Differential localizations of protein phosphatase 1 isoforms determine their physiological function in the heart

Figures

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  Figure 1.. Distinct subcellular localizations of PP1 isoforms in cardiac myocytes (A) Immunocytochemistry analysis of endogenous PP1 isoforms in neonatal rat cardiac myocytes. Scale bar, 10 μm. (B) Immunocytochemistry analysis of endogenous PP1 isoforms in adult rat cardiac myocytes. Magnification, 400×. (C) Quantification of relative fluorescence intensity of PP1 isoforms based on A using FIJI-ImageJ. *P < 0.05 vs cyto. Western blot analysis of PP1s in the cytoplasm (cyto) and nucleus (nucl) of neonatal (D) and adult (E) rat cardiac myocytes. Lamin A/C and GAPDH were used as controls for nuclear and cytosolic proteins respectively. (F) Quantification of PP1 isoforms based on E. *P < 0.05 vs cyto. All the experiments were repeated three times with similar results.

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  Figure 2.. PP1s differentially regulate substrate phosphorylation in neonatal cardiac myocytes Western blot analysis for the phosphorylation of PLB at serine 16 and threonine 17, total PLB, and PP1s from neonatal rat cardiac myocytes infected with adenoviruses expressing either β-gal (Con) or individual PP1 isoform. p-MLC2 was performed using ProQ-Diamond staining method. Quantification was based on individual western blot. N = 4 for each group. *P < 0.05 vs. Con 5 min.

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  Figure 3.. Deletion of PP1β from mouse heart leads to increased HDAC7 phosphorylation (A–C) Western blot analysis for phospho-HDAC7, HDAC7, PP1α, PP1β, PP1γ, and GAPDH from the hearts of the indicated mice at 2 months of age. Phosphorylation of MLC2 was detected using a ProQ-Diamond staining method [17]. (D) Quantification of western blots shown in A–C. N = 4 for each group. *P < 0.05 vs. NKX-Cre.

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  Figure 4.. PP1s are essential for the physiological cardiac function (A) Real-time PCR analysis of PP1 expression in cardiac myocytes and fibroblasts isolated from neonatal and adult mouse hearts. N = 4 for each group. *P < 0.05 vs PP1α from neonatal cells; #P < 0.05 vs PP1α from neonatal cells; $P < 0.05 vs PP1α from adult cells; %P < 0.05 vs PP1α from adult myocytes. (B) Western blot analysis for each PP1 isoform in the neonatal and adult mouse hearts. (C) Quantification of protein levels of PP1s in B. N = 4 for each group. *P < 0.05 vs neonatal hearts. (D,E) Fractional shortening (FS) and LVIDd of the aging mice. Mouse numbers for four groups were indicated in the bars. *P < 0.05 vs NKX-Cre; #P < 0.05 vs NKX-Cre; &P < 0.05 vs NKX-Cre. (F) Masson’s trichrome staining of paraffin-embedded heart sections from NKX-Cre controls and the indicated PP1-deleted mice at 3 weeks of age. Scale bar, 50 μm.

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  Figure 5.. Deletion of individual PP1 isoform does not influence cardiac stress responses (A–D) Echocardiographic measurements of 2-month-old mice after 2 weeks of TAC. Mouse numbers for each group were indicated in the bars. *P < 0.05 vs sham (A–E). ^P < 0.05 vs sham. In D, *P < 0.05 vs NKX-Cre. (E) Measurements of heart/body weight (HW/BW) ratios in the indicated groups of mice after 2 weeks of TAC. *P < 0.05 vs sham. (F) Representative histological sections stained with Masson’s trichrome (shows fibrosis in blue) from TAC hearts of indicated groups of mice. (G) Quantification of interstitial fibrosis based on F. Mouse numbers for each group were indicated in the bars.