Fig 1: Thbs3 reduces surface integrin levels and destabilizes the sarcolemma. a Representative Western blots of sarcolemma protein extracts from hearts of tTA control, Thbs3 DTG, and Thbs4 DTG mice, assayed for integrin proteins and ß-dystroglycan. Laminin2a2 and Cacna1c serve as loading controls. Quantitation is shown in Supplementary Figure 3a-k. The red box shows integrin proteins specifically reduced on the sarcolemma by Thbs3. b Western blots for input of microsomal protein extracts before immunoprecipitation for Thbs3 and ß1D integrin and Cacna1c from NRVMs infected with Adßgal or AdThbs3 (left panel). The microsomal fraction was then immunoprecipitated (IP) for ß1 integrin and blotted for the indicated proteins (right panel). Mouse IgG was used as a control for the IP. c Regimen of Evans blue dye (EBD) and isoproterenol (Iso, 300 mg/kg) injection to measure membrane permeability in the hearts of mice. d Representative histological heart sections from 3 tTA control and 3 Thbs3 DTG mice, imaged for WGA (green fluorescence) and leakage of EBD into the cardiomyocytes by red fluorescence subjected to the experimental regimen shown in c. Scale bars are 1 mm. e Regimen of Evans blue dye (EBD) injection after TAC surgery to measure membrane permeability in adult mice. Mice were sacrificed (Sac) 24 h after EBD injection, 1 week after TAC surgery. f, g Representative cardiac histological images of tTA control and Thbs3 DTG hearts 1 week after TAC surgery stained with WGA (green) and intracellular leakage of EBD (red) according to the experimental scheme shown in e, while g shows quantitation of EBD positive cells in the hearts of these mice. Scale bars are 100 µm. *P < 0.05 versus tTA control sham; two-tailed students T-test. Data are represented as percentage EBD positive cells (=3000 cells from =8 animals). Error bars are +/- standard error of the mean and number of mice used is shown in the graph as individual data points
Fig 2: Thbs3 is expressed in the diseased heart and induces an adaptive ER stress response. a Quantitation of relative protein expression from Western blots for Thbs3 from heart tissue of mice that were sham-operated, subjected to 2 weeks of TAC, that contain the activated calcineurin A transgene (CnA) or that lack the Csrp3 gene. *P < 0.05 vs sham by one-way ANOVA and Turkey multiple comparisons test. Results are from four experiments and error bars represent +/-SEM. b Immunohistochemistry for Thbs3 protein (green), desmin (red) and both merged with DAPI (blue) sham or TAC-operated hearts 12 weeks later. Scale bars are 10 µm. c Representative immunohistochemistry for Thbs3 (red) and PDI (green) from CnA transgenic hearts at 8 weeks of age. Scale bars are 10 µm. d Schematic diagram depicting the inducible bi-transgenic system regulated by tetracycline for inducible overexpression of Thbs3 in the heart. e Representative Western blots for Thbs3, the nuclear form of ATF6a, Armet, BiP, calreticulin (CRT) and Gapdh from hearts of tTA control and Thbs3 DTG mice. f Western blots for Thbs3 from neonatal rat ventricular myocytes (NRVM) infected with recombinant adenovirus expressing Myc-tagged ATF6a and ßgal or Thbs3. Input control and immunoprecipitation (IP) of Thbs3 with Myc-tagged ATF6a are shown. g Representative immunohistochemistry for Thbs3 (red), WGA (green) or PDI (green) and DAPI (blue) from hearts of tTA control and Thbs3 DTG mice. Scale bars are 10 µm. h Transmission electron microscopy (EM) of heart sections from tTA control and Thbs3 DTG mice. The white arrows show expanded ER and vesicles only in Thbs3 DTG hearts. Scale bars are 1 µm
