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. 2009 Dec 18;284(51):35839-49.
doi: 10.1074/jbc.M109.057273.

Cardiac-specific deletion of LKB1 leads to hypertrophy and dysfunction

Yasumasa Ikeda  1 , Kaori Sato, David R Pimentel, Flora Sam, Reuben J Shaw, Jason R B Dyck, Kenneth Walsh
Affiliations

Cardiac-specific deletion of LKB1 leads to hypertrophy and dysfunction

Yasumasa Ikeda et al. J Biol Chem. .

Abstract

LKB1 encodes a serine/threonine kinase, which functions upstream of the AMP-activated protein kinase (AMPK) superfamily. To clarify the role of LKB1 in heart, we generated and characterized cardiac myocyte-specific LKB1 knock-out (KO) mice using alpha-myosin heavy chain-Cre deletor strain. LKB1-KO mice displayed biatrial enlargement with atrial fibrillation and cardiac dysfunction at 4 weeks of age. Left ventricular hypertrophy was observed in LKB1-KO mice at 12 weeks but not 4 weeks of age. Collagen I and III mRNA expression was elevated in atria at 4 weeks, and atrial fibrosis was seen at 12 weeks. LKB1-KO mice displayed cardiac dysfunction and atrial fibrillation and died within 6 months of age. Indicative of a prohypertrophic environment, the phosphorylation of AMPK and eEF2 was reduced, whereas mammalian target of rapamycin (mTOR) phosphorylation and p70S6 kinase phosphorylation were increased in both the atria and ventricles of LKB1-deficient mice. Consistent with vascular endothelial growth factor mRNA and protein levels being significantly reduced in LKB1-KO mice, these mice also exhibited a reduction in capillary density of both atria and ventricles. In cultured cardiac myocytes, LKB1 silencing induced hypertrophy, which was ameliorated by the expression of a constitutively active form AMPK or by treatment with the inhibitor of mTOR, rapamycin. These findings indicate that LKB1 signaling in cardiac myocytes is essential for normal development of the atria and ventricles. Cardiac hypertrophy and dysfunction in LKB1-deficient hearts are associated with alterations in AMPK and mTOR/p70S6 kinase/eEF2 signaling and with a reduction in vascular endothelial growth factor expression and vessel rarefaction.

