Genetically induced moderate inhibition of the proteasome in cardiomyocytes exacerbates myocardial ischemia-reperfusion injury in mice - PubMed (original) (raw)

Genetically induced moderate inhibition of the proteasome in cardiomyocytes exacerbates myocardial ischemia-reperfusion injury in mice

Zongwen Tian et al. Circ Res. 2012.

Abstract

Rationale: Both cardiomyocyte-restricted proteasome functional enhancement and pharmacological proteasome inhibition (PSMI) were shown to attenuate myocardial ischemia/reperfusion (I/R) injury. The role of cardiac proteasome dysfunction during I/R and the perspective to diminish I/R injury by manipulating proteasome function remain unclear.

Objectives: We sought to determine proteasome adequacy in I/R hearts, create a mouse model of cardiomyocyte-restricted PSMI (CR-PSMI), and test CR-PSMI impact on I/R injury.

Methods and results: Myocardial I/R were modeled by ligation (30 minutes) and subsequent release of the left anterior descending artery in mice overexpressing GFPdgn, a validated surrogate proteasome substrate. At 24 hours of reperfusion, myocardial proteasome activities were significantly lower whereas total ubiquitin conjugates and GFPdgn protein levels were markedly higher in all regions of the I/R hearts than the sham controls, indicative of proteasome functional insufficiency. CR-PSMI in intact mice was achieved by transgenic (tg) overexpression of a peptidase-disabled mouse β5 subunit (T60A-β5) driven by an attenuated mouse mhc6 promoter. Overexpressed T60A-β5 can replace endogenous β5 and inhibits proteasome chymotrypsin-like activities in the heart. Mice with moderate CR-PSMI showed no abnormalities at the baseline but displayed markedly more pronounced structural and functional damage during I/R, compared with non-tg littermates. The exacerbation of I/R injury by moderate CR-PSMI was associated with significant increases in the protein level of PTEN and protein kinase Cδ (PKCδ), decreased Akt activation, and reduced PKCε.

Conclusions: Myocardial I/R causes proteasome functional insufficiency in cardiomyocytes and moderate CR-PSMI augments PTEN and PKCδ, suppresses Akt and PKCε, increases cardiomyocyte apoptosis, and aggravates I/R injury in mice.

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Figures

Figure 1

Figure 1. Myocardial ischemia/reperfusion (I/R) impairs proteasome peptidase activities in mouse hearts

GFPdgn tg mice were subject to myocardial I/R created by ligation (30min) and subsequent release of the ligation (24h) of the anterior descending artery. Crude protein extracts from myocardial tissues of the left ventricle (LV) of the sham surgery group or of the LV area at risk (AAR), border zone (BZ), and remote area (RA) of the I/R group were used for proteasomal peptidase activity assays (A) and western blot analysis for ubiquitinated proteins (B, C). n=6 mice/group; *p<0.05, **p<0.01 vs. sham.

Figure 2

Figure 2. Increase of GFPdgn protein levels in GFPdgn tg mouse hearts after in vivo focal I/R

A and B, Western blot analyses show increases in the myocardial GFPdgn protein level in the indicated zones (RA, BZ, and AAR) of the LV free wall after I/R. Representative images (A) and densitometry data (B) are presented. N=4 mice per group; *p<0.05, **p<0.01 vs. sham. C, Representative images of myocardial GFPdgn direct green fluorescence in the indicated zones of the LV. Nuclei were stained blue with DAPI. Scale bar=100μm. D and E, RT-PCR assays show that I/R did not increase GFPdgn mRNA levels in the area at risk and the remote area. N=6 mice/group.

