p38-Regulated/activated protein kinase plays a pivotal role in protecting heart against ischemia-reperfusion injury and preserving cardiac performance

Abstract

p38-Regulated/activated protein kinase (PRAK) plays a critical role in modulating cellular survival and biological function. However, the function of PRAK in the regulation of myocardial ischemic injury remains unknown. This study is aimed at determining the function of PRAK in modulating myocardial ischemia-reperfusion injury and myocardial remodeling following myocardial infarction. Hearts were isolated from adult male homozygous PRAK^−/− and wild-type mice and subjected to global ischemia-reperfusion injury in Langendorff isolated heart perfusion. PRAK^−/− mice mitigated postischemic ventricular functional recovery and decreased coronary effluent. Moreover, the infarct size in the perfused heart was significantly increased by deletion of PRAK. Western blot showed that deletion of PRAK decreased the phosphorylation of ERK1/2. Furthermore, the effect of deletion of PRAK on myocardial function and remodeling was also examined on infarcted mice in which the left anterior descending artery was ligated. Echocardiography indicated that PRAK^−/− mice had accelerated left ventricular systolic dysfunction, which was associated with increased hypertrophy in the infarcted area. Deletion of PRAK augmented interstitial fibrosis and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive myocytes. Furthermore, immunostaining analysis shows that CD31-postive vascular density and α-smooth muscle actin capillary staining decreased significantly in PRAK^−/− mice. These results indicate that deletion of PRAK enhances susceptibility to myocardial ischemia-reperfusion injury, attenuates cardiac performance and angiogenesis, and increases interstitial fibrosis and apoptosis in the infarcted hearts.

INTRODUCTION

In cardiomyocyte ischemic injury, a major role has been attributed to the mitogen-activated protein (MAP) kinase pathway, which is upregulated in response to a variety of stress stimuli (1). MAPKs are protein serine/threonine kinases that are divided into two categories, namely the conventional and atypical MAPKs. Each member of the conventional MAPKs functions within a three-tiered kinase cascade: each cascade comprises a MAPK kinase (MAP3K), a MAPK kinase (MAP2K), and the MAPK itself. Activation of the pathway is achieved through sequential phosphorylation of these kinases. Four conventional MAPK cascades have been described in mammals: extracellular signal-regulated kinases (ERKs) 1 and 2 (ERK1/2), p38 MAPK c-Jun NH₂-terminal kinases 1/2/3 (JNK1/2/3), and ERK5. Atypical MAPKs, in contrast, do not appear to be organized into three kinase cascades. Besides these cascade kinases, there is one functional group of conventional MAPK substrates consisting of protein kinases known as MAPK-activated protein kinases (MAPKAPKs) (11, 19). Eleven mammalian MAPKAPKs have been identified. Among those, p38-regulated/activated kinase (PRAK) or MAP kinase-activated protein kinase 5 (MK5), ubiquitously expressed in almost all human tissues, was originally identified as a p38 MAKP-activated protein, but afterward work found that it was also activated by atypical MAPKs, ERK3/4, indicating involvement of PRAK/MK5 in both conventional and atypical MAPK-mediated signal transduction pathways (13, 15, 16, 18). PRAK has been shown to phosphorylate several substrates including heat shock protein 27 (HSP27), forkhead box O1 (FoxO1), FoxO3, and Ras homolog enriched in the brain (Rheb), indicating the versatile biological role of PRAK in various environments (2, 10, 15, 30). p38 has been shown to induce the preconditioning effect to protect the heart against myocardial ischemia and reperfusion injury (4, 25, 26). Genetic knockouts of p38 developed significant levels of cardiac hypertrophy. In response to pressure overload to the left ventricle (LV), these knockouts also developed cardiac dysfunction and heart dilatation (17). However, the role of PRAK, as a downstream target of p38 in modulating myocardial ischemia and reperfusion injury, has not been determined. In the present study, we demonstrate that genetic deletion of PRAK increased the susceptibility of the heart to myocardial ischemia and reperfusion injury as evident by the promotion of postischemic functional recovery and reduction of infarct size in association with a reduction of ERK-1 and -2 phosphorylation. Moreover, deletion of PRAK also attenuated myocardial functional recovery, mitigated myocardial remodeling, decreased angiogenesis, and increased myocardial fibrosis and apoptosis. These data are the first demonstration to show that PRAK plays a critical role against myocardial ischemia and reperfusion injury and promoting cardiac performance following myocardial infarction (MI).

