Integrative Physiology

Lats2 Is a Negative Regulator of Myocyte Size in the Heart

Yutaka Matsui, Noritsugu Nakano, Dan Shao, Shumin Gao, Wenting Luo, Chull Hong, Peiyong Zhai, Eric Holle, Xianzhong Yu, Norikazu Yabuta, Wufan Tao, Thomas Wagner, Hiroshi Nojima, Junichi Sadoshima
https://doi.org/10.1161/CIRCRESAHA.108.180042
Circulation Research. 2008;103:1309-1318
Originally published November 21, 2008

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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 [3H]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.

The size of organs is controlled by the intricate coordination between cell growth and death.1 Increasing lines of evidence suggest that the heart is a renewing organ.2 However, hypertrophy of existing cardiac myocytes and cell death, including apoptosis, are still major, if not sole, determinants of the heart size. Although the signaling mechanisms controlling hypertrophy and apoptosis have been extensively studied, only a few mechanisms reported thus far can regulate both growth and death of cardiac myocytes in a coordinated fashion.

Mammalian sterile 20-like kinase (Mst)1 is a serine threonine kinase, which is activated by proapoptotic stimuli and potently induces apoptosis in cardiac myocytes. Interestingly, Mst1 not only induces apoptosis but also negatively regulates hypertrophy of cardiac myocytes.3 Stimulation of apoptosis and inhibition of compensatory hypertrophy could be detrimental for the heart because they could lead to progressive wall thinning and elevated wall stress, which further increases myocardial cell death and initiates a malignant spiral of heart failure.4

In Drosophila, hippo (Hpo), a homolog of human Mst1, also regulates the size of organs, such as eyes and wings.5,6 Using genetic approaches, a series of molecules associating with Hpo have been identified. Hpo physically interacts with Salvador (Sav), a scaffold protein, which in turn binds to Warts (Wts) (also known as lats).7,8 Hpo phosphorylates Wts and triggers cell cycle arrest and apoptosis.7,9,10

In mammals, the lats tumor suppressor family encodes Ser/Thr protein kinases.11 One family member, Lats2, negatively regulates cell growth in NIH3T3/v-ras and HeLa cells.12,13 Reduced expression of Lats2 has been shown to enhance the tumorigenesis.14,15 Lats2 initiates a positive-feedback mechanism with p53, thereby acting as a checkpoint mechanism for the maintenance of the proper chromosome number.16 Lats2 also regulates the mitotic machinery, thereby playing an important role in coordinating the mitotic fidelity and the genomic stability.17,18 Recent reports have shown that the effects of Lats2 may be mediated by Yki and YAP, nuclear transcription cofactors, in Drosophila and mammals, respectively.19,20

Northern blot analyses showed ubiquitous expression of Lats2 mRNA in several human tissues with the highest expression in the heart.21 Lats2 knockout mice are embryonic lethal and display atrial and ventricular defects, pericardial distension, and blood pooling, suggesting that Lats2 may play an important role in mediating cardiac development.17 Judging from the prominent proapoptotic and antigrowth functions of Mst1 in the heart,3 we expect that Lats2 should mediate some of the functions of Mst1 in the heart. However, the role of Lats2 in mediating growth and death of cardiac myocytes is not well understood.

Thus, the purposes of this study were (1) to clarify the cardiac function of Lats2 and (2) to determine whether the proapoptotic and antihypertrophic actions of Mst1 in the heart are mimicked by Lats2.

Materials and Methods

An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

cDNA

Dominant-negative form of Mst1 (Mst1K59R) was described.3 A dominant negative form of Lats2 (DN-Lats2) was generated by mutating Lys697 to alanine.

Antibodies

Mouse monoclonal antibody against N-terminal portion of human Lats2 (amino acids 78 to 256) and rabbit polyclonal antibody against N-terminal portion of human Lats2 (amino acids 79 to 257) have been described.18,21 Rat monoclonal antibody against human Lats1 has been described.22 Other antibodies include monoclonal anti-Myc 9E10 (Santa Cruz Biotechnology); anti-Mst1, anti-Bcl-2, anti-Bcl-xL (Pharmingen); anti-α-actin (Sigma); and polyclonal anti–cleaved caspase 3 (Cell Signaling).

Transgenic Mice

Lats2 and DN-Lats2 transgenic mice (Tg-Lats2 and Tg-DN-Lats2) were generated using the α-myosin heavy chain promoter (courtesy of J. Robbins, University of Cincinnati, Cincinnati, Ohio) to achieve cardiac-specific expression of transgene on an FVB background. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Medicine and Dentistry of New Jersey.

