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* Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany;
Medizinische Klinik und Poliklinik C (Kardiologie und Angiologie), Zentrale Projektgruppe Kleintierdiagnostik des Interdisziplinären Zentrums für Klinische Forschung Münster, Universitätsklinikum Westfälische Wilhelms-Universität Münster, Münster, Germany;
Medical Biosciences, School of Life Sciences, University of Bradford, Bradford, West Yorkshire, UK;
Lehrstuhl für Bioinformatik, Biozentrum, Am Hubland, Würzburg, Germany;
|| Research Institute for Environmental Medicine at the Heinrich Heine University Duesseldorf, Molecular Aging Research, Duesseldorf, Germany; and
¶ Institut für Klinische Chemie und Pathologische Biochemie, Bereich Pathologische Biochemie, Medizinische Fakultät der Otto-von-Guericke-Universität Magdeburg, Germany
2Correspondence: Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität Freiburg, Freiburg D-79104, Germany. E-mail: christoph.peters{at}mol-med.uni.freiburg-de
SPECIFIC AIM
Cathepsin L (CTSL) belongs to the papain-like family of lysosomal cysteine proteases. Unlike other cathepsin-deficient animals, CTSL knockout mice show a marked dilated cardiomyopathy (DCM) at 1 yr of age. The present study was initiated to investigate the pathogenic sequence that occurs in the heart of ctsl–/– mice. Specifically, we aimed to establish the time course of disease progression and to identify the cell biological processes that are altered by CTSL deficiency in the myocardium.
PRINCIPAL FINDINGS
1. Morphology and functional impairment of CTSL-deficient hearts
At 2 mo of age, hearts of CTSL knockout mice show no gross morphological alteration ex vivo and in vivo (Fig. 1
A, B). However, at 1 yr of age ctsl–/– mice show increased left-ventricular diameters by echocardiographic assessment (Fig. 1B
). Approximately 25% of the hearts will develop pronounced dilation of the left and right ventricles with only little hypertrophy at 12 mo of age (arrow, Fig. 1C
). The percentage of fractional shortening is an echocardiographic measure of myocardial contraction. At 2 months of age, ctsl–/– mice already show a small but significant impairment of fractional shortening (Fig. 1D
). This functional impairment progressed during aging reaching a 50% reduced contraction of hearts from 1-yr-old CTSL knockout mice (Fig. 1D
), even in absence of extreme dilation (arrow, Fig. 1C
). Transcriptional activation of the natriuretic peptides ANP and BNP in left-ventricular myocardium reflects an increased tension in heart walls. Consistent with the slightly reduced fractional shortening (Fig. 1D
), CTSL-deficient mice already show increased mRNA levels of ANP and BNP in ventricular myocardium of 2-month-old mice as compared with age matched controls (Fig. 1E
)
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2. Fibrosis and ultrastructural alterations in CTSL-deficient myocardium
Development of extensive interstitial fibrosis is a hallmark of cardiac remodeling in the pathogenesis of cardiomyopathies. We used a grading system on Masson’s Trichrome stained ventricular sections for semiquantitative assessment of age-dependent fibrosis in the hearts of ctsl+/+ and ctsl–/– mice. On aging, the wild-type mice show a slight increase in myocardial fibrosis. CTSL-deficient hearts do not reveal increased fibrosis at 2 months of age as compared with the wild type. However, starting at an age of 
4 months, ctsl–/– hearts develop increased fibrosis that can be severe, i.e., grade 4, at 1 yr of age. In contrast to fibrosis that appears relatively late in the pathogenesis of the cardiomyopathy, an aberrant myocardial ultrastructure, characterized in part by abnormal accumulations of enlarged lysosomes, is already observed in hearts of 3-day-old ctsl–/– animals. By 2 months of age, ctsl+/+ hearts showed normal tissue ultrastructure, with no vacuolation or accumulations of lysosomes apparent, whereas ctsl–/– hearts revealed extensive vacuolation, numerous end-stage lysosomes/residual bodies, and pleomorphic nuclei. Identical ultrastructural findings were observed in hearts of 12-month-old CTSL knockout mice (data not shown). Interestingly, these alterations appeared to be specific for the myocardium of ctsl–/– mice, since skeletal muscle of 1-yr-old wild-type and CTSL-deficient mice exhibited identical and normal ultrastructures.
