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. 2009 Summer;14(2):e57–e61.

Effect of a low dose of proteasome inhibitor on cell death and gene expression in neonatal rat cardiomyocyte cultures exposed to anoxia-reoxygenation

Olga V Surova 1,✉, Vasyl S Nagibin 1, Lesya V Tumanovskaya 1, Victor E Dosenko 1, Alexey A Moibenko 1
PMCID: PMC2722462  PMID: 19675822

Abstract

BACKGROUND:

Recent data suggest that low concentrations of proteasome inhibitors (PIs) are cytoprotective in models of ischemia-reperfusion injury, but the underlying mechanisms of this effect still remain unclear.

AIM:

To investigate the effect of 100 nM of clasto-lactacystin β-lactone on cell death and gene expression in neonatal rat cardiomyocytes exposed to anoxia-reoxygenation.

METHODS:

Fluorescent microscopy and real-time polymerase chain reaction were used to detect different types of cell death and gene expression, respectively, in neonatal rat cardiomyocyte cultures exposed to anoxia-reoxygenation.

RESULTS:

It was shown that a low dose of clasto-lactacystin β-lactone protected the cells against anoxia-reoxygenation injury by a reduction in the number of necrotic and apoptotic cells. The number of autophagic cells was greatly increased by proteasomal inhibition. The PI increased the heat shock protein 70 messenger RNA expression twofold and slightly reduced the expression of heat shock protein 90 gene. The expression of the FK506 binding protein 12-rapamycin associated protein gene was increased 1.57-fold on PI application. The B-cell lymphoma 2 gene expression was unaffected by the use of clasto-lactacystin β-lactone in low dose.

CONCLUSION:

Although PIs are injurious, they may be cardioprotective in low doses; ie, they do not result in cell death. Moreover, PIs initiate the protective mechanisms that prevent cell damage by changing the expression of several genes.

Keywords: Anoxia-reoxygenation, Cardiomyocytes, Cell death, mRNA expression, Proteasome inhibitor

Recent data suggest a cytoprotective effect of low concentrations of proteasome inhibitors (PIs) against ischemic and reperfusion injuries (1,2). The mechanisms of protection against cell damage induced by anoxia-reoxygenation (A-R), at specific PI concentrations, still remain unclear. It has been shown previously that PIs are the triggers of early pre- and postconditioning in neonatal cardiomyocyte cultures. It was also established that PIs abolish the protective effect of ischemic pre- and postconditioning (3,4). Thus, the proteasome degrades the proteins that provide cell resistance to ischemic damage, but on the other hand, the phenomenon of pre- and postconditioning is impossible without proteasome proteolysis. The differentiated effect of nontoxic doses of PIs MG132 or MG262 on gene expression in human endothelial cells was determined by Meiners et al (5). They found that low doses of PIs induced a defined dose-dependent transcriptional response – in particular, uniform upregulation of several antioxidative enzymes (glutathione peroxidase 3, heme oxygenase-1, super-oxide dismutase 1, glutathione S-transferase, etc) – and observed differential regulation of genes involved in endothelial function (endothelial NO synthase, interleukin-8, thrombomodulin, tissue plasminogen activator, monocyte chemoattractant protein-1, etc). Affymetrix GeneChip analysis revealed that among almost 300 genes, at least 50% of them had modified expressions in the presence of nontoxic doses of PIs. This adaptive transcriptional pattern was translated into a protective response of endothelial cells against H2O2-induced oxidative stress and into improvement of endothelial function of rat aortic rings. Previous studies (6–8) have established that PIs induce messenger RNA expression of heat shock proteins (HSPs) in neonatal rat cardiac myocytes. Thus, the protective effect of PIs can be related to alterations in proteasome-dependent control of messenger RNA expression and could provide an advanced mechanism by which cell response to injury is modulated.

PIs cause complex effects on programmed cell death, induce stress response in cardiac cells and signal upregulation of gene expression. In the present study, we established the effect of 100 nM of PI clasto-lactacystin β-lactone (cL) on different types of cell death (apoptotic, necrotic and autophagic cell death) in cultured cardiomyocytes. To elucidate whether the protective effects of proteasome inhibition are achieved via the modulation of gene expression, we measured the expression of two key regulators of apoptotic and autophagic cell death – B-cell lymphoma 2 (Bcl-2) and FK506 binding protein 12-rapamycin associated protein (FRAP) – in isolated neonatal cardiomyocyte cultures under A-R conditions. We also evaluated the expression of HSPs, which are shown to be protected in heart against ischemia-reperfusion and are substantially involved in the pre- and postconditioning of the heart.

