Journal of Molecular Medicine

, Volume 87, Issue 3, pp 249–260

Lysosomal cysteine peptidase cathepsin L protects against cardiac hypertrophy through blocking AKT/GSK3β signaling


    • Cardiovascular Research Institute of Wuhan University and Department of CardiologyRenmin Hospital of Wuhan University
  • Jun Cai
    • Cardiovascular Research CenterMassachusettes General Hospital, Harvard Medical School
    • Department of Cardiology, Beijing Chaoyang HospitalCapital Medical University
  • Difei Shen
    • Cardiovascular Research Institute of Wuhan University and Department of CardiologyRenmin Hospital of Wuhan University
  • Zhouyan Bian
    • Cardiovascular Research Institute of Wuhan University and Department of CardiologyRenmin Hospital of Wuhan University
  • Ling Yan
    • Cardiovascular Research Institute of Wuhan University and Department of CardiologyRenmin Hospital of Wuhan University
  • You-Xin Wang
    • Graduate School of Chinese Academy of Sciences
    • School of Public Health and Family MedicineCapital Medical University
  • Jie Lan
    • Graduate School of Chinese Academy of Sciences
    • School of Public Health and Family MedicineCapital Medical University
  • Guo-Qing Zhuang
    • Graduate School of Chinese Academy of Sciences
    • School of Public Health and Family MedicineCapital Medical University
  • Wen-Zhan Ma
    • Graduate School of Chinese Academy of Sciences
    • School of Public Health and Family MedicineCapital Medical University
  • Wei Wang
    • Graduate School of Chinese Academy of Sciences
    • School of Public Health and Family MedicineCapital Medical University
Original Article

DOI: 10.1007/s00109-008-0423-2

Cite this article as:
Tang, Q., Cai, J., Shen, D. et al. J Mol Med (2009) 87: 249. doi:10.1007/s00109-008-0423-2


The lysosomal cysteine peptidase cathepsin L (CTSL) is an important lysosomal proteinase involved in a variety of cellular functions including intracellular protein turnover, epidermal homeostasis, and hair development. Deficiency of CTSL in mice results in a progressive dilated cardiomyopathy. In the present study, we tested the hypothesis that cardiac overexpression of human CTSL in the murine heart would protect against cardiac hypertrophy in vivo. The effects of constitutive human CTSL expression on cardiac hypertrophy were investigated using in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding (AB) in CTSL transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by two-dimensional and M-mode echocardiography as well as by molecular and pathological analyses of heart samples. Constitutive overexpression of human CTSL in the murine heart attenuated the hypertrophic response, markedly reduced apoptosis, and fibrosis. Cardiac function was also preserved in hearts with increased CTSL levels in response to hypertrophic stimuli. These beneficial effects were associated with attenuation of the Akt/GSK3β signaling cascade. Our in vitro studies further confirmed that CTSL expression in cardiomyocytes blunts cardiac hypertrophy through blocking of Akt/GSK3β signaling. The study indicates that CTSL improves cardiac function and inhibits cardiac hypertrophy, inflammation, and fibrosis through blocking Akt/GSK3β signaling.


Cathepsin Lcardiac remodelingAKTFibrosisGSK3β


Human proteases are ubiquitously expressed in different tissues and play an essential role in numerous physiological and pathological processes. Cathepsins belong to the lysosomal cysteine proteinases, which were particularly emphasized for their activity in important biological functions, such as proteolytic processing of proenzymes, antigen presentation, inflammation, tissue remodeling, cell proliferation, differentiation, apoptosis and degradation of the extracellular matrix, facilitating wound healing, and invasion of tumor cells [13]. The human lysosomal cysteine cathepsins represent a family of 11 papain-like proteolytic enzymes with a principal subcellular localization in the endosomal and lysosomal compartment. Seven of these peptidases including cathepsins B, C, F, H, L, O, and X/Z exhibit ubiquitous expression in mammalian tissues [4]. Other papain-like cysteine peptidases exhibit cell type-specific expression. Cathepsin K is mainly found in osteoclasts, and cathepsin S is mainly expressed in peripheral antigen-presenting cells [4]. The functions of cysteine cathepsins have been identified in the cytoplasm, the nucleus, and in the mitochondrion.