3. Alterations of the acidic cellular compartment of CTSL-deficient myocardium
Labeling of primary cardiomyocytes isolated from newborn ctsl+/+ and ctsl–/– mice with an acidophilic dye revealed strong staining in the cardiomyocytes of both genotypes compared with "contaminating" cardiac fibroblasts also present in these cell cultures. These findings were further supported by sucrose-density fractionation of myocardial postnuclear supernatants, with identical total protein content for both genotypes. The activity of the lysosomal/endosomal enzyme ßbeta;-hexosaminidase was approximately twofold increased in the density fractions from 1.04 to 1.09 g/ml. The lysosome-associated membrane protein 1 (Lamp1) was detected by Western blotting in the fractions 1.08–1.10 g/ml in both genotypes, and, in comparison to the wild type, Lamp1 was more abundant in the ctsl–/– fractions. However, staining of myocardial histological sections of 1-yr-old mice with periodic acid Schiff’s reagent (PAS-staining; for the detection of carbohydrates), with Sudan III (intracellular lipid staining), and by the Mowry method for detection of glucosaminoglycan storage did not show lysosomal storage materials in CTSL-deficient hearts.
4. Cell death, cell proliferation, and vascular structure in CTSL-deficient myocardium
There are examples of cardiomyopathies for which an imbalance of apoptotic cell death and cell proliferation in the myocardium, as well as impairment of myocardial perfusion, has been shown to be an essential pathogenic factor. However, the myocardium of ctsl+/+ and ctsl–/– mice revealed no differences in incidence of cell death [terminal dUTP nick-end labeling (TUNEL) staining], proliferation (Ki67 immunohistochemistry), and capillary density (CD31 immunohistochemistry) during DCM progression.
5. Proteome analysis of ctsl+/+ and ctsl–/– myocardium
To obtain further data on the molecular pathogenesis of the DCM in CTSL knockout mice, we compared the myocardial protein pattern of ctsl+/+ and ctsl–/– mice by two-dimensional gel electrophoresis with mass spectrometric identification of the 30 proteins with significantly different expression levels. After functional classification of these proteins, the data reveal reduced levels of proteins involved in myocardial contraction, i.e., tropomyosin 1, calsarcin-1, and desmin, in the myocardium of ctsl–/– mice. Strikingly, the levels of many proteins involved in cellular and mitochondrial catabolism as well as in oxidative phosphorylation are altered in CTSL-deficient hearts. In addition, increased levels of peroxiredoxin 2 and oxidized peroxiredoxin 6 were associated with decreased levels of reduced peroxiredoxin 6, suggesting the presence of significant oxidative stress in the myocardium of CTSL-deficient mice.
6. Parameters of oxidative damage and mitochondrial respiration in CTSL-deficient myocardium
To assess the extent of oxidative damage in ctsl–/– hearts, which was suggested by the up-regulation of antioxidant proteins in our proteome screen, we measured malondialdehyde and protein-bound carbonyl-groups that represent stable end-products of lipid peroxidation and oxidative protein damage, respectively. There was no significant difference in either parameters of oxidative damage in samples from ctsl–/– vs. wild-type myocardium, indicating that the antioxidant system in ctsl–/– hearts is able to prevent extensive oxidative damage. Screening the proteome of CTSL-deficient hearts revealed reduced levels of respiratory chain proteins. Thus, we compared oxygen consumption in myocardial homogenates of 5-month-old ctsl–/– and ctsl+/+ mice. Measurement of citrate-synthase activity indicated similar amounts of mitochondria in ctsl–/– and ctsl+/+ homogenates. However, with the use of glutamate in combination with malate as respiratory substrates, a reduction of 50% in the oxygen consumption rates in resting and p-(trifluoromethoxy) phenylhydrazone (FCCP)-uncoupled respiration was observed in ctsl–/– homogenates compared with homogenates from wild-type mice. Respiration with succinate as substrate was revealed significant impairment in the ctsl–/– group, whereas this effect did not reach significance in the presence of FCCP.
CONCLUSIONS AND SIGNIFICANCE
We conclude that deficiency of CTSL in the heart primarily affects the lysosomal system, namely by increasing the number and changing the morphology of acidic organelles. Since lysosomal storage materials were not observed, the cardiomyopathy of CTSL knockout mice can be classified as a lysosomal cardiomyopathy without specific storage. The primary defects in the acidic cellular compartment of CTSL-deficient hearts result in complex biochemical and cellular alterations that are hallmarks of cardiomyopathies (Fig. 2
). These characteristics include the induction of an adaptive gene program, loss of cytoskeletal proteins, and mitochondrial impairment. Together, these interdependent cellular alterations lead to impaired cardiomyocyte function, cardiac remodeling, and progressive DCM. Our observation that CTSL plays an essential role in the structure and function of the lysosomal compartment in myocardium, but not apparently in skeletal muscle, requires further investigation.
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FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5517fje
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