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Exercise preconditioning-induced late phase of cardioprotection against exhaustive exercise: possible role of protein kinase C delta

The Journal of Physiological Sciences volume 64, pages 333–345 (2014)Cite this article

Abstract

The objective of this study was to investigate the late cardiac effect of exercise preconditioning (EP) on the exhaustive exercise-induced myocardial injury in rats and the role of protein kinase C (PKC) in EP. Rats were subjected to a run on the treadmill for four periods of 10 min each at 30 m/min with intervening periods of rest of 10 min as an EP protocol. The exhaustive exercise was performed 24 h after EP. PKC inhibitor chelerythrine (CHE) was injected before EP. The results showed that EP increased the running ability of rats, and alleviated the exhaustive exercise-induced injury in cardiomyocytes, but pretreatment with PKC inhibitor CHE did not abolish the late phase cardioprotection of EP. A significant increase of PKCδ, both at the protein level and the mRNA level in the left ventricular myocardium of rats, accompanied by its activated form (phosphorylated on Thr507, p-PKCδThr507) translocated to intercalated disks and was found in the late phase of EP. This circumstance was not attenuated by CHE. These results suggested that a high level of PKCδ might be involved in cardioprotection against myocardial damage, but if activated PKCδ at reperfusion took on a key role in cardioprotection was still an outstanding question.

Introduction

The cardiovascular benefits of regular physical exercise have been well documented. However, more and more evidence showed that impairments in myocardial function and elevations in highly specific cardiac biomarkers after prolonged exercise, suggested that strenuous physical exertion might also result in potential subclinical myocardial damage [1–4]. Therefore, developing a pragmatic countermeasure to reduce strenuous physical exercise-induced potential myocardial injury is vital [5, 6]. Recent studies demonstrated that, exercise preconditioning (EP), a single bout of moderately intense exercise, can render the heart more tolerant to a subsequent ischemic insult [7]. EP has two cardioprotective phases: an early phase, which occurs immediately after the exercise [8, 9], and a late phase, which peaks at 24 h postexercise. It has been proposed that the protective effect is initiated by activation of intracellular signaling pathways similar to those induced by ischemic preconditioning [10, 11]. Several studies suggest that protein kinase C (PKC) activation plays an important role in the mechanism of EP [12–15]. The PKC family of isozymes has three major subgroups: the conventional calcium dependent (α, βI, βII, and γ), the novel calcium independent (δ, ϵ, η, θ, and possibly μ), and the atypical PKCs (ζ and τ/λ). Translocation of PKC isoform (s) from the cytosolic to the particulate fraction results in it binding to a specific substrate [16], and phosphorylation of a threonine residue in the PKC activation loop has an essential role in the maturation of this enzyme [17]. Translocated and specific PKC isoforms during myocardial ischemia are believed to participate in several functions including the opening of mitoKATP channels or the induction of gene expression, which finally afford a cardioprotective phenotype [18, 19].

In our previous study, we have shown that EP attenuated the exhaustive exercise-induced myocardial injury in rats in the early phase. Moreover, p-PKCδThr507 in EP cardiomyocytes was primarily localized to intercalated disks and nuclei. This indicated that an appropriate activation and translocation of PKCδ may represent a mechanism whereby EP induced an early phase of cardioprotection [20]. However, whether EP can exert the same cardioprotective effect on exhaustive exercise-induced myocardial injury in the late phase is unknown. In the present study, we observed the cardioprotective effect of EP against exhaustive exercise-induced myocardial injury in the late phase according to the evaluation of serum cTnI, serum MDA, and myocardial HBFP staining. In addition, we observed the phosphorylation and translocation of PKCδ and PKCδ mRNA expression according to the immunohistochemistry, western blotting, in situ hybridization, and quantitative real-time PCR. In order to confirm the role of PKCδ in the late phase of EP, a PKC inhibitor chelerythrine (CHE) was used.

Methods

Animals

Adult (8-week-old) male Sprague–Dawley rats (Sippr BK, Shanghai, China) were housed in standard cages maintained at constant temperature and humidity with a 12:12 h lightdark cycle and were fed and watered ad libitum. All animal care and experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and approved by the Ethics Committee for Science Research of the Shanghai University of Sport.

Experimental protocol

All animals underwent a light exercise familiarization on the treadmill for five consecutive days. The velocity on the treadmill was 15 m/min and the exercise duration was 10–20 min/day. On the 7th day, animals were randomly assigned to five experimental groups:

  • Group C Control; rats were placed on the treadmill without belt movement.

  • Group EE Exhaustive exercise; rats ran to exhaustion on the treadmill (0 % grade) at a speed of 30 m/min and were sacrificed 0.5 h after the exhaustion. Exhaustion was defined as the rat being unable to upright itself when placed on its back

  • Group LEP Late exercise preconditioning; rats were allowed to run on the treadmill (0 % grade) for four periods of 10 min each at 30 m/min with intervening periods of rest of 10 min, and were sacrificed 24 h after exercise. Exercise began and ended with 5 min “warm up” and “cool down” periods at 15 m/min

  • Group LEP + EE Late exercise preconditioning plus exhaustive exercise; rats were treated as group LEP, but ran to exhaustion 24 h after exercise, and were sacrificed 0.5 h after the exhaustion

  • Group CHE + LEP + EE CHE plus late exercise preconditioning plus exhaustive exercise; rats were treated as group LEP + EE, but CHE was injected intraperitoneally 10 min before exercise

Animals were anesthetized with trichloroacetaldehyde monohydrate (400 mg/kg, intraperitoneally), and samples were taken as previously described [20]. Briefly, after the blood was drawn from the inferior caval vein, the heart was exposed for perfusion fixation for histology analysis, or rapidly excised for western blot and quantitative real-time PCR analysis.

In this study, we shared the same data sets in control (C) and exhaustive exercise (EE) group with our previous study [20], our previous study described the major finding of our systematic researches on the early cardiac effect of EP against myocardial injury.

Detection of serum cardiac biomarkers

Blood samples were centrifuged immediately after collection and the serum was separated. Serum cTnI levels were measured by automated immunochemiluminescence on an Access 2 immunoassay system (Beckman Coulter, USA). This assay is based on a single step sandwich principle with paramagnetic particles coated as the solid phase and two monoclonal cTnI antibodies [21]. The sensitivity threshold for cTnI was 0.01 g/L.

