Cell Type-specific Functions of the Lysosomal Protease Cathepsin L in the Heart*

Deficiency of the lysosomal cysteine protease cathepsin L (Ctsl) in mice results in a phenotype affecting multiple tissues, including thymus, epidermis, and hair follicles, and in the heart develops as a progressive dilated cardiomyopathy (DCM). To understand the role of Ctsl in the maintenance of regular heart morphology and function, it is critical to determine whether the DCM in Ctsl-/- mice is primarily because of the lack of Ctsl expression and activity in the cardiomyocytes or is caused by the additional extracardiac pathologies. Cardiomyocyte-specific expression of Ctsl in Ctsl-/- mice, using an α-myosin heavy chain promoter-Ctsl transgene, results in improved cardiac contraction, normal mRNA expression of atrionatriuretic peptide, normal heart weight, and regular ultrastructure of cardiomyocytes. Epithelial expression of cathepsin L2 (CTSL2) by a K14 promoter-CTSL2-transgene resulted in rescue of the Ctsl-/- hair loss phenotype. In these mice, cardiac atrionatriuretic peptide expression and end systolic heart dimensions were also significantly attenuated. However, cardiac contraction was not improved, and increased heart weight as well as the typical changes in lysosomal ultrastructure of Ctsl-/- hearts persisted. Myocardial fibrosis was detected in all Ctsl-/- mice irrespective of transgene-mediated cardiac Ctsl expression or extracardiac CTSL2 expression. Expression of collagen 1 was not enhanced in Ctsl-/- hearts, but a reduced collagenolytic activity suggests a role for Ctsl in collagen turnover by cardiac fibroblasts. We conclude that the DCM of Ctsl-/- mice is primarily caused by absence of the protease in cardiomyocytes, whereas the complex gross phenotype of Ctsl-deficient mice, i.e. the fur defect, results in additional stress to the heart.

Deficiency of the lysosomal cysteine protease cathepsin L (Ctsl) in mice results in a phenotype affecting multiple tissues, including thymus, epidermis, and hair follicles, and in the heart develops as a progressive dilated cardiomyopathy (DCM). To understand the role of Ctsl in the maintenance of regular heart morphology and function, it is critical to determine whether the DCM in Ctsl ؊/؊ mice is primarily because of the lack of Ctsl expression and activity in the cardiomyocytes or is caused by the additional extracardiac pathologies. Cardiomyocyte-specific expression of Ctsl in Ctsl ؊/؊ mice, using an ␣-myosin heavy chain promoter-Ctsl transgene, results in improved cardiac contraction, normal mRNA expression of atrionatriuretic peptide, normal heart weight, and regular ultrastructure of cardiomyocytes. Epithelial expression of cathepsin L2 (CTSL2) by a K14 promoter-CTSL2-transgene resulted in rescue of the Ctsl ؊/؊ hair loss phenotype. In these mice, cardiac atrionatriuretic peptide expression and end systolic heart dimensions were also significantly attenuated. However, cardiac contraction was not improved, and increased heart weight as well as the typical changes in lysosomal ultrastructure of Ctsl ؊/؊ hearts persisted. Myocardial fibrosis was detected in all Ctsl ؊/؊ mice irrespective of transgene-mediated cardiac Ctsl expression or extracardiac CTSL2 expression. Expression of collagen 1 was not enhanced in Ctsl ؊/؊ hearts, but a reduced collagenolytic activity suggests a role for Ctsl in collagen turnover by cardiac fibroblasts. We conclude that the DCM of Ctsl ؊/؊ mice is primarily caused by absence of the protease in cardiomyocytes, whereas the complex gross phenotype of Ctsl-deficient mice, i.e. the fur defect, results in additional stress to the heart.