Statistics

Statistical analyses between groups were done by 1-way ANOVA, and differences among group means were evaluated using the Fisher’s project least-significant difference post test procedure for group data, with a probability value less than 0.05 being considered significant.

Results

Lats2 Induces Apoptosis in Cardiac Myocytes

To examine the effect of Lats2 expression on growth and death of cardiac myocytes, cultured myocytes were transduced with Ad-Lats2 or Ad-LacZ. After 2 days, expression of Lats2 was significantly increased (Figure 1A). Because of differences in amino acid sequence in Lats2 between mice/rats and humans, antibodies raised against human proteins detected endogenous Lats2 in rat cardiac myocytes with lower efficiencies than the exogenous Lats2 encoded by human cDNAs, which did not allow us to precisely evaluate the extent of overexpression by adenovirus transduction. In any event, Ad-Lats2 dose-dependently increased cytoplasmic accumulation of mono- and oligonucleosomes, a sensitive indicator of apoptosis in cardiac myocytes, whereas Ad-LacZ failed to cause an increase (Figure 1B). Ad-Lats1 failed to induce accumulation of mono- and oligonucleosomes (Figure I in the online data supplement).

Figure1

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 mois. 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 oligonucleosomes was quantitated. Values are means±SEM obtained from 3 experiments. *P<0.05. C, NRCMs were transduced with Ad-LacZ and Ad-Lats2 at 30 mois. The result shown is representative of 3 experiments. D, Left, NRCMs were transduced with Ad-LacZ and Ad-Lats2 at 30 mois in the presence or absence of caspase inhibitor (C.I.) (100 μmol/L, ApoBlock, BD Biosciences). Right, NRCMs were transduced with Ad-LacZ or Ad-Bcl-xL at 10 mois. Twenty-four hours after transduction, NRCMs were transduced with Ad-LacZ or Ad-Lats2 at 30 mois. Forty-eight hours after the second transduction, cytoplasmic accumulation of mono- and oligonucleosomes was quantitated. Values are means±SEM obtained from 3 experiments. *P<0.05. Cell lysates were subjected to immunoblots for detection of Bcl-xL and α-actin.

The increase in the index of DNA fragmentation by Lats2 was accompanied by significant downregulation of Bcl-2/Bcl-xL and an increase in cleaved caspase 3 (Figure 1C), and it was abolished in the presence of a general caspase inhibitor or by inhibiting downregulation of Bcl-xL (Figure 1D). Expression of p53 was not significantly affected by Lats2 (supplemental Figure II). Taken together, these data suggest that Lats2 induces apoptosis in cardiac myocytes, possibly through downregulation of Bcl-xL.

We next examined whether Lats2 mediates Mst1-induced apoptosis in cardiac myocytes.3 To this end, we used Ad-DN-Lats2. We have shown recently that DN-Lats2 inhibits Mst1-induced increases in phosphorylation of YAP in cardiac myocytes, suggesting that DN-Lats2 inhibits endogenous Lats2.23 Although Ad-DN-Lats2 alone did not affect cell death, it significantly reduced Mst1-induced increases in apoptosis as determined by cytoplasmic accumulation of mono- and oligonucleosomes (Figure 2A). Downregulation of Lats2 by shRNA-Lats2 also inhibited Mst1-induced apoptosis (supplemental Figure III). On the other hand, increases in apoptosis by Ad-Lats2 were not inhibited by Ad-DN-Mst1 (Figure 2B). Ad-Lats2 did not exhibit additive effects on the induction of cell death by Ad-Mst1 (Figure 2C). Taken together, Lats2 partially mediates Mst1-induced increases in cardiac myocyte apoptosis.

Figure2

Figure 2. Lats2 partly mediates Mst1-induced apoptosis. A, NRCMs were transduced with Ad-LacZ or Ad-DN-Lats2 (DN-L2) at 30 mois. Twenty-four hours after transduction, myocytes were transduced with Ad-Mst1 at 30 mois. B, NRCMs were transduced with Ad-LacZ and Ad-DN-Mst1 at 30 mois. Twenty-four hours after transduction, myocytes were transduced with Ad-Lats2 (L2) at 30 mois. In A and B, 48 hours after the second transduction, cytoplasmic accumulation of mono- and oligonucleosomes was quantitated. Values are means±SEM obtained from 3 experiments. N.S. indicates not significant. C, NRCMs were transduced with Ad-LacZ, Ad-Lats2, or Ad-Mst1 with different combinations (total 60 mois). Forty-eight hours after the transduction, cytoplasmic accumulation of mono- and oligonucleosomes was quantitated. Values are means±SEM obtained from 3 experiments. *P<0.05.