Using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), the serum MDA levels were measured with a rat MDA ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Hematoxylin–basic fuchsin–picric acid (HBFP) staining

After the heart was postfixed, embedded in paraffin, sectioned and mounted, HBFP staining was performed as previously described [22]. The ischemic/hypoxic myocardial fibers stain a crimson red color under the light microscope (Olympus, Tokyo, Japan). For the histological analysis, five visual fields from each section, with five sections per group, totaling 25 visual fields, were randomly taken using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). The positive area and the integral optical density (IOD) were calculated.

Immunohistochemistry

For immunohistochemistry [20], sections were deparaffinized and rehydrated, immersed in hydrogen peroxide to block the endogenous peroxidase activity, and then digested with pepsin for antigen retrieval. After unspecific binding was blocked with serum, sections were incubated with primary antibodies specific to PKCδ or p-PKCδThr507 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted at 1:200 overnight at 4° C followed by detection using the strept actividin-biotin complex kit (Boster Bio-engineering, Wuhan, China) with diaminobenzidine/peroxidase substrate to produce a brown-colored signal, then counterstained with hematoxylin.

In situ hybridization

In situ hybridization was performed according to previously described approaches [23–25], and an in situ hybridization detection kit for PKCδ mRNA (Boster Bio-engineering, Wuhan, China) was used. Briefly, sections were deparaffinized and rehydrated, immersed in hydrogen peroxide and digested with pepsin, incubated with a prehybridization solution, and then incubated with the hybridization mixture containing digoxigenin-labelled oligonucleotide probe at 42° C for 48 h. The following probe sequences for PKCδ mRNA (Accession number NM_133307.1) detection were used (1:1:1 mixture): 5′-TGG AA G TCA ACA TTC GAC GCC CAC ATC TAT GAA GG-3′ (Location of nucleotides: 525–557), 5′-GAC ATG CCT CAC CGA TTC AAG GTC TAT AAC TAC AT-3′ (Location of nucleotides: 1044-1076), and 5′-AGG CTT CAC CCC TTT TTC AAG ACT ATC AAC TGG AA-3′ (Location of nucleotides: 2148-2180). Following hybridization, the sections were washed in standard saline citrate (SSC), incubated with a solution to block nonspecific binding sites, and then strept actividin-biotin complex/diaminobenzidine detection procedures were performed resulting in a brown staining product. As a negative control, the probe was omitted.

Western blotting

Western blotting was performed with a SDS-PAGE Electrophoresis System as described previously [20]. Briefly, 50 μg protein samples were resuspended in a reduced sample buffer, and then electrophoresed on 10 % SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes. They were then sequentially probed with primary antibodies against PKCδ or p-PKCδThr507 (1:3,000 dilution; Santa Cruze Biotechnology, Santa Cruz, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase antibody was used to normalize the loading. After washing, membranes were incubated with secondary horseradish peroxidase-conjugated antibodies. Results were visualized with the enhanced chemiluminescence method and evaluated by ImageJ software (NIH, Bethesda, MD, USA).

Quantitative real-time PCR

Total RNA was isolated using acid guanidinium isothiocyanate-phenol–chloroform extraction and ethanol precipitation [26]. RNA concentration and purity were determined using spectrophotometry, and the integrity was examined by agarose gel electrophoresis. Lack of genomic DNA contamination was confirmed by PCR amplification of RNA samples followed by gel electrophoresis analysis. First strand cDNA was synthesized for 60 min at 50° C in a 20 µl reaction containing total RNA, Oligo [27]18, dNTPs, 5 × first strand buffer, dithiothreitol (DDT), RNase inhibitor, Superscript III reverse transcriptase (Invitrogen, Carlsbad, USA) and PCR-grade water. The qRT-PCR was performed on an ABI 7,900 Realtime PCR system (Applied Biosystems, CA, USA) using SYBR Green I as previously described [28]. GAPDH was used as an internal reference. Primers used were: PKCδ, 5′-GAA GGA GGC ACT CAC CAC AGA-3′ (forward) and 5′-TGC AGG TCC AGC CAG AAC T-3′ (reverse); GAPDH, 5′-GGA AAG CTG TGG CGT GAT-3′ (forward) and 5′-AAG GTG GAA GAA TGG GAG TT-3′ (reverse). Reactions contained 2 µl cDNA, 0.5 µl of each of the 10 µM forward and reverse primers, 1 µl dNTPs, 0.6 µl MgCl2, 0.2 µl 2 × ROX Reference Dye, 1 µl 10 × PCR buffer, FastStart Taq DNA Polymerase, SYBR Green I, and PCR-grade water in a final volume of 10 µl. The protocol was initiated by incubation at 95° C for 3 min followed by 40 cycles (15 s at 95° C, 20 s at 59° C and 20 s at 72° C). Melting curve analysis after the final cycle confirmed the presence of a single PCR product. The mRNA content of PKCδ and GAPDH was calculated from the cycle threshold values by using a standard curve constructed from a serial dilution of aliquots of cDNA pooled from all the samples [29], and the relative amount of PKCδ mRNA was normalized to GAPDH.

Statistical analysis

All the results are expressed as mean ± SD, and values were compared using a one-way analysis of variance (SPSS 10.0; SPSS, Chicago, IL). Upon confirmation of a significant main effect, individual differences were determined with post hoc analysis. A value of P < 0.05 was considered significant.

Results

Late cardiac effect of EP on exhaustive exercise

The mean distance running to exhaustion in group EE, group LEP + EE, and group CHE + LEP + EE were 2657.37 ± 975.57 m (n = 19), 4,590.00 ± 1,582.82 m (n = 20), and 4,512.00 ± 1,467.93 m (n = 20), respectively. The running distance in group LEP + EE significantly increased compared with group EE (P < 0.05), and no significant differences were found between group LEP + EE and group CHE + LEP + EE (P = NS).