Fig 3: Integrin overexpression reduces Thbs3-mediated membrane instability and disease. a Schematic of the breeding used to generate combinatorial Thbs3 DTG, a7ß1D integrin TG mice. b Representative Western blots for Thbs3 and the indicated integrin proteins from heart sarcolemma protein extracts from tTA, Thbs3 DTG, a7ß1D integrin TG and Thbs3 DTG/ a7ß1D integrin TG mice. The a1Na+/K+-ATPase served as loading control. c Quantification of EBD positive area from cardiac histological sections after Iso (300 mg/kg) injection in Thbs3 DTG, a7ß1D integrin TG, Thbs4 DTG, and Thbs3 DTG/ a7ß1D integrin TG mice. d Representative histological images of heart sections from tTA, Thbs3 DTG and Thbs3 DTG/ a7ß1D integrin TG mice stained with WGA-FITC (green) after Iso injection (300 mg/kg). EBD is shown as red fluorescence. Scale bars are 300 µm. e Fractional shortening (FS) percentage as determined by echocardiography following 2 weeks of continuous Iso infusion (60 mg/kg/day) or PBS vehicle controls. Number of mice analyzed is shown within each histogram in c, e. *P < 0.05 versus vehicle treated; #<0.05 versus Thbs3 DTG with Iso. Statistical analysis was performed using one-way ANOVA and Turkey multiple comparisons test. Error bars are +/- standard error of the mean
Fig 4: Induction of endogenous Thbs3 is detrimental after cardiac injury. a Schematic diagram depicting the Thbs gene-deleted mice used to selectively analyze effects of endogenous Thbs3. b Representative Western blots from hearts of 1–3 day-old WT, Thbs1/2/3/4/5-/- and Thbs1/2/4/5-/- mice for the indicated Thbs proteins. Gapdh served as loading control. c Kaplan–Meier survival plot of shams, WT, Thbs1/2/3/4/5-/- and Thbs1/2/4/5-/- mice after TAC surgery in days. Number of mice used is shown in the graph for each group. P < 0.0001 analyzed by log-rank test WT versus Thbs1/2/4/5-/- and Thbs1/2/3/4/5-/- versus Thbs1/2/4/5-/-. d Experimental regimen of EBD and Iso injection into the groups of mice shown (e) to measure membrane permeability. e Quantification of EBD positive area in the hearts of the indicated groups of mice after Iso injection with the regimen shown in d. *P < 0.05 versus WT; #P < 0.05 versus Thbs1/2/4/5-/- mice. Statistical analysis was performed using one-way ANOVA and Turkey multiple comparisons test. Error bars are +/- standard error of the mean and number of mice used in each experiment are shown in the graphs. f Immunohistochemistry for ß1D integrin from heart sections of WT, Thbs3-/-, Thbs1/2/4/5-/- and Thbs1/2/3/4/5-/- mice 1 week after TAC surgery. Scale bars are 50 µm. g Representative Western blots for the indicated integrin proteins from hearts of WT, Thbs3-/-, Thbs1/2/4/5-/-, and Thbs1/2/3/4/5-/- mice subject to 1 week of TAC and processed for sarcolemma protein extracts. Cacna1c served as loading control. Quantitation of these results is shown in Supplementary Figure 4g–i
Fig 5: Loss of Thbs3 protects the heart from pressure overload pathology. a Low magnification cardiac histological images from WT and Thbs3-/- mice stained with Masson’s trichrome 12 weeks after TAC or sham surgery. Scale bar = 1 mm. b HW/BW ratios in the indicated groups of mice 12 weeks after TAC or sham surgery. c Echocardiography measured fractional shortening (FS) percentage and d LVIDD in the indicated groups of mice 12 weeks after a TAC or sham surgical procedure. e Pulmonary edema was analyzed by LW/BW ratio measurement in the indicated groups of mice 12 weeks after a TAC or sham surgical procedure. f Percentage of fibrotic area was quantified with heart sections stained with Masson’s trichrome in WT and Thbs3-/- mice after TAC. g Representative Western blots for integrin proteins from heart sarcolemma protein extracts from WT and Thbs3-/- mice 12 weeks after TAC or sham surgery. Cacna1c served as loading control. Quantitation of these results is shown in Supplementary Figure 4a-f. *P < 0.05 versus WT control sham; #P < 0.05 versus WT TAC. Statistical analysis was performed using one-way ANOVA and Turkey multiple comparisons test and two-tailed students T-test. Error bars are +/- standard error of the mean and number of mice used in each experiment are shown in the graphs
Supplier Page from R&D Systems, a Bio-Techne Brand for Thrombospondin-3 Protein
Available conjugates: Sizes Available: 50 ug