Lats2 Negatively Regulates Cardiac Myocyte Size In Vitro

Because the Drosophila Hpo pathway regulates both cell death and the organ size,6 we next examined the effect of Lats2 on cardiac myocyte size. Ad-Lats2 dose-dependently decreased the size of cardiac myocytes at baseline and in the presence of phenylephrine (PE), an agonist for the α1 adrenergic receptor (Figure 3A and supplemental Figure IV, A). The significant reduction in the myocyte size by Ad-Lats2 was observed at 10 multiplicities of infection (mois), which was lower than the dose required for the induction of apoptosis (see Figure 1B). A similar result was obtained regarding the effect of Ad-Lats2 on total protein content (Figure 3B) and [3H]phenylalanine incorporation (supplemental Figure IV, B) in cardiac myocytes. Lats2-induced decreases in cardiac myocyte size were observed even when apoptosis was completely suppressed by Bcl-xL, suggesting that Lats2 negatively regulates cell size independently of apoptosis (supplemental Figure V). Ad-DN-Lats2 significantly increased the total protein content (Figure 3B) and [3H]phenylalanine incorporation (supplemental Figure IV,C) at baseline and in response to PE, suggesting that endogenous Lats2 acts as a negative regulator of cardiac hypertrophy. Similar results were obtained in the presence of Ad-shRNA-Lats2 (supplemental Figure VI). Ad-Lats2 also significantly reduced PE-induced increases in expression of atrial natriuretic factor (ANF), a protein upregulated in many forms of cardiac hypertrophy (Figure 3C). On the other hand, Ad-DN-Lats2 significantly increased ANF expression at baseline (Figure 3C and supplemental Figure VII, A). Expression of Lats2 dose-dependently decreased, whereas that of DN-Lats2 dose-dependently increased, transcription of ANF, as determined by ANF-Luc assays (supplemental Figure VIIB). Lats2 significantly reduced phosphorylation of Akt and p70S6K, positive mediators of cardiac hypertrophy, whereas it increased phosphorylation of eEF2, a negative regulator of hypertrophy (supplemental Figure VIII). Conversely, DN-Lats2 increased phosphorylation of Akt (supplemental Figure IX). These results are consistent with the notion that Lats2 negatively regulates cardiac hypertrophy at least partially through suppression of protein synthesis.

Figure3

Figure 3. LATS2 inhibits cardiac hypertrophy. A through C, NRCMs were transduced with Ad-LacZ, Ad-Lats2, or Ad-DN-Lats2. Forty-eight hours after transduction, myocytes were stimulated with or without PE (30 μmol/L) for 48 hours. In A, myocyte surface area was determined. The cell surface area without PE was designated as 100%. Values are means±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 means±SEM obtained from 3 experiments. *P<0.05. In C, myocytes were stained with anti-ANF antibody. The percentage of myocytes expressing ANF, as determined by typical perinuclear staining, was counted. Values are means±SEM obtained from 3 experiments. *P<0.05. Scale bar=20 μm. D, NRCMs were transduced with or without Ad-LacZ (60 mois), Ad-Mst1 (30 mois)+Ad-LacZ (30 mois), and Ad-Mst1 (30 mois)+Ad-DN-Lats2 (30 mois). Forty-eight hours after transduction, myocytes were stimulated with or without PE (30 μmol/L) for 48 hours, and then myocyte surface was measured. Experimental values in myocytes transduced with Ad-LacZ without PE were designated as 1. Values are means±SEM obtained from 3 experiments. *P<0.05.

Expression of Mst1 decreases the cell size and total protein content in cardiac myocytes at baseline and in the presence of PE. Coexpression of DN-Lats2 reversed Mst1-induced decreases in the cell size and total protein content at baseline and in the presence of PE (Figure 3D and supplemental Figure X). These results suggest that Lats2 plays a significant role in mediating the antihypertrophic effect of Mst1.

Lats2 Negatively Regulates Ventricular Chamber Size In Vivo

We next examined the function of Lats2 in the heart in vivo. Although expression of Lats2 in the mouse heart was greater in the neonatal period than in the adult, Lats2 was expressed in the adult heart (supplemental Figure XI). Both mRNA and protein expression of Lats2 were significantly upregulated (6- and 4.5-fold, respectively) by pressure overload in the adult mouse heart (Figure 4A and supplemental Figure XII). To examine the function of Lats2 in vivo, we generated transgenic mice with cardiac-specific overexpression of Lats2 (Tg-Lats2). We made 2 lines and confirmed expression of the Lats2 transgene in the heart, where Lats2 was overexpressed up to 7-fold in line 8 and up to 5-fold in line 20 (Figure 4A and not shown). The level of transgene expression seems overestimated because the antibody detected human Lats2 with a greater sensitivity compared to endogenous Lats2. Thus, Lats2 expression in Tg-Lats2 (lines 8 and 20) appears to be comparable to that of endogenous Lats2 after pressure overload. In line 8, many mice exhibited enlargement of the right atrium (supplemental Figure XIII). At 5 months of age, both left ventricular (LV) weight/tibial length (LVW/TL) and right ventricular (RV) weight/tibial length (RVW/TL) were significantly smaller in Tg-Lats2 than in nontransgenic (NTg) mice (Figure 4B). RVW/TL was also significantly smaller in Tg-Lats2 (line 20) than in NTg mice (supplemental Table I).