Cardiomyopathies represent a heterogeneous group of heart diseases characterized by progressive myocardial remodeling that results in impaired pump function of the heart (1). Among other etiologic factors, abnormality of lysosomes and lysosomal hydrolases have been shown to cause myocardial heart disease. Thus, cardiomyopathies have been described in hereditary deficiencies of lysosomal glycosidases, like in mucopolysaccharidoses and glycogenoses (2,3). Deficiency of LAMP-2 (lysosomeassociated membrane protein-2) has been shown to induce accumulation of autophagic vacuoles and to cause Danon disease, leading to severe myopathy of cardiac and skeletal muscles (4,5). Furthermore, LAMP-2-deficient mice manifest a Danon disease-like vacuolar cardioskeletal myopathy (6). In hearts from cases of dilated cardiomyopathy (DCM), 2 increased activity of lysosomal enzymes was found (7), and autophagic degeneration was proposed to be an important mechanism (8). Among the many lysosomal protease types, "cathepsins" are expressed in the heart at considerable levels, although their specific roles in maintaining the cardiac form-function relationship have not been defined yet.
The human lysosomal cysteine cathepsins represent a family of 11 papain-like proteolytic enzymes (clan CA, family C1) with a principal subcellular localization in the endosomal/lysosomal compartment. Seven of these peptidases, the cathepsins B, C, F, H, L, O, and X/Z, exhibit ubiquitous but nevertheless differential expression in mammalian tissues. Other papain-like cysteine peptidases are known to exhibit cell type-specific expression, e.g. cathepsin K is mainly found in osteoclasts and cathepsin S is predominantly expressed in peripheral antigenpresenting cells (9,10). Traditionally, lysosomal cysteine peptidases are considered to execute unspecific bulk proteolysis inside the lysosome, which supports a homeostatic function of these enzymes (11). However, there is growing evidence for specific intra-and extracellular functions of these papain-like enzymes (12,13). For their extracellular actions, lysosomal peptidases are secreted in considerable amounts (14 -16). More recently, functions of cysteine cathepsins have been identified in the cytoplasm, the nucleus, and even in the mitochondrion (17)(18)(19)(20)(21).
Interestingly, it was shown that 1-year-old cathepsin L (Ctsl) knock-out mice develop heart disease that resembles many features of human dilated cardiomyopathy (22). Complete deficiency of Ctsl in these mice 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 at 12 months of age. Recently it was confirmed that deficiency of Ctsl in the heart primarily affects the lysosomal system, particularly by increasing the number and changing the morphology of acidic organelles, although without the accumulation of specific lysosomal storage materials (23). Furthermore, 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 (23). Interestingly, the skeletal muscles of Ctsl Ϫ/Ϫ mice are not pathologically altered. However, Ctsl-deficient mice develop spontaneous phenotypes in addition to DCM. Most prominent are periodic hair loss and epidermal thickening (24,25). It has been shown that these phenotypes are caused by a critical role of Ctsl in endosomal/lysosomal termination of growth factor signaling in keratinocytes (26). Furthermore, Ctsl-deficient mice show a reduced number of CD4 ϩ -T-cells because of a reduced positive selection in the thymus and decreased volume of trabecular bones (27)(28)(29).
Because of this complex phenotype of Ctsl knock-out mice, we set out to investigate whether their cardiomyopathy is primarily caused by defective Ctsl activity in the myocardium or secondary to the other extracardiac phenotypes. Here we address this question by transgenic expression of murine Ctsl in the heart of Ctsl Ϫ/Ϫ mice and by transgenic expression of human CTSL2 (the ortholog of mouse Ctsl) in keratin 14-expressing epithelia of Ctsl Ϫ/Ϫ mice.
Morphometry, Histology, and Immunohistochemistry-The body and heart weights were determined by weighing the body immediately after death and the heart after removal of both atria. The myocardium was washed in phosphate-buffered saline and fixed in 4% buffered formalin for 24 h. The fixed hearts were embedded in paraffin, and 7-m thick serial sections were cut, deparaffinized, and rehydrated, and extracellular matrix glucosaminoglycans were stained with Mowry staining (32). For immunohistochemistry of back skin, sections of 5-m thickness were deparaffinized and rehydrated. Mouse anti-human CTSL2 antibody (R & D Systems; 1:2000 dilution at 4°C for 16 h) was used for the detection of CTSL2. Detection of the primary antibody was performed using the EnVision TM kit (Dako Cytomation) according to the manufacturer's instructions.