Among all of cathepsins, cathepsin L (CTSL) has been of great interest owning to its numerous functions. CTSL is a ubiquitously expressed lysosomal cysteine proteinase and is primarily responsible for intracellular protein degradation [5]. Studies have demonstrated that CTSL plays an important role in hair formation and skin metabolism as well as T-cell selection [5]. CTSL-deficient mice were shown to develop epidermal hyperproliferation and periodic hair loss [5]. CTSL is also secreted by transformed mouse fibroblasts and converts interleukin (IL)-8 and urokinase-type plasminogen activator into active form [6]. Increased CTSL expression was found in cancer tissue compared with normal tissues and wasting skeletal muscle of septic rats, whereas decreased CTSL expression and enzyme activity were found in mesangial cells cultured with glucose and hypertrophied kidneys of streptozotocin-induced diabetic rats [79]. More importantly, recent studies revealed that CTSL-deficient mice develop heart disease that resembles many features of human dilated cardiomyopathy [10]. Complete inhibition of CTSL causes interstitial fibrosis in the myocardium and pleomorphism of cardiomyocyte nuclei, histological alterations characteristic of human cardiomyopathies, as well as cardiac chamber dilation, and impaired cardiac contraction in 1-year-old mice [10]. Petermann et al. [11] also demonstrated that CTSL-deficient mice show the lysosomal system disorder including increasing the number and changing the morphology of acidic organelles, although without the accumulation of specific lysosomal storage materials. These defects in the acidic compartments of CTSL-deficient cardiomyocytes result in complex biochemical and cellular alterations leading to loss of cytoskeletal proteins and mitochondrial impairment, which contribute to cardiomyocyte dysfunction. However, the skeletal muscles of CTSL-deficient mice are not pathologically altered. More recently, Spira et al. [12] reported that cardiac-specific expression of CTSL in CTSL-deficient mice results in improved cardiac contraction, normal mRNA expression of hypertrophic markers and heart weight, and regular ultrastructure of cardiomyocytes. Despite the potentially significant roles of CTSL in the function of heart, it has remained unclear whether CTSL could regulate cardiac hypertrophy under pathological conditions and whether targeted myocardial overexpression of CTSL is able to protect against cardiac hypertrophy and heart failure mediated by classical hypertrophic models. Thus, in the present study, our aim is to investigate the role of CTSL in cardiac hypertrophy mediated by pressure overload and Angiotensin II (Ang II) and to clarify the related molecular mechanisms.

Methods and materials

Transgenic mice and animal models

All experiments were approved by the Animal Care and Use Committee of Wuhan University. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). A CTSL cDNA construct containing full-length human CTSL cDNA (Addgene, Boston) was cloned downstream of the cardiac myosin heavy chain (MHC) promoter. Transgenic mice were produced by microinjection of the α-MHC-CTSL construct into fertilized mouse embryos (C57BL/6 background). Five independent transgenic lines were established and identified by polymerase chain reaction (PCR) analysis of tail genomic DNA. Cardiac function and gene expression were analyzed in pairs of α-MHC-CTSL transgenic mice (TG) and littermate nontransgenic (WT) male mice ranging in age from 8 to 10 weeks. Aortic banding (AB) was performed as described previously [13]. Ang II infusion models were established as described previously [13]. Briefly, Ang II (1.2 mg/kg/day dissolved in 0.9% NaCl) was subcutaneously infused for 4 weeks using an osmotic minipump (Alzet model 2004; Alza) implanted in each mouse. Saline-infused animals served as infusion controls and were subjected to the same procedures as the experimental animals with the exception of Ang II infusion.

Echocardiography and blood pressure measurement

Echocardiography was performed by SONOS 5500 ultrasound (Philips Electronics, Amsterdam) with a 15-MHz linear array ultrasound transducer. The left ventricle (LV) was assessed in both parasternal long-axis and short-axis views at a frame rate of 120 Hz. Left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were measured from the M-mode tracing with a sweep speed of 50 mm/s at the mid-papillary muscle level. End-systole and end-diastole were defined as the phase with the smallest or largest area of LV, respectively. Blood pressure and heart rate were measured as described previously [13].

Histological analysis and determination of apoptosis

Hearts were excised, washed with saline solution, and placed in 10% formalin. Hearts were cut transversely close to the apex to visualize the left and right ventricles. Several sections of heart (4-5 μm thick) were prepared and stained with hematoxylin and eosin (H&E) for histopathology or Picrosirius Red (PSR) for collagen deposition, then visualized by light microscopy. For myocyte cross-sectional area, sections were stained with H&E. Single myocyte was measured with an image quantitative digital analysis system (Image-Pro Plus 4.5). The outline of 300 myocytes was traced in each section. Cell death by apoptosis was evaluated by a TUNEL assay that was performed in sections using CardiaoTACS in situ Apoptosis Detection Kit (R&D Systems, Minneapolis, USA) according to the manufacturer’s recommendations. Caspase-3/8/9 activities and degradation were also used to examine the effects of CTSL on apoptosis as described previously [14].