HBFP staining of cardiomyocytes of the left ventricular free wall at the level of the near apex and image analysis are shown in Fig. 1a. The ischemic cardiomyocytes stained a vivid crimson red color in contrast to the light brown color of nonischemic tissue. Cardiomyocytes in group C and LEP displayed a light brown color while crimson red was seen in the majority of cases in group EE. Contrary to our hypothesis, EP aggravated the exhaustive exercise-induced ischemia and hypoxia in cardiomyocytes, resulting in markedly enhanced red staining in group LEP + EE, which was attenuated by pretreatment with CHE (group CHE + LEP + EE). The quantitative analysis (Fig. 1b) showed that the positive area and IOD of HBFP staining in group LEP + EE were significantly higher than those in group EE (69.09 ± 14.79 vs. 55.26 ± 6.91 µm2, 17,617.76 ± 3,770.28 vs. 14,092.39 ± 1,761.04, P < 0.05). Compared with group LEP + EE, the two values decreased significantly in group CHE + LEP + EE (43.65 ± 8.26 vs. 69.09 ± 14.79 µm2, 11,130.16 ± 2,107.04 vs. 17,617.76 ± 3,770.28, P < 0.05). Positive area and IOD of HBFP staining in group C and group LEP were both 0.

Fig. 1
figure 1

Late cardiac effect of EP on exhaustive exercise. a Myocardial ischemia detected by HBFP staining. Ischemic cardiomyocytes with crimson red dominated in group EE. The red staining was markedly increased in group LEP + EE (both in transection and longitudinal section) and decreased in group CHE + LEP + EE (original magnification, ×400). b Quantitative analysis of HBFP staining. The positive area and IOD of HBFP staining in group EE were significantly higher than those in group C (P < 0.05). The positive area and IOD of HBFP staining in group LEP + EE were significantly higher than those in group EE and group CHE + LEP + EE (P < 0.05). Values are mean ± SD, significant differences (P < 0.05) are indicated as follows: from group C (&), from group EE (*), and from group LEP + EE (#)

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Serum cTnI levels (Table 1) in group EE were significantly higher than those in group C (P < 0.05). Compared with group EE, serum cTnI levels in group LEP + EE decreased significantly (P < 0.05). Serum cTnI levels in group CHE + LEP + EE were lower than those in group LEP + EE; howeve, this reduction was not statistically significant (P = NS).

Table 1 Alterations of serum cTnI levels in the late cardiac effect of EP
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Serum MDA levels (Table 2) in group EE were significantly higher than those in group C (P < 0.05). Compared with group EE, serum MDA levels in group LEP + EE decreased significantly (P < 0.05). Serum MDA levels in group CHE + LEP + EE were higher than those in group LEP + EE; however, this reduction was not statistically significant (P = NS).

Table 2 Alterations of serum MDA levels in the late cardiac effect of EP
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Expression of PKCδ protein in the late cardiac effect of EP

Immunoreactivity for PKCδ was detected in cardiomyocytes from the left ventricular anterior free wall at the level of the near apex (Fig. 2a). PKCδ demonstrated a diffuse cytoplasmic staining in cardiomyocytes. Positive basal staining was noted in a diffuse cytoplasmic pattern in group C. Stronger staining has been observed in group EE, group LE, and group LEP + EE. Contrary to our hypothesis, immunoreactivity was elevated after CHE pretreatment in group CHE + LEP + EE. The semiquantitative analysis (Fig. 2b) showed that the positive area and IOD of the immunostaining in group EE and group LEP were significantly higher than those in group C (29.44 ± 12.40 and 44.55 ± 14.26 vs. 19.33 ± 11.28 µm2, P < 0.05; 7,419.66 ± 2,941.17 and 11,361.38 ± 3,637.49 vs. 4,929.62 ± 2,876.56, P < 0.05). Compared with group LEP + EE, the two values in group CHE + LEP + EE significantly increased (62.59 ± 16.65 vs. 34.60 ± 17.81 µm2, 15,961.34 ± 4,244.87 vs. 8,823.95 ± 4,541.68, P < 0.05). No significant differences were detected between group LEP + EE and group EE (34.60 ± 17.81 vs. 29.44 ± 12.40 µm2, 8,823.95 ± 4,541.68 vs. 7,419.66 ± 2,941.17, P = NS).

Fig. 2
figure 2

Alteration of PKCδ expression in the late cardiac effect of EP. a Immunohistochemistry staining of PKCδ in cardiomyocytes. PKCδ demonstrated diffuse cytoplasmic staining in cardiomyocytes in all the groups (original magnification, ×400). C–N demonstrated the negative result of none treated with PKCδ primary antibody in control group. b Semi-quantitative analysis of PKCδ immunostaining. The positive area and IOD of the immunostaining in group EE and group LEP were significantly higher than those in group C (P < 0.05). Compared with group LEP + EE, the two values in group CHE + LEP + EE significantly increased (P < 0.05). c Myocardial PKCδ levels determined by western blotting. Consistent with the results of immunostaining, total PKCδ levels in group EE and group LEP were also significantly higher than those in group C (P < 0.05). Compared with group LEP + EE, PKCδ levels in group CHE + LEP + EE significantly increased (P < 0.05). Values are mean ± SD, significant differences (P < 0.05) are indicated as follows: from group C (*), from group LEP + EE (&)

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Figure 2c shows total PKCδ levels in cardiomyocytes determined by western blotting. Total PKCδ levels in group EE (n = 9) and group LEP (n = 9) were significantly higher than those in group C (n = 9) (0.77 ± 0.18 and 0.89 ± 0.15 vs. 0.61 ± 0.12, P < 0.05). Compared with group LEP + EE, PKCδ levels in group CHE + LEP + EE (n = 9) significantly increased (1.15 ± 0.13 vs. 0.70 ± 0.14, P < 0.05). No differences were found between group LEP + EE (n = 9) and group EE (0.70 ± 0.14 vs. 0.77 ± 0.18, P = NS).

Expression of p-PKCδThr507 protein in the late cardiac effect of EP

The immunostaining of p-PKCδThr507 (Fig. 3a) demonstrated a granular, scattered cytoplasmic staining pattern in group C, and the intensity of staining was stronger in group EE. However, the pattern changed in group LEP such that p-PKCδThr507 was preferentially localized to intercalated disks (arrow), but not in group LEP + EE and group CHE + LEP + EE. The staining was almost completely lost in group LEP + EE but restored in group CHE + LEP + EE. The semiquantitative analysis (Fig. 3b) showed that the positive area and IOD in group EE were higher than those in group C, but not significantly (1.60 ± 0.74 vs. 1.28 ± 0.62 µm2, 406.89 ± 189.82 vs. 325.12 ± 158.97, P = NS). Compared with group EE, the two values in group LEP + EE were significantly decreased (0.76 ± 0.48 µm2 vs. 1.60 ± 0.74, P < 0.05; 194.30 ± 122.58 vs. 406.89 ± 189.82, P < 0.05). Compared with group LEP + EE, the two values in group CHE + LEP + EE were increased but not significantly (1.20 ± 0.82 vs. 0.76 ± 0.48 µm2, 305.33 ± 209.70 vs. 194.30 ± 122.58, P = NS). No differences were found between group LEP and group C (1.20 ± 0.54 vs. 1.28 ± 0.62 µm2, 306.29 ± 138.51 vs. 325.12 ± 158.97, P = NS). The p-PKCδThr507 semiquantitative analysis in LEP of intercalated disks showed that the positive area and IOD were 0.19 ± 0.09 µm2, 26.40 ± 12.14. The area analysis in LEP of p-PKCδThr507 localized to intercalated disks for total p-PKCδThr507 was 15.8 %.

