Protection against doxorubicin-induced myocardial dysfunction in mice by cardiac-specific expression of carboxyl terminus of hsp70-interacting protein

Carboxyl terminus of Hsp70-interacting protein (CHIP) is a critical ubiquitin ligase/cochaperone to reduce cardiac oxidative stress, inflammation, cardiomyocyte apoptosis and autophage etc. However, it is unclear whether overexpression of CHIP in the heart would exert protective effects against DOX-induced cardiomyopathy. Cardiac-specific CHIP transgenic (CHIP-TG) mice and the wild-type (WT) littermates were treated with DOX or saline. DOX-induced cardiac atrophy, dysfunction, inflammation, oxidative stress and cardiomyocyte apoptosis were significantly attenuated in CHIP-TG mice. CHIP-TG mice also showed higher survival rate than that of WT mice (40% versus 10%) after 10-day administration of DOX. In contrast, knockdown of CHIP by siRNA in vitro further enhanced DOX-induced cardiotoxic effects. Global gene microarray assay revealed that after DOX-treatment, differentially expressed genes between WT and CHIP-TG mice were mainly involved in apoptosis, atrophy, immune/inflammation and oxidative stress. Mechanistically, CHIP directly promotes ubiquitin-mediated degradation of p53 and SHP-1, which results in activation of ERK1/2 and STAT3 pathways thereby ameliorating DOX-induced cardiac toxicity.

Scientific RepoRts | 6:28399 | DOI: 10.1038/srep28399 CHIP is known to be a dual-function cochaperone/ubiquitin ligase that is highly expressed in the heart and other tissue cells. CHIP has ubiquitin ligase activity and targets chaperone-bound client proteins such as p53, tau, ErbB2, Ask1, Foxo1, and myocardin for the ubiquitin-mediated degradation [8][9][10][11][12][13] . Recently, CHIP was reported to play a critical role in cardioprotection during oxidative stress 14 . CHIP deficient mice result in markedly increased apoptosis in cardiomyocytes and endothelial cells after infarction injury 15 . In contrast, overexpression of CHIP inhibits ASK1-and p53-mediated apoptosis in cardiomyocytes and other cells 10,16,17 . However, the physiological in vivo role of CHIP overexpression in DOX-induced cardiac injury has not yet been investigated.
On the basis of previous findings, we therefore postulated that increased CHIP levels would ameliorate DOX-induced cardiotoxicity. To test this hypothesis, wild-type (WT) and CHIP transgenic mice (CHIP-TG) were administered with a single dose of DOX (20 mg/kg; i.p.) for 5 or 10 days. Cardiac function, histologic aspects, cytokine production, apoptosis, oxidative stress and survival were examined. Here we showed that cardiac-specific CHIP expression significantly improved cardiac function and prolonged survival in vivo by blocking DOX-induced apoptosis, inflammation and oxidative stress. The cardioprotective effects of CHIP against DOX toxicity were associated with ubiquitin-mediated degradation of p53, SHP-1 and preserved activation of ERK1/2 and STAT3 signaling pathways. These results suggest that CHIP may be a potential therapeutic target for the treatment of DOX-induced heart failure.

Results
DOX downregulates the expression of CHIP in neonatal rat cardiomyocytes and in the mouse heart. To determine the functional role of cardiac CHIP in response to DOX treatment, we first examined the expression of CHIP, HSP70 and HSP90 in neonatal rat cardiomyocytes with different doses of DOX as indicated. Western blot analysis showed that DOX treatment markedly decreased CHIP expression in a dose-dependent manner, whereas no significant change in HSP70 and Hsp90 expression was observed (see Supplementary Fig. S1a). Moreover, injection of DOX (20 mg/kg; i.p.) in mice significantly decreased CHIP expression compared with control mice (see Supplementary Fig. S1b,c). These results indicate that CHIP expression is reduced in cardiomyocytes in response to DOX.

Overexpression of CHIP improves DOX-induced cardiac dysfunction and mortality of mice.
