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Glycogen synthase kinase 3β inhibition reduces mitochondrial oxidative stress in chronic myocardial ischemia

Open ArchivePublished:February 03, 2018DOI:https://doi.org/10.1016/j.jtcvs.2017.12.127

      Abstract

      Objectives

      Glycogen synthase kinase 3β (GSK-3β) inhibition has been reported to increase microvascular density and improve myocardial blood flow in a porcine model of chronic myocardial ischemia and metabolic syndrome. Inhibition of GSK-3β can also be cardioprotective by modulating fibrosis signaling and mitochondrial-induced apoptosis. We hypothesized GSK-3β inhibition would have a beneficial effect on myocardial fibrosis and oxidative stress in a porcine model of chronic myocardial ischemia and metabolic syndrome.

      Methods

      Pigs were fed a high fat diet for 4 weeks followed by placement of an ameroid constrictor to the left circumflex coronary artery. Three weeks later animals received either no drug or a GSK-3β inhibitor. The diets and placebo/GSK-3β inhibition were continued for an additional 5 weeks, the pigs were then euthanized, and the myocardial tissue was harvested. Collagen expression was analyzed via Picrosirius staining. Oxidative stress was analyzed via Oxyblot analysis. Protein expression was analyzed via Western blot.

      Results

      GSK-3β inhibition was associated with decreased collagen expression and oxidative stress in the ischemic and nonischemic myocardial tissue compared with control. There was a decrease in profibrotic proteins transforming growth factor-β, p-SMAD2/3, and matrix metalloproteinase-9, and in proapoptotic and oxidative stress proteins, apoptosis inducing factor, the cleaved caspase 3/caspase 3 protein ratio and phosphorylated myeloid cell leukemia sequence-1 in the GSK-3β inhibited group compared with the control.

      Conclusions

      In the setting of metabolic syndrome and chronic myocardial ischemia, inhibition of GSK-3β decreases collagen formation and oxidative stress in myocardial tissue. GSK-3β inhibition might be having this beneficial effect by downregulating transforming growth factor-β/SMAD2/3 signaling and decreasing mitochondrial induced cellular stress.

      Graphical abstract

      Key Words

      Abbreviations and Acronyms:

      AIF (apoptosis inducing factor), CON (control group), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), GSK-3β (glycogen synthase kinase- 3β), GSK-3βI (glycogen synthase kinase-3β inhibitor), HSF-1 (heat shock factor-1), MCL-1 (myeloid cell leukemia sequence-1), MMP-9 (matrix metalloproteinase-9), mPTP (mitochondrial permeability transition pore), p-MCL-1 (phosphorylated myeloid cell leukemia sequence-1), TGF-β (transforming growth factor-β)
      Figure thumbnail fx2
      GSK-3β inhibition decreases fibrosis in ischemic myocardium in pigs with metabolic syndrome.
      GSK-3β inhibition might serve as a potential therapy to decrease myocardial fibrosis and oxidative stress in patients suffering from coronary artery disease and metabolic syndrome.
      In the setting of metabolic syndrome and chronic myocardial ischemia, inhibition of GSK-3β decreases collagen formation and oxidative stress in myocardial tissue. GSK-3β inhibition is associated with decreased profibrotic and mitochondrial-induced stress protein signaling. GSK-3β inhibition might serve as a therapy to inhibit myocardial fibrosis for patients suffering from coronary artery disease.
      See Editorial Commentary page 2504.
      Metabolic syndrome is a diagnosis encompassing a host of pathologic conditions observed in cardiovascular disease states including hypertension, obesity, hyperlipidemia, and glucose intolerance/diabetes mellitus type 2. The incidence of metabolic syndrome is approximately 24% in the United States.
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      Methods