Figure4

Figure 4. Cardiac phenotype of Tg-Lats2 mice. A, Heart homogenates were prepared from normal mice subjected to either sham operation (sham) or TAC for 2 weeks, Tg-Lats2 (line 20), or NTg littermates and then immunoblotted with anti-Lats2 and anti–α-actin antibodies. B, Postmortem measurements of LVW/TL (left) and RVW/TL (right) in Tg-Lats2 (line 8) and NTg. Values are means±SD. C, Echocardiographically measured LV end diastolic dimension (LVEDD) and LV ejection fraction (LVEF) in Tg-Lats2 (line 8) and NTg mice. Values are means±SEM. D, Representative wheat germ agglutinin (WGA) staining of the LV and RV myocardium obtained from Tg-Lats2 (line 8) and NTg mice (upper) and the relative myocyte cross sectional area (lower). Values are means±SEM obtained from 5 experiments. *P<0.05.

Echocardiography showed that LV end diastolic dimension was significantly greater and LV ejection fraction was slightly lower in Tg-Lats2 (line 8) than in NTg (Figure 4E and Table 1). Invasive hemodynamic measurements, using pressure–volume loop analysis, showed that LV end-systolic volume is significantly higher but LV dP/dtmax and the slope of the end-systolic pressure–volume relationship (end-systolic elastance) are significantly lower in Tg-Lats2 (line 20) than in NTg, indicating that Tg-Lats2 mice have significantly reduced LV systolic function (Table 2). The time constant of isovolumic pressure decay (τ) was higher in Tg-Lats2 but did not reach statistical significance. LV dP/dtmin was significantly lower in Tg-Lats2, and the slope of the end-diastolic pressure-volume relationship was significantly higher in Tg-Lats2 than in NTg (Table 2). These data suggest that Tg-Lats2 mice also have LV diastolic dysfunction. The indices of LV wall stress (LV end-diastolic wall stress, and LV end-diastolic pressure/end diastolic dimension) were greater in Tg-Lats2 than in NTg, suggesting that LV wall stress was elevated in Tg-Lats2 (supplemental Figure XIV).

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Table 1.

Table 1. Echocardiographic Analysis of Tg-Lats2 Lines 8 and 20 at Five Months of Age

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Table 2.

Table 2. PV Loop Measurements of Tg-Lats2 (Line 20)

The total myocyte number estimated by histological analyses was not significantly different between Tg-Lats2 and NTg (supplemental Figure XV, A). Myocyte cross-sectional area in LV and RV was significantly smaller in Tg-Lats2 (line 8) than in NTg. These results suggest that Lats2 decreases the size of ventricular myocytes (Figure 4D). Histologically determined fibrosis in the LV was significantly greater in Tg-Lats2 (both lines 8 and 20) than in NTg (supplemental Figure XV, B, and data not shown). Neither Tg-Lats2 line 8 nor 20 had increased numbers of TUNEL-positive cardiac myocytes at baseline (not shown here but see supplemental Figure XX).

Biochemical analysis showed that expression of L-type Ca2+ channels and sarcoplasmic reticulum Ca2+-ATPase (SERCA)2a and phosphorylated phospholamban are significantly reduced in Tg-Lats2 compared to NTg, whereas expression of Na+/Ca2+ exchanger did not differ significantly (supplemental Figure XVI).

Tg-DN-Lats2 Mice Exhibit Biventricular Hypertrophy at Baseline

To examine the function of endogenous Lats2, we generated 2 lines (27 and 31) of transgenic mice with cardiac specific overexpression of dominant negative Lats2 (Tg-DN-Lats2). Overexpression of DN-Lats2 in the heart was confirmed by immunoblot analyses (up to 9-fold in line 27 and 11-fold in line 31) (Figure 5A and data not shown). Both LVW/TL and RVW/TL were significantly greater in Tg-DN-Lats2 (line 31) than in NTg mice (Figure 5B and supplemental Table II). LVW/body weight and RVW/body weight were significantly greater in Tg-DN-Lats2 than in NTg mice in both lines 27 and 31 (supplemental Table II). LV and RV myocyte cross-sectional area was significantly greater in Tg-DN-Lats2 than in NTg (Figure 5C). These results suggest that inhibition of endogenous Lats2 induces hypertrophy in both RV and LV cardiac myocytes. Expression of ANF was elevated in Tg-DN-Lats2 hearts (Figure 5A). Taken together, Tg-DN-Lats2 hearts exhibited all features of biventricular hypertrophy at baseline. Echocardiographically determined chamber size and LV function were normal in Tg-DN-Lats2 at baseline, except that LV wall thickness was significantly greater in Tg-DN-Lats2 (line 31) than in NTg (supplemental Table III).

