Therapeutic Chemical Screen Identifies Phosphatase Inhibitors to Reconstitute PKB Phosphorylation and Cardiac Contractility in ILK-Deficient Zebrafish

Figures

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  Figure 1. Schematic illustration of the integrin-linked kinase-protein kinase B (ILK-PKB) signaling pathway. Integrin-linked kinase forms, together with PINCH (particularly interesting Cys-His-rich protein) and parvin, the ILK-PINCH-parvin (IPP) complex and mediates signals from the extracellular matrix (ECM) to the cytoplasm through integrins. The phosphorylated downstream target PKB facilitates the expression of stretch responsive genes such as the atrial natriuretic factor (anf), thereby effectively transducing signals from the cardiac stretch sensor. Reduced PKB phosphorylation in ILK deficient main squeeze mutant zebrafish hearts was demonstrated to lead to impaired cardiac contractility and heart failure [10]. In this context, the inhibition of protein phosphatases (PP) by small chemical compounds that results in an increase of PKB phosphorylation might be a promising therapeutic approach to treat ILK-associated cardiomyopathies.

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  Figure 2. msq mutants show cardiac edema and reduced contractile function. msq mutants can be distinguished from their wild-type siblings at 48 h post fertilization (hpf). (A–C) Starting from this developmental stage, msq show cardiac edema (B) compared to wild-type sibling (A) and significantly decreased ventricular fractional shortening compared to wild-type controls at 72 hpf (C).

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  Figure 3. Primary small compound screen in msq mutants revealed three compounds to reconstitute fractional shortening (FS). Impact of tested compounds on ventricular fractional shortening in wild-type siblings as well as msq mutants. The data for average fractional shortening are plotted only for the compounds where at least three embryos could be quantified. (A) None of the embryos showed any increase or decrease in the average fractional shortening in wild-type embryos, whereas (B) compound A1, A2 and A3 results in significantly increased ventricular FS in msq mutants (*: Statistically significant, **: Highly significant, +: Lethal). For the drugs’ names, please refer to the Table A1 in the Appendix A.

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  Figure 4. Calyculin A reconstitutes contractile force, PKB phosphorylation and anf expression in msq. (A) Ventricular FS of homozygous mutant msq embryos (msq−/−) in comparison to wild-type (WT) zebrafish. Homozygous msq embryos treated with 100 nmol/L calyculin A during 4–96 hpf displayed improved contractile force (25.4 ± 18.9% (p = 0.00015) compared to embryos incubated with DMSO (2.2 ± 5.3%). (n.s.: Not significant) (B) Treatment of homozygous msq−/− embryos with 100 nmol/L calyculin A displayed an increased PKB phosphorylation (pPKB) in comparison to controls. In situ hybridization revealed that calyculin A treated homozygous msq−/− embryos (E) show, in contrast to DMSO-treated wild-type zebrafish (C) and DMSO-treated msq−/− embryos (D), an almost indistinguishable anf expression.

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  Figure 5. Okadaic acid and cyclosporine A partially reconstitute the contractile force and PKB phosphorylation in msq. (A) After treatment with 0.15 µmol/L okadaic acid during 4–96 hpf homozygous msq−/− embryos showed an increased contractility from 2.0 ± 3.5% to 20.8 ± 19.9%. (B) Immunoblotting indicated that the phosphorylation status of PKB (pPKB) was also slightly increased in 0.15 µmol/L okadaic acid treated msq−/− embryos (incubation during 4–96 hpf). (C) Ventricular fractional shortening of msq−/− embryos treated with 0.75 µmol/L cyclosporine A during 4–96 hpf showed in comparison to DMSO-treated msq−/− embryos (7.9 ± 8.7%) a partially reconstituted contractile force (16.9 ± 16.2%). (D) Correspondingly to fractional shortening, immunoblotting revealed that treatment with 0.5 µmol/L cyclosporine A (incubation during 48–72 hpf) slightly increased the level of phosphorylated PKB in msq−/− embryos compared to controls. (n.s.: Not significant).