       Animal Model

      Sixteen Yorkshire swine (E.M. Parsons and Sons, Hadley, Mass) were fed a 500 g/d high fat/high cholesterol diet for 4 weeks (Sinclair Research, Columbia, Mo).
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      • et al.
      Overfed Ossabaw swine with early stage metabolic syndrome have normal coronary collateral development in response to chronic ischemia.
      This diet consists of 4% cholesterol, 2.3% corn oil, 17.2% coconut oil, 1.5% sodium cholate, and 75% regular chow.
      The swine received aspirin (10 mg/kg) 1 day preoperatively and 5 days postoperatively and cephalexin (30 mg/kg) 1 day preoperatively and 5 days postoperatively. Anesthesia was then induced with telazol (4.4 mg/kg) and xylazine (2.2 mg/kg) and maintained with isoflurane (0.75%-3%). A 72-hour fentanyl patch (4 μg/kg) was placed before surgery. A left mini thoracotomy was then performed, the left circumflex artery was identified and manually occluded for 2 minutes while gold microspheres (5 cc, BioPal, Worcester, Mass) were injected into the left atrium to allow for microsphere labeling of the nonischemic territory. Heparin (80 IU/kg) was injected before occlusion. To induce chronic myocardial ischemia, a titanium ameroid constrictor ring (Research Instruments SW, Escondido, Calif) was placed on the left circumflex coronary artery. The hydrophilic core of the ameroid constrictor enlarges gradually over the course of 10 to 20 days to reproduce a chronic model of ischemia. Amiodarone (10 mg/kg) was used as needed for arrhythmias that developed as a result of isolation and manipulation of the left circumflex artery at the time of ameroid placements.
      Two weeks later, the animals were split into 2 groups and received either a placebo (high cholesterol control group, 1.5 mg/kg/d of DMSO, termed as “CON” group; n = 8); or a GSK-3β inhibitor (GSK-3βI; 1.5 mg/kg/d dissolved in DMSO, termed as “GSK-3βI” group; n = 8). The high cholesterol diet and GSK-3βI was continued for 5 weeks (once a day for 6 of 7 days a week).
      Five weeks after the start of treatment, pigs were placed under general anesthesia and the terminal procedure was performed. The heart was exposed through a sternotomy, coronary angiography was performed, and microspheres were injected at rest (Lutetium, 5 cc; BioPal) and with pacing (150 beats per minute; Samarium, 5 cc; BioPal) in the left atrium while simultaneously withdrawing blood from a femoral artery catheter. Euthanasia was performed using exsanguination under deep anesthesia. Myocardial and peripheral organ tissue was collected. Tissue was collected from the chronically ischemic myocardium (circumflex artery territory) and the nonischemic (anterior descending artery territory) myocardium. Ischemic tissue was identified on the basis of the proximity to the left circumflex (distal to the ameroid constrictor) and nonischemic tissue was identified on the basis of the proximity to the left anterior descending artery. This tissue was either rapidly frozen in liquid nitrogen or placed in formalin. One pig died at the end of the ameroid placement procedure as a result of difficulty with anesthesia. The pig became bradycardic and hypocarbic before going into cardiac arrest during closing of the incision. This pig was excluded from the study. The Rhode Island Hospital Institutional Animal Care and Use Committee approved and supervised all experiments. The Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals were used to ensure proper care of all animals.
      A glucose tolerance test was performed before the start of the ameroid and harvest procedures. We administered 0.5 g/kg of 50% dextrose at the start of the surgery and glucose was measured at 30 and 60 minutes. The pigs were measured for length and weight before the start of the ameroid and harvest procedures. Blood taken at the time of the harvest procedure was sent to the Rhode Island Hospital chemistry lab for quantification of cholesterol parameters. There were no differences in glucose levels, weight, or cholesterol parameters between the control and GSK-3β inhibited groups.
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      • et al.
      Glycogen synthase kinase 3B inhibition improves myocardial angiogenesis and collateral-dependent perfusion in a swine model of metabolic syndrome.