METHODS

Animal model.

Male PRAK^−/− and wild-type mice of the C57BL/6J background and at the age of 3–4 mo were used in this study. PRAK^−/− mice were provided by Dr. P. Sun from Immunology and Microbial Science, The Scripps Research Institute (La Jolla, CA). The details for generation of PRAK^−/− mice are fully described (22), and homozygous PRAK^−/− were used in this study. All studies on animals were performed after a protocol approved by the Institutional Animal Care and Use Committee, which conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996); these animals are housed in an accredited facility at Roger Williams Medical Center (Providence, RI). All animal procedures were carried out in accordance with guidelines approved by the Institutional Animal Care and Use Committee of Roger Williams Medical Center.

Langendorff isolated perfused heart.

The methodology of Langendorff's perfused heart preparation and measurement of ventricular function has been described previously in detail (25, 26, 28). Briefly, male wild-type C57BL/J6 mice (n = 7) and PRAK^−/− mice (n = 11) were anesthetized with a lethal intraperitoneal injection of pentobarbital sodium (120 mg/kg). Hearts were rapidly excised and arrested in ice-cold Krebs-Henseleit buffer. They were then cannulated via the ascending aorta for retrograde perfusion by the Langendorff method using Krebs Henseleit buffer containing the following (in mM): 110 NaCl, 4.7 KCl, 1.2 MgSO₄·7H₂O, 2.5 CaCl₂·2H₂O, 11 glucose, 1.2 KH₂PO₄, 25 NaHCO₃, and 0.5 EDTA. The buffer, aerated with 95% O₂–5% CO₂ to give a pH of 7.4 at 37°C, was perfused at a constant pressure of 55 mmHg. For experiment protocol, hearts were subjected to 20 min of equilibration and 30 min of ischemia followed by 30 min of reperfusion. A water-filled latex balloon, attached to the tip of polyethylene tubing, was then inflated sufficiently to provide a left ventricular end-diastolic pressure (LVEDP) of ∼10 mmHg measured by means of a disposable Gould pressure transducer. LV functional analysis was recorded using software and a computer-based recording system (MP100A; BIOPAC Systems, Goleta, CA). These parameters included left ventricular systolic pressure (LVSP), LVEDP, heart rate, and cardiac contractile function. The rate pressure product (RPP) was calculated as the product of left ventricular developed pressure and heart rate, where developed pressure is systolic pressure minus LVEDP.

Measurement of MI.

The infarction size was measured with a modification as previously described with 10% triphenyltetrazolium chloride staining (25, 28). The infarct size was calculated and presented as the percentage of risk area, defined as the sum of total ventricular area minus cavities.

Myocardial infarction.

The mouse MI model was created following thoracotomy by applying permanent ligation to the left anterior descending (LAD) artery as previously described (25, 26). Briefly, both male wild-type C57BL/6J mice and PRAK^−/− mice (n = 4 per group) were anesthetized by an intraperitoneal injection of 50 mg/kg sodium pentobarbital. Mice received a subcutaneous injection of buprenorphine (0.03 mg/kg) 2 h before surgery and also every 12 h postsurgery for 3 days. Ventilation was achieved by connecting the endotracheal tube with a rodent ventilator (model 683; Harvard Apparatus, Holliston, MA). The chest was then opened, and coronary occlusion was induced by ligation with a nylon suture. In addition, male wild-type C57BL/6 J mice and PRAK^−/− mice in the sham group were anesthetized and underwent thoracotomy without coronary ligation.

Echocardiography.

Echocardiography of mice was serially conducted using an Acuson Sequoia C512 system equipped with a 15L8 linear array transducer to access LV function. Cardiac function in normal male wild-type C57BL/6J mice (n = 5 per group) and PRAK^−/− mice (n = 5 per group) at 2 mo old was compared. Briefly, mice were anesthetized with 1.5% isoflurane mixed with oxygen via a nose cone and then placed in supine position on a heating pad. After removal of hair, the precordial region was covered with prewarmed ultrasound transmission gel (Aquasonic; Parker Laboratory, Fairfield, NJ). The short axis of LV was chosen to capture two-dimensional B-mode images and M-mode tracing at the level of the papillary muscles. The signal depth was set at 25 mm. Three to six consecutive cardiac cycles were measured from M-mode tracings with accompanying software. The procedure was performed at weeks 0, 2, 4, and 6 after LAD ligation.