To evaluate whether increased CHIP expression protects against DOX-induced cardiac dysfunction, CHIP-TG mice were injected with a single dose of DOX (20 mg/kg) for one time. Five days after DOX injection, cardiac function was evaluated by echocardiography. Figure 1a showed representative echocardiograms after vehicle or DOX administration in WT and CHIP-TG mice (Fig. 1a). WT mice were found to exhibit a significant decrease of left ventricular posterior wall thickness at end-diastole (LVPWD) and left ventricular posterior wall thickness at end-systole (LVPWS), a markedly increase of the left ventricular end diastolic dimensions (LVEDD) and left ventricular end systolic dimensions (LVESD), whereas these alterations were markedly restored in CHIP-TG mice ( Fig. 1b-e). Moreover, DOX treatment caused a pronounced reduction in cardiac contractility reflected by fractional shortening (FS%) and ejection fraction (EF%) in WT mice, and this effect was significantly attenuated in CHIP-TG mice (Fig. 1f,g). In addition, the survival rate was significantly higher in CHIP-TG than in WT mice after DOX treatment (40% versus 10%) (Fig. 1h). No death was observed in saline-treated groups (Fig. 1h). Together, these results indicate that overexpression of CHIP ameliorates cardiac functional deterioration and survival of mice in response to DOX.
Cardiac CHIP overexpression protects DOX-induced cardiac injury, apoptosis, atrophy, inflammation and oxidative stress in mice. To test whether CHIP overexpression inhibits DOX-induced cardiac injury and apoptosis in vivo, we first examined heart section with H&E staining and TUNEL assay. DOX-treated WT mice exhibited marked focal cytoplasmic vacuolization (~31.7%), a hallmark of cardiomyocyte injury, which is consistent with previous reports 18 , whereas this effect was significantly reduced in CHIP-TG mice (~11.7%) (Fig. 2a). TUNEL-positive cardiomyocytes were barely detectable in the heart of mice with saline injection. DOX injection significantly increased the number of TUNEL-positive cardiomyocytes in WT mice (~20.9%). Conversely, this change was markedly attenuated in CHIP-TG mice (~11.4%) (Fig. 2b). In addition, DOX treatment resulted in a significant increase in Bax/Bcl-2 ratio in WT mice but not in CHIP-TG mice (Fig. 2c). Furthermore, we found that DOX injection significantly reduced the wall thickness and increase the diameter of ventricular. The ratio of heart weight/body weight (HW/BW) and cross-sectional area of cardiomyocytes were also decreased compared with saline group, indicating a ventricular dilation after DOX treatment. In contrast, these changes were reversed in CHIP-TG mice (Fig. 3a,b). Five days after DOX injection, the number of Mac-2-positive macrophages and the expression levels of IL-1β , IL-6 and TNF-α mRNA were significantly decreased in CHIP-TG hearts compared with WT hearts (Fig. 3c,d). In addition, malondialdehyde (MDA) level was significantly decreased but glutathione peroxidase (GPx) activity was markedly increased in CHIP-TG mice compared with WT mice (Fig. 3e,f). There was no difference in the number of infiltrated macrophages, the levels of the cytokines, the MDA level and GPx activity between two group hearts after saline injection ( Fig. 3c-f).
Knockdown of CHIP by siRNA increases DOX-induced apoptosis, inflammatory response and oxidative stress in neonatal rat cardiomyocytes. To further confirm the role of CHIP knockdown in DOX-triggered cardiac injury, the expression of endogenous CHIP was reduced in neonatal rat cardiomyocytes by infection of adenovirus siRNA-CHIP or siRNA-control. The infection efficiency reached more than 95% after 24 hours. Cardiomyocyte viability was significantly lower and the number of TUNEL-positive cell was higher in siRNA-CHIP-infected group than siRNA-control (Fig. 4a,b). qPCR analysis revealed that DOX treatment resulted in an increase in the Bax/Bcl-2 ratio, the expression of pro-inflammatory cytokines (IL-1β , IL-6 and TNF-α ) and the MDA level and a decreased in the GPx activity in siRNA-control than that in untreated group, and these Scientific RepoRts | 6:28399 | DOI: 10.1038/srep28399 effects were further enhanced in siRNA-CHIP-infected cells in response to DOX (Fig. 4c-f). No difference in these alteration was observed under the basal condition (Fig. 4).