High Resolution Light Microscopy and Transmission Electron Microscopy-The hearts were removed and immediately fixed in half-strength Karnovsky's fixative as 3-mm 3 tissue cubes. The tissues were postfixed in 2% osmium tetroxide and embedded in araldite resin. Semithin sections were stained with toluidine blue/borax, examined by light microscopy, and photographed (Leitz). Ultrathin sections were stained with uranyl acetate and lead citrate, examined, and photographed using a Jeol 1200EX transmission electron microscope (Jeol, Japan). Multiple blocks were examined from each heart, and 3-mm long and 1-m thick sections were examined by light microscopy from each block. These tissue blocks were further examined by transmission electron microscopy using ultrathin sections.
Measurement of Proteolytic Activities-Myocardial tissue was Dounce-homogenized in 150 mM NaCl, 50 mM Tris, 5 mM CaCl 2 (pH 7.6), and postnuclear supernatants (PNS) were obtained by centrifugation at 1000 ϫ g for 10 min. Assay for DQ-Collagen and DQ-Gelatin (both from Invitrogen) degradation in the PNS was according to the manufacturer's protocol. Organelles were isolated by centrifuging the PNS for 20 min at 17,000 ϫ g and resuspension of the organelle pellet in 100 mM sodium acetate, 1 mM EDTA, 0.05% Brij 35 (pH 5.5). Ctsl proteolytic activity was determined by hydrolysis of the fluorogenic dipeptide benzyloxycarbonyl-Phe-Arg-4-methylcoumarin-7amide (25 M; Bachem) in the presence of the CTSB-specific inhibitor CA-074 (150 nM; Bachem). The release of 7-amino-4methylcoumarin was continuously monitored for 20 min by spectrofluorometry at excitation and emission wavelengths of 370 and 460 nm, respectively.
Labeling of Cysteine Protease-active Sites with DCG-04-DCG-04 binds covalently to the active site of cysteine cathepsins and contains a biotin tag (33). 10 g of protein from heart organelles was incubated with DCG-04 (10 M; courtesy of M. Bogyo, University of California, San Francisco) for 1 h at room temperature. Subsequently, the lysates were diluted in SDS sample buffer and boiled for 10 min at 95°C. Samples were subjected to SDS-PAGE (15% gel) and blotted on Hybond-Ppolyvinylidene difluoride membrane (GE Healthcare). The membrane was incubated with streptavidin-peroxidase complex for 2 h, and protease-bound DCG-04 was visualized by the SuperSignal TM chemiluminescent substrate (Pierce).
Echocardiography and Cardiac Doppler Examination-Echocardiographic examination was performed under inhalation anesthesia with 0.8 -1.0% isoflurane. Transthoracic Doppler echocardiography was performed with a digital cardiac ultrasound machine equipped with a 15-MHz linear phased-array transducer and a 12-MHz short focal length-phased array transducer (SONOS 5500, C1 software package, Philips Medical Systems, The Netherlands). Both the parasternal long axis and short axis views were obtained. M-mode and Doppler recordings were performed at a sweep speed of 150 mm/s. Left ventricular septal and posterior wall thickness at the end of diastole as well as end diastolic and end systolic dimensions of the left ventricle were measured using leading edge to leading edge rule with the electronic caliper in M-mode (34).
The percentage of fractional shortening and ejection fraction was calculated with conversion formulas as described before (35). For determination of systolic outflow of the left ventricle and pulsed wave, Doppler signals were obtained by placing the sample volume parallel to flow during long axis view into the left ventricular outflow tract and the ascending aorta. Diastolic inflow was detected apical to the mitral valve within the left ventricle (35).