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Figures

FIGURE 1.
FIGURE 1.
Analysis of the cardiac phenotype in control and LKB1-KO mice at 4 and 12 weeks of age. A, gross appearance of the isolated hearts (top) and long axis section of four-chamber view with Masson's trichrome staining (middle). The mean atrial and ventricular weight to body weight ratios are also shown (bottom). B, histological sections of Masson's trichrome-stained left atrial and left ventricular tissue (upper). The mean atrial and ventricular myofibrillar transverse diameter in control (open bars) and LKB1-KO (filled bars) mice (lower) are shown. Values are expressed as mean ± S.E. **, p < 0.01; each group n = 5–7. C, mRNA level of collagen I and III expression in atria and ventricles of control and LKB1-KO mice. The real time PCR analyses were performed in control (open bars) and LKB1-KO (filled bars) at 4 and 12 weeks of age, respectively. Values are change relative to each control band (4-week-old control atria) and are expressed as mean ± S.E. **, p < 0.01; n = 6 in each group.
FIGURE 2.
FIGURE 2.
Direct and indirect measures of cardiac dysfunction in LKB1-KO mice at 4 and 12 weeks of age. A, representative echocardiogram. Serial changes of B-mode of parasternal long axis view and M-mode of left ventricular short axis view in control and LKB1-KO mice are shown. B, echocardiographic measurements of left atrial dimension (LA), left ventricular diastolic dimension (LVDd), and fractional shortening (FS) between 4- and 12-week-old control and LKB1-KO mice. *, p < 0.05; **, p < 0.01 versus age-matched control; n = 8 in each sample. NS, not significant. C, invasive analysis of cardiac function with Miller catheter in both 4- and 12-week-old control and LKB1-KO mice. Values are expressed as mean ± S.E. *, p < 0.05; **, p < 0.01. Each group n = 8. BP, blood pressure. D, mRNA and protein expression of phospholamban and SERCA2. The real time PCR or Western blot analyses were performed in control (open bars) and LKB1-KO (filled bars) at 4 and 12 weeks of age, respectively. Values are change relative to each control band (4-week-old control atria) and are expressed as mean ± S.E. *, p < 0.05; **, p < 0.01; n = 6–8 in each group.
FIGURE 3.
FIGURE 3.
Electrocardiographic assessment and survival of control and LKB1-KO mice. A, representative electrocardiogram showing control mice and LKB1-KO mice at 2, 3, and 4 weeks of age. Control mice presented normal sinus rhythm, whereas LKB1-KO mice presented Afib. Afib occurred in 80% LKB1-KO mice by 4 weeks of age. B, mortality rate of control mice and LKB1-KO mice. Mortality curves were created by the Kaplan-Meier method and compared by the log-lank test. n = 20 in each group.
FIGURE 4.
FIGURE 4.
mRNA and protein expression of connexin 40, 43, and 45. A, results of real time PCR analysis of connexin 40, 43, and 45. Values are change relative to each control (using atria at 4 weeks of age as control) and expressed as mean ± S.E. *, p < 0.05; **, p < 0.01, n = 6 in each group. B, upper, representative immunoblots of connexin 40, 43, and 45 in atria and ventricles of control mice and LKB1-KO mice. Lower, densitometric analysis of connexin 40, 43, and 45. The expression levels were quantified and are expressed as change relative to 4-week-old control atria. Values are change relative to each control (using atria at 4 weeks of age as control) and are expressed as mean ± S.E. *, p < 0.05; **, p < 0.01, n = 6 in each group.
FIGURE 5.
FIGURE 5.
Capillary density in both atria and ventricles from control and LKB1-KO mice at 4 and 12 weeks of age. A, top and middle panels, representative histological sections of CD31 immunohistochemistry staining in atria and ventricles. Bottom panel, mean capillary density in control (open bars) and LKB1-KO (filled bars) mice. Values are expressed as mean ± S.E. *, p < 0.05; **, p < 0.01; n = 4–6 in each group. B, mRNA (left) levels and protein levels with the representative blots (right) of VEGF expression in atria and ventricles. Values are expressed as mean ± S.E. *, p < 0.05; **, p < 0.01, n = 6–8 in each group.
FIGURE 6.
FIGURE 6.
Phosphorylation level of AMPK, ACC, CREB, and Akt in both atria and ventricles from control and LKB1-KO mice. A, left, representative blot of phosphorylated and total AMPK (pAMPK and tAMPK, respectively), phosphorylated and total ACC (pACC and tACC, respectively), phosphorylated and total Akt (pAKT and tAKT, respectively), and phosphorylated and total CREB (pCREB and tCREB, respectively). Right, results of densitometric analysis of ACC, AMPK, Akt, and CREB phosphorylation. B, phosphorylation level of mTOR, p70S6 kinase, and eEF2 in atria and ventricles from control and LKB1-KO mice. Upper, representative blot of phospho- and total mTOR, p70S6 kinase and eEF2. Lower, densitometric analysis of mTOR, p70S6 kinase, and eEF2 phosphorylation. The phosphorylation levels were quantified and are expressed as change relative to each control band (atria from control mice at 4 weeks of age). Values shown are mean ± S.E. *, p < 0.05; **, p < 0.01; n = 8 in each group.
FIGURE 7.
FIGURE 7.
Assessment of LKB1 siRNA-induced cardiac myocyte hypertrophy. LKB1 siRNA-induced cardiac myocyte hypertrophy was inhibited by caAMPK, not DCREB, in vitro. A, representative cell size of cardiac myocytes transfected with unrelated siRNA (control) or LKB1 siRNA for 48 h. After 24 h of siRNA transfection, cardiac myocytes were transfected with β-galactosidase (β-gal) or caAMPK adenovirus. Cardiac myocytes were stained with α-actinin for quantification of cell surface area. B, quantitative analysis of cell surface area after siRNA transfection with or without caAMPK adenovirus infection. *, p < 0.05; **, p < 0.01; n = 4 in each group. C, [3H]leucine incorporation for assessment of protein synthesis in cardiac myocytes. The measurement of radioactivity in cardiac myocytes after siRNA transfection with or without caAMPK adenovirus infection is shown. *, p < 0.05; **, p < 0.01; n = 8–12 in each group. D, results of real time PCR analysis of A-type natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and α-smooth muscle actin (α-SMA), expression with control or siRNA in the presence of β-galactosidase or caAMPK adenovirus. *, p < 0.05; **, p < 0.01; n = 6 in each group. E, representative cell size of cardiac myocytes transfected with unrelated siRNA (control) or LKB1 siRNA for 48 h. After 24 h of siRNA transfection, cardiac myocytes were treated with vehicle (DMSO) or rapamycin. Cardiac myocytes were stained with α-actinin for quantification of cell surface area. n = 4 in each group. F, quantitative analysis of cell surface area after siRNA transfection with or without rapamycin treatment. *, p < 0.05; **, p < 0.01; n = 4 in each group. G, [3H]leucine incorporation for assessment of protein synthesis in cardiac myocytes. The measurement of radioactivity in cardiac myocytes after siRNA transfection with or without rapamycin treatment is shown. *, p < 0.05; **, p < 0.01; n = 6–8 in each group. H, representative cell size of cardiac myocytes transfected with unrelated siRNA (control) or LKB1 siRNA for 48 h. Cardiac myocytes were stained with α-actinin for quantification of cell surface area. n = 4 in each group. I, quantitative analysis of cell surface area after siRNA transfection with or without DCREB adenovirus infection. **, p < 0.01; n = 4 in each group. J, [3H]leucine incorporation for assessment of protein synthesis in cardiac myocytes. The measurement of radioactivity in cardiac myocytes after siRNA transfection with or without DCREB adenovirus infection is shown. *, p < 0.05; **, p < 0.01; n = 8–12 in each group. In all panels, values are expressed as mean ± S.E.
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