Cardiac overexpression of insulin-like growth factor 1 attenuates chronic alcohol intake-induced myocardial contractile dysfunction but not hypertrophy: Roles of Akt, mTOR, GSK3beta, and PTEN

Abstract

Chronic alcohol intake leads to the development of alcoholic cardiomyopathy manifested by cardiac hypertrophy and contractile dysfunction. This study was designed to examine the effects of transgenic overexpression of insulin-like growth factor 1 (IGF-1) on alcohol-induced cardiac contractile dysfunction. Wild-type FVB and cardiac-specific IGF-1 mice were placed on a 4% alcohol or control diet for 16weeks. Cardiac geometry and mechanical function were evaluated by echocardiography and cardiomyocyte and intracellular Ca(2+) properties. Histological analyses for cardiac fibrosis and apoptosis were evaluated by Masson trichrome staining and TUNEL assay, respectively. Expression and phosphorylation of Cu/Zn superoxide dismutase (SOD1), Ca(2+) handling proteins, and key signaling molecules for survival including Akt, mTOR, GSK3beta, Foxo3a, and the negative regulator of Akt, phosphatase and tensin homolog on chromosome 10 (PTEN), as well as mitochondrial proteins UCP-2 and PGC1alpha, were evaluated by Western blot analysis. Chronic alcohol intake led to cardiac hypertrophy, interstitial fibrosis, reduced mitochondrial number, compromised cardiac contractile function and intracellular Ca(2+) handling, decreased SOD1 expression, elevated superoxide production, and overt apoptosis, all of which, with the exception of cardiac hypertrophy, were abrogated by the IGF-1 transgene. Immunoblotting data showed reduced phosphorylation of Akt, mTOR, GSK3beta, and Foxo3a; upregulated Foxo3a and PTEN; and dampened SERCA2a, PGC1alpha, and UCP-2 after alcohol intake. All these alcohol-induced changes in survival and mitochondrial proteins were alleviated by IGF-1. Taken together, these data favor a beneficial role for IGF-1 in alcohol-induced myocardial contractile dysfunction independent of cardiac hypertrophy.

Fig. 1

Echocardiographic properties of FVB and IGF-1 transgenic mice with or without chronic alcohol intake. (A): Left ventricular (LV) wall thickness; (B): Fractional shortening (%); (C): LV end systolic diameter (ESD); (D): LV end diastolic diameter (EDD); (E): Calculated LV mass; and (F): Normalized LV mass. Mean ± SEM, n = 8 mice per group, * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB mice consuming ethanol (FVB-ETOH).

Fig. 2

Effect of chronic alcohol intake on cell shortening/relengthening in cardiomyocytes from FVB and IGF-1 mice. (A): Resting cell length; (B). Peak shortening amplitude (% of resting cell length); (C): Maximal velocity of shortening (+ dL/dt); (D). Maximal velocity of relengthening (− dL/dt); (E). Time-to-peak shortening (TPS); and (F): Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 69 - 70 cells per group. * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB mice consuming ethanol (FVB-ETOH).

Fig. 3

Effect of chronic alcohol intake on intracellular Ca²⁺ transients and stimulus frequency-peak shortening (PS) response in cardiomyocytes from FVB and IGF-1 mice. (A): Baseline intracellular Ca²⁺ fura-2 fluorescent intensity (FFI); (B) Change of fura-2 fluorescence intensity in response to electrical stimuli (ΔFFI); (C): Single exponential fluorescence decay rate; (D): Bi-exponential fluorescence decay rate; and (F): Changes in PS in response to increased stimulus frequency (0.1 – 5.0 Hz). Each point represents PS normalized to that of 0.1 Hz of the same cell. Mean ± SEM, n = 53 cells (A-D) or 23-25 cells (E) per group, * p < 0.05 vs. FVB; # p<0.05 vs. FVB mice consuming ethanol (FVB-ETOH).

Fig. 4

Effect of chronic alcohol intake on myocardial fibrosis in FVB and IGF-1 mice. (A-D): Representative photomicrographs (400x) of myocardial sections stained with Masson trichrome. (A): FVB; (B): IGF-1; (C): FVB-ETOH; and (D): IGF-1-ETOH; (E): Pooled data of 15 fields from 3 mice, Mean ± SEM, * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB mice consuming ethanol (FVB-ETOH).

