Lats2 is a negative regulator of myocyte size in the heart

Abstract

Mammalian sterile 20-like kinase (Mst)1 plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Because Hippo, a Drosophila homolog of Mst1, forms a signaling complex with Warts, a serine/threonine kinase, which in turn stimulates cell death and inhibits cell proliferation, mammalian homologs of Warts, termed Lats1 and Lats2, may mediate the function of Mst1. We here show that Lats2, but not Lats1, dose-dependently increased apoptosis in cultured cardiac myocytes. Lats2 also dose-dependently reduced [(3)H]phenylalanine incorporation and cardiac myocyte size, whereas dominant negative Lats2 (DN-Lats2) increased them, suggesting that endogenous Lats2 negatively regulates myocyte growth. DN-Lats2 significantly attenuated induction of apoptosis and inhibition of hypertrophy by Mst1, indicating that Lats2 mediates the function of Mst1 in cardiac myocytes. Cardiac specific overexpression of Lats2 in transgenic mice significantly reduced the size of left and right ventricles, whereas that of DN-Lats2 caused hypertrophy in both ventricles. Overexpression of Lats2 reduced left ventricular systolic and diastolic function without affecting baseline levels of myocardial apoptosis. Expression of endogenous Lats2 was significantly upregulated in response to transverse aortic constriction. Overexpression of DN-Lats2 significantly enhanced cardiac hypertrophy and inhibited cardiac myocyte apoptosis induced by transverse aortic constriction. These results suggest that Lats2 is necessary and sufficient for negatively regulating ventricular mass in the heart. Although Lats2 is required for cardiac myocyte apoptosis in response to pressure overload, it was not sufficient to induce apoptosis at baseline. In conclusion, Lats2 affects both growth and death of cardiac myocytes, but it primarily regulates the size of the heart and acts as an endogenous negative regulator of cardiac hypertrophy.

Figure 1. Lats2 induces apoptosis in cardiac myocytes

(A) Neonatal rat cardiac myocytes (NRCMs) were transduced with Ad-LacZ (control) or Ad-Lats2 at 30 MOI. Immunoblot analyses were performed using anti-Lats2 antibody.. (B) NRCMs were transduced with Ad-LacZ, Ad-Lats2 or Ad-Mst1. Cytoplasmic accumulation of mono- and oligo-nucleosomes was quantitated. Values are mean ± SEM obtained from 3 experiments. *p<0.05. (C) NRCMs were transduced with Ad-LacZ and Ad-Lats2 at 30 MOI. The result shown is representative of 3 experiments.. (D) (Left) NRCMs were transduced with Ad-LacZ and Ad-Lats2 at 30 MOI in the presence or absence of caspase inhibitor (C.I.) (100μM, ApoBlock, BD Biosciences). (Right) NRCMs were transduced with Ad-LacZ or Ad-Bcl-xL at 10 MOI. Twenty-four hours after transduction, NRCMs were transduced with Ad-LacZ or Ad-Lats2 at 10 MOI. Forty-eight hours after the second transduction, cytoplasmic accumulation of mono- and oligo-nucleosomes was quantitated. Values are mean ± SEM obtained from 3 experiments. *p<0.05. Cell lysates were subjected to immunoblots for detection of Bcl-xL and α-actin.

Figure 2. Lats2 partly mediates Mst1-induced apoptosis

(A) NRCMs were transduced with Ad-LacZ or Ad-DN-Lats2 (DN-L2) at 30 MOI. Twenty-four hours after transduction, myocytes were transduced with Ad-Mst1 at 30MOI. (B) NRCMs were transduced with Ad-LacZ and Ad-DN-Mst1 at 30 MOI. Twenty-four hours after transduction, myocytes were transduced with Ad-Lats2 (L2) at 30 MOI. In A and B, 48 hours after the second transduction, cytoplasmic accumulation of mono- and oligo-nucleosomes was quantitated. Values are mean ± SEM obtained from 3 experiments. N.S., not significant. (C) NRCMs were transduced with Ad-LacZ, Ad-Lats2 or Ad-Mst1 with different combinations (total 60 MOI). Forty-eight hours after the transduction, cytoplasmic accumulation of mono- and oligo-nucleosomes was quantitated. Values are mean ± SEM obtained from 3 experiments. *p<0.05.

