Deficiency of LKB1 in heart prevents ischemia-mediated activation of AMPKalpha2 but not AMPKalpha1

Abstract

Recent studies indicate that the LKB1 is a key regulator of the AMP-activated protein kinase (AMPK), which plays a crucial role in protecting cardiac muscle from damage during ischemia. We have employed mice that lack LKB1 in cardiac and skeletal muscle and studied how this affected the activity of cardiac AMPKalpha1/alpha2 under normoxic, ischemic, and anoxic conditions. In the heart lacking cardiac muscle LKB1, the basal activity of AMPKalpha2 was vastly reduced and not increased by ischemia or anoxia. Phosphorylation of AMPKalpha2 at the site of LKB1 phosphorylation (Thr172) or phosphorylation of acetyl-CoA carboxylase-2, a downstream substrate of AMPK, was ablated in ischemic heart lacking cardiac LKB1. Ischemia was found to increase the ADP-to-ATP (ADP/ATP) and AMP-to-ATP ratios (AMP/ATP) to a greater extent in LKB1-deficient cardiac muscle than in LKB1-expressing muscle. In contrast to AMPKalpha2, significant basal activity of AMPKalpha1 was observed in the lysates from the hearts lacking cardiac muscle LKB1, as well as in cardiomyocytes that had been isolated from these hearts. In the heart lacking cardiac LKB1, ischemia or anoxia induced a marked activation and phosphorylation of AMPKalpha1, to a level that was only moderately lower than observed in LKB1-expressing heart. Echocardiographic and morphological analysis of the cardiac LKB1-deficient hearts indicated that these hearts were not overtly dysfunctional, despite possessing a reduced weight and enlarged atria. These findings indicate that LKB1 plays a crucial role in regulating AMPKalpha2 activation and acetyl-CoA carboxylase-2 phosphorylation and also regulating cellular energy levels in response to ischemia. They also provide genetic evidence that an alternative upstream kinase can activate AMPKalpha1 in cardiac muscle.

Fig. 1

Cardiac phenotype of muscle LKB1-decient mice. A: mice (2–3 mo old) were anesthetized, and left ventricular function was assessed by a 2-dimensional echocardiographic analysis. Left ventricular function was evaluated by measuring the percentage of anterior wall thickening (AWT) and the ejection fraction (EF) (fractional area shortening) from short-axis view. The percentage of AWT is [(AWT in end-systole − AWT in end-diastole)/AWT in end-systole] × 100. Left ventricular areas during end-diastole (LVED_area) and end-systole (LVES_area) were measured to calculate the percentage of EF {%EF = [(LVED_area − LVES_area)/LVED_area] × 100}. Results are presented as the average ± SE; n, no. of animals. BW, body weight; HW, heart weight; HW/BW, ratio of HW to BW; AWD, diastolic AWT; AWS, systolic AWT; HR, heart rate; bpm, beats/min. ^aP < 0.05 vs. LKB1^+/+; ^bP < 0.05 vs. LKB1^fl/fl. B: morphological and histological analysis of hearts from LKB1^+/+, LKB1^fl/fl, and LKB1^fl/fl Cre^+/− mice (∼3 mo old). The hearts were fixed in 10% formalin, embedded in wax and stained with hematoxylin and eosin. Top: image of entire heart. Middle: representative longitudinal sections with the horizontal bar representing 2 mm. Bottom: higher magnification of the image of cardiomyocytes present in the longitudinal section, with the horizontal bar representing 0.2 mm. EA, enlarged atrium; rv, right ventricle; lv, left ventricle.

Fig. 2

LKB1 activity in response to ischemia in cardiac muscle. Isolated heart derived from the indicated mice (2–3 mo of age) were perfused under normoxic (Norm) or no-flow ischemic (Ischem) conditions for 10 min, as described in experimental procedures. Equal amounts of protein (30 μg) from heart extracts were immunoblotted with LKB1 antibody. LKB1 was immunoprecipitated and assayed, employing the LKBtide peptide.

Fig. 3

Role of LKB1 in regulating ischemia- or anoxia-induced AMP-activated protein kinase (AMPK) activation in the heart. Isolated heart derived from the indicated mice (2–3 mo of age) were perfused under normoxic, no-flow ischemic (A, B, and C) or anoxic (D, E, and F) conditions for 10 or 15 min, as described in the experimental procedures. A and D: AMPKα2 was immunoprecipitated and assayed with the AMARA peptide. Assays were performed in duplicate from the heart tissue derived from 3–6 mice, and results are presented as the average specific activity ± SE. B and F: equal amounts of protein (20–40 μg) from heart extracts were immunoblotted with the indicated antibodies. The immunoblotting results are representative of independent experiments performed with heart from at least 3 mice. Immunoblot analysis of total AMPKα1 and AMPKα2 levels was performed by quantitative Li-Cor method. The data presented are the mean ± SE expression relative to expression in LKB1^+/+ muscle derived from 3–5 mice. C and E: as in A, except that AMPKα1 was assayed. ACC, acetyl-CoA carboxylase. *P < 0.05 basal vs. ischemia within each genotype; ^aP < 0.05 vs. LKB1^+/+ (normoxia); ^bP < 0.05 vs. LKB1^fl/fl Cre^−/− (normoxia); ^cP < 0.05 vs. LKB1^+/+ (ischemia); ^dP < 0.05 vs. LKB1^fl/fl Cre^−/− (ischemia).

Fig. 4

Activity of AMPKα1 and AMPKα2 in isolated cardiomyocytes from LKB1^+/+ or LKB1^fl/fl Cre^+/− mice. Cardiomyocytes were isolated from the 2- to 3-mo-old LKB1^+/+ or LKB1^fl/fl Cre^+/− mice, as described in experimental procedures, and total cell extracts were immediately generated. A: original image taken by laser confocal microscopy LSM-510 of cardiomyocytes isolated from the mouse heart, as described in experimental procedures. Magnification was ×10. Note a lack of noncardiac cells in this preparation. AMPKα1 (B) or AMPKα2 (C) was immunoprecipitated from the lysates and assayed with the AMARA peptide. Assays were performed from 3 mice per genotype, and results are presented as the average specific activity ± SE.

Fig. 5

Role of LKB1 in regulating ADP-to-ATP and AMP-to-ATP ratios during ischemia in cardiac muscle. Isolated heart derived from wild-type or LKB1 mutant mice (2–3 mo of age) was perfused under normoxic or no-flow ischemic conditions for 10 min. The ratios of ADP to ATP (A) and AMP to ATP (B) derived from 4–5 heart samples for each condition were measured. The results are shown as the average ± SE. *P < 0.05 normoxia vs. ischemia within each genotype; ^aP < 0.05 vs. LKB1^+/+ (ischemia).