Histological analysis.

Paraffin-embedded heart tissue slices were fixed with 10% neutral buffered formalin and washed twice with PBS-Tween. Myocyte cross-sectional area was measured from images captured from the sections obtained middistance from the base to the apex. The outline of myocytes was traced using NIH ImageJ software (NIH version 1.40g, https://imagej.nih.gov/ij/) to determine myocyte cross-sectional area. A value from each heart was calculated by the measurements of ∼400–600 cells in a remote area from infarction of an individual heart. For immunofluorescent staining, fixed slices were incubated with appropriate primary antibodies overnight at 4°C. Primary antibodies and lectin used in this study were as follows: anti-α-smooth muscle actin (anti-α-SMA; cat. no. A5228; Sigma-Aldrich, St. Louis, MO), FITC-conjugated wheat germ agglutinin (cat. no. L4895; Sigma-Aldrich), anti-CD31 (cat. no. CBL1337; Millipore), and anti-active caspase-3 (cat. no. ab49822; Abcam). All of the antibodies were validated in our previous publications (29). After washes with PBS-Tween (3 × 5 min), the slides for α-SMA and CD31 staining were incubated with Alexa Fluor 488 goat anti-mouse IgG (A11001; Invitrogen) secondary antibodies (1:2,000) for 2 h at room temperature in the dark. Then, the slides were washed (5 × 5 min), and DAPI containing mounting medium was applied. Fluorescent microscopy was performed using a Carl Zeiss LSM 700 laser scanning microscope equipped with intuitive ZEN 2009 software. Five to ten randomly selected high-power fields were selected for quantification using ImageJ software. The total number of vessels from each group was calculated and normalized to the tissue area. The stained numbers of each section were counted in ~10 randomized fields of the tissue sections, which were taken in the middle plane of each heart for both sham and MI in C57BL/6J (n = 3 per group) and PRAK^−/− mice (n = 4 per group). Cardiac interstitial fibrosis was quantified by picrosirius red staining of five to six sections from each heart (n = 3 per group). ImageJ software was used to quantify the red-stained area of each section.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay.

Terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) was carried out using a TACS 2-TdT-DAB In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD) following the manufacturer’s instructions, using a 1-h labeling reaction in the presence of cobalt. To verify that apoptosis occurred in the myocytes, immunohistochemical staining of α-sarcomeric actin was carried out with α-sarcomeric actin antibody (α-actin, monoclonal, 1:100 dilution; Sigma) at 4°C overnight. For each section, the number of TUNEL-positive myocyte nuclei was counted in five randomly selected regions; five to six sections were randomized to use for counting of signals, and three hearts were used for both sham and MI in C57BL/6J (n = 3 hearts per group) and PRAK^−/− (n = 3 hearts per group) mice. Approximately 400–600 cells were counted in each independent group. The index of apoptosis was then determined.

Electrophoresis and Western blot analysis.

Hearts tissues were homogenized in ice-cold RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktails (Calbiochem, Billerica, MA). Protein lysates were then obtained after centrifugation at 12,000 g for 15 min at 4°C. Protein concentrations were estimated using a Micro BCA Assay Kit (Thermo Scientific, Rockford, IL). Proteins (50 µg/lane) were separated on reducing SDS polyacrylamide gels and transferred to PVDF membranes. Nonfat dry milk (5%) was used to block the membranes at room temperature for 1 h followed by overnight incubation with primary antibody against phosphorylated ERK1/2 (cat. no. 4370; Cell Signaling Technology) and total ERK (cat. no. 4695; Cell Signaling Technology) at 4°C (23). The membranes were then incubated in the appropriate horseradish peroxidase-conjugated secondary antibody solution. The blots were incubated with their respective polyclonal antibody against PRAK (cat. no. 9772; Cell Signaling Technology), anti-phosphorylated PRAK antibody (cat. no. ab138668; Abcam), and β-actin (cat. no. 3700; Cell Signaling Technology; 1:1,000) for 2 h and visualized by incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat. no. 926-32211; LI-COR; 1:5,000) or anti-mouse horseradish peroxidase-conjugated secondary antibody (cat. no. 926-322110; LI-COR) for 1 h and developed with ECL Chemiluminescence detection reagent (Amersham Pharmacia Biotech).