Figure5

Figure 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 LVW/TL (mg/mm, left) and RVW/TL (mg/mm, right) in Tg-DN-Lats2 (line 31) and NTg. Values are means±SD. N=12 to 16. C, Representative WGA staining of the LV myocardium obtained from Tg-DN-Lats2 and NTg mice (upper) and the relative myocyte cross-sectional area (lower). Values are means±SEM obtained from 5 experiments.

Endogenous Lats2 Is a Negative Regulator of Pathological Hypertrophy in Response to Pressure Overload

Because expression of Lats2 is increased in response to pressure overload, we examined whether upregulation of endogenous Lats2 during pressure overload works as a negative-feedback mechanism for cardiac hypertrophy. To this end, Tg-DN-Lats2 mice were subjected to transverse aortic constriction (TAC). After 2 weeks of TAC, the pressure gradient was similar between NTg and Tg-DN-Lats2 (Figure 6A). Tg-DN-Lats2 mice exhibited significantly greater LVW/TL than NTg after TAC (Figure 6B and supplemental Table IV). Increases in LVW/TL after TAC (compared with mean LVW/TL in sham operated mice in each group) were significantly greater in Tg-DN-Lats2 than in NTg (+37% versus 27%, P<0.05). Increases in LV cross-sectional area after TAC were also greater in Tg-DN-Lats2 than in NTg (+25% versus +16%, P<0.05) (Figure 6C). TUNEL analyses indicated that TAC-induced increases in LV apoptosis were significantly smaller in Tg-DN-Lats2 than in NTg (Figure 6D). These results suggest that endogenous Lats2 negatively regulates LV hypertrophy and apoptosis by pressure overload.

Figure6

Figure 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 LVW/TL (mg/mm, left) after TAC. Right, Percentage increase in LVW/TL after TAC compared with the mean value of sham-operated mice. Values are means±SD. N=7. C, Representative WGA staining of the LV obtained from Tg-DN-Lats2 and NTg mice after TAC (left) and the relative myocyte cross-sectional area (right). Values are means±SEM obtained from 5 experiments. D, Percentage of TUNEL-positive myocytes. Values are means±SEM obtained from 5 experiments. #P<0.05 vs NTg sham.

Discussion

Appropriate cell numbers and the organ size in multicellular organisms are determined by coordination of cell growth, proliferation, and apoptosis.1 A recent burst of studies on Drosophila has demonstrated that a novel and evolutionarily conserved signaling cascade termed the “Hpo pathway” critically controls the size of cells and organs.6 Wts/Lats protein kinase is a key component of the pathway, which controls the coordination between cell proliferation and apoptosis.8–10 We demonstrated here that Mst1 and Lats2, the mammalian counterparts of the Hpo pathway components, play an important role in mediating both apoptosis and inhibition of growth of cardiac myocytes, thereby controlling the size of left and right ventricles in the adult mouse heart (supplemental Figure XVII).

Previous reports demonstrated that both Drosophila Wts and mammalian Lats1 induce apoptosis.9,24–26 However, overexpression of Lats1 failed to induce apoptosis in cardiac myocytes (supplemental Figure I). Although Lats1 is phosphorylated and activated by Mst2, no stable interaction could be observed between Lats1 and hWW45, a mammalian homolog of Sav and an adaptor protein in the Hpo pathway, or Mst2.27 On the other hand, Lats2, but not Lats1, interacts with hWW45, forms a complex with Mst1 through hWW45 in cardiac myocytes, and is phosphorylated by Mst1 in COS-7 cells (supplemental Figure XVIII and XIX). These results suggest that the function of Lats1 and Lats2 may be cell type–dependent in mammalian cells.