METHODS

Culture of rat neonatal cardiomyocytes

Neonatal cardiomyocytes were isolated from ventricular myocardium of two-day-old Wistar rats by enzymatic digestion according to Reinecke et al (9). The number of living and necrotic cells was determined by staining with 0.2% trypan blue solution, and normally ranged from 90% to 95% and from 5% to 10%, respectively. The resulting cardiomyocytes were plated in culture dishes covered with 2% gelatin solution with a density of 1.2×105 cells/cm2. The cells were cultivated for one to two days in Dulbecco’s modified eagle’s medium/medium 199 (4:1), containing 7% calf serum, 4.2 mM Na2CO3, 15 mM HEPES and antibiotics (streptomycin 100 g/mL, gentamycin 0.05 mg/mL and penicillin 100 U/mL) at 37°C in an atmosphere with 5% CO2, 20% O2 and 75% Ar (standard gas mixture).

Experimental treatments

There were four treatment groups: control, A-R, 100 nM cL, and 100 nM cL plus A-R. Twenty-four hours after plating, the cells were treated with 100 nM of cL and incubated for 24 h. Anoxia was attained with an airtight jar from which the O2 was flushed with a gas mixture containing 5% CO2 and 95% Ar for 30 min. Reoxygenation was achieved by exchanging fresh medium and by its aeration with the standard gas mixture for 1 h. The changing of medium was performed to initiate and expedite reoxygenation simultaneously with the recovery of oxygen supply. The substitution of buffer was performed in both the control and experimental groups to prevent differences in cell distribution. Each experiment was performed seven to 10 times.

Determination of gene expression

The cells were harvested for real-time polymerase chain reaction (PCR) before and after A-R. Total RNA was isolated from cells using a TRIzol-RNA preparation kit (Isogen, Russian Federation) according to the manufacturer’s protocol. RNA concentration was determined using the NanoDrop spectrophotometer ND1000 (NanoDrop Technologies Inc, USA). Reverse transcription was performed using a RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Lithuania) using 1.2 μg to 1.5 μg of total RNA and a random hexamer primer. Primers specific for rat HSP70 and HSP90 sequences were used: HSP70-F 5′-ATG CGC TCG AGT CCT ACG CCT T-3′, HSP70-R 5′-GCT GAT CTT GCC CTT GAG ACC CTC-3′, HSP90-F 5′-TCC AAT AGG CTT GTG TCT TCC CCC-3′ and HSP90-R 5′-AAT CCG TTC CAT GTT GGC TGT CC-3′. Messenger RNA expression was standardized to the 18S (ribosomal subunit 18) gene, as housekeeping gene, the transcription levels of which were not influenced by the experimental conditions. The primers used for this gene were 18S-F 5′-CTT AGA GGG ACA AGT GGC G-3′ and 18S-R 5′-GGA CAT CTA AGG GCA TCA CA-3′. PCR amplification for HSP70 and HSP90 was performed in 10 μL SYBR Green PCR Master Mix (Applied Biosystems, USA) containing 30 pM of each primer. The final volume of the reaction mixture was brought to 20 μL with deionized water. Thermal cycling conditions comprised an initial denaturation and AmpliTaq Gold DNA polymerase (Applied Biosystems, USA) activation step at 95°C for 10 min, followed by treatment at 95°C for 15 s, and at 59°C for 1 min and for 45 cycles, with a following dissociation step. Each PCR step was performed in duplicate. Calculations were performed using the 7500 Fast System SDS software provided (Applied Biosystems, USA). The cycle threshold is defined as the number of cycles required for the fluorescence signal to exceed the detection threshold. The expression of the target gene was calculated relative to the housekeeping gene as the difference between the threshold values of the two genes.

Gene expression values of FRAP (also known as mammalian target of rapamycin) and Bcl-2 were determined using the TaqMan Gene Expression Assay (Applied Biosystems, USA). The pairs of forward and reverse primers for the genes mentioned above and the TaqMan probes for the target messenger RNAs were designed based on the rat messenger RNA sequence by Applied Biosystems, USA. Gene expression in each probe was normalized by glyceraldehyde-3-phosphate dehydrogenase, as housekeeping gene, using TaqMan Rodent GAPDH Control Reagent (VIC probe). The thermal cycles of PCR amplification were as follows: initial denaturation step at 95°C for 20 s, followed by treatment at 95°C for 3 s, and at 60°C for 30 s and for 45 cycles with the use of 7500 Fast Real-Time PCR (Applied Biosystems, USA). Analysis of obtained data was carried out with 7500 Fast Real-Time PCR Software.