Fig. 3
figure 3

Alteration of p-PKCδThr507 expression in the late cardiac effect of EP. a Immunohistochemistry staining of PKCδ in cardiomyocytes. p-PKCδThr507 demonstrated a granular, scattered cytoplasmic staining pattern in group C and group EE while the pattern changed in group LEP such that p-PKCδThr507 was preferentially localized to intercalated disks (arrow), but not in group LEP + EE and group CHE + LEP + EE. C–N demonstrated the negative result of none treated with p-PKCδThr507 primary antibody in the control group, original magnification, ×1,000. b Semiquantative analysis of p-PKCδThr507 immunostaining. The positive area and IOD of the immunostaining in group LEP + EE were significantly lower than those in group EE (P < 0.05). Compared with group LEP + EE, the two values in group CHE + LEP + EE were increased but not significantly (P < 0.05). c Myocardial p-PKCδThr507 levels determined by western blotting. Consistent with the results of immunostaining, total p-PKCδThr507 levels in group EE were significantly higher than those in group C (P < 0.05). However, compared with group EE, total p-PKCδThr507 levels in group LEP + EE were significantly decreased (P < 0.05). Compared with group LEP + EE, p-PKCδThr507 levels in group CHE + LEP + EE were significantly increased. Values are mean ± SD, significant differences (P < 0.05) are indicated as follows: from group C (*), from group EE (#), and from group LEP + EE (&)

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Figure 3c shows that total p-PKCδThr507 levels determined by western blot in group EE (n = 9) were significantly higher than those in group C (n = 9) (0.92 ± 0.22 vs. 0.72 ± 0.08, P < 0.05). Compared with group EE, total p-PKCδThr507 levels in group LEP + EE (n = 9) were significantly decreased (0.56 ± 0.11 vs. 0.92 ± 0.22, P < 0.05). Compared with group LEP + EE, p-PKCδThr507 levels in group CHE + LEP + EE (n = 9) were significantly increased (0.71 ± 0.08 vs. 0.56 ± 0.11, P < 0.05). No differences were found between group LEP (n = 9) and group C (0.65 ± 0.10 vs. 0.72 ± 0.08, P = NS).

The activated ratio of PKCδ

The activated ratio of PKCδ was evaluated by phosphorylated PKCδThr507 to total PKCδ (data from western blot) while the activated ratio of PKCδ in group C was 24 %, group EE 24 %, group LEP 13 %, group LEP + EE 16 %, and group CHE + LEP + EE 12 %.

Expression of PKCδ mRNA in the late cardiac effect of EP

Distribution of PKCδ mRNA by ISH was detected in cardiomyocytes from the left ventricular anterior free wall at the level of the near apex (Fig. 4a). The positive signal of PKCδ mRNA demonstrated a granular, diffuse cytoplasmic distribution pattern in cardiomyocytes. No difference was found in the intensity of positive signal between group C and group EE while a markedly enhanced signal was observed in group LEP. The signal in group LEP + EE was diminished compared with group EE. The intensity of the reaction was downregulated by CHE pretreatment in group CHE + LEP + EE. The semiquantitative analysis (Fig. 4b) showed that the area and IOD of the positive signal of PKCδ mRNA in group LEP were significantly higher than those in group C (50.39 ± 8.16 vs. 42.28 ± 5.53 µm2, P < 0.05; 12,848.98 ± 2,080.83 vs. 10,780.88 ± 1,411.32, P < 0.05). Compared with group EE, the two values in group LEP + EE significantly decreased (37.34 ± 4.70 vs. 43.42 ± 9.14 µm2, 9,520.80 ± 1,199.10 vs. 11,071.33 ± 2,330.23, P < 0.05). No significant differences were detected between group C and group EE (42.28 ± 5.53 vs. 43.42 ± 9.14 µm2, 10,780.88 ± 1,411.32 vs. 11,071.33 ± 2,330.23, P = NS), and group LEP + EE and group CHE + LEP + EE (37.34 ± 4.70 vs. 35.29 ± 4.93 µm2, 9,520.80 ± 1,199.10 vs. 8,997.90 ± 1,258.05, P = NS).

Fig. 4
figure 4

Alteration of PKCδ mRNA expression in the late cardiac effect of EP. a Distribution of PKCδ mRNA in cardiomyocytes by in situ hybridization. The positive signal of PKCδ mRNA demonstrated a granular, diffuse cytoplasmic distribution pattern in cardiomyocytes. The intensity of the hybridization signal was markedly strong in group LEP. C–N, the negative result of none treated with PKCδ mRNA probes in the control group. b Semiquantative analysis of the positive signal of PKCδ mRNA. The area and IOD of the positive signal of PKCδ mRNA in group LEP were significantly higher than those in group C. The c PKCδ mRNA levels determined by quantitative real-time PCR. PKCδ mRNA levels in group LEP were significantly higher than those in group C (P < 0.05). Values are mean ± SD, significant differences (P < 0.05) are indicated as follows: from group C (*), from group EE (#)

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Figure 4c shows PKCδ mRNA levels in cardiomyocytes determined by quantitative real-time PCR. PKCδ mRNA levels in group LEP (n = 8) were significantly higher than those in group C (n = 8) (3.65 ± 1.93 vs. 2.55 ± 0.63 × 10−3, P < 0.05). No differences were found between group EE (n = 9) and group C (2.45 ± 0.58 vs. 2.55 ± 0.63 × 10−3, P = NS), group LEP + EE (n = 9) and group EE (2.96 ± 0.51 vs. 2.45 ± 0.58 × 10−3, P = NS), group CHE + LEP + EE (n = 8) and group LEP + EE (2.67 ± 1.06 vs. 2.96 ± 0.51 × 10−3, P = NS).