Our results suggest that Lats2 induces apoptosis in cardiac myocytes in vitro. Furthermore, Lats2 plays an important role in mediating Mst1-induced apoptosis. Downregulation of Bcl-2 or Bcl-xL might be partially responsible for Lats2-induced apoptosis in cardiac myocytes. Lats2 binds to Mdm2 and upregulates p53, which in turn upregulates Lats2, thereby constituting a positive-feedback loop for p53 activation in U2OS cells.16 However, Lats2 did not upregulate p53 in cardiac myocytes. The effect of Lats2 on apoptosis may be modulated by YAP, a cofactor of nuclear transcription factors, whose phosphorylation and nuclear localization are regulated by Lats2.28 YAP mediates c-Jun–induced apoptosis through a p73-dependent mechanism in MEF,29 whereas it inhibits apoptosis through activation of Tead, a transcription factor in embryonic tissues.30 The effect of YAP on cell death in the heart remains to be elucidated.

Tg-Lats2 mice displayed modest but significant LV dysfunction at baseline (Table 1) and in response to pressure overload (supplemental Table V). Apoptosis may not be responsible for basal LV dysfunction, because increases in TUNEL-positive cells were not significant in Tg-Lats2 at baseline. Downregulation of Ca2+ handling proteins, such as L-type Ca2+ channels and SERCA, and reduction of phosphorylated phospholamban levels, possibly through both transcriptional and posttranslational modifications initiated by increased activity of Lats2, may contribute to baseline LV dysfunction in Tg-Lats2. On the other hand, because TUNEL-positive myocytes were significantly greater in Tg-Lats2 than in NTg under pressure overload (supplemental Figure XX) and because increases in apoptosis by pressure overload are partially inhibited by DN-Lats2, Lats2 may sensitize myocytes to apoptosis under stress.

Our results from both gain- and loss-of-function experiments suggest that endogenous Lats2 acts as a negative regulator of the size of both left and right ventricles and cardiac myocytes therein in vivo. Although left and right ventricles in Tg-Lats2 were significantly smaller at baseline, the enhancement of apoptosis was observed only under stress. Thus, one of the most prominent functions of Lats2 in the heart is similar to that of Drosophila Wts, namely the regulation of the organ size.6 Furthermore, Mst1-induced decreases in cardiac myocyte size were significantly reduced by DN-Lats2 in vitro, suggesting that Lats2 plays an important role in mediating the antihypertrophic effects of Mst1.

Although Tg-Lats2 mice exhibited dilated hearts at baseline, the wall thickness was not increased, suggesting that the LV wall stress is elevated. Suppression of compensatory hypertrophy might have contributed to LV dysfunction in Tg-Lats2 hearts. Because the lack of compensatory hypertrophy despite LV dilation was also observed in Tg-Mst1,3 these results are consistent with the notion that Lats2 partly mediates the detrimental function of Mst1 in the adult heart. We speculate that upregulation of Lats2 observed during pressure overload is detrimental because it not only increases apoptosis but also prevents compensatory hypertrophy.

In Drosophila Hpo pathway, Wts negatively regulates cyclin E and diap, a homolog of XIAP and an antiapoptotic molecule.7 Lats2 induced significant reduction in expression of cyclin E in cardiac myocytes in vitro (supplemental Figure XXI). Although it is generally believed that postnatal cardiac myocytes are terminally differentiated and its growth is regulated primarily by hypertrophy rather than proliferation, recent evidence suggests that cyclins, mediators of cell proliferation, also play an important role in mediating hypertrophy.31 At present, the significance of cyclin E in the regulation of cardiac myocyte growth remains to be elucidated. Wts regulates nuclear localization of Yki,20 thereby regulating its transcriptional targets, such as microRNA bantam.32 Whether or not Lats2 regulates gene transcription through YAP and, if so, which genes are the targets of YAP would be important issues to address in the future. Recent evidence suggests that miRNA-327 and -373 downregulate Lats2, thereby neutralizing p53-mediated CDK inhibition in human testicular germ cell tumors.33 Our results suggest that Lats2 negatively regulates protein synthesis in cardiac myocytes. It remains to be elucidated how activation of Lats2 modulates the activity of signaling molecules that regulate cardiac protein synthesis, such as Akt, p70S6K, and eEF2.

In conclusion, our results suggest that Lats2 regulates both growth and death of cardiac myocytes, thereby mimicking the cellular function of Mst1 (supplemental Figure XVII). Although Lats2 partly mediates cardiac myocyte apoptosis in response to pressure overload in vivo, Lats2 has more prominent effects on regulation of the heart size and plays an essential role in inhibiting hypertrophy at baseline and in response to pressure overload.

Acknowledgments

Sources of Funding

This work was supported, in part, by US Public Health Service grants HL 59139, HL67724, HL67727, HL69020, and HL 73048 and by American Heart Association Grant 0340123N.

Disclosures

None.

Footnotes

  • ↵*Both authors contributed equally to this work.

  • Original received May 23, 2008; revision received September 18, 2008; accepted October 8, 2008.

