Exogenous ubiquitin reduces inflammatory response and preserves myocardial function 3 days post-ischemia-reperfusion injury

Abstract

β-Adrenergic receptor (β-AR) stimulation increases extracellular levels of ubiquitin (UB) in myocytes, and exogenous UB decreases β-AR-stimulated myocyte apoptosis and myocardial fibrosis. Here, we hypothesized that exogenous UB modulates the inflammatory response, thereby playing a protective role in cardiac remodeling after ischemia-reperfusion (I/R) injury. C57BL/6 mice infused with vehicle or UB (1 μg·g⁻¹·h⁻¹) were subjected to myocardial I/R injury. Functional and biochemical parameters of the heart were examined 3 days post-I/R. Heart weight-to-body weight ratios were similarly increased in I/R and UB + I/R groups. The area at risk and infarct size were significantly lower in UB + I/R versus I/R groups. Measurement of heart function using echocardiography revealed that I/R decreases percent fractional shortening and percent ejection fraction. However, the decrease in fractional shortening and ejection fraction was significantly lower in the UB + I/R group. The UB + I/R group displayed a significant decrease in inflammatory infiltrates, neutrophils, and macrophages versus the I/R group. Neutrophil activity was significantly lower in the UB + I/R group. Analysis of the concentration of a panel of 23 cytokines/chemokines in the serum using a Bio-Plex assay revealed a significantly lower concentration of IL-12 subunit p40 in the UB + I/R versus I/R group. The concentration of monocyte chemotactic protein-1 was lower, whereas the concentration of macrophage inflammatory protein-1α was significantly higher, in the UB+I/R group versus the sham group. Expression of matrix metalloproteinase (MMP)-2 and activity of MMP-9 were higher in the UB + I/R group versus the I/R group. Levels of ubiquitinated proteins and tissue inhibitor of metalloproteinase 2 expression were increased to a similar extent in both I/R groups. Thus, exogenous UB plays a protective role in myocardial remodeling post-I/R with effects on cardiac function, area at risk/infarct size, the inflammatory response, levels of serum cytokines/chemokines, and MMP expression and activity.

NEW & NOTEWORTHY Stimulation of β-adrenergic receptors increases extracellular levels of ubiquitin (UB) in myocytes, and exogenous UB decreases β-adrenergic receptor-stimulated myocyte apoptosis and myocardial fibrosis. Here, we provide evidence that exogenous UB decreases the inflammatory response and preserves heart function 3 days after myocardial ischemia-reperfusion injury. Further identification of the molecular events involved in the anti-inflammatory role of exogenous UB may provide therapeutic targets for patients with ischemic heart disease.

INTRODUCTION

Heart disease is a leading cause of morbidity and mortality worldwide. Coronary artery disease is the most common form of heart disease. Myocardial infarction is a serious outcome of coronary artery disease. The principal therapy for patients with myocardial infarction to limit infarct size is myocardial reperfusion. Timely reperfusion facilitates myocyte salvage. However, the process of myocardial reperfusion can itself induce myocyte death, a phenomenon known as myocardial reperfusion injury (28, 49).

Myocardial remodeling after ischemia-reperfusion (I/R) injury typically involves a period of cell death via necrosis and apoptosis. Necrosis and reperfusion of the ischemic area trigger an inflammatory response in the heart with infiltration of cells such as neutrophils and macrophages (monocytes) in the area of injury to clear dead cells and cellular debris (24). Next, fibroblasts proliferate and move into the infarcted area, depositing collagenous scar material to fill the void of irreplaceable myocytes. Cytokines/chemokines and growth factors play a role in the differentiation of fibroblasts into myofibroblasts (9). Myofibroblasts, a major producer of extracellular matrix (ECM) proteins, produce matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinase (TIMPs). A highly regulated balance between MMPs and TIMPs is essential to the maintenance of ECM homeostasis (21).

Ubiquitin (UB), found in all eukaryotic cells, is a highly conserved low-molecular-weight protein (~8.5 kDa) with 76 amino acid residues. Intracellularly, UB plays a vital role in regulating protein turnover and protecting cells from damaged or misfolded proteins via the UB-proteasome system (UPS) (27). The UPS has been proposed to regulate internalization of cell surface receptors, the hypertrophic response, apoptosis, and tolerance to I/R insults in myocytes (69). UB is a normal component of plasma. Elevated levels of circulating extracellular UB are found in patients with parasitic and allergic diseases (4), alcoholic liver disease (63), type 2 diabetes (1), and β₂-microglobulin amyloidosis (46) and in patients undergoing chronic hemodialysis (2). Increased levels of extracellular UB are also observed in the cerebrospinal fluid of patients with traumatic brain injury (40). Extracellular UB has been proposed to have multiple functions including immune response regulation, anti-inflammatory properties, and neuroprotective activities (38, 55) as well as a role in growth and apoptosis of hematopoietic cells (12). Previously, our laboratory provided evidence that stimulation of β-adrenergic receptors (β-ARs) increases extracellular levels of UB in adult rat ventricular myocytes and that UB plays a significant role in β-AR-stimulated myocardial remodeling with effects on left ventricular (LV) function, fibrosis, and myocyte apoptosis (14, 59). However, the role of exogenous UB after myocardial I/R injury, a clinically relevant model, has not yet been examined.

Here, we investigated the in vivo role of exogenous UB in myocardial remodeling after 3 days of I/R injury in mice. It was hypothesized that exogenous UB modulates the inflammatory response, thereby playing a protective role in cardiac remodeling after I/R injury. We report that UB treatment improves heart function and decreases infarct size 3 days post-I/R. It also affects the inflammatory response, expression and activity of MMP-2 and MMP-9 in the heart, and circulating levels of cytokines/chemokines.