Statistical analysis.

Data were expressed as an average ± SE of independent experiments. An unpaired, two-tailed Student t-test was used to determine significance between two groups. Multiple groups were analyzed using one-way ANOVA followed by Bonferroni post hoc test. Differences between groups were considered statistically significant when P < 0.05.

RESULTS

PRAK deletion deteriorates postacute ischemic ventricular functional recovery.

As shown in Figs. 1, A and B, deletion of PRAK shows the absence of PRAK protein in the myocardium, PRAK phosphorylation demonstrated a decline in an age-dependent pattern (n = 3 per group). In addition, echocardiography shows that deletion of PRAK suppressed myocardial function (n = 5 per group in C57BL/6J and PRAK^−/−, Fig. 1, C and F) but did not affect the heart size (n = 10 per group; Fig. 1, G–J, and other organs including liver and kidney not shown). As shown in Table 1, there is no significant difference in LVSP and left ventricular diastolic pressure (LVDP) at baseline. However, both LVdP/dt_max and LVdP/dt_min demonstrate a profound reduction as compared with wild-type control at baseline, indicating that depletion of PRAK resulted in deficiency in contractility and dilation (C57BL/6J, n = 7 per group; PRAK^−/−, n = 11 per group). Coronary effluents also display a reduction by deletion of PRAK, indicating a deficiency in vascular dilation or development. As shown in Fig. 2, A–H, following postischemia and reperfusion injury, wild-type mice demonstrate a better recovery of LVSP, LVDP, RPP, LVdP/dt_max, LVdP/dt_min, and coronary flow as compared with PRAK^−/− mice. At the end of reperfusion, LVDP was 60.3 ± 6.4 mmHg in the PRAK^−/− mice, which was significantly deteriorated as compared with the wild-type littermates (82.9 ± 7.2 mmHg, P < 0.01). In addition, PRAK/MK5 deletion also induced a deterioration of LVSP (from 96.0 ± 5.84 to 75.2 ± 5.0 mmHg, P < 0.01) and RPP (from 25.4 ± 2.3 to 18.4 ± 1.8 × 10⁻³ mmHg/s, P < 0.01). Likewise, a significant reduction was observed in LVdP/dt_max (from 2,231.1 ± 199.2 mmHg/s × 10⁻³ in wild types to 1,694.4 ± 204.5 mmHg/s × 10⁻³ in PRAK^−/−, P < 0.001) and LVdP/dt_min recovery (from 2,141.0 ± 206.3 mmHg/s × 10⁻³ in wild types to 1,500.5 ± 206.6 mmHg/s × 10⁻³ in PRAK^−/−, P < 0.001). Coronary flow was also decreased in PRAK^−/− mice (from 2.5 ± 0.4 ml/min in wild types to 1.7 ± 0.1 ml/min in PRAK^−/−, P < 0.05). Taken together, the postischemic recovery of ventricular function was significantly deteriorated in PRAK^−/− mice. Coronary effluents following ischemia and reperfusion are closely related to the function of vascular dilation. There is a notable difference in coronary effluent before ischemic insult, as the magnitude of reduction of coronary effluents was further expanded by deletion of PRAK as compared with wild-type control.

Fig. 1.

Depletion of p38-regulated/activated protein kinase (PRAK) suppresses cardiac performance, but morphology is normal. A: Western blot showing the absence of PRAK in PRAK−/− myocardium (n = 3 per group). B: PRAK phosphorylation decreases with an age increase in the myocardium (n = 3 per group). E, embroyonic day; P, postnatal day. C-F: cardiac function assessed by echocardiography: ejection fraction (EF; C), fractional shortening (FS; D), and left ventricular internal diameter in diastole (LVIDd; E) and systole (LVIDs; F) (n = 5 per group). G–J: body weight (BW; G), heart weight (HW; H), HW/BW (I), and HW/tibia length (J) (n = 10 per group). Values represent means ± SE. **P < 0.01 vs. wild type (WT). An unpaired, two-tailed Student t-test was applied.