Fig. 5

Transmission electron microscopic micrographs of left ventricular tissues from FVB or IGF-1 mice with or without chronic alcohol consumption. Panels A (FVB), B (IGF-1) and D (IGF-1-ETOH) display regular myofilament and regular mitochondrial structure whereas panel C (FVB-ETOH) displays regular myofilament associated with reduced mitochondrial number or density (10,000x). Panel E: mitochondrial density; Panel F: mitochondrial size. Mean ± SEM, n = 6-7 fields per mouse from 3 mice,* p < 0.05 vs. FVB group, # p < 0.05 vs. FVB mice consuming ethanol (FVB-ETOH) group.

Fig. 6

Effect of chronic alcohol intake on superoxide generation, Cu/Zn superoxide dismutase (SOD1) expression and caspase-3 activity in cardiomyocytes or myocardium from FVB and IGF-1 mice. (A): Representative DHE fluorescent images (400x) showing superoxide production in cardiomyocytes from FVB and IGF-1 mice with or without chronic alcohol intake; (B): Pooled data of superoxide production from 120-160 cells from 4 mice per group; (C): SOD1 expression. Inset: Representative gel bots of SOD1 and GAPDH (loading control) using specific antibodies; and (D): Caspase-3 activity using the colorimetric substrate Ac-DEVD-pNA. Mean ± SEM, n = 5-7 mice per group. * p < 0.05 vs. FVB; # p < 0.05 vs. FVB mice consuming ethanol (FVB-ETOH).

Fig. 7

Effect of chronic alcohol intake on apoptosis using TUNEL staining in myocardium. All nuclei were stained with DAPI shown in blue in panels A (FVB), D (IGF-1), G (FVB-ETOH) and J (IGF-1-ETOH). The TUNEL-positive nuclei were visualized with fluorescein (green) in panels B (FVB), E (IGF-1) H (FVB-ETOH) and K (IGF-1-ETOH). Panels C (FVB), F (IGF-1), I (FVB-ETOH) and I (IGF-1-ETOH) depict merged DAPI and TUNEL-positive nuclei staining. Original magnification = 400×. Quantified data are shown in panel M. Mean ± SEM, n = 15 fields from 3 mice per group, * p < 0.05 vs. FVB; # p<0.05 vs. FVB mice consuming ethanol (FVB-ETOH).

Fig. 8

Western blot analysis of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a), Na⁺-Ca²⁺ exchanger and phospholamban in myocardium from FVB and IGF-1 mice with or without ethanol consumption (ETOH). (A): Representative gel blots of SERCA2a, Na⁺-Ca²⁺ exchanger, phospholamban and GAPDH (loading control) using specific antibodies; (B): SERCA2a; (C): Na⁺-Ca²⁺ exchanger; and (D): Phospholamban. Mean ± SEM. n = 4 – 6, * p < 0.05 vs. FVB.

Fig. 9

Western blot analysis of pan and phosphorylated Akt and mTOR in myocardium from FVB and IGF-1 mice with or without chronic alcohol consumption. (A): Akt; (B): mTOR; (C): Phosphorylated Akt (pAkt); (D): Phosphorylated mTOR (pmTOR); (E): pAkt-to-pan Akt ratio; and (F): pmTOR-to-pan mTOR ratio. Insets: Representative gel blots depicting expression of Akt, mTOR, pAkt, pmTOR and GAPDH (used as loading control). Mean ± SEM. n = 6 – 7, * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB mice consuming ethanol (FVB-ETOH).

Fig. 10

Western blot analysis of pan and phosphorylated Foxo3a and GSK3β in myocardium from FVB and IGF-1 mice with or without chronic alcohol consumption. (A): Foxo3a; (B): GSK3β; (C): Phosphorylated Foxo3a (pFoxo3a); (D): Phosphorylated GSK3β (pGSK3β); (E): pFoxo3a-to-pan Foxo3a ratio; and (F): pGSK3β-to-pan GSK3β ratio. Insets: Representative gel blots depicting expression of Foxo3a, GSK3β, pFoxo3a, pGSK3β and GAPDH (loading control). Mean ± SEM. n = 6 – 8, * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB mice consuming ethanol (FVB-ETOH).

Fig. 11

Western blot analysis of PTEN, UCP-2 and PGC1α in myocardium from FVB and IGF-1 mice with or without chronic alcohol consumption. (A): Representative gel blots of PTEN, UCP-2, PGC1α and GAPDH (loading control); (B): PTEN; (C): UCP-2; and (D): PGC1α. Mean ± SEM. n = 6 – 7, * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB mice consuming ethanol (FVB-ETOH).