Western blot analysis, quantitative real-time RT-PCR, and Cathepsin L activity

For Western blot, protein extracts from different groups of myocardium (50 µg) or cultured cardiac myocytes were fractionated on a 10% polyacrylamide gel under reducing conditions, transferred to nitrocellulose membranes, and probed with various antibodies. Anti-human CTSL monoclonal antibody (reacts with human) was from Millipore and anti-mouse CTSL polyclonal antibody (reacts with mouse) was from R&D Systems. Polyclonal antibodies against Akt, mTOR, GSK3β, FOXO3A, FOXO1, p38, JNK1/2, and ERK1/2 as well as phosphorylated Akt, mTOR, GSK3β, FOXO3A, FOXO1, p38, JNK1/2, and ERK1/2, respectively, were purchased from Cell Signaling Technology. Rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase antibody was purchased from Sigma Chemical. The antibodies against CTGF, Collagen I, Collagen III, and TGFβ1 were ordered from Abcam. Other antibodies were purchased from Santa Cruz Biotechnology. After incubation with a secondary peroxidase-conjugated antibody, signals were visualized by Chemiluminescence Kit (Amersham, Sunnyvale, CA, USA). QRT-PCR was used to detect mRNA levels of hypertrophic, inflammatory, and fibrotic markers. Total RNA was extracted from frozen, pulverized mouse tissues using TRIzol (Invitrogen), and cDNA was synthesized using oligo (dT) primers with the Advantage RT-for-PCR Kit (BD Biosciences). We quantified PCR amplifications using SYBR Green PCR Master Mix (Applied Biosystems) and normalized results against glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene expression. Cathepsin L activity determination was performed as previously published [12].

Cultured neonatal rat cardiac myocytes and recombinant adenoviral vectors

Hearts from 1-day-old Sprague–Dawley neonatal rats were excised, and the ventricular myocardium was cut into small pieces in the dissociation buffer and then incubated on a shaker at 37°C for 20 min at 100 rpm. Tissue pieces were allowed to settle, and the supernatant was collected, suspended in 1 ml newborn calf serum and centrifuged at 1,000 rpm for 10 min. The cell pellet was resuspended in 1 ml newborn calf serum and stored at 37°C. The procedure was repeated until all tissue was digested. The cells were then resuspended in F10 medium supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. After 48 h, the culture medium was replaced with F10 medium containing 0.1% FCS and BrdU (100 μM). The cells were used for experiments after demonstrating rhythmic contractions. For construction of adenovirus containing CTSL, the human full-length human CTSL cDNA was subcloned into the shuttle plasmid pShuttle-cytomegalovirus (CMV). Recombinant adenovirus was generated by bacterial homologous recombination between pShuttle-CMV containing CTSL cDNA and the viral backbone vector pAdEasy-1. Recombinant adenovirus carrying the green fluorescent protein (AdGFP) gene was prepared as a control and titrated in the same way. Three rat shCTSL constructs were from SuperArray (Cat. No. KR42457) and then generated three Ad-shCTSL adenovirus, and one of them was selected that led to a significant decrease in CTSL levels for further experiments.

[3H]-Leucine incorporation and cell surface area measurement

Cardiac myocytes were infected with Ad-CTSL or Ad-shCTSL for 24 h and subsequently stimulated with Ang II (1 μM) and coincubated with [3H]-Leucine (1 μCi/mL) for the indicated time. At the end of the experiment, the cells were washed with Hanks’ solution, scraped off the well, and then treated with 10% trichloroacetic acid at 4°C for 60 min. The precipitates were then dissolved in NaOH (1 N) and subsequently counted with a scintillation counter. For surface areas, the cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS), permeabilized in 0.1% Triton X-100 in PBS, and stained with α-actinin (Sigma) at a dilution of 1:100 by standard immunocytochemical techniques.

Statistical analysis

All values are expressed as mean ± SEM. Differences between two groups were determined by a Student’s t test. Comparison between groups on Western blotting data was assessed by one-way analysis of variance followed by a Bonferroni correction. A value of P<0.05 was considered statistically significant.


