Hypercontractile cardiac phenotype in mice overexpressing the regulatory subunit PR72 of protein phosphatase 2A

Figures

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  Figure 1. Direct interaction between PR72 and PP2Ac. The expression of PR72 in different tissues is shown in the left panel (A) In the murine heart, PR72 is the more abundant PPP2R3A splice variant when compared to PR130, as shown in the right panel (A) The physical interaction between PR72 and PP2Ac was also demonstrated by pulldown of the catalytic subunit on immobilized 6xHis-PR72 (B) The recombinant expression of the 6xHis-tagged PR72 (lane 2) was performed in HEK293 cells and controlled using an RGS-6xHis control vector (lane 1). Note the expression of the endogenous PP2Ac in the cells (left panel). Elution of PR72 from the Ni-NTA column using imidazole identified PP2Ac as a binding partner of the regulatory B'' subunit (right panel). Subcellular fractions were prepared from pulverized heart tissue of WT mice by differential centrifugation (C) PR72 was found to be mainly expressed in the cytosol. The purity of the cytosolic fraction was tested by using a specific antibody directed against GAPDH.

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  Figure 2. 2.5-fold overexpression of PR72 in TG hearts. Overexpression of PR72 was confirmed using a specific antibody for immunoblotting (A) Additionally, the protein expression of the catalytic subunit of PP2A (Ca) and calsequestrin (CSQ) was determined (A), with representative immunoblots shown for heart homogenates of transgenic (TG) and wild-type (WT) mice (*P < 0.05 vs. WT, N = 7 hearts each, unpaired Student's t-test). Proteins were normalized to total protein content measured in Ponceau stainings. Protein phosphatase activities, including total, PP1, and PP2A, were measured in homogenates of Langendorff-perfused hearts under basal conditions and after β-adrenergic stimulation with isoprenaline (B) (#P < 0.05 vs. Basal, N = 10-12 hearts, 2-way-ANOVA and Holm–Sidak post-hoc test). The proportion of PP2A on total PP activity is depicted (C) (*P < 0.05 vs. WT, N = 10-12 hearts, Kruskal–Wallis One-way-ANOVA on Ranks and Dunn's post-hoc test).

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  Figure 3. Intracellular distribution of PR72 and marker proteins in TG and WT cardiomyocytes. Cardiomyocytes were isolated from TG and WT mice. Subsequently, they were fixed with formaldehyde on polylysine-coated glass slides and permeabilized using tergitol. Nonspecific binding was minimized by employing BSA and goat serum as blocking agents. Immunostaining procedures were conducted on the cardiomyocytes using anti-PR72 antibody in conjunction with either anti-sarcomeric α-actinin antibody or anti-GAPDH antibody. The primary antibodies were then detected using fluorescence-coupled secondary antibodies and analyzed through confocal laser microscopy. The expression of PR72 was visualized in green, while other marker proteins were visualized in red, resulting in a yellow coloration where they colocalized. White scale bars represented a 20-µm reference. For improved clarity, specific regions of interest were magnified, and adjustments to contrast and brightness were made (indicated by white framed boxes). Striated regions were exemplarily marked with white arrows. In the lower panel, negative controls demonstrated cardiomyocyte autofluorescence. In all staining experiments, 10 cells per heart…

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  Figure 4. Overexpression of PR72 results in moderate cardiac hypertrophy. To assess the development of cardiac hypertrophy in transgenic mice, the heart-body weight (HW/BW, N = 7-10 hearts) and ventricle-body weight (VW/BW, N = 9-13 hearts) ratios were determined (A) (*P < 0.05 vs. WT, unpaired Student's t-test). The cardiomyocyte diameter was determined on hematoxylin and eosin stained tissue sections (B) Exemplary sections for both genotypes are shown on the right hand side (N = 9-10 hearts). The black scale bars show a 50 µm distance. The degree of fibrosis was analyzed in ventricular sections stained with Masson-Goldner's Trichrome (C) (N = 9 hearts each). Black scale bars indicate a 1 mm distance. mRNA expression levels were determined for selected markers of a hypertrophic gene program (Acta1, skeletal muscle α-actin; Col1a1, collagen type I α1 chain; Col3a1, collagen type III α1 chain; Gja1, connexin 43; Myh7, myosin heavy chain beta (MHC-β) isoform; Nppa, atrial natriuretic peptide; Nppb, brain natriuretic peptide) (D) The mRNA expression levels were normalized to the reference genes Hypoxanthine-guanine phosphoribosyltransferase (Hprt) and Gapdh. RT-PCR data were analyzed using…

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  Figure 5. Increased left ventricular pressure in TG mice. The basal contractile parameters of catheterized transgenic (TG) and wild-type (WT) mice were monitored. The summarized data show the maximal ventricular pressure (A), heart rate (B), contraction velocity (C), and relaxation velocity (D) (*P < 0.05 vs. WT, N = 7-11 mice, unpaired Student's t-test).