Table 1.

Baseline data of ventricular function

Parameters	WT (n = 7)	PRAK−/− (n = 11)
LVSP, mmHg	110.3 ± 5.7	110.5 ± 5.4
LVDP, mmHg	100.3 ± 6.3	99.5 ± 5.9
LVEDP, mmHg	10.0 ± 1.3	11.0 ± 1.2
RPP, mmHg/min × 10−3	34.3 ± 1.9	34.6 ± 3.1
+dp/dtmax, mmHg/s × 10−3	2,656.3 ± 172.4	2,280.5 ± 96.5***
−dp/dtmin, mmHg/s × 10−3	2,613.1 ± 209.6	2,288.8 ± 172.4**
HR, beats/min	345.9 ± 17.7	346.8 ± 17.6
CF, mL/min	2.8 ± 0.3	2.5 ± 0.2*

WT, wild type; PRAK, p38-regulated/activated protein kinase; RPP, rate pressure product; LVSP, left ventricular systolic pressure; LVEDP, left ventricular diastolic pressure; LVEDP, left ventricular end-diastolic pressure; HR, heart rate; CF, coronary flow.

^*P < 0.05;

^**P < 0.01;

^***P < 0.001 vs. WT.

Fig. 2.

p38-Regulated/activated protein kinase (PRAK) deletion deteriorates postacute ischemic ventricular functional recovery. A-G: the effects of PRAK deletion on ventricular function in left ventricular systolic pressure (LVSP; A), left ventricular diastolic pressure (LVDP; B), left ventricular end-diastolic pressure (LVEDP; C), rate pressure product (RPP; D), LVdP/dt_max (E) LVdP/dt_min (F), and heart rate (HR; G) during ischemia and reperfusion. H: coronary effluents (CF) following myocardial ischemia and reperfusion injury. Values represent means ± SE n = 7 per group in wild type (WT); n = 11 per group in PRAK^−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. control ischemia and reperfusion. An unpaired, two-tailed Student t-test was applied.

Deletion of PRAK increased infarct size in the postischemic myocardium.

Myocardial infarct size was determined by using the triphenyltetrazolium chloride method. As shown in Fig. 3, as compared with wild-type mice, PRAK^−/− mice demonstrated a significantly larger necrotic area as compared with control wild-type mice (n = 5 per group). The statistical analysis shows that the infarct size was increased in the PRAK^−/− mice in response to myocardial ischemia and reperfusion injury (n = 5 per group). In addition, as shown in Fig. 4, there was a significant decrease in ERK phosphorylation in the myocardium of PRAK^−/− as compared with wild-type following myocardial ischemia and reperfusion (n = 4 per group).

Fig. 3.

p38-regulated/activated protein kinase (PRAK) deletion increases myocardial infarct size following myocardial ischemia-reperfusion (I/R) injury. The graph shows infarct size expressed as a percentage of area at risk. WT, wild type. Values represent means ± SE; n = 5 per group. *P < 0.05 vs. control I/R. An unpaired, two-tailed Student t-test was applied.

Fig. 4.

ERK1/2 phosphorylation is suppressed in p38-regulated/activated protein kinase deleted (PRAK^−/−) mice. Lysates from wild-type (WT) and PRAK^−/− mice hearts were subject to Western blotting with anti-phospho or total ERK1/2. The graph shows the densitometric scanning results (means ± SE; n = 4 per group). *P < 0.01. An unpaired, two-tailed Student t-test was applied.

PRAK^−/− exacerbated cardiac dysfunction in infarcted heart.

The role of PRAK on MI was examined on the infarction model. As shown in Fig. 5, A and B, MI increased LV dimensions as compared with sham control in both wild-type and PRAK^−/− mice (n = 4 per group). MI resulted in cardiac depression as shown by the reduced ejection fraction and fractional shortening (Fig. 5, C and D) However, infarcted mice with deletion of PRAK displayed a profound reduction of cardiac performance. As shown in Fig. 5E, echocardiography indicated that measurement of two-dimentional guided M-mode shows a decreased ejection fraction (55.43 ± 3.71 vs. 32.86 ± 1.54%, wild type vs. PRAK^−/−, P < 0.01, Fig. 5C), fractional shortening (30.97 ± 2.06 vs. 12.19 ± 1.27%, P < 0.01, Fig. 5D), and increased LV internal dimension (LVIDs, 2.77 ± 0.09 vs. 3.70 ± 0.10 mm, P < 0.01, Fig. 5, B and E; LVIDd, 3.91 ± 0.11 vs. 5.13 ± 0.15 mm, P < 0.01, Fig. 5, A and E). In addition, deletion of PRAK also showed a reduction of wall thickness and larger chamber dimension versus wild type following infarction (data not shown).

