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. 2015 Aug:85:273-81.
doi: 10.1016/j.yjmcc.2015.06.014. Epub 2015 Jun 25.

Genetically induced moderate inhibition of 20S proteasomes in cardiomyocytes facilitates heart failure in mice during systolic overload

Mark J Ranek  1 , Hanqiao Zheng  2 , Wei Huang  3 , Asangi R Kumarapeli  4 , Jie Li  5 , Jinbao Liu  6 , Xuejun Wang  7
Affiliations

Genetically induced moderate inhibition of 20S proteasomes in cardiomyocytes facilitates heart failure in mice during systolic overload

Mark J Ranek et al. J Mol Cell Cardiol. 2015 Aug.

Abstract

The in vivo function status of the ubiquitin-proteasome system (UPS) in pressure overloaded hearts remains undefined. Cardiotoxicity was observed during proteasome inhibitor chemotherapy, especially in those with preexisting cardiovascular conditions; however, proteasome inhibition (PsmI) was also suggested by some experimental studies as a potential therapeutic strategy to curtail cardiac hypertrophy. Here we used genetic approaches to probe cardiac UPS performance and determine the impact of cardiomyocyte-restricted PsmI (CR-PsmI) on cardiac responses to systolic overload. Transgenic mice expressing an inverse reporter of the UPS (GFPdgn) were subject to transverse aortic constriction (TAC) to probe myocardial UPS performance during systolic overload. Mice with or without moderate CR-PsmI were subject to TAC and temporally characterized for cardiac responses to moderate and severe systolic overload. After moderate TAC (pressure gradient: ~40mmHg), cardiac UPS function was upregulated during the first two weeks but turned to functional insufficiency between 6 and 12weeks as evidenced by the dynamic changes in GFPdgn protein levels, proteasome peptidase activities, and total ubiquitin conjugates. Severe TAC (pressure gradients >60mmHg) led to UPS functional insufficiency within a week. Moderate TAC elicited comparable hypertrophic responses between mice with and without genetic CR-PsmI but caused cardiac malfunction in CR-PsmI mice significantly earlier than those without CR-PsmI. In mice subject to severe TAC, CR-PsmI inhibited cardiac hypertrophy but led to rapidly progressed heart failure and premature death, associated with a pronounced increase in cardiomyocyte death. It is concluded that cardiac UPS function is dynamically altered, with the initial brief upregulation of proteasome function being adaptive; and CR-PsmI facilitates cardiac malfunction during systolic overload.

Keywords: Cardiac hypertrophy; Heart failure; Pressure overload; Proteasome inhibition; Transgenic mice; Ubiquitin–proteasome system.