Discussion

The key findings of this study can be summarized as follows: 1) EP significantly increased the running distance to exhaustion of rats, and alleviated the exhaustive exercise-induced injury in cardiomyocytes, but pretreatment with PKC inhibitor CHE did not abolish the late phase cardioprotection of EP. 2) After 24 h of EP, PKCδ was found to be markedly upregulated in left ventricular myocardium of rats. During the late phase cardioprotection of EP, p-PKCδThr507 was translocated to intercalated disks. Even though PKCδ was markedly upregulated during exhaustive exercise, but p-PKCδThr507 was mainly distributed in the cytoplasm. CHE injection before EP did not suppress the activation or translocation of PKCδ.

To our surprise, EP in the late phase markedly enhanced the exercise performance, which was usually improved by weeks or months of training. Several investigators have demonstrated that EP in the late phase significantly decreased infarct size induced by ischemia/reperfusion in animals, Domenech et al., had dogs take treadmill training after 1 h coronary artery ligation and 4.5 h reperfused. They found that exercise prior to a coronary occlusion induced early and late preconditioning of the infarct size, the early effect was mediated through mitochondrial ATP-sensitive potassium channels [8]. Yamashita et al., used a rat model to determine whether exercise can reduce ischemic injury to the heart and elucidate a mechanism for the cardioprotective effect of exercise. Results showed that exercise significantly reduced the magnitude of a myocardial infarction in a biphasic manner. The time course for cardioprotection resembled that of the change in manganese superoxide dismutase (Mn-SOD) activity [30]. Then Yamashita et al., had rats exerciseon a treadmill for 30 min at 23–27 m/min, then 24 h later, hearts were Langendorff perfused and subjected to 35 min left main coronary artery occlusion followed by 120 min reperfusion. They found that infarct size was reduced to 17.3 from 48.4. To examine the potential signalling pathway, and determine protein kinase C activation during exercise appears to be an important signal mediator of this protective response. Melling et al., found that that selected isoforms of PKC played an important role in exercise-mediated protection of the myocardium during ischemia–reperfusion injury [12]. Jimenez et al., investigated acute (swimming) exercise, and found it was sufficient such that sustained beneficial effects against cardiac functional decline were observed after high-dose isoproterenol administration [31]. In our previous study, EP in the late phase markedly attenuated the isoproterenol-induced myocardial injury in rats [22]. Studies also revealed that the underlying mechanisms might be involved in the increased manganese superoxide dismutase (Mn-SOD) activity and heat shock protein 70 (HSP70) expression in the late phase of EP [13, 30].

In this experiment, serum cTnI was chosen for evaluation of myocardial injury. The high serum cTnI levels may suggest membrane disruption of mechanical stress or irreversible myocyte degeneration during exercise [32]. In addition, serum MDA was chosen for evaluation of oxidative damage. The high serum MDA levels may suggest a myocardial injury occurred [33]. HBFP staining techniques were used in the present experiment to prove early myocardial ischaemia, and myocardial ischemic fiber staining produced a vivid crimson color [34]. Our results showed that the even though prolonged exercise finally resulted in more severe ischemia/hypoxia (HBFP staining), myocardial damage was still markedly attenuated (serum cTnI and MDA level). These results were similar with many investigations such that EP could attenuate myocardial injury. Quindry et al. [35] reported that the mitochondrial or sarcolemmal ATP-sensitive potassium channel mediates exercise-induced cardioprotection against post-IR cell death and apoptosis in rats. Lee et al. [36] reported that three to five consecutive days of exercise training promotes cardioprotection, at least in part, by increasing mitochondrial antioxidants, preventing mitochondrial release of reactive oxygen species, and protecting cardiac mitochondria against IR-induced oxidative damage and functional impairment. In addition, several observations found that HBFP staining was not a useful tool in the recognition of myocardial necrosis [37]. Therefore, EP could alleviate the exhaustive exercise-induced oxidative damage and myocardium injury, but prolonged exercise could aggravate ischemia/hypoxia of myocardial in rats.

We also found that PKCδ was significantly increased both at the protein and the mRNA levels in the late phase of EP. The changes of PKCδ mRNA expression may be sufficient to account for the elevation of PKCδ protein, although other factors operating on the protein level may also be involved [38]. Studies showed that a high level of PKCδ might be involved in cardioprotection against ischemic damage, such as enhanced ability to cardiac ischemic tolerance [39], increased NCX-mediated Ca2+ influx [40], and improved the recovery of left ventricular developed pressure and mechanical function [41]. For example, Kudo et al. [42] observed a significant protection against myocardial infarction 24 h after treatment with the selective adenosine A1 receptor agonist 2-chloro-N 6-cyclopentyladenosine (CCPA) in rats, PKCδ was upregulated 24 h after treatment with CCPA, and inhibition of PKCδ abolished the cardioprotective effect of CCPA. Neckár et al. [43] demonstrated that adaptation to chronic intermittent hypoxia (CIH) led to a marked upregulation of PKCδ and CIH-induced cardioprotection against ischemic insults in rats is partially mediated by PKCδ. It has been believed that the distinctive activation mechanism of PKCδ was involved in the coordination of phosphorylation and translocation events [44]. The beneficial role of PKCδ associated with its translocation to mitochondria and/or sarcolemma was confirmed in a variety of experiments on ischemic and pharmacological preconditioning [40, 45]. Melling et al. [14] showed that membrane levels of PKCδ in rat myocardium were significantly elevated in the late phase of EP. In our study, the activated ratio of PKCδ (data from western blot) in group C was 24 %, group EE 24 %, group LEP 13 %, group LEP + EE 16 %, and group CHE + LEP + EE 12 %. These results could not explain whether activated PKCδ had a key role in EP-induced cardioprotection. But our novel finding is that p-PKCδThr507 was preferentially localized to intercalated disks in the late phase of EP. The p-PKCδThr507 localized to intercalated disks in the LEP positive area to total p-PKCδThr507 was 15.8 %. These results demonstrated that components of intercalated disks such as connexin43 (C × 43) may represent a molecular target of PKCδ in EP induced cardioprotection [46]. However, there still exist several controversial opinions, which reported that the activated PKCδ at reperfusion is potential harmful [47–49]. Our results showed that, compared with exhaustive exercise alone, exhaustive exercise pretreated with EP led to a markedly downregulation of p-PKCδThr507, suggesting that after prolonged exercise, the activated PKCδ at reperfusion was downregulated in order to stabilize the intracellular environment and maintain cell survival in the myocardium. Taken together, EP promoted PKCδ localized to intercalated disks. A high level of PKCδ may enhance the ability of the myocardium to a subsequent exercise-induced myocardial injury, resulting in a marked increase in exercise capacity of rats. Nevertheless, after prolonged exercise, whether activated PKCδ at reperfusion has a key role in cardioprotection is still an outstanding question.

