Cathepsin K knockout alleviates pressure overload-induced cardiac hypertrophy

Abstract

Evidence from human and animal studies has documented elevated levels of lysosomal cysteine protease cathepsin K in failing hearts. Here, we hypothesized that ablation of cathepsin K mitigates pressure overload-induced cardiac hypertrophy. Cathepsin K knockout mice and their wild-type littermates were subjected to abdominal aortic constriction, resulting in cardiac remodeling (heart weight, cardiomyocyte size, left ventricular wall thickness, and end diastolic and end systolic dimensions) and decreased fractional shortening, the effects of which were significantly attenuated or ablated by cathepsin K knockout. Pressure overload dampened cardiomyocyte contractile function along with decreased resting Ca2+ levels and delayed Ca2+ clearance, which were partly resolved by cathepsin K knockout. Cardiac mammalian target of rapamycin and extracellular signal-regulated kinases (ERK) signaling cascades were upregulated by pressure overload, the effects of which were attenuated by cathepsin K knockout. In cultured H9c2 myoblast cells, silencing of cathepsin K blunted, whereas cathepsin K transfection mimicked phenylephrine-induced hypertrophic response, along with elevated phosphorylation of mammalian target of rapamycin and ERK. In addition, cathepsin K protein levels were markedly elevated in human hearts of end-stage dilated cardiomyopathy. Collectively, our data suggest that cathepsin K ablation mitigates pressure overload-induced hypertrophy, possibly via inhibition of the mammalian target of rapamycin and ERK pathways.

Keywords: cardiac hypertrophy; cathepsin K; contractile function; mammalian target of rapamycin.

Figure 1

Effect of pressure overload on myocardial expression of cathepsin K. A, Representative gel blots exhibiting cathepsin isoforms and GAPDH (loading control) in myocardial tissues from mice subjected to sham or abdominal aortic constriction (AAC) procedure. B, Densitometric quantification of cathepsin K, cathepsin B, cathepsin S, and cathepsin L. Mean±SEM, n=6 mice per group, *P<0.05 vs sham group.

Figure 2

Morphological changes in hearts from wild-type (WT) and cathepsin K knockout mice. A, Heart-to-body weight ratio for wild-type and cathepsin K knockout mice subjected to sham or abdominal aortic constriction (AAC). B, Quantitation of cardiomyocyte cross-sectional area. C, Quantitative analysis of cardiac interstitial fibrosis. D, Representative images using FITC-conjugated wheat germ agglutinin (WGA) staining for cardiac tissues from wild-type and cathepsin K knockout mice subjected to sham surgery of AAC. E, Representative images of Masson trichrome staining for cardiac tissues from wild-type and cathepsin K knockout mice subjected to sham surgery or AAC. Mean±SEM, n=100 cardiomyocytes for cross-sectional area quantification, n=20 sections for Masson trichrome staining, n=6 to 9 mice for heart weight-to-body weight evaluation per group. *P<0.05 vs WT-sham, &P<0.05 vs WT-AAC group.

Figure 3

Echocardiographic features in wild-type (WT) and cathepsin K knockout mice subjected to sham or abdominal aortic constriction (AAC). A, Left ventricular wall thickness. B, Left ventricular end-diastolic dimension (LVEDD). C, Left ventricular end systolic dimension (LVESD). D, Fractional shortening (FS). Mean±SEM, n=6 to 9 mice group. *P<0.05 vs WT-sham group, #P<0.05 vs Ctsk^−/−-sham group, &P<0.05 vs WT-AAC group.

Figure 4

Cardiomyocyte contractile properties in wild-type (WT) and cathepsin K knockout mice subjected to sham or abdominal aortic constriction (AAC). A, Resting cell length. B, Peak shortening (PS; normalized to cell length). C, Maximal velocity of shortening (+dL/dt). D, Maximal velocity of relengthening (−dL/dt). E, Time to PS (TPS). F, Time to 90% relengthening (TR₉₀). Mean±SEM, n=76 to 97 cells per group, *P<0.05 vs WT-sham group, &P<0.05 vs WT-AAC group.