       Picrosirius Red Staining

      Picrosirius red staining was performed on approximately 1 cm2 tissue sections using the Picrosirius Red Stain Kit (Polysciences, Inc, Warrington, Pa). Tissue slides containing paraffin sections were dewaxed and hydrated, nuclei were stained with Weigert's hematoxylin and the sections were then washed with running tap water. Picrosirius stain was applied to the tissue slides for 1 hour after which the slides were washed in 2 changes of acidified water. Water was removed and dehydration was performed using 3 changes of 100% ethanol. The tissue was cleared in xylene and mounted in a resinous medium. Images were captured at 20× magnification with a Nikon E800 Eclipse microscope (Nikon, Tokyo, Japan) at the same exposure in 5 random fields. Image J software (National Institutes of Health, Bethesda, Md) was used to measure collagen expression in a blinded fashion. Representative images are included (Figures 1 and 2).

       Oxidative Stress

      Oxyblot (Millipore, Billerica, Mass) was performed on the ischemic and nonischemic myocardial tissue as described by the manufacturer. Tissue was lysed in Radioimmunoprecipitation Assay buffer (Boston BioProducts, Ashland, Mass) and 2 aliquots of each specimen were analyzed (1 aliquot was subjected to a derivatization reaction, the other aliquot served as a negative control). For each pig, 5 μL of protein sample was divided into 2 Eppendorf tubes and then denatured with 12% sodium dodecyl sulfate. For the positive control, 2,4 dinitrophenylhydrazine solution was used to derivatize the samples. Derivatization control solution was used in the control samples. Both sets of samples were incubated at room temperature for 15 minutes. Neutralization solution was then added. Both samples were then loaded onto a polyacrylamide gel and imaged as described in Figure 3.

       Protein Expression

      Western blot analysis was conducted as previously described.
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      • Elmadhun N.Y.
      • Feng J.
      • Liu Y.
      • Mitchell H.
      • et al.
      Calpain inhibition decreases myocardial apoptosis in a swine model of chronic myocardial ischemia.
      Tissue was lysed in radioimmunoprecipitation assay buffer (Boston BioProducts); 40 μg was fractionated using sodium dodecyl sulfide polyacrylamide gel electrophoresis using 3% to 8% Tris-Acetate gels (NuPage Novex Mini Gel; Thermo Fisher, Waltham, Mass), and the protein was transferred to polyvinylidene difluoride membranes (Millipore) and incubated overnight at 4°C with primary antibodies against cleaved caspase 3, caspase 3, phosphorylated (serine 140) MCL-1, TGF-β, phosphorylated-SMAD2/3, matrix metalloproteinase-9 (MMP-9), apoptosis inducing factor (AIF), and heat shock factor-1 (all from Cell Signaling, Danvers, Mass). Twenty-four hours later, the membranes were incubated with the appropriate horseradish peroxidase-linked secondary antibody for 1 hour at room temperature (Jackson ImmunoResearch, West Grove, Pa). Immune complexes were visualized with enhanced chemiluminescence. Images were captured with a digital camera system (G-Box; Syngene, Cambridge, England). Image J software (National Institutes of Health) was used to quantify band densitometry as arbitrary light units. Loading error was controlled for by probing membranes with an antibody against glyceraldehyde 3-phosphate dehydrogenase.

       Data Analysis

      Results are reported as a mean ± standard error of the mean. GraphPad 5.0 Software (GraphPad Software Inc, San Diego, Calif) was used to perform unpaired Mann–Whitney U test between the GSK-3βI and CON groups. Protein expression was normalized to glyceraldehyde 3-phosphate dehydrogenase in all pigs and is reported as fold change compared with the CON group.

      Results

       GSK-3β Inhibition Decreases Fibrosis and Collagen Expression in Pigs With Metabolic Syndrome