MATERIALS AND METHODS

Experimental animals.

This study conformed with regulations provided in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). The animal protocols were approved by the University Committee on Animal Care of East Tennessee State University. Animals were anesthetized using a mixture of isoflurane (2.5%) and oxygen (0.5 l/min) when undergoing termination by exsanguination. The heart was excised through an incision in the diaphragm. The study used C57BL/6 male mice (22–27 g) aged 8–12 wk (Jackson Laboratory).

I/R surgery.

Myocardial I/R surgery was performed as previously described (56). Briefly, mice were anesthetized using a mixture of isoflurane (2.5%) and oxygen (0.5 l/min) inhalation and ventilated using a rodent ventilator (Harvard Apparatus). Body temperature was maintained for the duration of the surgery at ~37°C using a heating pad. Mice underwent myocardial I/R injury by ligation of the left anterior descending coronary artery (LAD) using 7-0 braided silk ligature, which was tied using a shoestring knot over a 1-mm polyethylene tube. After 45 min of occlusion, the LAD was reperfused by pulling out the polyethylene tube and releasing the knot. I/R was confirmed by myocardial color and electrocardiographic changes (56). Sham-operated (sham) mice underwent left thoracotomy without LAD ligation. At the 3 day post-I/R time point, hearts were isolated and used for either histology or protein extraction.

Treatment of mice.

Mice were randomly assigned to the following four different treatment groups: sham, UB, I/R, and UB + I/R. Mice were implanted with micro-osmotic pumps (Alzet) that released either normal saline (sham) or UB from bovine erythrocytes (1 μg·g⁻¹·h⁻¹, cat. no. U-6253, Sigma-Aldrich) dissolved in normal saline over a 3-day period. The dose of UB was selected on the basis of previously published reports (14, 17). Myocardial I/R surgery was performed ~16 h after pump implantation.

Echocardiography.

Transthoracic two-dimensional M-mode echocardiograms were obtained using a VEVO 1100 imaging system (VisualSonics, Fujifilm) equipped with a 22- to 55-MHz MS550D transducer (13, 14). Echocardiography was performed at baseline and 3 days after I/R surgery. During echocardiography, mice were anesthetized using a mixture of isoflurane (1.5%) and oxygen (0.5 l/min), and body temperature was maintained at ~37°C using a heating pad. Heart rates were sustained between 400 and 500 beats/min as recommended (35). Percent fractional shortening (FS) and percent ejection fraction (EF) were calculated by Fujifilm software on the VEVO 1100. All echocardiography measurements were performed by a single investigator and confirmed by a second investigator.

Area at risk and infarct size quantification.

A dual-staining technique using Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) is commonly used to identify the area at risk (AAR) and area of necrosis (AON) after myocardial I/R injury (5, 18). Reperfusion for 2 h has been suggested to be necessary for reliable infarct staining by TTC post-I/R (18, 34). To measure AAR and AON, saline- and UB-infused mice underwent 45 min of ischemia. The suture was left in situ to retie the knot to obtain the initial occlusion. The LAD was reperfused by pulling out the polyethylene tube and releasing the knot. After 2 h of reperfusion, the knot was retied and isolated hearts were perfused using Krebs-Henseleit buffer to clear the blood followed by perfusion with 1% Evans blue dye. Hearts were then wrapped in cling wrap and frozen at −80°C for 5 min. Hearts were sliced into five to six short-axis sections, and slices were incubated with freshly prepared 1% TTC for 15 min at 37°C (18). Sections were weighed and photographed from both sides using a scanner. The remote area (blue stained), AAR (red and white stained), and AON (white stained) were quantified by planimetry using NIS-Elements software (Nikon). Different areas of the heart were corrected for heart weight and calculated as previously described (5, 18). AAR is expressed as a percentage of the total heart area, whereas AON is expressed as a percentage of AAR.

Infarct size 3 days post-I/R was also measured using Masson’s trichrome staining. For this, mice were euthanized, and isolated hearts were perfused using Krebs-Henseleit buffer to clear the blood. Hearts were then arrested in diastole using KCl (30 mmol/l) followed by fixation with 10% buffered formalin and subsequent paraffin embedding. Cross sections of the heart (5-µm thick) were stained with Masson’s trichrome (Sigma-Aldrich). Infarct size was calculated as a percentage of the affected LV using NIS-Elements software (Nikon).

Hematoxylin and eosin staining.

Cross sections (5 μm) of the paraffin-embedded heart were stained with hematoxylin and eosin to quantify inflammatory infiltrates. Images were acquired using Nikon Eclipse TE-2000-S microscope (Nikon) equipped with an Andor Zyla sCMOS camera (Andor, Belfast, UK). Quantitative analysis of inflammatory infiltrates was performed using NIS-Elements software (Nikon). For this, three randomly chosen fields of the infarcted LV were analyzed per animal.

Immunohistochemistry.

Cross sections (5 μm) of the heart were deparaffinized and rehydrated using xylene and ethanol washes. Epitope retrieval was performed using proteinase-XXIV at 37°C (0.1% in PBS) for 10 min. Tissue sections were then incubated in anti-neutrophil antibody (1:50, catalog no. SC-59338, Santa Cruz Biotechnology) or anti-F4/80 antibody (1:200, catalog no. SC-52664, Santa Cruz Biotechnology) overnight to quantify the number of neutrophils or macrophages, respectively. Sections were then stained using an ABC DAB kit (Santa Cruz Biotechnology) for colorimetric analysis. Images were acquired using a Nikon Eclipse TE-2000-S microscope (Nikon) equipped with an Andor Zyla sCMOS microscope camera (Andor) and analyzed using NIS-Elements software. Data are expressed as the number of positively stained cells per 0.1 mm² of infarct area. Four randomly selected fields of the infarcted LV were analyzed per animal.