Characterization of CTSL transgenic mice

To examine the functional effects associated with CTSL induction, a cDNA encoding human full-length CTSL was subcloned into the α-MHC promoter vector to permit generation of transgenic mice with cardiac-specific overexpression. We analyzed CTSL protein levels in various tissues by Western blot analysis using anti-CTSL antibody (human specific). A robust expression of human CTSL protein was found in the heart, but not in other organs (Fig. 1A). Five independent transgenic lines were initially generated and characterized by Western blot. One high expression line (Tg3) was selected for in-depth analysis (Fig. 1B). Western blot analysis further demonstrated that the expression level of the mouse CTSL protein was not modified by expression of human CTSL gene (data not shown). The human CTSL transgene-induced overexpression of proCTSL protein in the heart resulted in the significant elevation of CTSL activity in the heart (Fig. 1C). However, the levels of CTSL protein in the hearts of TG mice had not different compared with that in wild-type mice (Fig. 1A and B). To investigate whether CTSL expression is regulated by pressure overload, WT mice were subjected to AB for different durations. CTSL expression in the LV was markedly increased compared to basal levels after 2 weeks of AB and reached maximum level after 4 weeks of AB. However, the expression of proCTSL was significantly decreased after 8 weeks of AB (Fig. 1D). Importantly, the protein levels of transgenic pro CTSL were not significantly affected by AB, and this pattern of expression persisted in hearts for 8 weeks after surgery (Fig. 1D). CTSL transgenic mice showed no pathological alterations in ventricular chamber dimensions, ventricular wall thicknesses, fractional shortening, or heart weight normalized to body-weight (data not shown). Extensive histological analysis also revealed no gross morphological alterations or fibrosis (data not shown).
Fig. 1

Characterization of human CTSL transgenic mice. A Representative Western blot of human proCTSL and CTSL proteins from different tissue of TG mice as indicated. B Representative Western blots of human pro CTSL and CTSL proteins in the heart tissue from five lines of both TG and WT mice. C The activity of CTSL in the hearts of WT and TG mice (n = 5). *P < 0.01 was obtained for the WT mice values. D Representative Western blots of mouse and human proCTSL and CTSL proteins in the heart tissue from TG mice after aortic banding at time points indicated (raw data of the Western blots can be found as electronic supplementary material)

Forced CTSL expression attenuates pathological cardiac hypertrophy

To investigate the role of CTSL in biomechanical stress in the heart, we performed aortic banding (AB) surgery on 8- to 10-week-old TG and WT mice. Cardiac function was examined by echocardiography after 8 weeks of surgery. As shown in Table 1, the heart weight/body weight (HW/BW) and lung weight/body weight (LW/BW) ratios as well as cardiomyocyte cross-sectional area were significantly decreased in TG mice. No significant differences were observed in the sham-operated TG and WT mice. The increase in left ventricle (LV) chamber dimensions and thickness induced by pressure overload were also markedly reduced during both systole and diastole in TG mice compared with WT littermates (Table 1). We further examined the potential effect of CTSL expression on cardiac hypertrophy in an Ang II-infusion model. Analyses were performed on the TG and WT mice after 4 weeks of chronic administration of Ang II using implanted osmotic minipumps. Consistent with AB results, the ratios of HW/BW and LW/BW were significantly decreased in the TG mice compared with WT littermates (Table 2). LV chamber dimensions and thickness were also significantly decreased during both systole and diastole in TG mice compared to control littermates (Table 2). The reduced chamber dilation along with the decrease in heart weight was consistent with attenuated hypertrophy in the TG mice after Ang II stimulation. Gross hearts and H&E staining further confirmed the inhibitory effect of CTSL on cardiac remodeling in response to AB and Ang II stimulation (Fig. 2A–D). We examined the expression of several cardiac hypertrophy markers in TG and WT mice after AB surgery and Ang II infusion by real-time PCR analysis. Expression levels of ANP, BNP, and Myh7 were induced to a higher level in WT mice after AB or Ang II infusion, and such increases were markedly attenuated in TG mice, whereas expression of Myh6 was unchanged under all tested conditions (Fig. 2E and F). These results indicate that forced expression of CTSL in the heart decreases the expression of cardiac hypertrophy markers ANP, BNP, and Myh7, and results in attenuated cardiac hypertrophy induced by pressure overload or Ang II stimulation.
Fig. 2

The effects of CTSL on cardiac hypertrophy in vivo (AD) Gross hearts and HE staining of sham and AB mice at 8 weeks postsurgery or 4 weeks of Ang II-infused mice. E and F Analysis of hypertrophic markers. Total RNA was isolated from hearts of mice of the indicated groups, and expression of transcripts for ANP, BNP, Myh7, and Myh6 induced by AB or Ang II infusion were determined by real-time PCR analysis. Data represent typical results of three to four different experiments as mean ± SEM

Table 1

Echocardiographic data showed the effects of CTSL on cardiac hypertrophy induced by aortic banding model


WT-sham mice

TG-sham mice

WT-AB mice

TG-AB mice


n = 10

n = 10

n = 8

n = 9

BW, g

24.9 ± 1.2

25.3 ± 2.0

27.1 ± 1.4

26.3 ± 1.5

HW/BW (mg/g)