      Inhibition of GSK-3β decreased myocardial fibrosis assessed using Picrosirius red staining compared with the control group. GSK-3β inhibition reduced fibrosis in the ischemic (Figure 1) as well as nonischemic myocardial tissue compared with the control (Figure 2).
      Figure thumbnail gr1
      Figure 1Glycogen synthase kinase 3β (GSK-3β) inhibition decreases fibrosis and collagen expression in ischemic myocardium in metabolic syndrome pigs. GSK-3β inhibition decreased collagen formation in the ischemic myocardium compared with the high cholesterol control group (CON; n = 7). Representative images for Picrosirius, hematoxylin and eosin (H&E), and Trichrome staining for each pig group staining shown to right of graph. GSK-3βI, GSK-3β inhibited group (n = 8). *P < .05, Mann–Whitney U test.
      Figure thumbnail gr2
      Figure 2Glycogen synthase kinase 3β (GSK-3β) inhibition decreases fibrosis and collagen expression in nonischemic myocardium in metabolic syndrome pigs. GSK-3β inhibition decreased collagen formation in the ischemic myocardium compared with the high cholesterol control group (CON; n = 6). Representative images for Picrosirius, hematoxylin and eosin (H&E), and Trichrome staining for each pig group; staining shown to right of graph. GSK-3βI, GSK-3β inhibited group (n = 7). *P < .05, Mann–Whitney U test.

       GSK-3β Inhibition Decreases Oxidative Stress in Metabolic Syndrome Pigs

      Inhibition of GSK-3β decreased oxidative stress in the GSK-3β inhibited pigs assessed using measurement of protein carbonyls with Oxyblot. GSK-3β inhibition reduced tissue oxidative stress in the ischemic as well as nonischemic myocardial tissue in the GSK-3β inhibited group compared with the control group (Figure 3).
      Figure thumbnail gr3
      Figure 3Glycogen synthase kinase 3β (GSK-3β) inhibition decreases oxidative stress in ischemic and nonischemic myocardium in metabolic syndrome pigs. GSK-3β inhibition decreased oxidative stress in the ischemic and nonischemic myocardium compared with the high cholesterol control group (CON; n = 8). Representative image from Oxyblot shown below graph. GSK-3βI, GSK-3β inhibited group (n = 8). *P < .05, Mann–Whitney U test.

       GSK-3β Inhibition Decreases Profibrotic Signaling in Ischemic Myocardial Tissue in Metabolic Syndrome Pigs

      Inhibition of GSK-3β decreased expression of the profibrotic cytokine TGF-β and phosphorylation of its downstream effector protein p-SMAD2/3. Inhibition of GSK-3β also decreased expression of extracellular matrix remodeling protein MMP-9 in the ischemic myocardial tissue compared with the control (Figure 4 and Table 1).
      Figure thumbnail gr4
      Figure 4Glycogen synthase kinase 3β (GSK-3β) inhibition decreases profibrotic signaling in ischemic myocardial tissue in metabolic syndrome pigs. GSK-3β inhibition decreased transforming growth factor-β (TGF-β), p-SMAD2/3, and MMP-9 in the ischemic myocardium compared with the high cholesterol control group (CON; n = 7). Representative images from Western blot shown below graph. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; MMP-9, matrix metalloproteinase-9; GSK-3βI, GSK-3β inhibited group (n = 7). *P < .05, Mann–Whitney U test.
      Table 1GSK-3β inhibition downregulates TGF-β/SMAD2/3 signaling and mitochondrial-induced cellular stress
      Ischemic myocardial tissueNonischemic myocardial tissue
      ProteinCONGSK-3βIP valueProteinCONGSK-3βIP value
      TGF-β1.0 ± 0.200.43 ± 0.13<.03TGF-β1.0 ± 0.130.29 ± 0.07.0006
      p-SMAD2/31.0 ± 0.060.69 ± 0.23.007p-SMAD2/31.0 ± 0.140.43 ± 0.10.006
      MMP-91.0 ± 0.070.45 ± 0.07.006MMP-91.0 ± 0.270.44 ± 0.11.054
      AIF1.0 ± 0.120.70 ± 0.21<.01AIF1.0 ± 0.271.07 ± 0.29.96
      Cleaved/total caspase 31.0 ± 0.130.62 ± 0.07.01Cleaved caspase 31.0 ± 0.250.58 ± 0.20<.007
      p-MCL-11.0 ± 0.190.33 ± 0.03.0003p-MCL-11.0 ± 0.150.31 ± 0.05<.006
      HSF-10.88 ± 0.160.75 ± 0.09.09
      Results are reported as a mean ± standard error of the mean. GraphPad 5.0 Software (GraphPad Software Inc, San Diego, Calif) was used to perform unpaired Mann–Whitney U test between the GSK-3βI (n = 7) and CON (n = 7) groups. Protein expression is normalized to GAPDH in all pigs and is reported as fold change compared with CON group. *P < .05, Mann–Whitney U test. CON, High-cholesterol control group; GSK-3βI, glycogen synthase kinase-3β inhibitor; TGF-β, transforming growth factor-β; MMP-9, matrix metalloproteinase-9; AIF, apoptosis inducing factor; p-MCL-1, phosphorylated myeloid cell leukemia sequence-1; HSF-1, heat shock factor-1.