Neutrophil activity assay.

Cross sections (5 μm) of the heart were stained using a naphthol AS-D chloroacetate esterase kit, also known as Leder stain, for the measurement of enzymatic activity of neutrophils according to the manufacturer’s instructions (Sigma-Aldrich). The areas stained pink were considered to be positive for neutrophil activity. Data are presented as average percentages of the positive-stained area to the total infarct area per image. Three randomly selected fields of the infarcted LV were analyzed per animal.

Serum cytokines and chemokines.

Concentrations of a group of cytokines/chemokines in the serum 3 days post-I/R were measured using a Bio-Plex Pro Mouse Cytokine 23-Plex assay according to the manufacturer’s instructions (Bio-Rad). The kit consisted of the following cytokines/chemokines: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 subunit p40 [IL-12(p40)], IL-12(p70), IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, keratinocyte chemoattractant (KC), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, macrophage inflammatory protein (MIP)-1β, regulated upon activation, normal T cell expressed, and secreted (RANTES), and TNF-α.

Western blot analysis.

LV tissue was snap frozen in liquid nitrogen and pulverized using a mortar and pestle. The tissue was then suspended in RIPA buffer [1% Triton X-100, 150 mmol/l NaCl, 10 mmol/l Tris (pH 7.4), 1 mmol/l EDTA, 1 mmol/l EGTA, 0.2 mmol/l PMSF, 0.2 mmol/l sodium orthovanadate, and 0.5% Nonidet P-40]. After centrifugation, equal amounts of total protein (10–50 μg) from the supernatant were resolved on SDS-polyacrylamide gels and transferred onto PVDF membranes. Serum samples (1 µl) were also analyzed by Western blot to examine levels of ubiquitinated proteins. Membranes were probed with primary antibodies directed against MMP-2 (1:1,000 dilution, catalog no. MAB-3308, Millipore), TIMP-2 (1:1,000 dilution, catalog no. MAB-3310, Millipore), or UB (1:5,000 dilution, catalog no. SC-8017, Santa Cruz Biotechnology) followed by the corresponding horseradish peroxidase-conjugated secondary antibodies. Antibody dilutions, catalog numbers, and sources are provided as recommended (6). Band intensities were quantified using ImageQuant LAS 500 imaging system (GE Healthcare). GAPDH immunostaining (Santa Cruz Biotechnology) or Ponceau S staining was used to normalize protein loading.

In-gel zymography.

Gelatin in-gel zymography using LV lysates (75 µg) was performed as previously described (68). Digested clear bands representing the activity of MMP-9 were quantified using ImageJ software.

Statistical analysis.

Data are expressed as means ± SE. Data were analyzed using one-way ANOVA followed by a Student-Newman-Keuls test or Student’s t-test where appropriate. Cytokine data were additionally analyzed using two-way ANOVA followed by a Newman-Keuls comparison test using GraphPad Prism 8 software. Exogenous UB has been suggested to modulate expression of TNF-α in response to lipopolysaccharide (39, 41, 44); therefore, TNF-α data were also analyzed using Welch’s t-test. P values of <0.05 were considered significant.

RESULTS

Survival and morphometric measurements.

The study used a total of 79 animals. Fourteen of the 79 animals died during the course of the experiment. One animal died in the UB-alone group. Seven of 33 mice died in I/R group, whereas 6 of 33 mice died in the UB + I/R group. The mortality rate was not significantly different between the two I/R groups. All deaths can be attributed to known and recorded surgical errors. Four animals were excluded from the study because of lack of infarction or lack of reperfusion. There were no significant changes in heart weights or body weights in any of the groups during the course of the study. I/R injury led to an increased lung wet weight-to-dry weight ratio with no significant difference between the two I/R groups (Table 1).

Table 1.

Morphometric measurements

Parameter	Sham	UB	I/R	UB + I/R
Body weight, g	24.1 ± 0.6	23.7 ± 1.0	23.5 ± 0.4	23.2 ± 0.5
Heart weight, mg	109.2 ± 5.8	106.8 ± 6.3	114.7 ± 3.6	108 ± 3.1
Heart weight/body weight, mg/g	4.54 ± 0.17	4.49 ± 0.11	4.89 ± 0.16	4.66 ± 0.13
Lung wet weight-to-dry weight ratio	1.02 ± 0.18	1.28 ± 0.02	2.77 ± 0.55*	2.86 ± 0.6†

Values are means ± SE; n = 4–5 mice in the sham and ubiquitin (UB) groups and 14–15 mice in the ischemia-reperfusion (I/R) and UB + I/R groups.

^*P < 0.05 vs. the sham group;

^†P < 0.05 vs. the UB group.

AAR and infarct size.

Analysis of AAR and AON using Evans blue/TTC staining showed that UB infusion significantly decreases AAR 2 h post-I/R surgery compared with the IR group 9AAR: 48.1 ± 4.7% in the I/R group vs. 20.1 ± 3.7% in the UB + I/R group, n = 4–5, P < 0.05 vs. the I/R group; Fig. 1, A and B). However, AON remained unchanged between the two I/R groups (AON: 11.3 ± 5.3% in the I/R group vs. 11.9 ± 5.0% in the UB + I/R group, n = 4–5).