Fig. 5.

Echocardiographic assessment of the chronic effect of p38-regulated/activated protein kinase (PRAK) deletion on left anterior descending (LAD) ligated heart. A: measurements of left ventricular internal diameter in diastole (LVIDd). B: left ventricular internal diameter in systole (LVIDs). C and D: measurements of ejection fraction (EF; C) and fractional shortening (FS; D) from M-mode tracing at 6 wk after LAD ligation. E: representative image of M-mode tracing echocardiography of 4 groups of mice at 6 wk. WT, wild-type mice; PRAK, PRAK^−/− mice; sham, sham-operated mice; MI, myocardial infarction LAD ligation mice. Values represent means ± SE; n = 4 per group. ***P < 0.001, WT-MI vs. PRAK^−/−-MI. A one-way ANOVA followed by Bonferroni post hoc test was applied.

Vascularization is attenuated by the deletion of PRAK in post-MI heart.

To assess the angiogenic response in post-MI hearts, we measured the vascular density by immunofluorescent staining of the capillary density using CD31 to identify vascular endothelial cells. The baseline number of CD3-positive vessels was decreased in PRAK^−/− mice (n = 4 per group) in comparison to the wild-type (n = 3 per group) counterparts (sham operated: 352.63 ± 12.75 vs. 232.56 ± 6.00; MI: 378.46 ± 28.49 vs. 201.49 ± 8.45 capillary density/mm², wild type vs. PRAK^−/−, P < 0.01), while showing no significant difference between sham and MI mice in the both group of mice (Fig. 6, A and B). In addition, to assess the angiogenesis in post-MI hearts, we performed immunofluorescent staining using anti-α-SMA antibody to identify capillary density. As shown in Fig. 6, C and D, PRAK^−/− showed a lower density of capillaries in the infarcted area as compared wild-type mice both in sham-operated (8.63 ± 0.58 vs. 5.32 ± 0.10/mm², wild type vs. PRAK^−/−, P < 0.01) and MI mice (7.91 ± 0.37 vs. 5.65 ± 0.57/mm², wild type vs. PRAK^−/−, P < 0.05).

Fig. 6.

p38-Regulated/activated protein kinase deleted (PRAK^−/−) mice showed a reduction of angiogenesis following myocardial infarction (MI). A: immunofluorescent staining of CD31 (an endothelial cell marker)-positive cells in hearts. B: statistical analysis showing CD31-positive vessels in wild-type and PRAK^−/− myocardium. C: immunofluorescent staining for α-smooth muscle actin (α-SMA)-positive cells in the hearts, D: statistical analysis showing α-SMA-positive capillaries in wild-type (n = 3 per group) and PRAK^−/− myocardium (n = 4 per group). Values represent means ± SE. *P < 0.05, ***P < 0.001. A one-way ANOVA followed by Bonferroni post hoc test was applied. Scale bar = 50 μm for CD31; scale bar = 200 μm for α-SMA.

Deletion of PRAK enhanced cardiac remodeling and increased apoptotic cells in the post MI heart.

The ratio of heart weight to tibia length or body weight was assessed to evaluate the hypertrophic response. Wheat germ agglutinin staining was performed to assess the cross-sectional cardiomyocyte size. PRAK^−/− mice showed an increase in the cardiomyocyte size as compared with wild-type mice (187.63 ± 22.46 vs. 276.16 ± 39.15 relative cross-sectional size, wild type vs. PRAK^−/−, n = 4 per group, P < 0.05, Fig. 7, A and B). PRAK^−/− MI mice demonstrated an increased heart weight/tibia length ratio as compared with wild-type MI mice (16.50 ± 0.40 vs. 19.63 ± 0.76 mg/mm, wild type vs. PRAK^−/−, n = 4 per group, P < 0.05) while the ratio to body weight shows no significant change between the both MI mice, but the ratio of heart weight and tibia length was increased by deletion of PRAK (Fig. 7, C and D). Interstitial fibrosis in the infarcted hearts was evaluated by picrosirius red staining. As shown in Fig. 7, E and F, area of interstitial fibrosis was significantly larger in the PRAK^−/− Mice as compared with wild-type littermates (wild type vs. PRAK^−/−, 12.648 ± 0.299 vs. 21.585 ± 1.051% area, n = 3 per group, P < 0.01).