       GSK-3β Inhibition Reduces Apoptotic Signaling Pathways in Ischemic Myocardial Tissue in Pigs With Metabolic Syndrome

      Inhibition of GSK-3β reduced levels of the proapoptotic protein AIF compared with control pigs. Inhibition of GSK-3β also decreased the ratio of cleaved/total caspase 3 compared with the control. Inhibition of GSK-3β was also associated with decreased expression of the phosphorylated, inactive, form of antiapoptotic MCL-1 in the ischemic myocardial tissue compared with the control. There was no significant difference in the expression levels of heat shock factor-1 (P = .093; Figure 5 and Table 1).
      Figure thumbnail gr5
      Figure 5Glycogen synthase kinase 3β (GSK-3β) inhibition decreases mitochondrial proapoptotic signaling in ischemic in myocardial tissue in metabolic syndrome pigs. GSK-3β inhibition decreased apoptosis inducing factor, phosphorylated myeloid cell leukemia sequence-1 (Ser 140; p-MCL-1), and the ratio of cleaved caspase 3/caspase 3 in the ischemic myocardium compared with the high cholesterol control group (CON; n = 8). Representative images from Western blot shown below graph. HSF-1, Heat shock factor-1; GSK-3βI, GSK-3β inhibited group (n = 8); AIF, apoptosis inducing factor; p-MCL-1, phosphorylated myeloid cell leukemia sequence-1. *P < .05, Mann–Whitney U test.

       GSK-3β Inhibition Decreases Profibrotic Signaling in Nonischemic Myocardial Tissue in Metabolic Syndrome Pigs

      Inhibition of GSK-3β resulted in decreased expression of the profibrotic cytokine TGF-β and phosphorylation of its downstream effector protein SMAD2/3 compared with the control pigs (Figure 6 and Table 1). There was no significant difference in the expression level of extracellular matrix remodeling protein MMP-9 between the 2 groups (P = .054).
      Figure thumbnail gr6
      Figure 6Glycogen synthase kinase 3β (GSK-3β) inhibition decreases profibrotic signaling in nonischemic myocardial tissue in metabolic syndrome pigs. GSK-3β inhibition decreased transforming growth factor-β (TGF-β) and p-SMAD 2/3 in the nonischemic myocardium compared with the high cholesterol control group (CON; n = 7). Representative images from Western blot shown below graph. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; MMP-9, matrix metalloproteinase 9; GSK-3βI, GSK-3β inhibited group (n = 7). *P < .05, Mann–Whitney U test.

       GSK-3β Inhibition Reduces Apoptotic Signaling Pathways in Nonischemic Myocardial Tissue in Metabolic Syndrome Pigs

      Inhibition of GSK-3β was associated with a decreased expression of proapoptotic protein cleaved caspase 3 compared with the control. Inhibition of GSK-3β was also associated with decreased expression of the phosphorylated, inactive, form of antiapoptotic MCL-1 in the ischemic myocardial tissue compared with the control. There was no significant difference in AIF (P = .96; Figure 7 and Table 1).
      Figure thumbnail gr7
      Figure 7Glycogen synthase kinase 3β (GSK-3β) inhibition decreases mitochondrial proapoptotic signaling in the nonischemic myocardial tissue in metabolic syndrome pigs. GSK-3β inhibition decreased phosphorylated myeloid cell leukemia sequence-1 (Ser 140; p-MCL-1) and cleaved caspase 3 in the ischemic myocardium compared to the high cholesterol control group (CON; n = 7). Representative images from Western blot shown below graph. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GSK-3βI, GSK-3β inhibited group (n = 7). *P < .05, Mann–Whitney U test.