4.72 ± 0.11

4.56 ± 0.12

7.25 ± 0.26*

5.11 ± 0.37**

LW/BW (mg/g)

4.78 ± 0.21

4.69 ± 0.18

8.14 ± 0.35*

5.36 ± 0.18**

CSA (μm2)

277 ± 24

259 ± 27

436 ± 34*

295 ± 32**

SBP, mmHg

115.7 ± 2.1

116.3 ± 1.9

151.0 ± 3.2*

146.2 ± 3.3*

HR, beats/min

463 ± 24

458 ± 22

443 ± 15

471 ± 24

PWT (mm)

1.21 ± 0.02

1.23 ± 0.02

3.31 ± 0. 03*

1.82 ± 0.04**

LVEDD (mm)

3.61 ± 0.03

3.64 ± 0.04

5.81 ± 0.05*

4.09 ± 0.03**

LVESD (mm)

2.41 ± 0.04

2.39 ± 0.04

3.54 ± 0.03*

2.54 ± 0.05**

IVSd (mm)

0.63 ± 0.04

0.63 ± 0.04

1.67 ± 0.05*

0.93 ± 0.04**

LVPWd (mm)

0.64 ± 0.02

0.64 ± 0.04

1.30 ± 0.02*

0.85 ± 0.05**

FS (%)

54.2 ± 2.1

56.3 ± 3.3

22.3 ± 2.5*

45.4 ± 1.7**

CSA cardiomyocyte cross-sectional area, SBP systolic blood pressure, HR heart rate, BW body weight, HW heart weight, LW lung weight, PWT posterior wall thickness, LVEDD left ventricular end-diastolic diameter, LVESD left ventricular end-systolic diameter, IVSd left ventricular septum, diastolic; LVPWd left ventricular posterior wall, diastolic. FS=fractional shortening. All values are mean ± SEM

*P < 0.01 was obtained for the WT-sham values

**P < 0.01 was obtained for the WT-AB values after AB

Table 2

Echocardiographic data showed the effects of CTSL on cardiac hypertrophy induced by Ang II infusion


WT-Saline mice

TG-Saline mice

WT-Ang II mice

TG-Ang II mice


n = 12

n = 10

n = 8

n = 12

BW, g

24.9 ± 1.0

25.1 ± 1.2

26.4 ± 1.2

26.1 ± 1.4

HW/BW (mg/g)

4.77 ± 0.21

4.79 ± 0.14

6.13 ± 0.11*

4.93 ± 0.23§

LW/BW (mg/g)

4.79 ± 0.13

4.72 ± 0.11

5.45 ± 0.21*

4.81 ± 0.18§

CSA (μm2)

256 ± 28

259 ± 31

394 ± 27*

287 ± 32§

SBP, mmHg

115.2 ± 4.3

117.4 ± 3.1

143.2 ± 3.1*

141.2 ± 5.1*

HR, beats/min

456 ± 24

459 ± 20

471 ± 21

468 ± 34

PWT (mm)

1.20 ± 0.04

1.22 ± 0.03

2.61 ± 0. 04*

1.63 ± 0.03§

LVEDD (mm)

3.62 ± 0.04

3.64 ± 0.05

4.79 ± 0.04*

3.83 ± 0.02§

LVESD (mm)

2.43 ± 0.04

2.42 ± 0.04

3.53 ± 0.03*

2.55 ± 0.04§

IVSd (mm)

0.63 ± 0.04

0.65 ± 0.05

1.38 ± 0.05*

0.83 ± 0.06§

LVPWd (mm)

0.62 ± 0.06

0.64 ± 0.03

1.35 ± 0.06*

0.83 ± 0.05§

FS (%)

55.4 ± 3.4

54.5 ± 2.1

38.5 ± 1.3*

45.7 ± 1.9*§

*P < 0.01 was obtained for the WT-Saline values

**P < 0.01 was obtained for the WT-Ang II infusion values after Ang II infusion

CTSL attenuates hypertrophy in cultured cardiomyocytes

The results observed in response to AB or Ang II stimulation were extended using an in vitro model of cellular growth in rat neonatal cardiac myocytes cultured under serum-free conditions. At the beginning, we screened three shCTSL and found that no. 1 shCTSL markedly inhibited CTSL expression in cardiac myocytes (Fig. 3A). Therefore, no. 1 shCTSL was chosen for the following experiments. Cardiac myocytes were infected with Ad-CTSL, or Ad-GFP as a control, and Ad-shCTSL or Ad-shRNA, and allowed to incubate for 24 h before stimulation with the combination of Ang II (1 µM). CTSL overexpression significantly reduced the increase [3H]-Leucine incorporation and cell surface area induced by Ang II treatment compared with Ad-GFP infection, whereas inhibition of CTSL promoted these effects of Ang II (Fig. 3B). Western blot further confirmed that CTSL expression markedly reduced ANP, BNP, and β-MHC protein expression levels induced by Ang II (Fig. 3C). These findings indicate that CTSL expression inhibits cardiac hypertrophy in vitro.
Fig. 3