In addition, we found that the increase at protein level was enhanced by CHE treatment, but the response was not apparent at the mRNA level. This was consistent with the results from a study by Carson where the research showed that significant decreases were found in myocardial PKCε mRNA levels immediately and 24 h after one bout of acute exercise [15]. This and the assessments of PKCδ protein expression, which showed protein PKCδ to be highly expressed by CHE treatment, showed that PKCδ mRNA undergoes downregulation upon sustained stimulation. The molecular mechanisms underlying the downregulation of PKC are still unclear, but studies have shown that chronic activation of PKC eventually results in the complete dephosphorylation and degradation of the enzyme by an ubiquitin/proteasome-dependent mechanism, which we here refer to as downregulation [50, 51].

It was observed that administration of CHE suppressed the membrane translocation of myocardial PKCδ in the late phase of EP and attenuated the exercise mediated reduction of myocardial infarct size during I/R injury [14]. However, our study showed that CHE treatment before EP neither prevented the translocation of PKCδ nor accelerated the exhaustive exercise-induced ischemia/hypoxia and damage in cardiomyocytes. On the contrary, it promoted the expression of PKCδ and provided a strong resistance to the myocardial injury. These results were consistent with our previous study that CHE treatment before EP did not attenuate the protective effect of EP against ISO-induced myocardial injury, and unexpectedly provided a more prominent cardioprotective effect [22]. In fact, the influence of CHE on the cardiac effect varied depending on the dosage and the experimental model [52–54], and an independent role of CHE on Na+-K+-ATPase activity was also reported [55]. Thus, its influence on cardioprotection should be interpreted with caution with respect to the role of PKC.

Conclusions

The present work demonstrated that EP enhanced the exercise capacity of rats, and attenuated exhaustive exercise-induced myocardial injury in the late phases of cardioprotection. A significant increase of PKCδ in the late phase of EP, both at the protein level and the mRNA level in the left ventricular myocardium, was accompanied with p-PKCδThr507 translocation to intercalated disks. Compared with exhaustive exercise alone, exhaustive exercise pretreated with EP led to a markedly downregulation of p-PKCδThr507. These results suggested that a high level of PKCδ might be involved in cardioprotection against ischemic damage, but whether activated PKCδ at reperfusion took a key role in cardioprotection was still an outstanding question. CHE injection during exercise might not be an appropriate means of demonstrating the role of PKCδ.

Abbreviations

CHE:

Chelerythrine

cTnI:

Cardiac troponin I

Cx43:

Connexin 43

EP:

Exercise preconditioning

HBFP:

Hematoxylin–basic fuchsin–picric acid

I/R:

Ischemia/reperfusion

ISH:

In situ hybridization

LV:

Left ventricle

PKC:

Protein kinase C

References

  1. Shave R, Oxborough D (2012) Exercise-induced cardiac injury: evidence from novel imaging techniques and highly sensitive cardiac troponin assays. Prog Cardiovasc Dis 54:407–415

    Article  PubMed  Google Scholar 

  2. Vidotto C, Tschan H, Atamaniuk J, Pokan R, Bachl N, Muller MM (2005) Responses of N-terminal pro-brain natriuretic peptide (NT-proBNP) and cardiac troponin I (cTnI) to competitive endurance exercise in recreational athletes. Int J Sports Med 26:645–650

    Article  CAS  PubMed  Google Scholar 

  3. Neilan TG, Januzzi JL, Lee-Lewandrowski E, Ton-Nu TT, Yoerger DM, Jassal DS et al (2006) Myocardial injury and ventricular dysfunction related to training levels among nonelite participants in the Boston marathon. Circulation 114:2325–2333

    Article  PubMed  Google Scholar 

  4. Neumayr G, Pfister R, Mitterbauer G, Eibl G, Hoertnagl H (2005) Effect of competitive marathon cycling on plasma N-terminal pro-brain natriuretic peptide and cardiac troponin T in healthy recreational cyclists. Am J Cardiol 96:732–735

    Article  CAS  PubMed  Google Scholar 

  5. Takeshita D, Tanaka M, Mitsuyama S, Yoshikawa Y, Zhang GX, Obata K et al (2013) A new calpain inhibitor protects left ventricular dysfunction induced by mild ischemia-reperfusion in in situ rat hearts. J Physiol Sci 63:113–123

    Article  CAS  PubMed  Google Scholar 

  6. Redington KL, Disenhouse T, Li J, Wei C, Dai X, Gladstone R et al (2013) Electroacupuncture reduces myocardial infarct size and improves post-ischemic recovery by invoking release of humoral, dialyzable, cardioprotective factors. J Physiol Sci 63:219–223

    Article  PubMed  Google Scholar 

  7. Frasier CR, Moore RL, Brown DA (2011) Exercise-induced cardiac preconditioning: how exercise protects your achy-breaky heart. J Appl Physiol 111:905–915

    Article  CAS  PubMed  Google Scholar 

  8. Domenech R, Macho P, Schwarze H, Sanchez G (2002) Exercise induces early and late myocardial preconditioning in dogs. Cardiovasc Res 55:561–566

    Article  CAS  PubMed  Google Scholar 

  9. Sanchez G, Escobar M, Pedrozo Z, Macho P, Domenech R, Hartel S et al (2008) Exercise and tachycardia increase NADPH oxidase and ryanodine receptor-2 activity: possible role in cardioprotection. Cardiovasc Res 77:380–386

    Article  CAS  PubMed  Google Scholar 

  10. de Waard MC, van der Velden J, Bito V, Ozdemir S, Biesmans L, Boontje NM et al (2007) Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction. Circ Res 100:1079–1088