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Figures

Figure 1
Figure 1. Temporal changes in myocardial UPS performance in mice subject to moderate trans-aortic constriction (mTAC)
NTG and GFPdgn TG mice were subject to mTAC at ~10 weeks of age. A, Representative images (upper panel) and pooled densitometry data of western blot analyses for LV myocardial GFPdgn at the indicated time points. GFPdgn was detected using antibodies against GFP and α-actinin (Actn) was probed for loading control. For each time point, n=4~6/group; T=TAC; S=sham. B~D, Temporal changes in myocardial 20S proteasome chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) peptidase activities after mTAC. AU, arbitrary unit; n=8~12/group. E and F, Western blot analyses for total ubiquitinated proteins in the 1% Triton X-100 soluble and insoluble fractions of myocardium at 12 weeks after mTAC. *p<0.05, **p<0.01 vs. Sham.
Figure 2
Figure 2. CR-PsmI does not prevent cardiac hypertrophic responses to mTAC
B> Western blot analyses for endogenous (endo) proteasome β5 subunit and Myc-tagged protease-disabled β5 (Myc-T60A-β5) in LV myocardium of T60A-β5 Tg and Ntg littermate mice. B, The impact of T60A-β5 expression on myocardial chymotrypsin-like peptidase activity. C ~ E, Effects of CR-PsmI on cardiac hypertrophic responses to mTAC. Both gravimetric analyses (C) and RNA dot blot assessment of the fetal gene program (D, E) at 6 days after mTAC show comparable hypertrophic responses between T60A-β5 Tg and Ntg mice (p>0.05). *p<0.05, **p<0.01 vs. Ntg sham.
Figure 3
Figure 3. Effects of CR-PsmI on cardiac hypertrophy and function at 24 weeks after mTAC
T60A-β5 Tg and Ntg littermate mice of 10-week-old were subject to mTAC or sham surgery. A, Gravimetric analyses. For each parameter shown, the difference between Ntg-mTAC and Tg-mTAC groups is not statistically significant (p>0.05). **p<0.01 vs. Ntg sham; n=8 mice/group. B, LV hemodynamics assessments. *p<0.05 vs. Ntg sham and Ntg TAC; n=5 mice/group.
Figure 4
Figure 4. The impact of TG expression of T60A-β5 on proteasome functional changes during sTAC
The T60A-β5 Ntg and Tg littermate mice were subject to sTAC or sham surgery. At 6 days after the surgery, the LV tissue was sampled for extraction of crude myocardial proteins. A and B, Representative images (A) and pooled densitometry data (B) of western blot analyses of the indicated proteins. C, Proteasomal peptidase activity assays. D and E, Western blot analysis for LV myocardial total ubiquitinated (Ub’n) proteins. *p<0.05, #p<0.01, ##p<0.001 vs. Ntg sham; $p<0.01; n=4 mice/group.
Figure 5
Figure 5. Effects of CR-PsmI on cardiac responses to sTAC
A, Changes in the mRNA expression of the indicated genes at 6 days after surgery. GAPDH was probed as a house keeping gene for RNA quantification control. *p<0.05 vs. NTG sham; $p<0.05; n=4 mice/group. B, Kaplan-Meier survival curve. n=10 mice/group; p < 0.01, log-rank test. C & D, Prevalence of cardiomyocyte necrosis. Cryosections from LV myocardium collected 6 days after sTAC were immuno-stained for mouse IgG (red) using a Fluor-568 conjugated anti-mouse IgG antibody and for desmin using rabbit primary antibodies for desmin and Fluor-488 conjugated anti-rabbit IgG secondary antibodies. Desmin- and mouse IgG positive (IgG+) cardiomyocytes are considered necrotic. Nuclear DNA was stained blue with DAPI. Representative images (C) and pooled data from 3 mice per group (D) are shown.
Figure 6
Figure 6. CR-PsmI suppresses cell survival signaling and activates caspase 3
LV crude proteins collected as described in Figure 4 were used. A and B, Representative images (A) and pooled densitometric data (B) of western blot analyses for total Akt, Ser473-phosphorylated Akt (p-Akt), and PTEN. GAPDH was probed as loading control. C and D, Representative images (C) and pooled densitometry data (D) of western blot analyses for total and cleaved (CL) caspase 3. *p<0.05, #p<0.01 vs. Ntg sham; δ, p<0.05; $, p<0.01; n=6 mice/group.
Figure 7
Figure 7. Central illustration
Maintaining cardiac proteostasis requires timely removal of misfolded proteins whereas increased production of misfolded proteins tilts the balance toward PQC inadequacy. In response to acute systolic overload, the heart undergoes hypertrophy, which inevitably increases protein synthesis and by-production of misfolded proteins. To counter the increased proteotoxic stress during hypertrophy, cardiomyocytes adaptively mobilizes proteasomal reserve and thereby elevates proteasome (Psm) activities and UPS performance; however, when the stress sustains, proteasome function and UPS performance are impaired by unknown factors, which diminishes the capability of cardiomyocytes to maintain proteostasis and suppresses cell survival signaling, leading to cardiomyocyte malfunction and cell death. Cardiomyocyte death, especially in the form of necrosis, triggers inflammatory responses which could further fuel cardiomyocyte death and stimulate proliferation of interstitial cells (e.g., fibroblasts), leading to fibrosis. Both loss of cardiomyocytes and fibrosis are main pathogenic factors of heart failure. CR-PsmI prevents the initial adaptive proteasomal activation and hastens UPS impairment, thereby facilitating heart failure during systolic overload. Systemic PsmI inhibits UPS-mediated proteolysis in both cardiomyocytes and non-cardiomyocyte compartments. The non-cardiac PsmI could potentially attenuate inflammatory responses and the proliferation of fibroblasts, thereby being anti-fibrotic; hence, systemic PsmI might be beneficial at the certain stage of systolic overload. The dashed lines denote other potential pathways that are directly tested by the present study.
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