The effects of CTSL on cardiac hypertrophy in vitro. A The protein expression level of CTSL after infection with Ad-CTSL or Ad-shCTSL. Upper, quantitative results. Bottom representative blots. Values are mean ± SEM. *P < 0.01 for difference from Ad-GFP group values. B The effects of CTSL on Ang II-induced [3H]-leucine incorporation and cell area enlargement. C CTSL blunted Ang II-induced ANP, BNP, and β-MHC protein expression levels. Cardiomyocytes were infected with Ad-CTSL or Ad-shCTSL for 24 and then incubated with 1 µM Ang II for indicated time. [3H]-Leucine incorporation and Western blot were measured as described under the “Materials and Methods” section. The results were reproducible in three separate experiments. *P < 0.01 vs exposed to control. (raw data of the Western blots can be found as electronic supplementary material)

Forced CTSL expression attenuated Akt/GSK3β signaling

Mitogen-activated protein kinase (MAPK) pathways are activated by a variety of hypertrophic stresses, including neurohormonal stimuli and hemodynamic overload, and play an important role in hypertrophy [15]. Thus, the activation of the MAPK pathway was investigated in our two hypertrophic models. We found that the phosphorylated levels of p38, JNK1/2, and ERK1/2 were significantly increased by both AB and Ang II-infusion in WT hearts. However, these phosphorylation were not significantly affected in TG hearts (data not shown). Akt/GSK3β signaling also plays a crucial role in the regulation of cardiac remodeling and apoptosis. We also examined Akt/GSK3β activation between WT and TG mice. Our results demonstrated that CTSL expression markedly blocked Akt and GSK3β phosphorylation induced by AB or Ang II infusion (Fig. 4A). Levels of phospho-mammalian target of rapamycin (mTOR), forkhead box O3A (FOXO3A) and forkhead box O1 (FOXO1) were also reduced in CTSL transgenic mice, although total levels of these kinases were unchanged, which is consistent with downregulation of the Akt pathway (Fig. 4A), whereas levels of phospho-protein kinase A (PKA), protein kinase C (PKC), and integrin-linked kinase (ILK) were unchanged (data not shown). PI3K is an upstream activator of the Akt/GSK3β pathway and the phosphatase, and tensin homolog (PTEN) opposes PI3K-mediated activation of the pathway. However, no change was seen in the phosphorylation status of the p85 subunit of PI3K and PTEN activation (data not shown). Collectively, these data suggest that forced expression of CTSL suppresses the activation of Akt/GSK3β, although it has no effects on p38, JNK1/2, and ERK1/2 activation as well as PTEN/PI3K and ILK activation in hearts subjected to AB or Ang II stimulation. In vitro studies also demonstrated that Akt and GSK3β phosphorylation levels were enhanced following the reduction of CTSL expression by infection with Ad-shCTSL in response to hypertrophic stimuli (Fig. 4B). In contrast, Akt and GSK3β phosphorylation were almost completely blocked by increased CTSL expression (Fig. 4B). These findings suggest that Akt/GSK3β signaling is critical to the influence of CTSL on cardiac hypertrophy.
Fig. 4

The effect of CTSL on AKT/GSK3β signaling pathway. A Representative blots of AKT, GSK3β, mTOR, FOXO3A, and FOXO1 phosphorylation and their total protein expression at 8 weeks post-AB surgery or at 4 weeks post-Ang II infusion in WT and TG mice. B Representative blots of AKT and GSK3β phosphorylation and their total protein expression after treated with Ang II for indicated time in different adenovirus-infected primary cardiac myocytes. The results were reproducible in three separate experiments (raw data of the Western blots can be found as electronic supplementary material)