Fig. 1.

Ubiquitin (UB) infusion decreases the percent area at risk (%AAR) and infarct size post-ischemia-reperfusion (I/R). A: representative images (both sides) of the heart stained with Evans blue and 2,3,5-triphenyltetrazolium chloride. AAR was defined by the red and white-stained areas, whereas the blue staining indicates the remote myocardium. B: average data representing %AAR; n = 4–5. C: cross sections of the heart stained with Masson’s trichrome stain. Collagen fibers are stained blue, nuclei are stained dark purple, and cytoplasm is stained red/pink. Arrows indicate areas of infarct. D: average data representing percent infarct size; n = 5. #P < 0.05 vs. I/R.

Analysis of infarct size using Masson’s trichrome staining showed that UB infusion significantly decreases infarct size 3 days post-I/R surgery (LV infarct: 28.92 ± 4.61% in the I/R group vs. 12.97 ± 2.73% in the UB + I/R group, n = 5, P < 0.05 vs. the I/R group; Fig. 1, C and D).

Echocardiographic measurements.

M-mode echocardiographic parameters were not significantly different between sham and UB-alone groups. I/R significantly reduced heart function, as evidenced by decreased FS and EF versus the sham group. UB infusion significantly improved I/R-mediated decreases in FS [sham: 37.85 ± 2.77%, UB: 34.88 ± 1.32%, and I/R: 27.66 ± 1.05% (P < 0.05 vs. the respective sham group), and UB + I/R: 32.95 ± 1.83% (P < 0.05 vs. the I/R group), n = 4–6; Fig. 2, A and B] and EF [sham: 68.50 ± 3.56%, UB: 64.85 ± 1.63%, I/R: 54.62 ± 1.74% (P < 0.05 vs. the respective sham group), and UB-I/R: 62.35 ± 2.56% (P < 0.05 vs. the I/R group), n = 4–6; Fig. 2, A and C].

Fig. 2.

Ubiquitin (UB) infusion improves heart function 3 days post-ischemia-reperfusion (I/R). Indexes of heart function, percent fractional shortening (%FS) and ejection fraction (%EF), were calculated using echocardiographic images 3 days post-I/R. A: M-mode images. B: %FS. C: %EF. n = 4 in the sham and UB-alone groups; n = 6 in the I/R groups. *P < 0.05 vs. the sham and UB-alone groups; #P < 0.05 vs. the I/R group.

Inflammatory infiltration.

Hematoxylin and eosin staining of myocardial cross sections showed a significant presence of inflammatory cells in the infarct LV regions of both I/R groups. Quantitative analysis revealed a significant decrease in the number of infiltrates in the UB + I/R versus I/R group (infiltrates per 0.1 mm²: 106.63 ± 15.95 in the I/R group and 46.52 ± 6.80 in the UB + I/R group, n = 3, P < 0.05 vs. the I/R group; Fig. 3).

Fig. 3.

Ubiquitin (UB) infusion decreases inflammatory infiltrates. Heart sections were stained with hematoxylin and eosin. The number of inflammatory infiltrates was quantified using NIS-Elements software. Top: hematoxylin and eosin-stained images of infarct left ventricular regions from ischemia-reperfusion (I/R) and UB + I/R groups. Blue staining represents nuclei; pink staining represents cytoplasm. Bottom: quantitative analysis of inflammatory infiltrates 3 days post-I/R; n = 3. #P < 0.05 vs. the I/R group.

Neutrophil number and activity.

Sham and UB-alone groups showed the presence of only a few neutrophils in the LV region with no significant difference between the two groups. I/R increased the number of neutrophils in the infarct LV regions of both groups. However, the number of neutrophils was significantly lower in UB + I/R versus I/R group [neutrophils per 0.1 mm²: 0.46 ± 0.34 in the sham group, 0.58 ± 0.32 in the UB group, 71.13 ± 6.83 in the I/R group (P < 0.05 vs. the sham and UB groups), and 20.27 ± 7.55 in the UB + I/R group (P < 0.05 vs. the sham and UB groups and P < 0.05 vs. the I/R group), n = 5; Fig. 4A].

Fig. 4.

Ubiquitin (UB) infusion decreases neutrophil number and activity 3 days post-ischemia-reperfusion (I/R). A: cross sections of the heart were immunostained using anti-neutrophil antibody. Top: images obtained from left ventricular (LV) regions of sham and UB-alone groups and infarct LV regions of I/R and UB + I/R groups. Brown staining represents positive immunostaining using anti-neutrophil primary antibodies. Bottom: quantitative analysis of the number of neutrophils in the heart; n = 5. B: cross sections of the heart were stained using a naphthol AS-D chloroacetate esterase kit. Top: images obtained from infarct LV regions of I/R and UB + I/R groups. Red staining indicates neutrophil activity. Bottom: quantitative analysis of neutrophil activity; n = 5. *P < 0.05 vs. sham and UB-alone groups; #P < 0.05 vs. the I/R group.

Measurement of neutrophil activity using a naphthol AS-D chloroacetate esterase kit showed the presence of active neutrophils in the infarct LV region post-I/R. Neutrophil activity was significantly lower in UB + I/R versus I/R group (neutrophil activity as a percentage of the infarct LV area: 9.95 ± 2.18 in the I/R group and 4.33 ± 2.18 in the UB + I/R group, n = 5, P < 0.05 vs. the I/R group; Fig. 4B).

Macrophage number.