      Discussion

      In this study, we examined the role of GSK-3β on the development of myocardial fibrosis and oxidative stress in the setting of chronic myocardial ischemia and metabolic syndrome. We found that inhibition of GSK-3β decreases collagen formation and oxidative stress in ischemic as well as nonischemic myocardial tissue. GSK-3β inhibition might mediate this beneficial effect via downregulation of TGF-β/SMAD2/3 signaling and decreasing mitochondria-induced oxidative stress and apoptosis (Figure 8).
      Figure thumbnail gr8
      Figure 8Glycogen synthase kinase 3β (GSK-3β) inhibition decreases myocardial fibrosis and oxidative stress in a pig model of chronic myocardial ischemia and metabolic syndrome. GSK-3β inhibition decreases mitochondrial proapoptotic and transforming growth factor-β (TGF-β)/SMAD2/3 signaling in the myocardium of metabolic syndrome pigs. AIF, Apoptosis inducing factor; mPTP, mitochondrial permeability transition pore; MCL-1, myeloid cell leukemia sequence-1.
      Accumulation of extracellular matrix proteins including collagen leads to aberrant myocardial architecture ultimately resulting in cardiac dysfunction and heart failure.
      • Asbun J.
      • Villarreal F.J.
      The pathogenesis of myocardial fibrosis in the setting of diabetic cardiomyopathy.
      Hyperglycemia and ischemic disease are both known to contribute to the development of cardiac fibrosis.
      • Asbun J.
      • Villarreal F.J.
      The pathogenesis of myocardial fibrosis in the setting of diabetic cardiomyopathy.
      • Gyöngyösi M.
      • Winkler J.
      • Ramos I.
      • Do Q.
      • Firat H.
      • McDonald K.
      • et al.
      Myocardial fibrosis: biomedical research from bench to bedside.
      In our study, inhibition of GSK-3β decreased fibrosis and collagen deposition in the ischemic and nonischemic myocardial tissue. This is consistent with previous research showing that downregulation of GSK-3β is cardioprotective, associated with decreased infarct size myocardial fibrosis, and hypertrophy in small animal models of myocardial infarction and cardiac hypertrophy.
      • Zhao X.
      • Hua Y.
      • Chen H.
      • Yang H.
      • Zhang T.
      • Huang G.
      • et al.
      Aldehyde dehydrogenase-2 protects against myocardial infarction-related cardiac fibrosis through modulation of the Wnt/β-catenin signaling pathway.
      • Nishihara M.
      • Miura T.
      • Miki T.
      • Sakamoto J.
      • Tanno M.
      • Kobayashi H.
      • et al.
      Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3.
      In our study, GSK-3β inhibition decreased expression of the profibrotic cytokine TGF-β and phosphorylation of its downstream transcription factor SMAD2/3 in the ischemic myocardial tissue. This suggests a mechanism through which GSK-3β might be inducing enhanced fibrosis in the setting of chronic myocardial ischemia and metabolic syndrome.
      Interestingly, inhibition of GSK-3β with lithium in rats can lead to increased cardiac hypertrophy.
      • Lal H.
      • Ahmad F.
      • Zhou J.
      • Zhou J.
      • Yu J.E.
      • Ronald J.
      • Guo Y.
      Cardiac fibroblast GSK-3β regulates ventricular remodeling and dysfunction in ischemic heart.
      • Tateishi A.
      • Matsushita M.
      • Asai T.
      • Masuda Z.
      • Kuriyama M.
      • Kanki K.
      • et al.
      Effect of inhibition of glycogen synthase kinase-3 on cardiac hypertrophy during acute pressure overload.
      However, the role of GSK-3β in fibrosis is controversial. In a mouse model of renal fibrosis induced by ischemia/reperfusion injury, GSK-3β inhibition was shown to suppress renal fibrosis by reducing TGF-β1/SMAD-3 signaling.
      • Singh S.P.
      • Tao S.
      • Fields T.A.
      • Webb S.
      • Harris R.C.
      • Rao R.
      Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia/reperfusion in mice.
      In a mouse model of myocardial infarction, GSK-3β was thought to inhibit fibrosis and left ventricular remodeling by inhibiting TGF-β1/SMAD-3 signaling via interaction with SMAD-3. However, the exact mechanism of this interaction was undetermined.
      • Lal H.
      • Ahmad F.
      • Zhou J.
      • Zhou J.
      • Yu J.E.
      • Ronald J.
      • Guo Y.
      Cardiac fibroblast GSK-3β regulates ventricular remodeling and dysfunction in ischemic heart.
      Nevertheless, in our large animal model of diet-induced metabolic syndrome, we observe a reduction in fibrosis and profibrotic signals in the ischemic as well as nonischemic myocardium.