Figure 5

Intracellular Ca²⁺ transients in cardiomyocytes in wild-type (WT) and cathepsin K knockout mice subjected to sham surgery or abdominal aortic constriction (AAC). A–C, Resting fura-2 fluorescence intensity (FFI), electrically stimulated rise in FFI ( FFI), single exponential intracellular Ca²⁺ decay rate, respectively. Mean±SEM, n=94 to 127 cardiomyocytes for intracellular Ca²⁺ transient detection.

Figure 6

Effect of cathepsin K knockout on abdominal aortic constriction (AAC)–induced changes in GATA-4, phospho-Akt, phospho-GSK3 , phospho-AMPK, phospho-ACC, and phospho-p38 MAPK in mouse hearts. A, Representative Western blot image of GATA4, phosphor-Akt, Akt, phosphor-GSK3 , GSK3 , phosphor-AMPK, AMPK, phosphor-ACC, ACC, phosphor-p38 MAPK, p38 MAPK, and GAPDH (loading control). B–D, Densitometric quantitation of GATA4, p-Akt-to-Akt ratio, and p-AMPK-to-AMPK ratio, respectively. Mean±SEM, n=6 to 9 mice group. *P<0.05 vs WT-sham group, #P<0.05 vs Ctsk^−/−- sham group, &P<0.05 vs WT-AAC group.

Figure 7

Effect of cathepsin K knockout on extracellular signal-regulated kinases (ERK), mammalian target of rapamycin (mTOR), 4E-BP1, p70 S6K, Raptor phosphorylation, and Rictor expression in mouse hearts. A, Representative gel blots depicting levels of total or phosphorylated ERK, mTOR, 4E-BP1, p70S6K, Raptor, Rictor, and GAPDH (loading control). B–D, pERK-to-ERK ratio, phospho–mTOR-to-mTOR ratio and p–4E-BP1-to-4E-BP1 ratio, respectively. Mean±SEM, n=6 to 9 mice pre-group, *P<0.05 vs WT-sham group, &P<0.05 vs WT-AAC group. AAC indicates abdominal aortic constriction; and WT, wild-type.

Figure 8

Cathepsin K knockdown alleviates phenylephrine (PE)–induced hypertrophy and protein synthesis, whereas cathepsin K overexpression facilitates hypertrophic response in cultured H9c2 cells. A, Representative images of α-actin staining exhibiting H9c2 cells transfected with cathepsin K small interfering RNAs (siRNA) in the presence or absence of phenylephrine (PE, 100 μmol/L, 24 h). B, Quantitative analysis of cell area from α-actin staining images. C, ³H-leucine incorporation in H9c2 cells transfected with cathepsin K siRNA in the presence or absence of phenylephrine. D, Representative images of α-actin staining in H9c2 cells transfected with the cathepsin K plasmid in the presence or absence of rapamycin. E, Pooled data from D. F, ³H-leucine incorporation in H9c2 cells transfected with cathepsin K plasmid in the presence or absence of rapamycin. *P<0.05 vs NT-siRNA group, #P<0.05 vs catK siRNA group, &P<0.05 vs NT-siRNA + PE group in B and C. *P<0.05 vs CONT group, &P<0.05 vs catK cDNA group in E and F.

Figure 9

Overexpression of cathepsin K upregulates mammalian target of rapamycin (mTOR) signaling. A, Plasmid–mediated expression of cathepsin K upregulates GATA4 and phosphorylation of mTOR and 4E-BP1. B, Pharmacological inhibitor of extracellular signal-regulated kinases (ERK) attenuates cathepsin K–induced mTOR and 4E-BP1 phosphorylation.

Figure 10

Myocardial expression of cathepsin K is elevated in human failing hearts. Top, Representative gel blots of cardiac cathepsin K expression in human nonfailing and failing hearts. Bottom, Densitometric quantification of cathepsin K. n=6 nonfailing and 6 failing hearts, P<0.05 between the groups. The horizontal bar represents average ratio of the density of the cathepsin K blot to that of the corresponding GAPDH blot.