Sham and UB-alone groups showed the presence of a few macrophages in the infarct LV region with no significant difference between the two groups. I/R significantly increased the number of macrophages in both groups. However, the number of macrophages was significantly lower in the UB + I/R versus I/R group [macrophages per 0.1 mm²: 0.84 ± 0.54 in the sham group, 0.63 ± 0.30 in the UB group, 91.33 ± 26.32 in the I/R group (P < 0.05 vs. the sham and UB groups), and 28.84 ± 5.22 in the UB + I/R group (P < 0.05 vs. the sham and UB groups and P < 0.05 vs. the I/R group), n = 3–5; Fig. 5].

Fig. 5.

Ubiquitin (UB) infusion decreases the number of macrophages in the infarct left ventricular (LV) region. Cross sections of the heart were immunostained using anti-F4/80 (macrophage) antibody. Top: images obtained from LV regions of sham and UB-alone groups and infarct LV regions of ischemia-reperfusion (I/R) and UB + I/R groups. Brown staining represents positive immunostaining using anti-F4/80 antibodies. Bottom: quantitative analysis of the number of macrophages in the heart; n = 5. *P < 0.05 vs. sham and UB-alone groups; #P < 0.05 vs. the I/R group.

Serum cytokine/chemokine levels.

Serum concentrations of the cytokines/chemokines remained unchanged between the sham and UB-alone groups (Table 2). However, myocardial I/R injury significantly decreased circulating concentration of IL-12(p40) in both groups compared with sham and UB-alone groups, respectively. Interestingly, the decrease in IL-12(p40) concentration was significantly greater in the UB + I/R versus I/R group (Table 2 and Fig. 6A). The serum concentration of MCP-1 was significantly lower in the UB+I/R group versus the sham group (Table 2 and Fig. 6B). On the other hand, the serum concentration of MIP-1α was significantly higher in the UB + I/R group versus the sham and UB-alone groups (Table 2 and Fig. 6C). For all other cytokines/chemokines, no significant differences were observed among the four groups. Analysis of TNF-α levels using Welch’s t-test revealed a significant decrease in TNF-α concentration in the UB-alone group versus the sham group (P = 0.02; Table 2). TNF-α levels tended to be higher in the I/R group versus the sham group with a P value equal to 0.08. However, no significant difference was found between the two I/R groups.

Table 2.

Serum cytokine/chemokine concentrations

Cytokine/Chemokine	Sham	UB	I/R	UB + I/R
IL-1α	15.4 ± 4.9	9.6 ± 1.3	12.7 ± 1.0	17.6 ± 3.5
IL-1β	33.8 ± 2.4	31.0 ± 6.2	32.1 ± 2.2	31.6 ± 9.4
IL-2	8.1 ± 1.5	5.5 ± 0.7	10.4 ± 1.3	9.8 ± 1.9
IL-3	12.8 ± 1.3	10.9 ± 0.8	14.9 ± 1.3	17.6 ± 3.8
IL-4	13.0 ± 2.2	9.4 ± 1.1	16.0 ± 1.5	19.1 ± 4.8
IL-5	16.1 ± 2.9	12.2 ± 1.1	15.3 ± 1.7	14.2 ± 2.4
IL-6	26.7 ± 3.7	23.0 ± 2.6	26.6 ± 6.3	32.2 ± 5.7
IL-9	47.1 ± 3.9	42.1 ± 2.7	54.8 ± 4.2	53.8 ± 11.5
IL-10	108.6 ± 11.8	100.4 ± 6.4	117.2 ± 9.1	121.8 ± 21.2
IL-12(p40)	871.4 ± 43.2	777.4 ± 92.2	574.3 ± 34.9a	349.3 ± 100.9a,b,c
IL-12(p70)	539.6 ± 67.1	450.4 ± 37.0	662.2 ± 63.5	612.8 ± 144.6
IL-13	198.2 ± 10.7	164.1 ± 14.0	200.4 ± 19.0	192.8 ± 35.2
IL-17A	139.0 ± 15.6	132.4 ± 8.5	183.8 ± 17.9	141.4 ± 45.4
Eotaxin	1,138.9 ± 102.9	1,010.0 ± 47.9	1,174.8 ± 127.8	1,227.3 ± 40.3
G-CSF	444.8 ± 54.3	356.7 ± 40.1	448.5 ± 33.5	495.9 ± 105
GM-CSF	64.9 ± 7.0	59.1 ± 4.4	73.0 ± 4.5	71.1 ± 12.2
IFN-γ	45.1 ± 4.8	41.6 ± 4.0	53.1 ± 5.2	51.0 ± 10.3
KC	180.7 ± 24.6	174.9 ± 27.9	109.5 ± 8.9	109.4 ± 10.5
MCP-1	486.6 ± 39.5	390.9 ± 16.3	412.0 ± 14.0	355.7 ± 39.7a
MIP-1α	6.7 ± 0.3	5.6 ± 0.5	8.5 ± 0.5b	9.8 ± 1.3a,b
MIP-1β	46.8 ± 4.9	38.3 ± 0.8	54.3 ± 2.3	57.5 ± 9.6
RANTES	177.7 ± 10.1	161.6 ± 6.7	197.2 ± 4.1	185.9 ± 16.0
TNF-α	207.9 ± 10.3	168.6 ± 9.0d	233.9 ± 7.5e	218.4 ± 47.1

All values are means ± SE (in pg/ml); n = 5 mice/group. UB, ubiquitin; I/R, ischemia-reperfusion; IL, interleukin; IL-12(p40), IL-12 subunit p40; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; KC, keratinocyte chemoattractant; MCP-1, monocyte chemotactic protein-1; MIP, macrophage inflammatory protein; RANTES, regulated upon activation, normal T cell expressed, and secreted.