Forced CTSL expression attenuates fibrosis in vivo

Heart sections were stained with PSR to detect fibrosis. In both groups, collagen continued to accumulate in the heart after 8 weeks of AB and 4 weeks of Ang II stimulation. As shown in Fig. 5A and B, increased collagen deposition was observed in WT mice, but this was markedly reduced in TG mice. Quantitative analysis also showed reduced collagen volume in the myocardium of TG mice compared to WT mice (Fig. 5C). Reduced fibrosis in TG mice may represent increased collagen degradation or decreased collagen synthesis in response to tissue damage. We therefore examined the synthesis of collagen by examining the expressions of mRNA and protein of CTGF, collagen I, collagen III, and TGF-β1, known to be involved in the proliferation of cardiac fibroblasts and the biosynthesis of ECM proteins. The results showed that both mRNA and protein expressions of CTGF, collagen I, collagen III, and TGF-β1 were significantly lower in TG mice than that in WT mice in response to hypertrophic stimuli (Fig. 5D–F).
Fig. 5

The effects of CTSL on fibrosis in vivo. A and B PSR staining on histological sections of the LV was performed on indicated groups 8 weeks after AB or 4 weeks after Ang II infusion. C Fibrotic areas from histological sections were quantified using an image-analyzing system. (D and E) Real-time PCR analyses of Tgfβ1, Col1α1, Col3α1, Ctgf were performed to determine mRNA expression levels in indicated groups. GAPDH was used as the sample loading control. *P< 0.05 was obtained for the WT/sham or WT/saline values. Data represent typical results of three different experiments as mean ± SEM (n = 4 to 6 mice/per group). F Western blot analysis of TGFβ1, Collagen I, Collagen III, and CTGF protein expression of the myocardium was obtained from indicated animals (n = 4)

CTSL expression inhibits apoptosis and inflammatory response induced by aortic banding

CTSL has been shown to promote cell survival by protection against apoptosis. We therefore performed TUNEL assays on heart sections from TG and WT mice after 8 weeks of AB. Apoptotic cells were detected in TG and control mice, and the fraction of apoptotic versus total cells was significantly lower in TG mice than in WT mice (Fig. 6A). To determine whether TG mice are resistant to apoptotic signals, we examined the activity and cleavage of caspase-3/8/9. As expected, TG mice displayed a significant decrease of the activity and delay of cleavage of caspase-3/8/9 in response to AB (Fig. 6A and B). Thus, the inhibitory effect of CTSL on cardiomyocyte apoptosis is primarily due to the inhibition of apoptotic signals. Further study also revealed that CTSL expression reversed the increased Bax, Bad, Bik and Nip3, and the decreased Bcl-2 and Bcl-XL proteins expression in response to chronic pressure overload (Fig. 6C). To determine whether expression of CTSL prevents the inflammatory responses in the hearts, cytokine induction was characterized by Western blot analyses. Compared with WT mice, TG mice had significantly lower TNF-α, IL-6, and MCP-1 protein levels in cardiac tissue after 8 weeks AB (Fig. 6D). To determine the molecular mechanisms by which CTSL attenuated cytokine induction in vivo, we analyzed NF-κB signaling pathways. NF-κB DNA-binding acivity, IκBα phosphorylation, and IκBα degradation were clearly detected after 8 weeks AB in WT mice, but evidently blocked in TG mice (Fig. 6D and E).
Fig. 6

The effects of CTSL on apoptosis and inflammatory response. A The results of quantitative analysis on TUNEL assay and caspase-3/8/9 activities. *P < 0.01 was obtained for the WT/sham. B The effects of CTSL on caspase-3/8/9 cleavages in response to chronic pressure overload. C Western blot analysis of Bax, Bad, Bik, Nip3, Bcl-2, and Bcl-XL protein expression in the myocardium obtained from indicated groups. D Western blot analysis of TNF-α, IL-6, and MCP-1 protein expression as well as IκBα degradation and IKKβ and IκBα phosphorylation in the myocardium obtained from indicated groups. E EMSA assay of NF-κB DNA binding activity from indicated groups


Our current study demonstrates that the transgeneic overexpression of CTSL in the heart protects against cardiac hypertrophy. The cardioprotection of CTSL is mediated by interruption of AKT/GSK3β-dependent signaling pathways, which protects the host from the combined deleterious effects of cardiac hypertrophy, apoptosis, inflammation, and fibrosis. The ability of CTSL to prevent cardiac dysfunction and hypertrophy mediated by sustained pressure overload and Ang II-infusion suggests that CTSL may be an effective therapeutic candidate.