Statistics-All data are reported as arithmetic mean Ϯ S.D. Statistical analysis was performed by using the U test of Mann and Whitney. p values Յ0.05 were considered as statistically significant.
Interstitial Fibrosis-Interestingly, interstitial fibrosis was not markedly improved by cardiac or extracardiac expression of Ctsl. However, the transcription of collagen I, which represents the major fibrotic collagen in the myocardium, was not increased in Ctsl Ϫ/Ϫ hearts (Fig. 6A). Thus to address this, we decided to measure gelatinolytic and collagenolytic activities in the myocardial homogenates by degradation of fluorescencequenched DQ-gelatin and DQ-collagen to assess whether fibrosis could be caused by impaired turnover of extracellular matrix proteins (Fig. 6B). When compared with wild-type mice, the proteolytic cleavage of both substrates was significantly reduced in Ctsl Ϫ/Ϫ hearts and in hearts of Tg(K14-CTSL2); Ctsl Ϫ/Ϫ mice. Interestingly, transgenic re-expression of Ctsl in cardiomyocytes normalized the cleavage of DQ-gelatin, but degradation of DQ-collagen remained at the level of Ctsl Ϫ/Ϫ hearts. Thus, the fibrosis observed in Ctsl-deficient hearts might be caused by defects of collagen turnover in cardiac fibroblasts.

DISCUSSION
Ctsl is known as a highly potent endoprotease of the cysteine cathepsin family. It is ubiquitously expressed; however, Ctsl expression levels vary considerably among different cell types. Furthermore, Ctsl executes critical functions in specific cell and tissue types as has been revealed by analysis of Ctsl-deficient mouse models (12,24,37). In addition to the cardiomyopathy addressed here (22,23), a delayed and reduced tumor development in the Rip1-Tag2 model for pancreatic islet cell carcinomas has been detected in Ctsl-deficient mice (38). Ctsl contributes to maturation and release of enkephalin and thyroid hormone (39,40). Important cell type-specific functions of Ctsl include antigen presentation and maturation of major histocompatibility class II complexes in thymic cortex epithelial cells (27,41), and periodic hair loss as well as epidermal hyperproliferation and thickening (24,25). The most convincing evidence for cell autonomous functions of Ctsl has been provided by the rescue of the immune and skin phenotypes by transgenic mice with specific re-expression of Ctsl in thymic cortex epithelial cells and keratinocytes, respectively (26, 31).  Here we determined whether the cardiomyopathy in Ctsldeficient is primarily caused by the cathepsin defect in cardiomyocytes is secondary to the other phenotypes. Cardiomyocyte-specific expression of murine Ctsl by the ␣MHC promoter in Ctsl Ϫ/Ϫ mice results in improvements of all parameters of heart ultrastructure and heart function with the notable exception of myocardial fibrosis ( Table 1). Expression of CTSL2 (the human ortholog of Ctsl with about 75% amino acid identity) under control of the human cytokeratin 14 (K14) promoter in Ctsl Ϫ/Ϫ mice rescues the skin phenotype (i.e. periodic hair loss and epidermal thickening) and normalizes of CD4-T-cell counts (31). Here we show that the K14-CTSL2 transgene is not expressed in the heart. Interestingly, K14-CTSL2 expression also results in a significant improvement of left ventricular end systolic diameters in Ctsl Ϫ/Ϫ mice. In support of the more favorable heart dimensions is the significantly reduced mRNA expression of ANP, a marker for sheer stress in the myocardial wall (42)(43)(44). However, myocardial contraction/fractional shortening and ultrastructure are not normalized in the Tg(K14-CTSL2);Ctsl Ϫ/Ϫ mice. Thus, metabolic stress exerted by heat loss because of the hair loss phenotype of Ctsl Ϫ/Ϫ mice may support cardiomyopathy development but is apparently not causing the initiation of cardiac pathogenesis. We have previously reported an impairment of cardiomyocyte ultrastructure in Ctsl Ϫ/Ϫ hearts in newborn mice as the earliest detectable pathogenic event in development of cardiomyopathy in