^aP < 0.05 vs. the sham group;

^bP < 0.05 vs. the UB group;

^cP < 0.05 vs. the I/R group;

^dP = 0.02 vs. the sham group (as analyzed by Welch’s t-test);

^eP = 0.08 vs. the sham group (as analyzed by Welch’s t-test).

Fig. 6.

Serum cytokine levels. Serum cytokine levels were quantified using a Bio-Plex Pro Mouse Cytokine 23-Plex assay kit. A: IL-12 subunit p40. B: monocyte chemotactic protein (MCP)-1. C: macrophage inflammatory protein (MIP)-1α. I/R, ischemia-reperfusion; UB, ubiquitin. n = 5.

Protein ubiquitination.

Protein degradation via the UPS involves covalent linkage of UB onto proteins to mark them for degradation (69). Myocardial I/R injury increases levels of ubiquitinated proteins in the heart (48). To investigate whether UB infusion affects protein ubiquitination in the heart or circulation with or without I/R injury, levels of ubiquitinated proteins in LV lysates and serum samples were analyzed using Western blots. This analysis showed that UB alone had no effect on the levels of ubiquitinated proteins in LV lysates versus the sham group. Interestingly, I/R significantly increased levels of ubiquitinated proteins in LV lysates versus the sham and UB-alone groups. However, protein ubiquitination was not found to be significantly different between the two I/R groups (Fig. 7A). Levels of ubiquitinated proteins in serum samples remained unchanged among the four groups (Fig. 7B).

Fig. 7.

Protein ubiquitination. Total left ventricular lysates (10 µg; A) or serum samples (1 µl; B) were analyzed by Western blot using anti-ubiquitin (UB) antibodies. Top: UB immunostaining and Ponceau S staining. Bottom: quantitative data for the levels of ubiquitinated proteins normalized to Ponceau S staining (entire lanes). I/R, ischemia-reperfusion; Std, standard. n = 3–4. *P < 0.05 vs. sham and UB-alone groups.

Expression of MMP-2 and activity of MMP-9.

MMPs (MMP-2 and MMP-9) are key players in myocardial fibrosis and remodeling processes (60). Western blot analysis of LV lysates revealed that I/R increases MMP-2 protein (72 kDa) levels in both groups. However, MMP-2 protein levels were significantly higher in the UB + I/R versus I/R group (Fig. 8A). In-gel zymography showed no difference in MMP-9 activity between the sham and UB-alone groups (data not shown). I/R increased MMP-9 activity in both groups versus the sham group. However, the increase in MMP-9 activity was significantly greater in the UB + I/R versus I/R group (Fig. 8B).

Fig. 8.

A: matrix metalloproteinase (MMP)-2 protein levels. Total left ventricular (LV) lysates (50 µg) were analyzed by Western blot using anti-MMP-2 antibodies. Top: MMP-2 and GAPDH immunostaining. Bottom: quantitative analysis of MMP-2 normalized to GAPDH; n = 3. *P < 0.05 vs. sham and ubiquitin (UB)-alone groups; #P < 0.05 vs. the ischemia-reperfusion (I/R) group. B: MMP-9 activity. Total LV lysates (75 μg) were analyzed by gelatin in-gel zymography to measure MMP-9 activity (~90 kDa). Sham in the bar graph represents the pooled data from sham and UB-alone groups (n = 2 in each group); n = 3 for I/R and UB + I/R groups. *P < 0.05 vs. the sham group; #P < 0.05 vs. the I/R group.

Expression of TIMP-2.

MMP, specifically MMP-2, activity is regulated by TIMP-2 (33). Western blot analysis of LV lysates showed no difference in TIMP-2 expression between the sham and UB-alone groups (data not shown). I/R increased TIMP-2 protein levels in the heart. However, no significant difference in TIMP-2 protein levels was observed between the two I/R groups (Fig. 9).

Fig. 9.

Tissue inhibitor of metalloproteinase (TIMP)-2 protein levels. Total left ventricular lysates (50 µg) were analyzed by Western blot using anti-TIMP-2 antibodies. Top: TIMP-2 and GAPDH immunostaining. Bottom: quantitative analysis of TIMP-2 normalized to GAPDH. I/R, ischemia-reperfusion; UB, ubiquitin. n = 4–6. *P < 0.05 vs. the sham group.

DISCUSSION

Cardiovascular disease resulting from myocardial I/R injury is a leading cause of death worldwide. Previously, our laboratory reported that sympathetic stimulation (β-AR) increases extracellular levels of UB in myocytes and that extracellular UB plays a protective role in β-AR-stimulated myocyte apoptosis and myocardial remodeling (14, 59). This study provides evidence showing, for the first time, that exogenous UB plays a cardioprotective role in response to myocardial I/R injury. The major findings of the study are that preischemic UB treatment 1) decreases percent AAR and infarct size; 2) improves heart function, as evidenced by improved FS and EF; 3) decreases the inflammatory response, as evidenced by decreased numbers of infiltrates, number and activity of neutrophils, and number of macrophages; 4) affects circulating levels of cytokines/chemokines; and 5) increases expression of MMP-2 and activity of MMP-9.

