CD44 regulates Epac1-mediated β-adrenergic-receptor-induced Ca2+-handling abnormalities: implication in cardiac arrhythmias

Figures

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  Fig. 1. Expression of osteopontin, CD44, CaMKII and Ca2+-handling proteins in β-AR-activated CD44−/− mice at different time points during treatment with β-AR agonist. A Representative examples and mean ± SE analysis western blot for osteopontin, CD44, ox-CaMKII, (p-)CaMKII, (p-)RyR2, SERCA, NCX and (p-)PLN. The relative expression of each protein was quantified to GAPDH by densitometry and normalized to the control. N = 4 for each group. B Representative examples and mean ± SE analysis for co-immunoprecipitation of CD44 and control IgG with Epac1 and Epac2. C Representative examples and mean ± SE analysis for co-immunoprecipitation of CD44 and PKAc and PKAIIα. In B and C, the pictures are representation of blots from 3 independent experiments for each. the mean ± SE analysis was for precipitated protein-bound CD44 and Epac1 to precipitated protein ratio and cell input, both of which were quantified to GADPH and normalized to the control (WT without ISO) level, which was set at 1.0. N = 3 for each group. For A–C *P < 0.05 versus control (WT and CD44−/− mice without ISO) by one-way ANOVA with Bonferroni’s post hoc test. #P < 0.05 between groups by unpaired Student’s t-test. D…

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  Fig. 2. Crosstalk between β-AR/Epac and osteopontin/CD44 signaling on Ca2+-handling proteins. A–C Representative examples and mean ± SE analysis western blot of (p-)CaMKII, (p-)RyR2 and (p-)PLN in HL-1 myocytes treated with (A) isoproterenol (B) 8-CPT-cAMP (C) osteopontin and with or without Epac1 or Epac2 siRNA. D and E Representative examples and mean ± SE analysis western blot of CD44, Epac1, (p-)CaMKII, (p-)RyR2 and (p-)PLN in HL-1 myocytes treated with (D) osteopontin or (E) 8-CPT-cAMP and with or without CD44 siRNAs or apocynin. F Representative examples and mean ± SE analysis western blot of CD44, Epac1, (p-)CaMKII, (p-)RyR2 and (p-)PLN in HL-1 myocytes treated with isoproterenol + ICI-118,551 with or without CD44 siRNAs or Rp-8-CPT-cAMPS. The relative expression of each protein was quantified to GAPDH by densitometry and normalized to the control. For all A–F, n = 4 per group. *P < 0.05 versus control by one-way ANOVA with Bonferroni’s post hoc test. ISO = isoproterenol

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  Fig. 3. Association between CD44 and Epac1. A Representative examples and mean ± SE analysis for co-immunoprecipitation, reverse co-immunoprecipitation and cell input of CD44 and Epac1, Epac2, PAKIIα and PKAc in HL-1 myocytes treated with osteopontin, isoproterenol or 8-CPT-cAMP. B Representative examples and mean ± SE analysis for co-immunoprecipitation and cell input of His-tagged CD44 and Epac1 in HL-1 myocytes treated with 8-CPT-cAMP. In A and B the mean ± SE analysis was for precipitated protein-bound Epac1, Epac2, PAKIIα or PKAc or CD44 to precipitated protein ratio and cell input, both of which were quantified to GADPH and normalized to the control. ISO = isoproterenol. The picture is a representation of blots from 3 independent experiments. In A and B, n = 3 per group. C Representative examples and mean ± SE confocal images of proximity ligation assay in HL-1 myocytes. The red color (arrows) indicated the linkage between CD44 and Epac1. The blue color indicated nuclei. The relative fluorescence was normalized to control as 1.0. n = 4 images per group. For A–C, *P < 0.05 versus control by one-way ANOVA with Bonferroni’s post hoc test. ISO = isoproterenol