      Cardiac fibrosis is associated with dysregulation of the extracellular matrix. Matrix metalloproteinase 2 and 9 are known to process a number of collagens.
      • Fan D.
      • Takawale A.
      • Lee J.
      • Kassiri Z.
      Cardiac fibroblasts, fibrosis and extracellular matrix remodeling in heart disease.
      Interestingly, reduced matrix metalloproteinase 2 activity has been shown to contribute to ischemic heart disease in diabetic mice.
      • Van Linthout S.
      • Seeland U.
      • Riad A.
      • Eckhardt O.
      • Hohl M.
      • Dhayat N.
      • et al.
      Reduced MMP-2 activity contributes to cardiac fibrosis in experimental diabetic cardiomyopathy.
      Tissue inhibitor of metalloproteinase-2 is associated with cardiac fibrosis and dysfunction in chronically pressure-overloaded human hearts.
      • Heymans S.
      • Schroen B.
      • Vermeersch P.
      • Milting H.
      • Gao F.
      • Kassner A.
      • et al.
      Increased cardiac expression of tissue inhibitor of metalloproteinase-1 and tissue inhibitor of metalloproteinase-2 is related to cardiac fibrosis and dysfunction in the chronic pressure-overloaded human heart.
      Additionally, acute inhibition of matrix metalloproteinases has been shown to prevent cardiac rupture after an acute myocardial infarction but long-term administration was associated with impaired angiogenesis and cardiac failure.
      • Heymans S.
      • Luttun A.
      • Nuyens D.
      • Theilmeier G.
      • Creemers E.
      • Moons L.
      • et al.
      Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.
      In our study of chronic myocardial ischemia, we showed that GSK-3β inhibition was associated with decreased expression of the matrix matalloproteinase-9 in the ischemic myocardial tissue.
      GSK-3β inhibition was also associated with decreased oxidative stress in the ischemic and nonischemic myocardial tissue. GSK-3β is thought to be cardioprotective by reducing oxidative stress and modulating various mitochondrial pathways including modulation of the mPTP.
      • Das S.
      • Wong R.
      • Rajapakse N.
      • Murphy E.
      • Steenbergen C.
      Glycogen synthase kinase 3 inhibition slows mitochondrial adenine nucleotide transport and regulates voltage-dependent anion channel phosphorylation.
      • Juhaszova M.
      • Zorov D.B.
      • Yaniv Y.
      • Nuss H.B.
      • Wang S.
      • Sollott S.J.
      Role of glycogen synthase kinase-3β in cardioprotection.
      MCL-1 inhibits mitochondrial cell stress and apoptosis. MCL-1 inhibits mitochondrial proapoptotic protein Bak, preventing it from oligomerizing and thus blocking complete formation of mPTP, which ultimately leads to the release of AIF and activation of the caspase apoptotic cascade. GSK-3β phosphorylates and inhibits MCL-1 at serine 140 and inactivates it.
      • Sutherland C.
      What are the bona fide GSK3 substrates? .
      • Miura T.
      • Miki T.
      GSK-3β, a therapeutic target for cardiomyocyte protection.
      • Wagman A.S.
      • Maurer U.
      • Dejardin E.
      • Green D.R.
      Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1.
      Thus, inhibition of GSK-3β should promote antiapoptotic activity of MCL-1. We found decreased phosphorylation of MCL-1 (Ser 140) in the GSK-3β inhibited group compared with the control group. This suggests a mechanism through which GSK-3β inhibition is antiapoptotic and confirms its decreased GSK-3β activity. In addition, AIF released from mitochondria and cleaved caspase 3 are both downstream effector proteins and function to induce cellular apoptosis. We found decreased expression of AIF and cleaved caspase 3 (to total caspase 3) in the GSK-3β inhibited group compared with the control group.
      Finally, we saw a decrease in MMP-9 and AIF in the ischemic myocardial tissue of the GSK-3β inhibited group compared with the control; however, we saw no change in expression levels of these 2 proteins in the nonischemic tissue between groups. This difference is likely associated with the difference in cellular conditions between the 2 areas of the myocardium.
      Using this model, we have previously shown that inhibition of GSK-3β increases blood flow and vessel density in the ischemic and nonischemic myocardial tissue. In this study, we show inhibition of GSK-3β also decreases collagen formation and oxidative stress in the ischemic territory as well as nonischemic remote myocardial tissue. Our research suggests that inhibition of GSK-3β is cardioprotective by increasing myocardial perfusion, increasing vascular density, and decreasing myocardial fibrosis and oxidative stress.