Cell viability test

The number of living, necrotic, apoptotic and autophagic cells was determined by staining with 8.75 μM bisbenzimide (Hoechst 33342), propidium iodide (10) and 50 μM monodan-sylcadaverine (11), and examined by fluorescent microscopy (Nikon Eclipse E200, filter B-2E/C). Five hundred cells were counted in each experiment.

Statistical analysis

All values are presented as mean ± SEM. Statistical analysis was performed using Student’s t test. P<0.05 was considered statistically significant. The software used was Origin 7.0 (OriginLab Corporation, USA) and Excel 2000 (Microsoft, USA).

RESULTS

Cell death at A-R and PI application

The application of A-R and PI significantly influenced the ratio of living, necrotic, apoptotic and autophagic cells (Figures 1 and 2).

Figure 1).

Figure 1)

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The number of living (A), necrotic (B) and apoptotic (C) cardiomyocytes in control, anoxia-reoxygenation (A-R), proteasome inhibitor (100 nM clasto-lactacystin β-lactone [cL]) and its combination (100 nM cL + A-R). Data are expressed as the percentage of the general number of cells. *P<0.05 compared with control; **P<0.05 compared with the A-R group. A-R injury led to a twofold and threefold increase of necrotic and apoptotic cell numbers, respectively. The 100 nM PI pretreatment for 24 h (100 nM cL + A-R) decreased the number of apoptotic cells compared with the A-R group by 1.6-fold and slightly reduced the number of necrotic cells. The PI attenuated the effect of A-R on the cultured cells and decreased A-R-induced apoptotic and necrotic cell death.

Figure 2).

Figure 2)

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The number of the cells with autophagic features in control, anoxia-reoxygenation (A-R), proteasome inhibitor (100 nM clasto-lactacystin β-lactone [cL]) and its combination (100 nM cL + A-R). Autophagy was increased in A-R and A-R + 100nM cL groups 2.6-fold and 3.3-fold, respectively. cL alone increased the number of cells with autophagic features by 3.7-fold. Data are expressed as the percentage of the general number of cells. *P<0.05 compared with control; **P<0.05 compared with the A-R group

The dose-response curve of cL was estimated (data not shown). The cardiomyocytes were treated with cL in concentrations ranging from 100 nM to 500 nM for 24 h. The 100 nM PI concentration was shown to be the most moderate dose that did not cause a dramatic increase of dead cells during 24 h of incubation, revealing its preconditioning effect.

The PI concentration of 100 nM protected the cell culture from A-R injury. A-R injury led to a twofold (from 5.98±0.66% to 11.88±1.90%; P<0.05) and threefold (from 2.59±0.61% to 7.85±1.96%; P<0.05) increase of necrotic and apoptotic cells, respectively. The PI pretreatment (100 nM cL + A-R) resulted in a 1.6-fold decrease in the number of apoptotic cells compared with the A-R group (7.85±1.96% to 4.98±1.80%; P<0.05) and slightly reduced the number of necrotic cells by 2% (from 11.88±1.99% to 10.14±1.86%; P<0.05). The PI attenuated the effect of A-R on the cultured cells and decreased A-R-induced apoptotic and necrotic cell death. cL alone increased the number of cells with autophagic features by 3.7-fold (from 6.45±1.47% to 23.8±2.33%; P<0.005). The number of autophagic cells was increased in the A-R and A-R + 100 nM cL groups 2.6-fold (6.45±1.47% to 16.6±1.66%; P<0.005) and 3.3-fold (6.45±1.47% to 21.5±3.22%; P<0.05), respectively (Figure 2).

Gene expression in neonatal rat cardiomyocytes treated with PI

The treatment with 100 nM PI for 24 h led to a twofold increase in HSP70 gene expression and a slightly reduced expression of the HSP90 gene (Figure 3). The expression of the FRAP gene was increased 1.57-fold after the application of PI (Figure 4). The Bcl-2 gene expression was unaffected by the use of cL in a low dose.

Figure 3).

Figure 3)

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Changes in heat shock protein (HSP) 70 (A) and HSP90 (B) gene expression in control and proteasome inhibitor (100 nM clasto-lactacystin β-lactone [cL]) group. *P<0.05 compared with control. mRNA Messenger RNA

Figure 4).