Fig. 7.

A and B: p38-regulated/activated protein kinase deleted (PRAK^−/−) mice showed inhibition of improved cardiac remodeling and increased apoptosis representing quantitative analyses of myocyte cross-sectional area. Representative images of wheat germ agglutinin (WGA) staining in hearts (n = 4 per group). C and D: assessment of cardiac hypertrophy of myocardial infarction (MI) mice by heart weight (HW) to body weight (BW) ratio (C) and HW to tibia length (TL) ratio (D). Values represent means ± SE (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar = 50 μm (A). E and F: interstitial fibrosis was evaluated by picrosirius red staining in MI hearts from wild-type (WT) and PRAK^−/− mice (n = 3 per group). G and H: extents of apoptosis in MI hearts were evaluated by and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay (n = 3 per group). I: Western blot shows that PRAK^−/− myocardium increased active caspase-3 content as compared with WT-MI heart (n = 4 per group). Scale bar = 100 μm (E) and 50 μm (G). Graphs represent quantitative analyses of fibrosis and apoptosis in cross-sectional area. Values represent means ± SE. *P < 0.05. A one-way ANOVA followed by Bonferroni post hoc test (B, C, and D) and an unpaired, two-tailed Student t-test (F and H) were applied.

PRAK^−/− mice demonstrate increased apoptotic cardiomyocytes in post-MI hearts.

Apoptotic cardiomyocytes in the infarcted area were detected by fluorescence-based TUNEL assay. As shown in Fig. 7, G and H, TUNEL-positive apoptotic cardiomyocytes in the infarcted area were significantly increased in the PRAK^−/− mice as compared with wild type. Likewise, PRAK^−/− myocardium displayed an elevated signaling of active caspase-3 protein as compared with wild-type MI mice (Fig. 7I).

DISCUSSION

Salient findings.

This is the first demonstration to show that deletion of PRAK decreases myocardial contractility and dilation as compared with wild-type mice, which is accompanied by the suppression of vascular development. Second, depletion of PRAK increased the susceptibility of the heart to ischemia and reperfusion injury as evident by reduction of myocardial functional recovery, increase in myocardial infarct, and decrease in coronary effluents. Third, depletion of PRAK resulted in a decrease in phosphorylation of ERK in the postischemic heart. Fourth, deletion of PRAK exacerbated cardiac dysfunction following MI and promoted cardiac remodeling and cardiac hypertrophy. Fifth, knockout of PRAK attenuated vascular density, angiogenesis and increased apoptotic cardiomyocytes.

Early studies have indicated that p38 plays an essential role in the induction of preconditioning effects against myocardial ischemia and reperfusion injury (5, 6). One of the protein kinases that was activated in response to cardiac injury in isolated hearts is PRAK/MK5, and hence, PRAK/MK5 may be implicated in regulating cardioprotective effects during stress (3, 20). Deletion of PRAK was shown to increase the susceptibility of tumor growth development in response to carcinogen stimulation and increased mortality (22). However, it remains unclear whether deletion of PRAK would modulate myocardial ischemia and reperfusion injury and cardiac dysfunction in the post-MI heart.

Our results indicated that deletion of PRAK resulted in a decrease in cardiac performance as compared with wild type at the normal condition, which was accompanied with less vessel growth. This is consistent with our previous observations in which deletion of MKK3 decreased myocardial functional recovery and increased cardiac remodeling in the infarcted heart (27), which is associated with the stimulation of Akt1 signaling. In addition, we have demonstrated that ERK phosphorylation decreased in myocardial ischemia and reperfusion injury, which indicates ERK phosphorylation is related to PRAK in the production of myocardial protection. It will be interesting to further determine the physiological function of ERK signaling attributable to the beneficial effect of PRAK using additional ERK overexpression models in the future.