CHIP overexpression attenuates DOX-induced cardiac injury by regulating multiple mechanisms in the heart. To investigate the molecular events of cardiac contraction improvement in CHIP-TG mice after DOX injection, we performed microarray assay to examine the effects of CHIP overexpression on the global gene expression profile of hearts after saline and DOX injection. We found that cardiac CHIP overexpression resulted in significant regulation of 1938 genes compared with WT mice after DOX-treatment. Among them, 657 genes were significantly upregulated, and 1281 genes markedly downregulated. The hierarchical clustering analysis showed differentially expressed genes in the four groups (Fig. 5a). We then analyzed the functional bias of these differentially expressed genes according to Gene Ontology (GO) classifications. A total of 511 GO items associated with up-regulated genes and 477 GO items associated with down-regulated genes were significantly altered between CHIP-TG and WT mice after DOX injection. Moreover, the majority of enriched GO terms among the differentially expressed genes were associated with apoptosis, immune/inflammation, cell growth  Table 1). Some of the significantly up-regulated genes such as Cyp2b10, Mthfd2, Postn, Col3a1, Btg2, Igf1, and down-regulated genes such as Cyr61 and Lcn2 were also verified by qPCR analysis (Fig. 5c,d). Furthermore, analysis of pathways of genes with significantly differences between CHIP-TG and WT  mice showed that 113 pathways were significantly up-regulated and 140 pathways were down-regulated, which are mainly involved in regulating metabolic pathways, cytokine-receptor interaction, p53, insulin signaling, mTOR, MAPKs, and ubiquitin-mediated proteolysis, etc. (see Supplementary Fig. S3).
Effect of CHIP on the p53, ERK and STAT3 signaling pathways. To further determine which signaling pathways were involved in the possible mechanism of CHIP cardioprotection against DOX, we selectively assessed the activation of several major signaling pathways, including p53, IGF1R/AKT, MAPKs (ERK, JNK) and gp130/STAT3, which play important roles in regulating cardiac hypertrophy, myocyte apoptosis, inflammation, and oxidative stress 19 . We found that DOX-treatment did not significantly reduce the protein levels of receptors (e,f) MDA levels and GPx activities were measured in cardiomyocytes homogenates from siRNA-control and siRNA-CHIP infected groups. * P < 0.05 versus siRNA-control + saline. # P < 0.05 versus siRNA-control + DOX.
IGF1R and gp130, but markedly increased the levels of p53 and phosphorylated JNK1/2, decreased the levels of phosphorylated AKT, ERK1/2, Jak and STAT3 in WT hearts compared with saline-treated group, whereas the levels of p53 and phosphorylated ERK1/2, Jak and STAT3 rather than phosphorylated AKT and JNK1/2 were significantly reversed in CHIP-TG mice ( Fig. 6a and Supplemental Fig. S2a,b). In contrast, knockdown of CHIP by siRNA markedly increased the level of p53 protein and decreased ERK1/2 and STAT3 phosphorylation in neonatal rat cardio-myocytes compared with DOX-treated siRNA-control (see Supplementary Fig. S2c). These results suggest that the protective effect of CHIP on DOX-induced cardiotoxicity was partially mediated by p53, ERK1/2 and STAT3 signaling pathways.

CHIP promotes ubiquitin-mediated degradation of SHP-1. Previous study have proved that CHIP
was responsible for p53 degradation in the heart 20 . To investigated how CHIP mediates activation of Jak/STAT3 and ERK1/2, we first assessed whether CHIP affected the tyrosine phosphatase SHP-1 that was known to suppress Jak/STAT3 and ERK1/2 pathways in H9c2 cells 21,22 . Our co-immunoprecipitation assays revealed that SHP-1 were precipitated by antibody against CHIP, but not by control rabbit IgG (Fig. 6b), indicating that CHIP directly interacted with SHP-1 in cardiomyocytes. We then sought to determine whether CHIP downregulates SHP-1 in heart tissues, and found that the SHP-1 protein level was significantly increased in DOX-treated wild-type mice, whereas this effect was abrogated in CHIP-Tg hearts (Fig. 6a). To establish whether CHIP mediated SHP-1 ubiquitylation, we immuneprecipitated SHP-1 from Ad-GFP-or Ad-CHIP-infected H9c2 cells and immunoblotted for ubiquitylated species. We found that increased expression of CHIP significantly increased SHP-1 ubiquitylation compared with Ad-GFP control (Fig. 6c). To further determine whether CHIP promotes SHP-1 degradation through proteasomes, we used proteasome inhibitor MG132 to treat H9c2 cells. Consistent with the results from animal experiments, overexpression of CHIP also significantly decreased the protein levels of SHP-1 compared with Ad-GFP control (Fig. 6d, lane 2 vs 1), and this effect was markedly reversed MG132 (Fig. 6d, lane 4 vs 2),  indicating that CHIP targets SHP-1 protein for proteasome degradation. To further study the crucial role of CHIP in regulating SHP-1 degradation and activation of Jak/STAT3 and ERK1/2 pathways, H9c2 cells were transfected with siRNA-CHIP, siRNA-SHP-1 or siRNA control. We found that knockdown of CHIP significantly upregulated SPH1 protein level but decreased the phosphorylation of Jak, STAT3 and ERK1/2 (Fig. 6e, lane 2 vs 1), whereas depletion of SHP-1 markedly reversed this effect (Fig. 6e, lane 3 vs 2), indicating that CHIP promotes Jak/STAT3 and ERK1/2 activation through degradation of SHP-1.