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  Figure 6. Enhanced contraction and hastened relaxation in TG cardiomyocytes. Myocellular contraction parameters were determined in electrically stimulated (0.5 Hz) cardiomyocytes of both WT and TG mice. Representative tracings of myocellular contractions are shown (A), and summarized data of maximal sarcomere length (SL) shortening under basal conditions, in the presence of 3 nM okadaic acid (OA), or after stimulation with 1 μM isoprenaline (ISO) are presented (B) Additionally, the time to 50% relengthening of cardiomyocytes is shown for each of the study conditions described above (C) (basal conditions (left panels): n/N = 120 cardiomyocytes/12 hearts, *P < 0.05 vs. WT, Mann–Whitney test; ISO (right panels) and OA (central panels): n/N = 60 cardiomyocytes/6 hearts, respectively, *P < 0.05 vs. WT, #P < 0.05 vs. Basal, Kruskal–Wallis One-way-ANOVA on Ranks and Dunn's post-hoc test).

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  Figure 7. Increased peak amplitude and faster decay of Ca2+ transients in TG cardiomyocytes. Intracellular Ca2+ transients were determined in TG and WT cells by loading them with indo-1. Representative tracings of intracellular Ca2+ transients are shown (A) The peak amplitude of Ca2+ transient ratios was measured under basal conditions and after the application of okadaic acid (OA) or isoprenaline (ISO). Summarized data are provided (B), and summarized data of the time to 50% decay are presented for each of the study conditions described above (C) (basal conditions (left panels): n/N = 120 cardiomyocytes/12 hearts, *P < 0.05 vs. WT, Mann–Whitney test; ISO (right panels) and OA (central panels): n/N = 60 cardiomyocytes/6 hearts, respectively, *P < 0.05 vs. WT, #P < 0.05 vs. Basal, Kruskal–Wallis One-way-ANOVA on Ranks and Dunn's post-hoc test).

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  Figure 8. Unchanged SR Ca2+ load but enhanced Ca2+ spark parameters in TG cardiomyocytes. Caffeine-induced Ca2+ transients were measured in isolated myocytes to estimate the SR Ca2+ load. The quantitative analysis of peak amplitude is shown for TG and WT in the absence and presence of 1 μM isoprenaline (A) (n/N = 15-23 cardiomyocytes/3 hearts, Kruskal–Wallis One-way-ANOVA on Ranks and Dunn's post-hoc test). Additionally, Ca2+ spark parameters were determined using laser scanning microscopy. Characteristic X-T scans of WT and TG cardiomyocytes after 1 Hz prestimulation are illustrated (B) The white scale bars indicate horizontally a 10-µm width and vertically a 1-sec duration. The quantified data for Ca2+ spark frequency (CaSpF), amplitude, full duration and full width, full duration and width at half maximum (FDHM and FWHM, respectively), and decay kinetics are shown (C) (*P < 0.05 vs. WT, n/N = 48-50 cardiomyocytes/5 hearts, Mann–Whitney test).

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  Figure 9. Higher phosphorylation of phospholamban at threonine-17 and lower expression of NCX in TG hearts. Shown are representative immunoblots of cardiac proteins involved in calcium regulation and contractility, for both TG and WT mice (A) Protein phosphorylation was measured in the presence and absence of isoprenaline (ISO). Molecular weight markers were included on the left-hand side of the blots. Protein phosphorylation for sarcoplasmic reticulum (PLN, phospholamban; RyR2, ryanodine receptor type 2) and myofilament proteins (MyBP-C, myosin-binding protein C; TnI, troponin inhibitor) was quantified under both basal and stimulated (ISO) conditions (B) Phosphorylation levels were normalized to the nonphosphorylated form of the individual protein and subsequently adjusted to WT levels under basal conditions (*P < 0.05 vs. WT, #P < 0.05 vs. Basal, N = 8-10 hearts, 2-way-ANOVA and Holm–Sidak post-hoc test or, for pS2808/RyR, Kruskal–Wallis One-way-ANOVA on Ranks and Tukey post-hoc test). The β-adrenergic signaling was assessed by measurement of cAMP levels (N = 10-11 hearts) and the protein expression level of the catalytic subunit of cAMP-dependent protein kinase (PKA-Cα, N = 9-10 hearts)…