Figure 3

Figure 3. Baseline characterization of a tg mouse model of moderate cardiomyocyte-restricted proteasome inhibition (CR-PSMI)

Tg mouse lines with a moderate expression of Myc-tagged T60A-β5 precursor under the control of an attenuated mhc6 promoter were created. A, Schematic illustration of the transgenic (TG) construct used for fertilized egg microinjections to create the T60A-β5 Tg mouse founders. B, Sample images of western blot analyses for Myc (upper panel) and the β5 subunit (lower panel) of the 20S proteasome in ventricular myocardium of mice from 2 independent tg lines (Line 1, TG-1; Line 2, TG-2) at 8 weeks. C, A summary of myocardial endogenous β5 (Endo-β5) protein expression in T60A-β5 TG and NTG littermate mice at 8 weeks. D, Representative images of immunoblot analyses (IB) of the indicated proteins in immunoprecipitated (IP) 20S proteasomes from the ventricular myocardium of Myc-T60A-β5 TG and NTG littermate mice at the baseline condition. Antibodies againstα3 subunit of the 20S were used for IP 20S proteasomes from crude protein extracts from ventricular myocardium. E, Expression of T60A-β5 suppressed the chymotrypsin-like activity of 20S proteasomes in the heart. **p<0.01 vs. Ntg; n=4 mice/group.

Figure 4

Figure 4. Effects of T60A-β5 tg expression in cardiomyocytes on endogenous β5 expression and proteasomal peptidase activities of I/R and sham hearts

T60A-β5 tg and Ntg littermate mice were subject to myocardial I/R as described in Figure 1. Crude protein extracts from the indicated zones of the LV were used for western blot analyses of endogenous β5 and tg T60A-β5 (A, B) or for proteasomal chymotrypsin-like (C) and caspase-like (D) activity assays. A and B, Representative images (A) and a summary of densitometric data (B) of the western blot analyses for the endogenous β5 expressions. n=4. C and D, Changes in the indicated proteasomal peptidase activities. n=6. *p< 0.05, **p<0.01, ***p<0.005 vs. Ntg sham; #p< 0.05, ##p<0.01.

Figure 5

Figure 5. Moderate CR-PSMI significantly increases the infarct size in I/R mice

T60A-β5 Tg and Ntg littermates at 10~12 weeks of age were subject to LAD ligation and release as described in Figure 1. Infarct size was determined at 24h after reperfusion. A, Representative comparison of infarct size between a Tg mouse heart and a littermate Ntg mouse heart. B and C, Morphometric analysis of area at risk (B) and infarct size (C) in T60A-β5 Tg and Ntg mouse hearts. n=6, *p<0.05 vs. Ntg.

Figure 6

Figure 6. Evaluation of cardiomyocyte apoptosis in Ntg and T60A-β5 Tg mice undergone myocardial I/R

A, Representative images of TUNEL staining (green) in the sham and the indicated zones of the LV of I/R hearts. Nuclei were stained blue with DAPI. Cardiomyocytes are identified by the green fluorescence resulting from Alexa-568-phalloidin staining. Scale bar=50μm. B, The number of TUNEL-positive nuclei is expressed as a percentage of total nuclei detected by DAPI staining. n=6 mice/group, *P<0.05 vs. Ntg. C and D, Western blot analysis myocardial levels of the cleaved (i.e., activated) caspase-3 in all zones of the T60A-β5 Ntg and Tg mouse hearts. N=4; *p< 0.05, **p<0.01, ***p<0.005 vs. Ntg sham; #p< 0.05, ##p<0.01.

Figure 7

Figure 7. Effects of CR-PSMI on Akt activation and PKCδ protein expression in sham and I/R hearts

A ~ C, Representative images (A) and a summary of densitometry data of western blot analyses for total Akt and S473-phosphorylated Akt (p-S473-Akt, B) and PTEN (C) in sham hearts and in the indicated zones of I/R hearts. GAPDH was probed for loading control. D ~ F, Representative images (D) and a summary of densitometry data of western blot analyses for PKCδ (E) and PKCε (F) in sham hearts and in the indicated zones of I/R hearts. β-Tubulin was probed for loading control. n=4 mice/group; *p< 0.05, **p<0.01, ***p<0.005 vs. Ntg sham; #p< 0.05, ##p<0.01, ###p<0.005.

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