Discussion
CHIP, as a chaperone and ubiquitin E3 ligase, has been widely explored in protection against ischemic injury and other stress stimuli 7,10,13-15,17,18 . However, its possible protective effects on DOX-induced cardio-toxicity and underlying mechanisms are not well defined. In this report, we provide the first evidence that CHIP in vivo and in vitro protects against DOX-induced cardiac apoptosis, atrophy, inflammatory and oxidative stress resulting in prevention of cardiac dysfunction and improvement of mouse survival. These effects were associated with alteration of multiple signaling pathways, including decreased p53 and increased activation of ERK1/2 and STAT3 signaling pathways. DOX is one of the most important anticancer agents. However, clinical use of DOX is limited by its cardiotoxicity [1][2][3] . Although the precise mechanisms whereby DOX induces myocardial injury have not been fully elucidated, it is widely accepted that the DOX induces cardiac injury via several mechanisms, including activation of ubiquitin-proteasome system, sarcomere reorganization, induction of proinflammatory cytokines, free radical generation and apoptotic cell death that are the typical changes in DOX-induced heart failure 23 . Several recent findings indicate that cardiomyocyte apoptosis is a leading cause of cardiac dysfunction in DOX-induced cardiomyopathy 23 . DOX evokes oxidative stress and expression of pro-apoptotic protein p53 and Bax, which activates apoptotic signaling leading to cardiomyocyte apoptosis in the heart and in isolated cardiomyocytes 3,24,25 . Moreover, overexpression of antioxidant genes such as manganese superoxide dismutase and catalase in the heart protects mice against DOX-induced heart failure 26,27 . CHIP can activate HSF1 and protect against apoptosis and cellular stress 14 . CHIP also promotes ASK1 degradation leading to inhibition of cell apoptosis 10 . In contrast, CHIP knockout aggregates ischemia/reperfusion-induced myocardial apoptosis and dysfunction 15,16 . To seek evidence of the protective effect of CHIP in DOX-induced apoptosis, we conducted a series of TUNEL assays, analyses of Bax and Bcl-2 expression and measurement of oxidative stress in vivo and in vitro. Our results demonstrated that increased CHIP expression markedly preserved cardiac dysfunction and mouse survival (Fig. 1), effectively decreased TUNEL-positive cardiomyocytes, Bax/Bcl-2 ratio, MDA level but enhanced the activity of antioxidant enzyme GPx (Figs 2 and 3). These results suggest that CHIP may play a critical role in protecting cardiomyocyte apoptosis evoked by DOX partially through inhibition of oxidative stress. . Quantitative analysis of relative protein levels (right). (e) Western blot analysis of protein levels of SHP-1, p-Jak/Jak, p-STAT3/STAT3 and p-ERK1/2/ERK1/2 in H9c2 cells after transfected with siRNA-control, siRNA-CHIP or siRNA-SHP-1 (left). Quantitative analysis of relative protein levels (right). * P < 0.05, * * P < 0.01, * * * P < 0.001 versus Ad-GFP/siRNA-control; # P < 0.05, ### P < 0.001 versus Ad-GFP + MG132/siRNA-CHIP. Studies have demonstrated that DOX induces cardiac dysfunction, which was accompanied by marked cardiac atrophy 28,29 and infiltration of inflammatory cells 30 . It has been reported that DOX can lead to cardiac atrophy 30,31 , which was also confirmed in our study (Fig. 3a,b). Our new finding is that cardiac-specific overexpression of CHIP exerts an anti-atrophic effect on heart caused by DOX (Fig. 3a,b). Oxidative stress can directly trigger cytokine expression, which was markedly increased in the early time and 5 days after DOX injection 32 . Recently, our results indicate that CHIP is involved in Ang II-induced expression of pro-inflammatory cytokines through inactivation of NF-κ B in the mouse heart and neonatal rat cardiomyocytes 18 . Consistent with these observations, the present results showed that DOX-induced accumulation of macrophages and the expression of pro-inflammatory cytokines such as IL-1β , IL-6 and TNF-α were markedly suppressed in CHIP-TG mice (Fig. 3c,d). In contrast, knockdown of CHIP had opposite effect (Fig. 4).
