Y-27632 was purchased from Tocris Cookson, Ltd. (Bristol, UK). All primary antibodies used in the present study, including anti-RhoA, -ROCK1, -c-Jun N-terminal kinase (JNK), -phosphorylated (P)-JNK, -extracellular signal-regulated kinase (ERK), -P-ERK, -P-P38, -P38, -P-NF-κB, -NF-κB, -inhibitor of NF-κB (IκB), -P-IκB, -B-cell lymphoma 2 (Bcl-2), -Bcl-2-associated X (Bax), -Caspase-3, -Caspase-9 and -GAPDH, were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). ELISA kits for the detection of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-1β were purchased by R&D Systems, Inc. (Minneapolis, MN, USA). Creatine kinase (CK) and lactate dehydrogenase (LDH) assay kits were obtained from MultiSciences (Lianke) Biotech Co., Ltd. (Hangzhou, China).

Male Sprague-Dawley rats (8 weeks old; ~250-280 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed together under a 12-h light/dark cycle, with free access to food and water, at a constant temperature of 22-24°C and relative humidity of 50±5%. All animal treatments in the present study were performed humanely and following the institutional and national guidelines for ethical animal research. All experimental procedures were conducted according to the guidelines and following approval from the Ethical Committee of Experimental Animal Care at Wenzhou Medical University (Wenzhou, China).

The rats were anesthetized via 2% isoflurane inhalation prior to establishing the I/R rat model by ligation of the left descending coronary artery, as previously described (9). The rats were anesthetized and restrained; the trachea was then incised and wrapped in catgut for later use. An incision was then made in the skin at the position of the heart, and the underlying ribs were exposed by blunt dissection to provide convenient access during the heart procedure. Following exposure of the heart, the left anterior descending coronary artery was blocked with a specific suture. A positive pressure respirator was applied to provide respiratory function for the experimental rats during the procedure. Elevation of the ST segment was used to confirm successful implementation of the surgical model. Recovery of a strong, regular heart rhythm indicated that the thoracic cavity was ready to be sutured. The suture of rats in the sham group was not tied. The rats were randomly divided into four groups: i) Sham; ii) I/R; iii) I/R + diltiazem (Dil; I/R + 10 mg/kg Dil); and iv) I/R + Y-27632 (I/R + 5 mg/kg Y-27632). The rats in the Y-27632 and Dil groups were treated continuously with Y-27632 or Dil for 5 days following I/R. At 24 h following the final administration of Y-27632 or Dil, the rats were sacrificed by CO₂ asphyxiation with 20% volume displacement/min; 8-ml blood samples and myocardial tissues were then collected.

Under anesthesia, the rats were administered with heparin (250 U/kg) intraperitoneally and the heart was excised following retrograde perfusion with complex Krebs-Henseleit (K-H) buffer at a pressure of 70 cm H₂O using the Langendorff system. The heart and perfusate were maintained at 37°C with a water jacket. Subsequently, K-H buffer was applied for the perfusion of the isolated hearts under 80-90 mmHg. The rats were then randomly divided into four groups: i) Control, hearts were perfused for a stabilization period of 80 min; ii) I/R, hearts were equilibrated for 20 min followed by global ischemia for 30 min. K-H buffer was used for reperfusion for 30 min; iii) I/R + Dil; and iv) I/R + Y-27632, hearts were processed according to the method of the I/R group; however, reperfusion was conducted using Y-27632 (0.5 mg/kg) or Dil (0.2 mg/kg) dissolved K-H buffer for 30 min.

Following reperfusion for various durations, the K-H effluent flow was measured as the coronary flow; a flow rate of >8 ml/min during the stabilization period was selected. Coronary effluent was collected following downstream analysis. A portable heart clip was used to record the contractile force of the myocardium during the experiment.

In the present study, an ECG was generated using needle electrodes and the BL-420 S biological function experiment system (Chengdu TME Technology, Co., Ltd., Chengdu, China). ST segment elevation on the ECG was considered to be an indicator of ischemia.

Triphenyltetrazolium (TTC) staining (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was conducted to determine the myocardial infarct size. Briefly, the hearts were incubated at -20°C for 15 min. The hearts were cut into five parallel pieces and placed in 1% TTC for 15 min at 37°C in the dark. The myocardial infarct ratio [(infarct area/whole heart area) x percentage] was calculated using image analysis software (Image-Pro Plus version 4.1; Media Cybernetics, Inc., Rockville, MD, USA).

The levels of inflammatory markers, including IL-6, IL-1β and TNF-α, in the serum were examined using mice ELISA kits according to the manufacturer’s protocols.

The serum levels of CK and LDH were measured using the commercial kits according to the manufacturer’s protocols.

The morphology of the myocardial tissues was examined using hematoxylin and eosin (H&E) staining. Following dehydration with 80, 90 and 100% ethanol and n-butanol, the myocardial tissue was waxed in a 60°C wax box and then embedded in paraffin. Tissue sections (5-µm) were dried at 45°C and obtained from each paraffin block. The sections were heated at 60°C for 1 h and dewaxed with xylene. Following hydration, the sections were stained with H&E, dehydrated with gradient ethanol, cleared with xylene and mounted with neutral gum. Images were captured using a light microscope (BX41; Olympus Corporation, Tokyo, Japan).