    Article  PubMed  Google Scholar 

  11. Yellon DM, Downey JM (2003) Preconditioning the myocardium: from cellular physiology to clinical cardiology. Physiol Rev 83:1113–1151

    CAS  PubMed  Google Scholar 

  12. Yamashita N, Baxter GF, Yellon DM (2001) Exercise directly enhances myocardial tolerance to ischaemia-reperfusion injury in the rat through a protein kinase C mediated mechanism. Heart 85:331–336

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Melling CW, Thorp DB, Noble EG (2004) Regulation of myocardial heat shock protein 70 gene expression following exercise. J Mol Cell Cardiol 37:847–855

    Article  CAS  PubMed  Google Scholar 

  14. Melling CW, Thorp DB, Milne KJ, Noble EG (2009) Myocardial Hsp70 phosphorylation and PKC-mediated cardioprotection following exercise. Cell Stress Chaperones 14:141–150

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Carson LD, Korzick DH (2003) Dose-dependent effects of acute exercise on PKC levels in rat heart: is PKC the heart’s prophylactic? Acta Physiol Scand 178:97–106

    Article  CAS  PubMed  Google Scholar 

  16. Mochly-Rosen D (1995) Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science 268:247–251

    Article  CAS  PubMed  Google Scholar 

  17. Seki T, Matsubayashi H, Amano T, Shirai Y, Saito N, Sakai N (2005) Phosphorylation of PKC activation loop plays an important role in receptor-mediated translocation of PKC. Genes Cells 10:225–239

    Article  CAS  PubMed  Google Scholar 

  18. Wang Y, Hirai K, Ashraf M (1999) Activation of mitochondrial ATP-sensitive K(+) channel for cardiac protection against ischemic injury is dependent on protein kinase C activity. Circ Res 85:731–741

    Article  CAS  PubMed  Google Scholar 

  19. Kawata H, Yoshida K, Kawamoto A, Kurioka H, Takase E, Sasaki Y et al (2001) Ischemic preconditioning upregulates vascular endothelial growth factor mRNA expression and neovascularization via nuclear translocation of protein kinase C epsilon in the rat ischemic myocardium. Circ Res 88:696–704

    Article  CAS  PubMed  Google Scholar 

  20. Shen YJ, Pan SS, Ge J, Hao Z (2012) Exercise preconditioning provides early cardioprotection against exhaustive exercise in rats: potential involvement of protein kinase C delta translocation. Mol Cell Biochem 368:89–102

    Article  CAS  PubMed  Google Scholar 

  21. Bertinchant JP, Robert E, Polge A, Marty-Double C, Fabbro-Peray P, Poirey S et al (2000) Comparison of the diagnostic value of cardiac troponin I and T determinations for detecting early myocardial damage and the relationship with histological findings after isoprenaline-induced cardiac injury in rats. Clin Chim Acta 298:13–28

    Article  CAS  PubMed  Google Scholar 

  22. Shen YJ, Pan SS, Zhuang T, Wang FJ (2011) Exercise preconditioning initiates late cardioprotection against isoproterenol-induced myocardial injury in rats independent of protein kinase C. J Physiol Sci 61:13–21

    Article  CAS  PubMed  Google Scholar 

  23. Chotteau-Lelievre A, Dolle P, Gofflot F (2006) Expression analysis of murine genes using in situ hybridization with radioactive and nonradioactively labeled RNA probes. Methods Mol Biol 326:61–87

    CAS  PubMed  Google Scholar 

  24. Ashurst HL, Varro A, Dimaline R (2008) Regulation of mammalian gastrin/CCK receptor (CCK2R) expression in vitro and in vivo. Exp Physiol 93:223–236

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Silverman AP, Kool ET (2007) Oligonucleotide probes for RNA-targeted fluorescence in situ hybridization. Adv Clin Chem 43:79–115

    Article  CAS  PubMed  Google Scholar 

  26. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  CAS  PubMed  Google Scholar 

  27. Lum MA, Pundt KE, Paluch BE, Black AR, Black JD (2013) Agonist-induced down-regulation of endogenous protein kinase C alpha through an endolysosomal mechanism. J Biol Chem 288:13093–13109

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Meyer K, Zhang H, Zhang L (2009) Direct effect of cocaine on epigenetic regulation of PKCepsilon gene repression in the fetal rat heart. J Mol Cell Cardiol 47:504–511

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193

    Article  CAS  PubMed  Google Scholar 

  30. Yamashita N, Hoshida S, Otsu K, Asahi M, Kuzuya T, Hori M (1999) Exercise provides direct biphasic cardioprotection via manganese superoxide dismutase activation. J Exp Med 189:1699–1706

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Jimenez SK, Jassal DS, Kardami E, Cattini PA (2011) A single bout of exercise promotes sustained left ventricular function improvement after isoproterenol-induced injury in mice. J Physiol Sci 61:331–336

    Article  CAS  PubMed  Google Scholar 

  32. Shave R, Baggish A, George K, Wood M, Scharhag J, Whyte G et al (2010) Exercise-induced cardiac troponin elevation evidence, mechanisms, and implications. J Am Coll Cardiol 56:169–176

    Article  CAS  PubMed  Google Scholar 

  33. Bermudez Pirela VJ, Bracho V, Bermudez Arias FA, Medina Reyes MT, Nunez Pacheco M, Amell De Diaz A et al (2000) Malondialdehyde and nitric oxide behaviour in patients with myocardial infarction. Rev Esp Cardiol 53:502–506

    Article  CAS  PubMed  Google Scholar 

  34. Li S, Zhong S, Zeng K, Luo Y, Zhang F, Sun X et al (2010) Blockade of NF-kappaB by pyrrolidine dithiocarbamate attenuates myocardial inflammatory response and ventricular dysfunction following coronary microembolization induced by homologous microthrombi in rats. Basic Res Cardiol 105:139–150

    Article  CAS  PubMed  Google Scholar 

  35. Quindry JC, Miller L, McGinnis G, Kliszczewicz B, Irwin JM, Landram M et al (2012) Ischemia reperfusion injury, KATP channels, and exercise-induced cardioprotection against apoptosis. J Appl Physiol (1985) 113:498–506

    Article  CAS  Google Scholar 

  36. Lee Y, Min K, Talbert EE, Kavazis AN, Smuder AJ, Willis WT et al (2012) Exercise protects cardiac mitochondria against ischemia-reperfusion injury. Med Sci Sports Exerc 44:397–405