Ctsldeficient mice (23). Specifically an increased number and size of "acidic" vesicles of the endosomal/lysosomal compartment has been observed. Similar observations have been made in keratinocytes, which are the critical cells for the Ctsl Ϫ/Ϫ skin phenotype (25,26). This suggests an alteration of the endosomal/ lysosomal compartment by Ctsl deficiency as a common cause of the prominent skin and heart phenotypes of Ctsl-deficient mice. In the case of Ctsl Ϫ/Ϫ keratinocytes it has been shown that an imbalance of degradation and recycling of growth factors (e.g., epidermal growth factor) causes a sustained mitogenic signaling and, hence, hyperproliferation in the epidermis and hair follicles of affected mice (25,26). Extensive proteome comparison of Ctsl-deficient and wild-type hearts revealed decreased levels of the sarcomere-associated proteins ␣-tropomyosin, desmin, and calsarcin 1, as well as changes in levels of metabolic enzymes and components of the vesicular transport system (23). Bioinformatics pathway analysis suggested a switch to anaerobic catabolism and the impairment of mitochondrial respiration. This interpretation was supported by a 50% reduction in resting state oxygen consumption and impaired respiration capacity in Ctsl Ϫ/Ϫ myocardial homogenates (23). Thus, Ctsl deficiency in hearts results in metabolic and sarcomeric alterations that promote DCM development.
Strikingly the Ctsl Ϫ/Ϫ -associated interstitial fibrosis is not rescued when expression of Ctsl activity is returned to cardiomyocytes (by the ␣MHC-Ctsl transgene) or to keratinocytes and thymic cortex epithelium (by the K14-CTSL2 transgene). Thus, fibrosis appears to be independent of the specific Ctsl functions in cardiomyocytes, epidermis, hair follicles, and the immune system. Fibrosis is an important pathologic hallmark of cardiomyopathies resulting in conduction alterations and cardiac valve defects (1,45,46). Both arrhythmias and valve insufficien-cies have been detected in Ctsl Ϫ/Ϫ hearts (22). Because we show that collagen 1 expression is not enhanced in Ctsl-deficient myocardium, it is most likely that accumulation of collagen in the extracellular matrix is because of a defective collagen turnover. Although Ctsl is mainly located in the endosomal/lysosomal compartment, about 10% of the zymogen is physiologically secreted, can be extracellularly activated, and has been reported to process extracellular matrix proteins such as fibronectin, laminin, and type I, IV, and XVIII collagen (47)(48)(49). Supporting this hypothesis is the marked decrease in collagenolytic and gelatinolytic activities in the affected myocardium (Fig. 6B). Interestingly, transgenic re-expression of Ctsl in cardiomyocytes normalized the cleavage of DQ-gelatin, but degradation of DQ-collagen remained at the level of Ctsl Ϫ/Ϫ hearts (Fig. 6B). We interpret this finding to mean that DQ-gelatin, which presents a highly denatured collagen preparation, is readily cleaved by a number of potent endoproteases, with Ctsl being a major representative. In contrast, DQ-collagen is a more complex native collagen preparation requiring the action of specific collagenases. Based on our present genetic studies, these collagenases are likely to be activated either directly or indirectly (i.e. by inhibitor degradation) by Ctsl derived from the cardiac fibroblasts. These in vivo results are in line with the recent observation that calvarial bone osteoclasts do not use matrix metalloproteinases for bone matrix resorption in the absence of Ctsl (29).
In summary, Ctsl exerts multiple cell type-specific functions in the heart. Its deficiency causes structural and functional alterations within cardiomyocytes and affects the collagen turnover of cardiac fibroblasts. In addition, the complex gross phenotype of Ctsl-deficient mice, i.e. the fur defect, results in additional stress for the heart. Taken together, pathogenic cardiac remodeling is initiated that leads to manifest cardiac dysfunction in 1-year-old mice.