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  Fig. 4. Effect of CD44 and β-AR signaling on the Ca2+ sparks and Ca2+ waves in myocytes. Representative examples and mean ± SE analysis for the frequency and amplitude of A Ca2+ sparks (arrow) in HL-1 myocytes transfected with CD44 siRNA or wild-type CD44 cDNA -containing plasmids. n = 17–28 cells for each group. B Ca2+ sparks (arrow) in ventricular myocytes from wild-type mice (WT) treated with CD44 blocking antibody and C Ca2+ waves in ventricular myocytes from WT after 3-Hz electrical stimulation. In B and C, n = 4–20 cells for each group. In A–C, all were obtained before and after isoproterenol (1 μmol/L) infusion with and without treatment with CD44 blocking antibody. *P < 0.05 versus control and between ISO and ISO + CD44 siRNA or CD44 blocking antibody by one-way ANOVA with Bonferroni’s post hoc test. CaSpF = Ca2+ spark frequency, ISO = isoproterenol, WT = wild-type mice

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  Fig. 5. Optical mapping in CD44−/− mice. A Representative examples (left panel) and mean ± SE analysis (right panel) of optical action potential duration (APD) and calcium transient duration (CaTD) maps of wild-type (WT) and CD44−/− mice at different pacing cycle length (PCLs). B Representative examples (left panel) and mean ± SE analysis (right panel) of ventricular tachyarrhythmia (VT) and delayed after depolarizations (DADs) in WT and CD44−/− mice. In A and B, both were before and after isoproterenol (10 nmol/L) infusion. N = 13–14 mice for each group. In B, *P < 0.05 between groups by Fisher’s exact test. CD44−/− = CD44 knock-out mice, ISO = isoproterenol, WT = wild-type mice, APD90 = APD at 80% repolarization, CaTD80 = CaTD at 80 repolarization, COV = coefficient of variance, PCL = pacing cycle length, VT = ventricular tachyarrhythmia, DAD = delayed afterdepolarization, n = number of mice for each group

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  Fig. 6. Increased expression of osteopontin, CD44 and Epac and inducibility of ventricular arrhythmias in heart failure mice. A–C ISO (30 mg/kg per day subcutaneously for 15 days) led to cardiac contractile dysfunction in mice. A Representative examples and mean ± SE analysis western blot for osteopontin, CD44, Epac1 and Epac2 in WT and CD44−/− mice treated with ISO. N = 4 for each group. *P < 0.05 versus control (WT without ISO) by one-way ANOVA with Bonferroni’s post hoc test. B Representative examples and mean ± SE analysis for co-immunoprecipitation of CD44 and control IgG with Epac1 and Epac2. N = 3 for each group. C Representative examples and mean ± SE analysis for co-immunoprecipitation of CD44 and control IgG with PKAc and PKAIIα. N = 3 for each group. In B and C, the pictures are representation of blots from 3 independent experiments for each. The mean ± SE analysis was for precipitated protein-bound CD44 and Epac1 to precipitated protein ratio and cell input, both of which were quantified to GADPH and normalized to the control level, which was set at 1.0. *P < 0.05 versus control by one-way ANOVA with Bonferroni’s post hoc test. D and E Inducibility of ventricular arrhythmias…

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  Fig. 7. Increased association between CD44 and Epac1 in compromised human heart.1. A Representative examples and mean ± SE analysis western blot for osteopontin, CD44, Epac1 and Epac2 in the left ventricle (LV) from patients with preserved and compromised LV contractility. The relative expression of each protein was quantified by densitometry and normalized to GAPDH. The relative expression of each protein was quantified to GAPDH by densimetry and normalized to preserved LV contractility. B Representative examples and mean ± SE analysis for co-immunoprecipitation of CD44 and Epac1/Epac2 in human heart. The input reported in B is a re-presentation of the 4 middle lanes of the western blot reported in A. The mean ± SE analysis was for precipitated protein-bound Epac1 and Epac2 to precipitated protein (CD44) ratio and cell input, both of which were quantified to GADPH and normalized to the group of preserved LV. In A and B, N = 4 for each group. *P < 0.05 between preserved LV and compromised LV by unpaired Student’s t-test. LV left ventricle

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  Fig. 8. Schema of mechanism for CD44 regulating cardiac β1-adrenergic Epac1-dependent abnormal Ca2+ release from SR