The present study demonstrates that deficiency of PRAK/MK5 results in decelerated recovery of infarcted heart from MI induced by LAD ligation, suggesting a protective role of PRAK/MK5 under the circumstance of the ischemic stress. However, it is not clear whether PRAK deletion-induced detrimental effects are related to Akt-1 signaling in the MI heart. Recently, Nawaito et al. (14) reported attenuated cardiac hypertrophy and preserved LV function in the PRAK/MK5 haplodeficient mice under increased chronic pressure overload created by transverse aortic constriction. This is likely due to the difference in the magnitude of stress and model used, indicating that PRAK potentially responds differently to different stresses. This may reflect the possibility that PRAK may play a different function in the modulation of myocardial ischemia/reperfusion injury and pressure overload-induced hypertrophy. The explanation of this discrepancy cannot be provided instantly and requires further investigation. Presumably, differences in the experimental design might be a cause of the contradictory effects of the PRAK deficiency. In addition, at normal baseline, we found that deletion of PRAK displays a lower cardiac performance as compared with control, suggesting that while PRAK is essential for maintaining normal cardiac performance, it is not clear if PRAK deletion will lead to a change in myocardial structure, which merits further investigation. In addition, we also found the deletion of PRAK resulted in the reduction of low coronary effluent as compared with control, suggesting the possibilities of vascular developmental deficiency or abnormal vessel dilation that led to the depression in cardiac performance.

PRAK/MK5 was first characterized as a p38-regulated/activated protein kinase (15, 16). In addition to p38 MAPK, PRAK/MK5 has been revealed to be phosphorylated by the atypical MAPKs, ERK3, and ERK4 (13, 18). Subsequent work indicated that both the activity and the subcellular localization of PRAK/MK5 could be regulated by interaction with these conventional and atypical MAPKs to evoke various physiological functions (9, 12, 21). In our studies, we found that deletion of PRAK induced a significant reduction of phosphorylated ERK, which was identified to act as a protective effect against myocardial ischemia and perfusion injury (7, 8). Therefore, the detrimental effect of deletion of PRAK myocardial ischemia and reperfusion injury may be associated with the attenuation of ERK activation following myocardial ischemia and reperfusion injury. Stimulation of p38 was reported to be linked to an increase cardiac remodeling in the hypertrophic model (24), but deletion of p38 was shown to increase cardiac remodeling and attenuate cardiac performance (17). Our study showed that deletion of PRAK increased cardiac fibrosis, apoptotic myocyte frequency, and apoptotic signal, indicating that PRAK plays a role in attenuating cardiac remodeling in the infarcted heart. Our current findings demonstrate a critical and novel role for PRAK in promoting functional recovery from myocardial ischemia and reperfusion injury and MI. This study the first to describe the cardioprotective effects of PRAK to maintain cardiac function and limit aberrant postacute MI remodeling, and it identifies a potentially novel target to treat postischemic injury in the myocardium. However, a detailed role of the PRAK pathway and conventional or atypical MAPK in cardiac pathophysiology remains to be determined. In addition, it remains unknown whether deletion of PRAK^−/− could change the signaling of HSP27, FoxO3, and/or Rhed in the myocardium and its correlation with PRAK. It will be interesting to examine their physiological interactions in the heart in the future. Furthermore, it will also be interesting to examine PRAK signaling using a p38 activation model in the future.

GRANTS

The work is supported by National Heart, Lung, and Blood Institute Grants R01 HL089405 and R01 HL115265.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

P.D., G.Q., S.Z., P.Y.L., Y.E.C., and T.C.Z. conceived and designed research; Y.T.Z., J.C.D., N.Y., H.C.W., J.W., and L.X.Z. performed experiments; J.C.D., H.C.W., J.W., L.X.Z., and Y.E.C. analyzed data; P.D., G.Q., S.Z., and T.C.Z. interpreted results of experiments; H.C.W. prepared figures; Y.T.Z., N.Y., and T.C.Z. drafted manuscript; S.Z. and P.Y.L. edited and revised manuscript; Y.T.Z., J.C.D., N.Y., H.C.W., J.W., P.D., G.Q., L.X.Z., S.Z., P.Y.L., Y.E.C., and T.C.Z. approved final version of manuscript.