Evidence for a role of immunoproteasomes in regulating cardiac muscle mass in diabetic mice

Abstract

The ubiquitin-proteasome system plays an important role in regulating muscle mass. Inducible immunoproteasome subunits LMP-2 and LMP-7 are constitutively expressed in the heart; however, their regulation and functions are poorly understood. We here investigated the hypothesis that immunoproteasomes regulate cardiac muscle mass in diabetic mice. Type 1 diabetes was induced in wildtype mice by streptozotocin. After hyperglycemia developed, insulin and the proteasome inhibitor epoxomicin were used to treat diabetic mice for 6weeks. Isolated mouse hearts were perfused with control or high glucose solution. Catalytic proteasome beta-subunits and proteolytic activities were analyzed in the heart by immunoblotting and fluorogenic peptide degradation assays, respectively. Insulin and epoxomicin blocked loss of heart weight and improved cardiac function in diabetic mice. LMP-7 and its corresponding chymotryptic-like proteasome activity were increased in diabetic hearts and high glucose-treated hearts. Myosin heavy chain protein was decreased in diabetic hearts, which was largely reversed by epoxomicin. High glucose decreased LMP-2 protein levels in perfused hearts. In diabetic hearts, LMP-2 expression was downregulated whereas expression of the phosphatase and tensin homologue deleted on chromosome ten (PTEN) and the muscle atrophy F-box were upregulated. Moreover, mice with muscle-specific knockout of PTEN gene demonstrated increased cardiac muscle mass, while mice with LMP-2 deficiency demonstrated PTEN accumulation, muscle mass loss, and contractile impairment in the heart. Therefore, we concluded that high glucose regulates immunoproteasome subunits and modifies proteasome activities in the heart, and that dysregulated immunoproteasome subunits may mediate loss of cardiac muscle mass in experimental diabetic mice.

FIG. 1

Diabetic cardiomyopathy is attenuated by EPX in STZ mice. Diabetes was induced in wildtype C57BL6 mice with STZ. Insulin and epoxomicin (EPX) were used to treat diabetic mice for 6 weeks. Vehicle (CON) and EPX only treatment were controls. Representative left ventricular pressure recording and echocardiography. Each picture was chosen from 4–5 animal studies.

FIG. 2

LMP-7 is up-regulated and chymotryptic-like proteasome activity is increased in STZ-induced diabetic hearts and normal hearts treated with high glucose. A: STZ (S) and CON (C) hearts were collected for Western blot analysis of LMP-7 and β5 subunit. GAPDH was used as a loading control. Isolated hearts from wildtype mice were exposed to high glucose (HG) and control glucose (CG) for 4 hrs. LMP-7 and β5 subunit protein levels were examined. B: Chymotryptic-like proteasome activity was measured by fluorogenic assay in STZ and CON hearts and in HG and CG hearts (D). N = 3–4. *: p < 0.01vs. CON. ^#: p < 0.01 vs. CG.

FIG. 3

MHC protein levels are reduced in STZ-induced diabetic hearts and this is inhibited by EPX. Diabetic mice were treated with or without EPX for 6 weeks. MHC was analyzed by Western blot analysis. N = 3, *: p < 0.01 vs. CON or EPX. B: Diabetic mice were treated with EPX or insulin and normal mice were treated with EPX for 6 weeks.

FIG. 4

LMP-2 and MECL-1 protein levels and tryptic-like proteasome activity are decreased in STZ-induced diabetic hearts. A: LMP-2, β1, MECL-1, and β2 were measured by Western blot analysis in STZ (S, solid bar) and CON (C, open bar) hearts and in HG and CG hearts. B: Tryptic-like and caspase-like proteasome activities were measured in STZ and CON hearts. N = 3–4, *: p < 0.01 vs. CON. **: p < 0.05 vs. CON. ^#: p < 0.01 vs. CG.

FIG. 5

PTEN/Akt/FOXO3a/MAFbx signaling axis is regulated in STZ-induced diabetic hearts. PTEN, p-Akt and Akt, p-FOXO3a and FOXO3a, MAFbx, and MyoD1 were analyzed in STZ (S, solid bar) and CON (C, open bar) hearts. N = 3, *: p < 0.01 vs. CON.

FIG. 6

Cardiac muscle mass is increased in muscle-specific Pten knockout mice. A: PTEN immunohistochemistry. PTEN was expressed in cardiac muscle in Pten ^lp/lp; mck-Cre^+/− control mice (red arrows), but undetectable in Pten ^lp/lp; mck-Cre ^−/− control mice. B: PTEN, p-Akt, and total Akt protein levels in the heart, liver, and skeletal muscle. C: Whole hearts of Pten control and knockout mice. The data from A–C are representative of 3 Pten ^lp/lp; mck-Cre^+/− mice and 3 Pten ^lp/lp; mck-Cre ^−/− mice. D: MHC was increased in Pten ^lp/lp; mck-Cre^+/− mice. N = 3, *: p < 0.01vs. Pten ^lp/lp; mck-Cre^−/−.

FIG. 7

LMP-2 deficiency leads to loss of cardiac muscle mass and a decrease in cardiac function in mice. A: Western blots of LMP-2, PTEN, p-Akt and Akt, and MHC in Lmp-2^+/+ (open bar) and Lmp-2^−/− (solid bar) mice. N = 4, *: p < 0.05 vs. Lmp-2^+/+. B: Whole hearts and middle sections from Lmp-2^+/+ and Lmp-2^−/− mice. The thickness of left ventricular muscle (red) was decreased. Fibrotic tissue (white) formed in the endomyocardium. The pictures are representative of 4 Lmp-2^+/+ mice and 4 Lmp-2^−/− mice. C and D: LVM and FS were decreased in Lmp-2^−/− mice. N = 4, *: p < 0.01 vs. Lmp-2^+/+.

FIG. 7

LMP-2 deficiency leads to loss of cardiac muscle mass and a decrease in cardiac function in mice. A: Western blots of LMP-2, PTEN, p-Akt and Akt, and MHC in Lmp-2^+/+ (open bar) and Lmp-2^−/− (solid bar) mice. N = 4, *: p < 0.05 vs. Lmp-2^+/+. B: Whole hearts and middle sections from Lmp-2^+/+ and Lmp-2^−/− mice. The thickness of left ventricular muscle (red) was decreased. Fibrotic tissue (white) formed in the endomyocardium. The pictures are representative of 4 Lmp-2^+/+ mice and 4 Lmp-2^−/− mice. C and D: LVM and FS were decreased in Lmp-2^−/− mice. N = 4, *: p < 0.01 vs. Lmp-2^+/+.