Cardiac injury including apoptosis, atrophy, inflammation and oxidative stress induced by DOX ultimately leads to cardiomyopathy and congestive heart failure 1 . The mechanisms leading to DOX-induced myocardial damage may involve multiple signaling pathways, including p53, IGF1R/AKT, NF-kB, MAPKs (ERK, JNK), and gp130/Jak/STAT3 23 . For example, the p53 null mice show reduced cardiomyocyte apoptosis and concomitant improvements in cardiac function 33 . Inhibition of AKT and ERK pathways is associated with DOX-induced cardiotoxicity, which is prevented by the administration of cardio-protective reagents, such as oleylethanolamide, dexrazoxane, granulocyte colony-stimulating factor (G-CSF) that increased ERK1/2 activity 5,19,34 . Moreover, several studies have demonstrated that CHIP interacts with HSP90 and can regulate both ERK1/2 and Jak/STATs pathways in various cell types [35][36][37][38][39][40][41] . To further explore the protective mechanisms of CHIP in DOX-induced cardiac injury, we first performed microarray assay, and identified several signaling pathways were associated with cardiac injury, including ECM-receptor interaction, cytokine-receptor interaction, p53, insulin signaling, MAPKs, and ubiquitin-mediated proteolysis (Supplementary Fig. S3). Western blot further demonstrated that CHIP did not affect IGF1R/AKT, JNK1/2, gp130 ( Supplementary Fig. S2A,B), but can interacted with and promoted SHP-1 degradation by proteasome thereby leading to activation of Jak/STAT3 and ERK1/2 (Fig. 6). A study has demonstrated that CHIP plays a role in mediating p53 degradation in the ischemic heart 20 . Gp130/ Jak/STAT3 signaling pathway also participates in cardiac injury. Overexpression of STAT3 in the heart protects against DOX-induced cardiomyopathy, thus resulting in an improved survival rate by preventing progression of heart failure 42 . SHP-1 is known to serve as an important phosphatase of the Jak/STAT signaling pathway 21,43,44 . Loss of SHP-1 enhances the stability of Jak3 and activates Jak3/STAT3 signaling 21 . In addition, SHP-1 also interacts with ERK1/2 and negatively regulated MAPK/ERK1/2 signaling pathway 22,45,46 . Together, above data indicate that CHIP protects against DOX-induced cardiac injury at least in part through ubiquitin-mediated degradation of p53 and SHP-1, and activation of Jak/STAT3 and ERK1/2 pathways.
In conclusion, this study demonstrates that cardiac-specific overexpression of CHIP ameliorated DOX-induced cardiac injury and dysfunction. The mechanisms underlying its protection at least in part were associated with the attenuation of DOX-triggered oxidative stress, the stability of p53 and SHP-1 proteins, and the activation of ERK1/2 and Jak/STAT3 signaling pathways, leading to inhibition of DOX-induced cardiomyocyte apoptosis, atrophy and inflammation. Consequently, left ventricular contractile function and mouse survival was improved after DOX injury. Thus, increased CHIP expression might be a promising therapeutic target for the treatment of DOX-triggered cardiac injury and heart failure.

Materials and Methods
Antibodies and reagents. Dulbecco's modified Eagle's medium, medium supplements, and fetal bovine serum was purchased from Invitrogen (Carlsbad, CA). Doxorubicin (DOX) was obtained from Sigma-Aldrich. Antibodies were purchased from Cell Signaling Technology (Beverly, MA) and (Abcam, Inc. (Cambridge, UK) (Supplemental Materials and methods).