References

  1. ↵
    Stanger BZ. Organ size determination and the limits of regulation. Cell Cycle. 2008; 7: 318–324.
    OpenUrlCrossRefPubMed
  2. ↵
    Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003; 114: 763–776.
    OpenUrlCrossRefPubMed
  3. ↵
    Yamamoto S, Yang G, Zablocki D, Liu J, Hong C, Kim SJ, Soler S, Odashima M, Thaisz J, Yehia G, Molina CA, Yatani A, Vatner DE, Vatner SF, Sadoshima J. Activation of Mst1 causes dilated cardiomyopathy by stimulating apoptosis without compensatory ventricular myocyte hypertrophy. J Clin Invest. 2003; 111: 1463–1474.
    OpenUrlCrossRefPubMed
  4. ↵
    Morisco C, Sadoshima J, Trimarco B, Arora R, Vatner DE, Vatner SF. Is treating cardiac hypertrophy salutary or detrimental: the two faces of Janus. Am J Physiol Heart Circ Physiol. 2003; 284: H1043–H1047.
    OpenUrlPubMed
  5. ↵
    Saucedo LJ, Edgar BA. Filling out the Hippo pathway. Nat Rev Mol Cell Biol. 2007; 8: 613–621.
    OpenUrlCrossRefPubMed
  6. ↵
    Pan D. Hippo signaling in organ size control. Genes Dev. 2007; 21: 886–897.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Harvey KF, Pfleger CM, Hariharan IK. The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis. Cell. 2003; 114: 457–467.
    OpenUrlCrossRefPubMed
  8. ↵
    Pantalacci S, Tapon N, Leopold P. The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila. Nat Cell Biol. 2003; 5: 921–927.
    OpenUrlCrossRefPubMed
  9. ↵
    Udan RS, Kango-Singh M, Nolo R, Tao C, Halder G. Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway. Nat Cell Biol. 2003; 5: 914–920.
    OpenUrlCrossRefPubMed
  10. ↵
    Wu S, Huang J, Dong J, Pan D. hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts. Cell. 2003; 114: 445–456.
    OpenUrlCrossRefPubMed
  11. ↵
    Xu T, Wang W, Zhang S, Stewart RA, Yu W. Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase. Development. 1995; 121: 1053–1063.
    OpenUrlAbstract
  12. ↵
    Li Y, Pei J, Xia H, Ke H, Wang H, Tao W. Lats2, a putative tumor suppressor, inhibits G1/S transition. Oncogene. 2003; 22: 4398–4405.
    OpenUrlCrossRefPubMed
  13. ↵
    Kamikubo Y, Takaori-Kondo A, Uchiyama T, Hori T. Inhibition of cell growth by conditional expression of kpm, a human homologue of Drosophila warts/lats tumor suppressor. J Biol Chem. 2003; 278: 17609–17614.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Dijkman R, van Doorn R, Szuhai K, Willemze R, Vermeer MH, Tensen CP. Gene-expression profiling and array-based CGH classify CD4+CD56+ hematodermic neoplasm and cutaneous myelomonocytic leukemia as distinct disease entities. Blood. 2007; 109: 1720–1727.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Takahashi Y, Miyoshi Y, Takahata C, Irahara N, Taguchi T, Tamaki Y, Noguchi S. Down-regulation of LATS1 and LATS2 mRNA expression by promoter hypermethylation and its association with biologically aggressive phenotype in human breast cancers. Clin Cancer Res. 2005; 11: 1380–1385.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Aylon Y, Michael D, Shmueli A, Yabuta N, Nojima H, Oren M. A positive feedback loop between the p53 and Lats2 tumor suppressors prevents tetraploidization. Genes Dev. 2006; 20: 2687–2700.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    McPherson JP, Tamblyn L, Elia A, Migon E, Shehabeldin A, Matysiak-Zablocki E, Lemmers B, Salmena L, Hakem A, Fish J, Kassam F, Squire J, Bruneau BG, Hande MP, Hakem R. Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity. EMBO J. 2004; 23: 3677–3688.
    OpenUrlCrossRefPubMed
  18. ↵
    Yabuta N, Okada N, Ito A, Hosomi T, Nishihara S, Sasayama Y, Fujimori A, Okuzaki D, Zhao H, Ikawa M, Okabe M, Nojima H. Lats2 is an essential mitotic regulator required for the coordination of cell division. J Biol Chem. 2007; 282: 19259–19271.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Edgar BA. From cell structure to transcription: Hippo forges a new path. Cell. 2006; 124: 267–273.