Myocardial I/R injury induces an amalgam of patterns in which the stretched and dilated infarcted tissue increases LV volume with a combined volume and pressure load on noninfarcted areas (47). Neurohumoral activation, cytokine release, and oxidative stress have been suggested to play pivotal roles in myocardial remodeling post-I/R. Myocardial I/R injury is associated with myocardial cell death resulting in LV dilation and systolic dysfunction (62). Cardiac dysfunction post-I/R injury is related to infarct size (58). Previously, our laboratory provided evidence that UB infusion decreases myocardial fibrosis 7 days after chronic β-AR-stimulation in mice (14). Similar to I/R, chronic β-AR stimulation is known to induce cardiac myocyte death, create infarct-like lesions, and increase myocardial fibrosis in rodent models (7, 51). Prolonged β-AR stimulation also leads to the development of heart failure and increased mortality in animals and human patients (10, 67). This study provides evidence that UB treatment significantly decreases percent AAR and infarct size and improves heart function 3 days post-I/R injury. UB + I/R hearts exhibited a lesser degree of impairment in systolic function compared with I/R hearts. In fact, cardiac function in the UB + I/R group was not found to be significantly different from that in the sham or UB-alone groups. The observed improvement in heart function in UB-treated mice 3 days post-I/R injury may relate to decreased infarct size. In a rat model of lung I/R injury, UB treatment has been shown to decrease lung edema formation while improving lung function (26). Myocardial I/R injury is also associated with lung edema formation, as observed by an increased lung wet weight-to-dry weight ratio. However, UB treatment with or without myocardial I/R injury had no effect on lung edema formation. These differential effects of UB on lung edema formation may relate to the model system (pulmonary vs. cardiogenic I/R model) and/or observation time point.

Myocardial I/R injury initiates an inflammatory response in the heart (23). Infiltration of neutrophils occurs within hours and peaks ~1–2 days after I/R in rodents (16, 36). Neutrophils facilitate the post-I/R repair process by engulfing dead cells and tissue debris. Neutrophils then undergo apoptosis and are then cleared by recruited macrophages (monocytes). This activates an anti-inflammatory program in macrophages (22), ending the inflammatory phase of infarct healing. During the proliferative phase (~2–5 days post-I/R), activated fibroblasts (myofibroblasts) produce ECM proteins aiding in scar formation (16). Activated neutrophils release cytotoxic oxidants and enzymes to exacerbate myocardial injury at the time of reperfusion (31). In a canine myocardial I/R injury model, depleting the number of neutrophils in circulation using antiserum decreased infarct size (50). Consistent with previously published reports, we observed an increased number of neutrophils and macrophages in the infarct LV region 3 days post-I/R. Interestingly, the inflammatory response in the heart was significantly lower in UB-treated mice 3 days post-I/R, suggesting an anti-inflammatory role for UB in myocardial I/R injury. Recruitment of neutrophils to the site of myocardial injury is a highly organized event involving the generation of damage-associated molecular patterns by necrotic cells, matrix fragments, chemokine induction, and adhesion molecules such as VCAM-1 and intracellular adhesion molecules (ICAM-1 and ICAM-2) (23). Although UB has been proposed to function as an endogenous opponent of damage-associated molecular patterns (38), future investigations are needed to understand the mechanism by which UB decreases the inflammatory response in the heart post-I/R.

Exogenous UB has been suggested to play a role in the modulation of the immune response after tissue injury. In a swine model of femur fracture and hemorrhage, intravenous delivery of UB was associated with reduced endotoxin-evoked TNF-α production (39). In vitro, UB treatment inhibited the expression of TNF-α in response to LPS in whole blood and peripheral blood mononuclear cell cultures (41). In contrast, UB treatment enhanced LPS-induced TNF-α mRNA expression and secretion in the RAW264.7 macrophage cell line (44). Here, TNF-α levels were significantly lower in the serum of the UB-alone group versus the sham group using Welch’s t-test, suggesting that UB treatment decreases basal circulating levels of TNF-α. However, no significant difference in the circulating levels of TNF-α was observed between the two I/R groups.

In a rat model of lung I/R injury, intravenous administration of UB enhanced concentrations of IL-4, IL-13, and IL-10 in lung homogenates (26). IL-10 is an anti-inflammatory cytokine (25), which could potentially affect UB-mediated remodeling by reducing the severity of the inflammatory response. In this study, circulating levels of IL-4, IL-10, and IL-13 remained unchanged 3 days post-I/R injury with or without UB treatment. However, it should be emphasized that Garcia-Covarrubias et al. (26) used the rat as their model system, whereas this study used the mouse model of I/R injury. In the lung I/R model, cytokine/chemokine concentrations were measured in lung homogenates after 90 min of ischemia and 60 min after reperfusion. The present study measured cytokine/chemokine concentrations in the serum after 45 min of ischemia and 3 days after reperfusion. In addition, UB (1.5 mg/kg body wt) was administered intravenously in the lung I/R model 5 min before reperfusion, whereas the present study used continuous UB infusion using osmotic pumps and UB infusion was started ~16 h before myocardial I/R injury.