Animals and treatments. Cardiac-specific CHIP transgenic mice were created as described previously 18 .
CHIP transgenic (TG) mice (male, C57BL/6, 10-12-week-old) and the wild-type (WT) littermates and were randomly assigned to either the control group or the DOX-treated group. DOX was dissolved in saline and administered by intra-peritoneal injection (i.p) at a single dose of 20 mg/kg for one time 48 . Five days after DOX injection, we sacrificed mice and performed additional experiments. Control mice received injections of saline of comparable volume. Hearts were removed from mice anesthetized with a mixture of 80 mg/kg ketamine and 30 mg/kg xylazine intra-peritoneally. Histological and immunohistochemical analysis. Histological analyses of hearts from WT and CHIP-TG mice were performed according to standard protocols. Heart sections were stained with hematoxylin and eosin (H&E), Masson's trichrome and wheat germ agglutinin-TRITC conjugate as described previously 48 . Macrophage populations in the heart sections were detected by with anti-Mac-2 antibody or isotype control.
Cell viability and TUNEL assay. Cell viability was determined by Trypan blue exclusion assay as described [48][49][50] . Cardiomyocte apoptosis was evaluated by the Dead End TM Fluorometric TUNEL System (Promega) according to manufacturer's instructions 51 . In the heart sections, cardiomyocytes were identified with α -actinin immunostaining, and nuclei were counterstained with DAPI. The percentage of TUNEL-positive cardiomyocytes was determined by counting 10 random fields per section under a microscope (magnification, x 400).

RNA analysis.
Hearts from WT and CHIP-TG mice were excised, rinsed in PBS, frozen in liquid nitrogen.

Measurement of maleic dialdehyde and glutathione peroxidase.
Maleic dialdehyde (MDA) as indicator of lipid peroxidation 52 was measured using the commercially available colorimetric assay kit (Nanjing Jiancheng Bioengineering Institute, China). Antioxidant enzyme was measured by Glutathione Peroxidase (GPx) Assay Kit (Calbiochem). Detailed operation procedure according to manufacturer's instructions as described previously 48 .
Microarray gene expression analysis. Total RNA was extracted with Trizol reagent (Invitrogen) from the hearts of saline and DOX-treated WT and CHIP TG mice (n = 3 per group) at day 5 after injection. The microarray assay was performed as described previously 51,53,54 . The bioinformatics of Gene Ontology (GO) and Pathway analysis were performed by using the Capital Bio Molecule Annotation System plate analysis as described before 55 . The gene expression data are available at the Gene Expression Omnibus (GEO) website: http:// www.ncbi.nlm.nih.gov/geo/ (accession number GSE59672) (Supplemental Materials and methods).
Western blot analysis. Protein samples were extracted from neonatal rat cardiomyocytes, or heart tissues which are collected on the fifth day after DOX injection. Western blot analyses were performed using indicated primary antibodies as described previously 18

. (Supplemental Materials and methods).
Immunoprecipitation. Immunoprecipitations were performed as described before 47 . Briefly, H9c2 cells were maintained in 10% DMEM complete medium. Cell lysates were immunoprecipitated with anti-CHIP or anti-IgG antibody for 2 h at 4 °C, then beads were washed and analyzed by immunoblotting with anti-SHP-1 and anti-CHIP antibodies.
Ubiquitination Assays. H9c2 cells were transfected with Ad-GFP and Ad-CHIP adenovirus for 24 h in serum-free medium. Then treated with 0.5 μ M DOX/saline for 24 h in 10% DMEM complete medium. Cell lysates were immunoprecipitated with anti-SHP-1 antibody, and analyzed by immunoblotting using anti-ubiquitin, anti-SHP-1 and anti-CHIP antibodies as previously described 47 . Statistical analysis. Data are presented as means ± SEM. Differences between WT and CHIP-TG mice were evaluated for statistical significance using Student's t test or by two-way ANOVA. Survival curves after DOX injection (20 mg/kg; i.p.) were created in WT (n = 20) and CHIP-TG (n = 20) mice by the Kaplan-Meier method and the log-rank test. The two-side Fisher's exact test and χ 2 test were used to classify the GO and Pathway category, and FDR was calculated to correct the P-value. A P < 0.05 was regarded as significant.