Figure 4)

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Changes in B-cell lymphoma 2 (Bcl-2) (A) and FK506 binding protein 12-rapamycin associated protein (FRAP) (B) gene expression in control and proteasome inhibitor (100 nM cL). *P<0.05 compared with control

DISCUSSION

PIs have been demonstrated to cause complex effects on programmed cell death, and represent a novel approach for the treatment of inflammatory, hyperproliferative and other diseases including reperfusion injuries (12–15). To narrow the therapeutic window for cytoprotective effects, it is critical to elucidate the complex cellular effects of different doses of PIs. Our analysis revealed that proteasome inhibition induced a stress response in cardiac cells and signalled upregulation of gene expression when nontoxic doses of PIs were used.

According to data by Meiners et al (16), low doses of PIs resulted in changes in the expression of approximately 300 genes, increased the expression of 40 genes and at the same time suppressed the expression of 81 genes. It is not a trivial task to predict the result of this influence, but the functional investigations performed by German researches on endothelial cells (16), as well as our experiments on cardiomyocytes, indicate a protective effect of low doses of PIs. In our opinion, the explanation of this observation can be attributed to the effect of proteasomal proteolysis on the gene transcription system.

The most studied system of transcription that works under the control of proteasomal proteolysis is the system involving the nuclear factor kappa-B (NF-κB), because the proteasome is responsible for degrading NF-κB after it has performed its function in the nucleus (17). Moreover, it has been established that the processing of one of the NF-κB subunits is also provided by ubiquitin-dependent proteolysis (18). Thus, on the one hand, the suppression of proteasomal activity can stimulate the active formation of NF-κB and, on the other hand, can prevent the formation of the heterodimer of this protein. Based on the fact that there were some transcriptional factor NF-κB molecules in the cells before exposing to a PI, we can conclude that the resulting effect of proteasomal inhibition will reveal a decrease in NF-κB-dependent gene expression. Some of these genes encode several proapoptotic proteins (Fas, interleukin-1β, death receptor 5 [tumour necrosis factor-related apoptosis-inducing ligand-RII], Bcl-extra large, and others), immune response proteins (tumour necrosis factor), molecules of cell adhesion, inducible NO synthase, monocyte chemoattractant protein 1 and other ischemia-reperfusion risk factors (19–22).

Furthermore, Zimmermann et al (23) showed that PIs lactacystin, lactacystin-β-lactone and MG132 induce the over-expression of transcriptional regulators activating transcription factor 3, GADD153 and mitotic arrest deficiency 1 in proliferating cells that leads to cell cycle arrest and apoptosis induction. However, in nondividing cells, such as cardiomyocytes, overexpression of these genes may have other consequences. In particular, it has been shown that GADD proteins take part in cell endoplasmic reticulum stress response through the expression of genes that protect cells from the accumulation of unfolding proteins (unfolding protein response) (24). When the unfolding proteins exceed a threshold, autophagy is activated (second endoplasmic reticulum-associated degradation system). In the present study and our other previous works, it was established that autophagy has protective effects against A-R or myocardial ischemia-reperfusion (25). Thus, the increase of cells with autophagic features after administering a PI can exert a protective effect in our experiments. In our opinion, this effect can be due to macroautophagy of mitochondria, which are the major source of free radicals and proapoptotic factors in cells exposed to A-R. According to our previous data, the inhibitors of autophagy decrease the number of mitochondria with reduced mitochondrial potential, causing mitochondrial permeability disturbances and secretion of proapoptotic factors into the cytosol (unpublished data). In the present experiment, we could not explain why there was an increase in FRAP gene expression, a key antiautophagic protein, at a low dose of PI. However, this finding obviously indicates that there is a significant use of this protein in the cytoplasm, which explains why the compensatory increase of FRAP gene expression takes place. Unfortunately, a complete absence of data regarding the role of FRAP messenger RNA expression warrants caution in the interpretation the evidence.

In the present study, HSP gene expression (mainly HSP70) can also explain the prevention of apoptotic cell death of cardiomyocytes after administering a PI and exposure to A-R. Recent reports have indicated that some HSPs are antiapoptotic and directly inhibit caspase activation. HSP70, HSP27, alpha A-crystallin and alpha B-crystallin are reported to be antiapoptotic (26–28). The role of HSP90 in controlling apoptosis is ambiguous and depends on the apoptotic stimulus, but the effect is usually antiapoptotic (29).

Thus, PIs are actually injurious agents; however, in low doses they do not result in cell death but initiate the protective mechanisms preventing cell damage. The concept of moderate injury is based on the phenomenon of pre- and postconditioning. In our previous studies, we postulated that the PIs are the factors of early pre- and postconditioning (4). In present work, we obtained some evidence demonstrating that the programing of late preconditioning is followed by several changes in gene expression and is also initiated through proteasome inhibition.

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