Figure 3. LATS2 inhibits cardiac hypertrophy

(A-C) NRCMs were transduced with Ad-LacZ, Ad-Lats2 or Ad-DN-Lats2. Forty-eight hours after transduction, myocytes were stimulated with or without phenylephrine (PE, 30 μM) for 48 hours. In A, myocyte surface area was determined. The cell-surface area without PE was designated as 100%. Values are mean ± SEM obtained from 3 experiments. *p<0.05. In B, total protein content was determined. The protein content in myocytes transduced with Ad-LacZ without PE was designated as 1. Values are mean ± SEM obtained from 3 experiments. *p<0.05. In C, myocytes were stained with anti-ANF antibody. The percent of myocytes expressing ANF, as determined by typical perinuclear staining, was counted. Values are mean ± SEM obtained from 3 experiments. *p<0.05. N.S, not significant. Bar = 20 μm. (D) NRCMs were transduced with or without Ad-LacZ (60MOI), Ad-Mst1 (30MOI) + Ad-LacZ (30MOI) and Ad-Mst1 (30MOI) + Ad-DNLats2 (30MOI). Fort-eight hours after transduction, myocytes were stimulated with or without PE (30 μM) for 48 hours and then myocyte surface measured. Experimental values in myocytes transduced with Ad-LacZ without PE were designated as 1. Values are mean ± SEM obtained from 3 experiments. * p<0.05

Figure 3. LATS2 inhibits cardiac hypertrophy

(A-C) NRCMs were transduced with Ad-LacZ, Ad-Lats2 or Ad-DN-Lats2. Forty-eight hours after transduction, myocytes were stimulated with or without phenylephrine (PE, 30 μM) for 48 hours. In A, myocyte surface area was determined. The cell-surface area without PE was designated as 100%. Values are mean ± SEM obtained from 3 experiments. *p<0.05. In B, total protein content was determined. The protein content in myocytes transduced with Ad-LacZ without PE was designated as 1. Values are mean ± SEM obtained from 3 experiments. *p<0.05. In C, myocytes were stained with anti-ANF antibody. The percent of myocytes expressing ANF, as determined by typical perinuclear staining, was counted. Values are mean ± SEM obtained from 3 experiments. *p<0.05. N.S, not significant. Bar = 20 μm. (D) NRCMs were transduced with or without Ad-LacZ (60MOI), Ad-Mst1 (30MOI) + Ad-LacZ (30MOI) and Ad-Mst1 (30MOI) + Ad-DNLats2 (30MOI). Fort-eight hours after transduction, myocytes were stimulated with or without PE (30 μM) for 48 hours and then myocyte surface measured. Experimental values in myocytes transduced with Ad-LacZ without PE were designated as 1. Values are mean ± SEM obtained from 3 experiments. * p<0.05

Figure 4. Cardiac Phenotype of Tg-Lats2 mice

(A) Heart homogenates were prepared from normal mice subjected to either sham operation (sham) or transverse aortic constriction for 2 weeks (TAC), Tg-Lats2 (line 20) or non-transgenic (NTg) littermates, and then immunoblotted with anti-Lats2 and anti-α-actin antibodies. (B) Postmortem measurements of left ventricular weight/tibial length (LVW/TL, left) and right ventricular weight/tibial length (RVW/TL, right) in Tg-Lats2 (line 8) and NTg. Values are mean ± SD. (C) Echocardiographically measured LV end diastolic dimension (LVEDD) and LV ejection fraction (LVEF) in Tg-Lats2 (line 8) and NTg mice. Values are mean ± SEM. (D) Representative WGA staining of the LV and RV myocardium obtained from Tg-Lats2 (line 8) and NTg mice (upper panel) and the relative myocyte cross sectional area (lower panel). Values are mean ± SEM obtained from 5 experiments. *p<0.05.

Fig. 5. Cardiac phenotype of Tg-DN-Lats2 mice

(A) Immunoblot analyses of heart homogenates from Tg-DN-Lats2 (line 31) and NTg mice with anti-Lats2, anti-ANP, and anti-α actin antibodies. (B) Postmortem measurements of left ventricular weight/tibial length (LVW/TL, mg/mm, left) and right ventricular weight/tibial length (RVW/TL, mg/mm, right) in Tg-DN-Lats2 (line 31) and NTg. Values are mean ± SD. N=12-16. (C) Representative WGA staining of the LV and RV myocardium obtained from Tg-DN-Lats2 and NTg mice and the relative myocyte cross sectional area (lower panel). Values are mean ± SEM obtained from 5 experiments.

Fig. 6. Effects of TAC on cardiac hypertrophy in Tg-DN-Lats2 mice

Tg-DN-Lats2 (line 31) and NTg were subjected to either TAC (2 weeks) or sham operation. (A) The pressure gradient was measured after 2 weeks of TAC. (B) (left) Postmortem measurements of left ventricular weight/tibial length (LVW/TL, mg/mm, left) after TAC. (right) Percent increase in LVW/TL after TAC compared with the mean value of sham operated mice. Values are mean ± SD. N=7. (C) Representative WGA staining of the LV obtained from Tg-DN-Lats2 and NTg mice after TAC (left panel) and the relative myocyte cross sectional area (right panel). Values are mean ± SEM obtained from 5 experiments. (D) Percent TUNEL positive myocytes. Values are mean ± SEM obtained from 5 experiments. #p<0.05 vs NTg sham.