    OpenUrlCrossRefPubMed
  20. ↵
    Huang J, Wu S, Barrera J, Matthews K, Pan D. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the drosophila homolog of YAP. Cell. 2005; 122: 421–434.
    OpenUrlCrossRefPubMed
  21. ↵
    Yabuta N, Fujii T, Copeland NG, Gilbert DJ, Jenkins NA, Nishiguchi H, Endo Y, Toji S, Tanaka H, Nishimune Y, Nojima H. Structure, expression, and chromosome mapping of LATS2, a mammalian homologue of the Drosophila tumor suppressor gene lats/warts. Genomics. 2000; 63: 263–270.
    OpenUrlCrossRefPubMed
  22. ↵
    Tao W, Zhang S, Turenchalk GS, Stewart RA, St John MA, Chen W, Xu T. Human homologue of the Drosophila melanogaster lats tumour suppressor modulates CDC2 activity. Nat Genet. 1999; 21: 177–181.
    OpenUrlCrossRefPubMed
  23. ↵
    Nakano N, Matsui Y, Sadoshima J. Yap is a nuclear target of Mst1/Lats2 regulating cell size in cardiac myocytes. Circulation. 2007; 116 (suppl II): II-128 Abstract.
    OpenUrl
  24. ↵
    Kuninaka S, Nomura M, Hirota T, Iida S, Hara T, Honda S, Kunitoku N, Sasayama T, Arima Y, Marumoto T, Koja K, Yonehara S, Saya H. The tumor suppressor WARTS activates the Omi / HtrA2-dependent pathway of cell death. Oncogene. 2005; 24: 5287–5298.
    OpenUrlCrossRefPubMed
  25. ↵
    Xia H, Qi H, Li Y, Pei J, Barton J, Blackstad M, Xu T, Tao W. LATS1 tumor suppressor regulates G2/M transition and apoptosis. Oncogene. 2002; 21: 1233–1241.
    OpenUrlCrossRefPubMed
  26. ↵
    Yang X, Li DM, Chen W, Xu T. Human homologue of Drosophila lats, LATS1, negatively regulate growth by inducing G(2)/M arrest or apoptosis. Oncogene. 2001; 20: 6516–6523.
    OpenUrlCrossRefPubMed
  27. ↵
    Chan EH, Nousiainen M, Chalamalasetty RB, Schafer A, Nigg EA, Sillje HH. The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene. 2005; 24: 2076–2086.
    OpenUrlCrossRefPubMed
  28. ↵
    Dong J, Feldmann G, Huang J, Wu S, Zhang N, Comerford SA, Gayyed MF, Anders RA, Maitra A, Pan D. Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell. 2007; 130: 1120–1133.
    OpenUrlCrossRefPubMed
  29. ↵
    Danovi SA, Rossi M, Gudmundsdottir K, Yuan M, Melino G, Basu S. Yes-Associated Protein (YAP) is a critical mediator of c-Jun-dependent apoptosis. Cell Death Differ. 2008; 15: 217–219.
    OpenUrlCrossRefPubMed
  30. ↵
    Sawada A, Kiyonari H, Ukita K, Nishioka N, Imuta Y, Sasaki H. Redundant roles of Tead1 and Tead2 in notochord development and the regulation of cell proliferation and survival. Mol Cell Biol. 2008; 28: 3177–3189.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Zhong W, Mao S, Tobis S, Angelis E, Jordan MC, Roos KP, Fishbein MC, de Alboran IM, MacLellan WR. Hypertrophic growth in cardiac myocytes is mediated by Myc through a Cyclin D2-dependent pathway. EMBO J. 2006; 25: 3869–3879.
    OpenUrlCrossRefPubMed
  32. ↵
    Thompson BJ, Cohen SM. The Hippo pathway regulates the bantam microRNA to control cell proliferation and apoptosis in Drosophila. Cell. 2006; 126: 767–774.
    OpenUrlCrossRefPubMed
  33. ↵
    Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R, Liu YP, van Duijse J, Drost J, Griekspoor A, Zlotorynski E, Yabuta N, De Vita G, Nojima H, Looijenga LH, Agami R. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell. 2006; 124: 1169–1181.
    OpenUrlCrossRefPubMed
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  • Lats2 Is a Negative Regulator of Myocyte Size in the Heart
    Yutaka Matsui, Noritsugu Nakano, Dan Shao, Shumin Gao, Wenting Luo, Chull Hong, Peiyong Zhai, Eric Holle, Xianzhong Yu, Norikazu Yabuta, Wufan Tao, Thomas Wagner, Hiroshi Nojima and Junichi Sadoshima
    Circulation Research. 2008;103:1309-1318, originally published November 21, 2008
    https://doi.org/10.1161/CIRCRESAHA.108.180042
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