Deficiency of insulin-like growth factor 1 reduces vulnerability to chronic alcohol intake-induced cardiomyocyte mechanical dysfunction: role of AMPK

Abstract

Circulating insulin-like growth factor I (IGF-1) levels are closely associated with cardiac performance although the role of IGF-1 in alcoholic cardiac dysfunction is unknown. This study was designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on chronic alcohol-induced cardiomyocyte contractile and intracellular Ca(2+) dysfunction. Adult male C57 and LID mice were placed on a 4% alcohol diet for 15 weeks. Cardiomyocyte contractile and intracellular Ca(2+) properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-relengthening (TR(90) ), change in fura-fluorescence intensity (ΔFFI) and intracellular Ca(2+) decay. Levels of apoptotic regulators caspase-3, Bcl-2 and c-Jun NH2-terminal kinase (JNK), the ethanol metabolizing enzyme mitochondrial aldehyde dehydrogenase (ALDH2), as well as the cellular fuel gauge AMP-activated protein kinase (AMPK) were evaluated. Chronic alcohol intake enlarged myocyte cross-sectional area, reduced PS, ± dL/dt and ΔFFI as well as prolonged TR(90) and intracellular Ca(2+) decay, the effect of which was greatly attenuated by IGF-1 deficiency. The beneficial effect of LID against alcoholic cardiac mechanical defect was ablated by IGF-1 replenishment. Alcohol intake increased caspase-3 activity/expression although it down-regulated Bcl-2, ALDH2 and pAMPK without affecting JNK and AMPK. IGF-1 deficiency attenuated alcoholism-induced responses in all these proteins with the exception of Bcl-2. In addition, the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside abrogated short-term ethanol incubation-elicited cardiac mechanical dysfunction. Taken together, these data suggested that IGF-1 deficiency may reduce the sensitivity to ethanol-induced myocardial mechanical dysfunction. Our data further depicted a likely role of Caspase-3, ALDH2 and AMPK activation in IGF-1 deficiency induced 'desensitization' of alcoholic cardiomyopathy.

Fig 1

Effect of LID on chronic ethanol intake-induced cardiomyocyte contractile defects. (A) Resting cell length; (B) PS (normalized to resting cell length); (C) Maximal velocity of shortening (+dL/dt); (D) Maximal velocity of relengthening (–dL/dt); (E) TPS and (F) TR₉₀. Mean ± S.E.M., n = 144–147 cells from five mice per group, *P < 0.05 versus C57 control (C57-Con) group, ^#P < 0.05 versus C57-Ethanol group.

Fig 2

Effect of LID on chronic ethanol intake-induced intracellular Ca²⁺ homeostasis and SR Ca²⁺ store evaluated by frequency (0.1–5.0 Hz) dependent shortening response in murine cardiomyocytes. (A) Resting FFI; (B) Peak FFI; (C) ΔFFI in response to electrical stimuli; (D) Intracellular Ca²⁺ transient decay rate; and (E) Frequency (0.1–5.0 Hz) response. Change in PS at each stimulus frequency was made in reference to that of 0.1 Hz from the same cell. Mean ± S.E.M., n = 91–95 cells (A–D) or 22 cells (E) from five mice per group, *P < 0.05 versus C57 control (C57-Con) group, ^#P < 0.05 versus C57-Ethanol group.

Fig 3

Histological analyses of hearts from C57 and LID mice with or without chronic ethanol treatment. (A) Representative haematoxylin and eosin stained micrographs showing transverse sections of left ventricular myocardium (×400) from C57 and LID mice with or without ethanol treatment; (B) Quantitative analysis of cardiomyocyte cross-sectional (transverse) area from approximately 200 cells from three to five mice per group. Mean ± S.E.M., *P < 0.05 versus C57 control (C57-Con) group, ^#P < 0.05 versus C57-Ethanol group.

Fig 4

Effect of IGF-1 replenishment on acute ethanol exposure-induced cardiac contractile response in myocytes from LID mice. Freshly isolated cardiomyocytes from LID mice were incubated with ethanol (240 mg/dl) in the presence or absence of IGF-1 (5 nM) for 4 hrs. (A) Resting cell length; (B) PS (normalized to resting cell length); (C) Maximal velocity of shortening (+dL/dt); (D) Maximal velocity of relengthening (–dL/dt); (E) TPS and (F) TR₉₀. Mean ± S.E.M., n = 34–39 cells from three mice, *P < 0.05 versus LID control (LID-Con) group, ^#P < 0.05 versus LID-Ethanol group.

Fig 5

Caspase-3 activity (A) and protein expression of cleaved caspase-3 (B), Bcl-2 (C) and JNK (D) in myocardium from C57 and LID mice with or without chronic ethanol treatment. Insets: Representative gel blots of caspase-3, Bcl-2, JNK and α-tubulin (loading control) using specific antibodies against caspase-3, Bcl-2, JNK and α-tubulin. Mean ± S.E.M., n = 4–6 per group, *P < 0.05 versus C57 control (C57-Con) group.

Fig 6

Protein expression of ALDH2 (B), AMPK (C) and pAMPK (D) in myocardium from C57 and LID mice with or without chronic ethanol treatment. (A) Representative gel blots of ALDH2, AMPK, pAMPK and α-tubulin (loading control) using specific antibodies against ALDH2, AMPK, pAMPK and α-tubulin. Mean ± S.E.M., n = 5–6 per group, *P < 0.05 versus C57 control (C57-Con) group, ^#P < 0.05 versus C57-Ethanol group.

Fig 7

Effect of the AMPK activator AICAR on ethanol-induced cardiomyocyte contractile defects. Freshly isolated cardiomyocytes from normal mice were incubated with ethanol (240 mg/dl) in the presence or absence of AICAR (500 μM) for 4 hrs. (A) Resting cell length; (B) PS (normalized to resting cell length); (C) Maximal velocity of shortening (+dL/dt); (D) Maximal velocity of relengthening (–dL/dt); (E) TPS and (F) TR₉₀. Mean ± S.E.M., n = 64–66 cells from three mice per group, *P < 0.05 versus control group, ^#P < 0.05 versus ethanol group.