Cathepsin K knockout protects against cardiac dysfunction in diabetic mice

Abstract

Diabetes is a major risk factor for cardiovascular disease and the lysosomal cysteine protease cathepsin K plays a critical role in cardiac pathophysiology. To expand upon our previous findings, we tested the hypothesis that, knockout of cathepsin K protects against diabetes-associated cardiac anomalies. Wild-type and cathepsin K knockout mice were rendered diabetic by streptozotocin (STZ) injections. Body weight, organ mass, fasting blood glucose, energy expenditure, cardiac geometry and function, cardiac histomorphology, glutathione levels and protein levels of cathepsin K and those associated with Ca²⁺ handling, calcineurin/NFAT signaling, insulin signaling, cardiac apoptosis and fibrosis were determined. STZ-induced diabetic mice exhibited distinct cardiac dysfunction, dampened intracellular calcium handling, alterations in cardiac morphology, and elevated cardiomyocyte apoptosis, which were mitigated in the cathepsin K knockout mice. Additionally, cathepsin K knockout mice attenuated cardiac oxidative stress and calcineurin/NFAT signaling in diabetic mice. In cultured H9c2 myoblasts, pharmacological inhibition of cathepsin K, or treatment with calcineurin inhibitor rescued cells from high-glucose triggered oxidative stress and apoptosis. Therefore, cathepsin K may represent a potential target in treating diabetes-associated cardiac dysfunction.

Figure 1

Metabolic properties of wildtype (WT) and cathepsin K knockout (Ctsk ^−/−) mice challenged with vehicle or streptozotocin (STZ) during light and dark cycles. (A–B) volume of O₂ consumption (VO₂) in the light cycle; (C–D) volume of CO₂ production (VCO₂) in the light cycle; (E) respiratory exchange ratio (RER) in the light cycle; (F) energy expenditure (heat) in the light cycle; (G–H) physical activity in the light cycle; (I,J) physical activity in the dark cycle; (K) RER in the dark cycle; (L) VO₂ during an entire light-dark cycle; (M) VCO₂ during an entire light-dark cycle; (N) RER during an entire light-dark cycle; (O) physical activity during an entire light-dark cycle; (P) heat during an entire light-dark cycle. Mean ± SEM, n = 5–7 mice per group, ^*p < 0.05 vs. WT group, ^†p < 0.05 vs. WT-STZ group

Figure 2

Echocardiographic properties in WT and Ctsk ^−/− mice treated with or without streptozotocin. (A) heart rate; (B) wall thickness; (C) left ventricular (LV) end-diastolic diameter; (D) LV end-systolic diameter; (E) LV mass normalized to body weight; (F) fractional shortening. Mean ± SEM, n = 6–8 mice per group. ^*p < 0.05 vs. WT group, ^†p < 0.05 vs. WT-STZ group.

Figure 3

Cardiomyocyte contractile properties in WT and Ctsk ^−/− mice treated with or without streptozotocin. (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 = 92–97 cells from three mice per group. ^*p < 0.05 vs. WT group, ^†p < 0.05 vs. WT-STZ group.

Figure 4

Cardiomyocyte intracellular Ca²⁺ handling properties in WT and Ctsk ^−/− mice treated with or without streptozotocin. (A) Resting fura-2 fluorescence intensity (FFI); (B) electrically-stimulated rise in FFI (ΔFFI); (C) intracellular Ca²⁺ decay rate (single exponential); Mean ± SEM, n = 105–120 cells from four mice per group. ^*p < 0.05 vs. WT group, ^†p < 0.05 vs. WT-STZ group.

Figure 5

Western blot analysis exhibiting levels of Ca²⁺ regulatory proteins as well as calcineurin A-NFAT signaling in myocardium from WT and Ctsk ^−/− mice treated with or without streptozotocin. (A) Representative gel blots of SERCA2a, phosphorylation of phospholamban (p-PLB), calcineurin A, phosphorylation of NFATc3, NFATc1 and GAPDH (loading control) using specific antibodies; (B) SERCA2a/GAPDH; (C) p-PLB/GAPDH; (D) calcineurin A/GAPDH; (E) p-NFATc3/GAPDH; (F) NFATc1/GAPDH. Mean ± SEM, n = 6 to 7 mice per group. ^*p < 0.05 vs. WT group, ^†p < 0.05 vs. WT-STZ group.

Figure 6

Phosphorylation of AKT, Phosphorylation of GSK3β, total AKT, total GSK3β and TGF-β in myocardium from WT and Ctsk ^−/− mice treated with or without streptozotocin. (A) Representative gel blots of p-AKT, AKT, p-GSK3β, GSK3β, TGF-β and GAPDH (loading control) using specific antibodies; (B) p-AKT/GAPDH; (C) AKT/GAPDH; (D) p-AKT/AKT Ratio; (E) p-GSK3β/GAPDH; (F) GSK3β/GAPDH; (G) p-GSK3β/GSK3β Ratio (H) TGF-β/GAPDH. Mean ± SEM, n = 6 to 7 mice per group. ^*p < 0.05 vs. WT group, ^†p < 0.05 vs. WT-STZ group.

Figure 7

Apoptosis markers Bax, Bcl-2 and cleaved caspase-3 expression in myocardium from WT and Ctsk ^−/− mice treated with or without streptozotocin. (A) Representative gel blots of Bax, Bcl-2, cleaved caspase-3 and GAPDH (loading control) using specific antibodies; (B) Bax/GAPDH; (C) Bcl-2/GAPDH; (D) cleaved caspase-3/GAPDH; Mean ± SEM, n = 6 to 7 mice per group. ^*p < 0.05 vs. WT group, ^†p < 0.05 vs. WT-STZ group.

Figure 8

Western blot analysis of Bax, Bcl-2, cleaved caspase-3, phosphorylation of AKT, AKT and GAPDH (loading control) in H9c2 cells treated with 25 mM high glucose in the present or absent of cathepsin K inhibitor CatK I-II or calcineurin inhibitor CsA. (A) Representative gel blots of Bax, Bcl-2, cleaved caspase-3, p-AKT, ATK and GAPDH (loading control) using specific antibodies; (B) cleaved caspase-3/GAPDH; (C) Bax/GAPDH; (D) Bcl-2/GAPDH; (E) Bax/Bcl-2 ratio; (F) p-AKT/GAPDH; (G) AKT/GAPDH; (H) p-AKT/AKT ratio. ^*p < 0.05 vs. LG group, ^†p < 0.05 vs. HG group.

Figure 9

Reactive oxygen species (ROS) levels in H9c2 cells treated with 25 mM high glucose in the present or absent of cathepsin K inhibitor CatK I-II or calcineurin inhibitor CsA. ^*p < 0.05 vs. LG group, ^†p < 0.05 vs. HG group.