The underlying mechanism for the inhibitory effects of CTSL on cardiac hypertrophy remained unknown. It has been reported that numerous hypertrophic stimuli induce adaptive hypertrophic growth in the heart that temporarily augments cardiac function [16]. Although this initial hypertrophic response may be beneficial, sustained hypertrophy often leads to heart failure, which is characterized by progressive deterioration in cardiac function [16]. The signaling mechanisms leading to cardiac hypertrophy have been extensively investigated in the past decade. It is important to dissect the pathways counteracting these pro-hypertrophic signaling mechanisms and thereby alleviating the hypertrophic responses. The role of the PI3-kinase/Akt pathway in cardiac hypertrophy is well established [17]. Targeted overexpression of constitutively active PI3-kinase in the heart increased the organ size while expression of a dominant-negative mutant has the opposite outcome [17]. The hypertrophic response following PI3-kinase activation is apparently associated with PI3-kinase downstream Akt, since activation of Akt in the heart lead to increased cardiac hypertrophy and heart failure [18]. Akt downstream target Glycogen synthase kinase 3β (GSK3β) is the first negative regulator of cardiac hypertrophy. Increasing evidence showed that this kinase blocks cardiomyocyte hypertrophy in response to endotelin 1, Ang II, phenylephrine, and isoproterenol [19]. AKT signaling pathway target mTOR appears critical for cardiac hypertrophy and also promotes the transition to heart failure during chronic pressure overload. AKT can increase the activation of mTOR indirectly by reducing tuberin activity, and AKT is also a direct target of mTOR kinase activity [20]. Activation of mTOR and its downstream targets results in increased cell size and is associated with cardiac hypertrophy. Furthermore, overexpression of p70S6 kinase results in cardiac hypertrophy, whereas inhibition of mTOR signaling with rapamycin attenuates the development of ventricular hypertrophy in mice exposed to pressure overload [21]. An important finding of the present study is that the increase in AKT, GSK3β, mTOR, and 70S6K phosphorylation levels in response to hypertrophic stimuli was largely blocked in TG mice. The phosphorylations of MAPKs, PTEN, and PKC in myocytes were not affected by CTSL expression. Further in vitro studies showed that inhibition of CTSL expression significantly enhanced the activation of Akt and GSK3β, but not that of p38, ERK1/2, and JNKs. To our knowledge, this is the first time to report that CTSL regulates Akt/GSK3β signaling pathway. These findings indicate that CTSL specifically downregulates AKT/GSK3β-dependent signaling pathway.

Cardiac fibrosis is another classical feature of pathological hypertrophy, which is characterized by the accumulation of collagen [22]. It is important to understand the mechanisms that stimulate collagen deposition in the heart, and find ways to inhibit such processes. We found that CTSL blocks cardiac fibrosis induced by chronic pressure overload and Ang II stimulation. The finding is consistent with a recent report that CTSL-deficient mice develop increased interstitial fibrosis. Cardiac myocyte apoptosis plays an important role in the transition of cardiac hypertrophy to heart failure [23]. Our results showed a correlation between an increase in the frequency of apoptosis and the extent of cardiac remodeling. Consistent with previous reports, CTSL expression in the heart markedly decreases the number of apoptotic cells in response to long-term pressure overload. In addition, forced expression of CTSL attenuates myocardial apoptosis and is associated with abrogated cleavages and activities of caspase-3/8. The effects of CTSL on these signaling molecules may explain the protection from apoptosis observed in transgenic hearts subjected to AB. In addition to apoptosis, there evidence that proinflammatory cytokines play a role in pathological cardiac hypertrophy and heart failure [24, 25]. We found a marked induction of cytokine expression in the heart in response to hypertrophic stimuli that was observably attenuated by cardiac forced expression of CTSL. One possible mechanism for such a protective effect is that CTSL expression blocks NF-κB activation, resulting in attenuation of the inflammatory response and subsequent myocardial hypertrophy. These findings suggest that CTSL abrogates NF-κB activation by phosphorylation and degradation of IκBα. By blocking NF-κB signaling, CTSL may inhibit the early steps of inflammation and modulate the amplification of multiple cytokine signaling cascades. Treatment that specifically targets upstream NF-κB activation may be a conceptually superior approach to blocking each respective cytokine individually.

In summary, the present work demonstrates that CTSL protects against cardiac remodeling and heart failure in response to hypertrophic stimuli. The mechanism underlying the protective effects of CTSL appears to involve the inhibition of Akt/GSK3β signaling pathway. The potential for CTSL as a therapeutic target should be considered in future studies.

Sources of funding

National Natural Science Foundation of China (30771193), Research Fund for the Doctoral Program of Higher Education of China (no. 20050025002) and Beijing Natural Science Foundation (5072007) to Wei Wang. National Natural Science Foundation of China (30670216), Research Fund for the Doctoral Program of Higher Education of China (no. 2007042086103) to Qizhu Tang.

Conflict of interests

None declared.

Supplementary material

109_2008_423_MOESM1_ESM.pdf (1.3 mb)
ESM Fig. 1 (PDF 1.31 MB)

Copyright information

© Springer-Verlag 2008