    Article  PubMed  Google Scholar 

  37. Ribeiro-Silva A, CC SM, Rossi MA (2002) Is immunohistochemistry a useful tool in the postmortem recognition of myocardial hypoxia in human tissue with no morphological evidence of necrosis? Am J Forensic Med Pathol 23:72–77

    Article  PubMed  Google Scholar 

  38. Greenbaum D, Colangelo C, Williams K, Gerstein M (2003) Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biol 4:117

    Article  PubMed Central  PubMed  Google Scholar 

  39. Kolar F, Jezkova J, Balkova P, Breh J, Neckar J, Novak F et al (2007) Role of oxidative stress in PKC-delta upregulation and cardioprotection induced by chronic intermittent hypoxia. Am J Physiol Heart Circ Physiol 292:H224–H230

    Article  CAS  PubMed  Google Scholar 

  40. Bouwman RA, Salic K, Padding FG, Eringa EC, van Beek-Harmsen BJ, Matsuda T et al (2006) Cardioprotection via activation of protein kinase C-delta depends on modulation of the reverse mode of the Na +/Ca2 + exchanger. Circulation 114:I226–I232

    Article  PubMed  Google Scholar 

  41. Xu P, Wang J, Kodavatiganti R, Zeng Y, Kass IS (2004) Activation of protein kinase C contributes to the isoflurane-induced improvement of functional and metabolic recovery in isolated ischemic rat hearts. Anesth Analg 99:993–1000

    Article  CAS  PubMed  Google Scholar 

  42. Kudo M, Wang Y, Xu M, Ayub A, Ashraf M (2002) Adenosine A(1) receptor mediates late preconditioning via activation of PKC-delta signaling pathway. Am J Physiol Heart Circ Physiol 283:H296–H301

    CAS  PubMed  Google Scholar 

  43. Neckar J, Markova I, Novak F, Novakova O, Szarszoi O, Ost’adal B et al (2005) Increased expression and altered subcellular distribution of PKC-delta in chronically hypoxic rat myocardium: involvement in cardioprotection. Am J Physiol Heart Circ Physiol 288:H1566–H1572

    Article  CAS  PubMed  Google Scholar 

  44. Steinberg SF (2004) Distinctive activation mechanisms and functions for protein kinase Cdelta. Biochem J 384:449–459

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Marinovic J, Bosnjak ZJ, Stadnicka A (2005) Preconditioning by isoflurane induces lasting sensitization of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel by a protein kinase C-delta-mediated mechanism. Anesthesiology 103:540–547

    Article  CAS  PubMed  Google Scholar 

  46. Srisakuldee W, Jeyaraman MM, Nickel BE, Tanguy S, Jiang ZS, Kardami E (2009) Phosphorylation of connexin-43 at serine 262 promotes a cardiac injury-resistant state. Cardiovasc Res 83:672–681

    Article  CAS  PubMed  Google Scholar 

  47. Kawamura S, Yoshida K, Miura T, Mizukami Y, Matsuzaki M (1998) Ischemic preconditioning translocates PKC-delta and -epsilon, which mediate functional protection in isolated rat heart. Am J Physiol 275:H2266–H2271

    CAS  PubMed  Google Scholar 

  48. Hahn HS, Yussman MG, Toyokawa T, Marreez Y, Barrett TJ, Hilty KC et al (2002) Ischemic protection and myofibrillar cardiomyopathy: dose-dependent effects of in vivo deltaPKC inhibition. Circ Res 91:741–748

    Article  CAS  PubMed  Google Scholar 

  49. Sivaraman V, Hausenloy DJ, Kolvekar S, Hayward M, Yap J, Lawrence D et al (2009) The divergent roles of protein kinase C epsilon and delta in simulated ischaemia-reperfusion injury in human myocardium. J Mol Cell Cardiol 46:758–764

    Article  CAS  PubMed  Google Scholar 

  50. Lu Z, Liu D, Hornia A, Devonish W, Pagano M, Foster DA (1998) Activation of protein kinase C triggers its ubiquitination and degradation. Mol Cell Bio 18:839–845

    CAS  Google Scholar 

  51. Goode NT, Hajibagheri MA, Parker PJ (1995) Protein kinase C (PKC)-induced PKC down-regulation. Association with up-regulation of vesicle traffic. JBC 270:2669–2673

    Article  CAS  Google Scholar 

  52. Rorabaugh BR, Ross SA, Gaivin RJ, Papay RS, McCune DF, Simpson PC et al (2005) alpha1A- but not alpha1B-adrenergic receptors precondition the ischemic heart by a staurosporine-sensitive, chelerythrine-insensitive mechanism. Cardiovasc Res 65:436–445

    Article  CAS  PubMed  Google Scholar 

  53. Vrba J, Dvorak Z, Ulrichova J, Modriansky M (2008) Conventional protein kinase C isoenzymes undergo dephosphorylation in neutrophil-like HL-60 cells treated by chelerythrine or sanguinarine. Cell Biol Toxicol 24:39–53

    Article  CAS  PubMed  Google Scholar 

  54. Liu Y, Cohen MV, Downey JM (1994) Chelerythrine, a highly selective protein kinase C inhibitor, blocks the anti-infarct effect of ischemic preconditioning in rabbit hearts. Cardiovasc Drugs Ther 8:881–882

    Article  CAS  PubMed  Google Scholar 

  55. Lundmark JL, Ramasamy R, Vulliet PR, Schaefer S (1999) Chelerythrine increases Na-K-ATPase activity and limits ischemic injury in isolated rat hearts. Am J Physiol 277:H999–H1006

    CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 31071031).

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The authors declare no conflict of interest.

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  1. Department of Sports Anatomy, School of Sports Science, Shanghai University of Sport, 399 Changhai Road, Shanghai, 200438, China

    Zhe Hao, Shan-Shan Pan, Yu-Jun Shen & Jun Ge

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Correspondence to Shan-Shan Pan.

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Zhe Hao and Yu-Jun Shen contribute to this article equally.

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Hao, Z., Pan, SS., Shen, YJ. et al. Exercise preconditioning-induced late phase of cardioprotection against exhaustive exercise: possible role of protein kinase C delta. J Physiol Sci 64, 333–345 (2014). https://doi.org/10.1007/s12576-014-0323-x

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