       Limitations

      It is unknown if the antifibrotic and antiapoptotic effects in this study are because of improved blood flow or potentially other direct targets in fibroblasts and the myocardium. For instance, hypoxia is a known driver of oxidative stress and reactive oxygen species production; improved blood flow should limit reactive oxygen species and reduce apoptosis and fibrosis. Additional investigation will be required to sort out the specific targets and mechanisms of action in each of the many cell types present in the myocardium. The GSK-3βI was given over 5 weeks and the tissue was analyzed at the end of the study, at only 1 time point. Future research should include performing a dose response curve to identify the optimal time and duration of drug treatment.

      Conclusions

      We examined the role of GSK-3β inhibition on myocardial fibrosis and oxidative stress in the setting of chronic myocardial ischemia and metabolic syndrome. We found that inhibition of GSK-3β decreases collagen formation and oxidative stress in the ischemic territory as well as nonischemic remote myocardial tissue. GSK-3β might have this beneficial effect by downregulating TGF-β/SMAD2/3 signaling and decreasing mitochondrial-induced cellular stress. Myocardial fibrosis and oxidative stress leads to aberrant myocardial architecture, resulting cardiac dysfunction and heart failure.
      • Asbun J.
      • Villarreal F.J.
      The pathogenesis of myocardial fibrosis in the setting of diabetic cardiomyopathy.
      Hyperglycemia as well as ischemic disease contribute to the development of cardiac fibrosis.
      • Asbun J.
      • Villarreal F.J.
      The pathogenesis of myocardial fibrosis in the setting of diabetic cardiomyopathy.
      • Gyöngyösi M.
      • Winkler J.
      • Ramos I.
      • Do Q.
      • Firat H.
      • McDonald K.
      • et al.
      Myocardial fibrosis: biomedical research from bench to bedside.
      Therefore, the results of this research might provide a potential mechanism for medical therapy of patients suffering from coronary artery disease and metabolic syndrome (Video 1).

       Conflict of Interest Statement

      Authors have nothing to disclose with regard to commercial support.
      Figure thumbnail fx3
      Video 1Brittany A. Potz describing the potential significance of glycogen synthase kinase 3β inhibition as a therapy to inhibit myocardial fibrosis for patients suffering from coronary artery disease. Video available at: http://www.jtcvsonline.org/article/S0022-5223(18)30268-X/fulltext.

      Supplementary Data

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