Table 1.
List of sequence specific primer used for q-PCR.
Fig 1.
Cardiac expression of GRK2 in patients with HF.
(A) Haematoxylin and eosin (H&E) staining and (B) GRK2 IHC staining of human cardiac tissue (10X).
Fig 2.
Effect of gallein on BW, HW and echocardiographic parameters.
(A-G) BW, body weight; HW, heart weight; LVDd, left ventricular dimension in diastole; LVDs, left ventricular dimension in systole; FS, fractional shortening; EF, ejection fraction; group Normal, age matched untreated rats; group EAM, rats with experimental autoimmune myocarditis treated with vehicle; group EAM+G10, rats with EAM treated with gallein given orally at 10 mg/kg/day. ***p<0.001, **p<0.01 and *p<0.05 vs Normal; ##p<0.01 and #p<0.05 vs EAM. Results are presented as mean ± SEM, n = 4–5.
Fig 3.
Effect of gallein on myocardial histological changes.
(A) H&E staining of left ventricular sections depicting infiltration of inflammatory cells, interstitial edema, vacuolization, and degeneration of cardiac fibers. (B) Masson’s trichrome (MT) staining for fibrosis (blue area) in the cross sectional tissue sections of left ventricle. (C-C1) Toluidine blue (TB) staining for mast cells of the cross sectional slices of heart and their quantification. Scale bar = 20 μm. Each bar represents mean ± SEM, n = 4–5. Normal, age matched normal rats; EAM, rats with experimental autoimmune myocarditis treated with vehicle; EAM+G10, rats with EAM treated with gallein (10 mg/kg/day). ***p<0.001 and *p<0.05 vs Normal; ###p<0.001 vs EAM.
Fig 4.
Gallein inhibits GRK2, CD80 and CD68 expression in myosin induced EAM rat heart.
Western blots show specific band for the expression of (A) GRK2 (ratio with GAPDH). IHC staining for (B-B1) CD80 and (C-C1) CD68 positive cells and their quantification. Scale bar = 20 μm. Each bar represents mean ± SEM, n = 4–5. Normal, age matched normal rats; EAM, rats with experimental autoimmune myocarditis treated with vehicle; EAM+G10, rats with EAM treated with gallein. ***p<0.001, and **p<0.01 vs Normal; ###p<0.001, ##p<0.01 vs EAM.
Fig 5.
Small molecule Gβγ-GRK2 inhibitor gallein suppressed M1-like macrophage phenotype marker expression in the hearts of EAM rats.
Western blots show specific bands for the expression of (A-E) IFNγ, COX2, HMGB1, TLR4 (ratio with GAPDH) and ERK1/2 (ratio with total ERK1/2). Each bar represents mean ± SEM, n = 3–4. Normal, age matched normal rats; EAM, rats with experimental autoimmune myocarditis treated with vehicle; EAM+G10, rats with EAM treated with gallein. ***p<0.001, **p<0.01 and *p<0.05 vs Normal; #p<0.05 vs EAM.
Fig 6.
Effect of gallein on M2-like macrophage phenotype in the heart of EAM rats.
IHC staining for (A-A1) CD163, (B-B1) CD36, (C-C1) IL-10 and their quantification data. Scale bar = 20 μm. Each bar represents mean ± SEM, n = 4–5. Normal, age matched normal rats; EAM, rats with experimental autoimmune myocarditis treated with vehicle; EAM+G10, rats with EAM treated with gallein. ***p<0.001 vs Normal; ###p<0.001 and ##p<0.01 vs EAM.
Fig 7.
Effect of gallein on human M1 and M2 macrophages in vitro.
(A-D) Changes in gene expression of macrophage phenotype markers after gallein treatment (qRT-PCR from n = 3 experimental replicates). Data are normalized to expression of GAPDH. Control groups in each phenotype are cells without gallein treatment. Data are shown as Mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 vs Control.