each condition. Results are means ± SE of 3 independent experiments (^*,**,***p versus untransfected/unstimulated control cells).