Interestingly, we observed a significant decrease in the serum concentration of IL-12(p40) in the UB + I/R versus I/R group. The IL-12 family consists of heterodimeric cytokines and includes members such as IL-12, IL-23, IL-27, and IL-35. Proinflammatory cytokine IL-12, also referred to as IL-12(p70), is produced predominantly by dendritic cells and macrophages. IL-12(p40) is a component of IL-12(p70) and IL-23. Multifunctional cytokine IL-12(p70) has been suggested to play a key role in the regulation of cell-mediated immune responses through the differentiation of naïve CD4⁺ T cells into type 1 helper T cells. On the other hand, IL-12(p40), when produced in excess, can act as an antagonist for IL-12(p70). It has also been shown to act as a chemotactic factor for the recruitment of macrophages (19, 66). In this regard, it is interesting to note that the levels of MCP-1 were lower, whereas levels of MIP-1α were higher, in the UB + I/R group versus the sham group. MCP-1 recruits monocytes to the site of inflammation. Increased serum levels of MCP-1 have been shown to be associated with an increased risk of death or myocardial infarction in patients with acute coronary syndrome (15). MIP-1α, also known as chemokine (C-C motif) 3, acts as a chemoattractant for CD8 T lymphocytes and B cells (54, 64). It also inhibits hematopoietic stem and progenitor cell proliferation (8). Myocardial I/R injury increases MIP-1α expression in the heart (37, 45). However, its role in myocardial I/R injury remains to be explored. In our study, the number of macrophages was lower in the UB + I/R group versus the I/R group. However, future investigations are needed to delineate the correlation between serum cytokine/chemokine levels and macrophage numbers in the heart post-I/R in the presence of exogenous UB.

Myocardial I/R injury is associated with increased levels of ubiquitinated proteins and decreased activities of the 20S and 26S proteasomes in the heart (48). Enhancement of proteasome proteolytic function has been shown to protect against I/R injury (32), whereas inhibition of the proteasome in cardiac myocytes exaggerates myocardial I/R injury in mice (65). Here, we observed increased levels of ubiquitinated proteins in the LV lysates 3 days post-I/R, suggesting that I/R injury induces impairment of UPS. UB infusion had no effect on the levels of ubiquitinated proteins post-I/R, suggesting that the protective effects of UB infusion may occur independent of the UPS. The G protein-coupled receptor chemokine (C-X-C motif) receptor type 4 (CXCR4) has been identified as a receptor for extracellular UB in the THP-1 leukemia cell line (52, 53). We have provided evidence that exogenous UB has the potential to modulate phenotype and function of cardiac microvascular endothelial cells and fibroblasts via its interaction with CXCR4 (57, 61). Therefore, it is likely that the protective effects of exogenous UB in the heart post-I/R occur via its interaction with CXCR4. However, future investigations are warranted to clarify the involvement of CXCR4 in UB signaling in the heart post-I/R.

The infarct healing process post-I/R is accomplished via a series of events that ultimately result in the formation of a fibrotic scar (29). ECM homoeostasis is controlled by MMPs and their inhibitors, TIMPs. Gelatinases (MMP-2 and MMP-9) are of particular interest as they are suggested to cleave several ECM substrates, including gelatin and collagen types I, IV, V, VII, and X (11). Myocardial I/R injury has been known to increase expression and activity of MMP-2 and MMP-9 in the human heart (30) as well as in animal models (3, 20). Previously, we have shown that UB infusion increases expression of MMP-2, MMP-9, and TIMP-2 in the heart in response to β-AR stimulation (14). Here, we observed that myocardial I/R injury increases expression of MMP-2 and TIMP-2 and activity of MMP-9 in the heart. However, the expression of MMP-2 and activity of MMP-9 were greater in the UB + I/R group versus the I/R group. The increase in expression and activity of MMPs may indicate enhanced degradation of the ECM in the presence of UB, which may ultimately contribute to reduced infarct size and/or deposition of fibrosis post-I/R injury.

In conclusion, the data presented here provide evidence, for the first time, of a cardioprotective role of exogenous UB after myocardial I/R injury in mice. UB treatment decreased AAR/infarct size and resulted in improved heart function, as observed by improved FS and EF. It also decreased the inflammatory response and led to interesting changes in circulating levels of cytokines/chemokines. These results, together with our previous findings (14), suggest that UB has the potential to modulate the myocardial remodeling process of the heart in response to an injury. However, the study has several limitations. First, all the observations were made 3 days post-I/R, and UB treatment was started ~16 h before myocardial I/R injury. This is important to point out since the infarct healing process post-I/R involves three overlapping phases (inflammation, proliferation, and maturation) and the remodeling process can last up to 28 days post-I/R in rodents (16). Therefore, the study time point should be extended beyond 3 days to determine the long-term beneficial impact of UB in the healing processes of the heart post-I/R. Investigations are also needed to determine the effect of postischemic UB treatment in myocardial remodeling. Second, concentrations of cytokines/chemokines should be measured in the serum as well as myocardial tissue at an earlier time point post-I/R to delineate the cause-and-effect relationship. Third, the study used male mice with a C57BL/6 background. The incidence of cardiovascular disease differs between sexes because of differences in risk factors and hormones (42, 43). Therefore, the study should be expanded to age-matched female mice.

GRANTS

This work was supported by Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Merit Review Awards BX002332 and BX000640 (to K. Singh), National Institutes of Health (NIH) Grants R15-HL-129140 (to M. Singh) and R15-HL-141947 (to K. Singh), and funds from the Institutional Research and Improvement account (to K. Singh) and NIH Grant C06-RR-0306551.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

S.L.C.S., S.D., M.S., and K.S. conceived and designed research; S.L.C.S., S.D., K.A.L., P.R.T., C.R.D., and J.M.P. performed experiments; S.L.C.S., S.D., K.A.L., and J.M.P. analyzed data; S.L.C.S., S.D., K.A.L., M.S., and K.S. interpreted results of experiments; S.L.C.S. and SD prepared figures; S.L.C.S. drafted manuscript; S.L.C.S., S.D., and K.S. edited and revised manuscript; S.L.C.S., S.D., K.A.L., P.R.T., C.R.D., J.M.P., M.S., and K.S. approved final version of manuscript.