Total protein was extracted from heart samples and analyzed by western blotting with the specific antibodies described. Briefly, the samples were collected and homogenized, and then lysed in extraction buffer for 1 h on ice. The total protein content was quantified using a Bicinchoninic Acid protein assay kit (Beyotime Institute of Biotechnology, Nanjing, China). Equal quantities of protein (20 µg) were separated by 8-12% SDS-PAGE and then electrotransferred onto polyvinylidene difluoride (PVDF) membranes, which were blocked with skim milk at room temperature for 2 h. Following blocking, the PVDF membranes were incubated with specific antibodies against RhoA (1:1,000; cat. no. 2117), ROCK1 (1:1,000; cat. no. 4035), P-JNK (1:1,000; cat. no. 9255), JNK (1:1,000; cat. no. 9252, P-ERK (1:1,000; cat. no. 8544), ERK (1:1,000; cat. no. 4348), P-P38 (1:1,000; cat. no. 4511), P38 (1:1,000; cat. no. 8690), P-NF-κB (1:1,000; cat. no. 3033), NF-κB (1:1,000; cat. no. 8242), P-IκB (1:1,000; cat. no. 2859) IκB (1:1,000; cat. no. 4812), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 2764), Caspase-3 (1:1,000; cat. no. 9662), Caspase-9 (1:1,000; cat. no. 9508) and GAPDH (1:2,000; cat. no. 5174; all from Cell Signaling Technology, Inc.) overnight at 4°C. On the second day, the PVDF membranes were incubated with a secondary antibody [horseradish peroxidase-labeled mouse anti-rabbit immunoglobulin (Ig)-G (1:5,000; cat. no. 5127; Cell Signaling Technology, Inc.); and horseradish peroxidase-labeled rabbit anti-mouse IgG (1:5,000; cat. no. 58802; Cell Signaling Technology, Inc.)], at room temperature for 1 h following three washes with tris-buffered saline with Tween-20. Subsequently, the immunoreactive bands were visualized with a gel imaging system (Tanon 5200; Tanon Science and Technology Co., Ltd., Shanghai, China).

The data are presented as the mean ± standard deviation, and were analyzed by one-way analysis of variance followed by Tukey’s post hoc test using GraphPad Prism software 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine whether Y-27632 exhibits myocardial protective effects, the infarct size of rat hearts was analyzed by TTC staining. As presented in Fig. 1A, compared with the sham group, I/R markedly increased the infarct size, which was significantly reduced following treatment with Y-27632 or Dil.

To investigate the effects of Y-27632 on myocardial injuries, histopathological alterations of the myocardial tissues were analyzed. As presented in Fig. 1B, extensive inflammatory cells and the notable destruction of myocardial cellular structure were observed in the I/R group compared with the sham group. Additionally, treatment with Y-27632 or Dil markedly attenuated the histological alterations induced by I/R.

As presented in Fig. 2A, the I/R group exhibited significant decreases in the ratio of coronary flow compared with that in the sham group (P<0.01). By contrast, treatment with Y-27632 or Dil led to notable increases in the ratio of coronary flow.

Analysis of the ratio of myocardial contractility of isolated rat hearts revealed that the model group exhibited significantly a reduced ratio of contractility following the inhibition of K-H flow compared with that in the sham group (P<0.01). By contrast, treatment with Y-27632 or Dil led to a notable increase in the myocardial contractility (Fig. 2B).

As presented in Fig. 3, an increase in ST-segment and a decrease in R-amplitude were observed in the I/R group compared with the sham group; however, these effects were reversed following treatment with Y-27632 or Dil.

To investigate the potential anti-inflammatory effects of Y-27632, the serum levels of IL-6, TNF-α and IL-1β were measured by ELISA. As presented in Fig. 4A, the levels of IL-6, TNF-α and IL-1β were significantly upregulated in the I/R model group compared with those in the sham group. Treatment with Y-27632 or Dil significantly reduced the levels of inflammatory cytokines compared with those in the I/R group.

As representative indicators of myocardial injury, the levels of CK and LDH in the serum were analyzed. As presented in Fig. 4B, the serum levels of CK and LDH were significantly upregulated in the I/R group compared with the those in the sham group (P<0.01, respectively). By contrast, treatment with Y-27632 or Dil effectively suppressed the levels of CK and LDH.

To determine the intracellular signaling pathway underlying the myocardial protective effects of Y-27632 in I/R, the RhoA/ROCK and MAPK/NF-κB signaling pathways, and apoptosis were investigated. Upregulated expression levels of RhoA and ROCK1 (Fig. 5A and B) and increased phosphorylation of ERK1/2, JNK and P38 proteins (Fig. 6A and B) were observed in the I/R group; however, treatment with Y-27632 or Dil treatment notably reversed the effects of I/R on the expression of the aforementioned proteins.

To further investigate the downstream mechanism underlying the effects of Y-27632 on the I/R-induced heart in rats, the expression levels of P-IκB and P-NF-κB were determined (Fig. 7A and B). Western blot analysis demonstrated upregulated expression of P-IκB and P-NF-κB in the model group compared with the sham group. Following treatment with Y-27632 or Dil, significantly suppressed expression levels of P-IκB and P-NF-κB were detected. These results indicated that the inhibition of ROCK1 may prevent the activation of NF-κB.

As presented in Fig. 8A and B, the expression levels of apoptosis-associated proteins were detected to investigate the mechanism underlying the effect of Y-27632 on cardiomyocyte apoptosis. I/R injury markedly increased the expression of Bax, Caspase-3 and Caspase-9, which was associated with reduced expression levels of Bcl-2. Treatment with Y-27632 or Dil markedly reversed the effects of I/R injury on the expression of the aforementioned proteins. These results indicated that the inhibition of ROCK1 may suppress apoptosis.