Cardiac function is regulated by B56α-mediated targeting of protein phosphatase 2A (PP2A) to contractile relevant substrates

Abstract

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of -5 mV, the ICa,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser(16) was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca(2+) sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation.

Keywords: Adrenergic Receptor; Cardiomyocyte; Contractile Protein; Protein Phosphatase 2 (PP2A); Protein Phosphorylation.

FIGURE 1.

Detection of protein phosphatase subunits and analysis of PP2A activity. A, ventricular tissue of nonfailing human heart (NF), as well as of WT and B56α-overexpressing TG mice was homogenized and then subjected to gel electrophoresis and subsequent immunoblotting (left panel). Blots were probed with antibodies specific for B56α and the catalytic subunits of PP2A and PP1 as described under “Experimental Procedures.” In addition, the B56α signal was identified by immunoprecipitation (IP), preincubating the affinity-purified rabbit B56α antibody (ab) with or without a blocking peptide (pep.) encoding the human full-length B56α (lower panel). Shown is the quantitation of PP subunits by densitometric scanning of immunoblots (right panel). B, total PP activity was measured in extracts of either ventricular (Ventr.) homogenates (left panel) or isolated cardiomyocytes (right panel) using ³²P-labeled phosphorylase a as substrate. Samples were also assayed in the presence of 3 nm okadaic acid, allowing the discrimination between PP2A and PP1 activities. C, PP activity was also determined in cardiac extracts incubated with increased concentrations of okadaic acid. Shown is the PP activity expressed as a percentage of control (Ctr.), which is the phosphatase activity in the absence of okadaic acid. *, p < 0.05 versus WT.

FIGURE 2.

Immunohistochemical detection of B56α and PP2A_C. Sections from paraffin-embedded ventricular tissue of WT and TG mice were immunoreacted either with a rabbit polyclonal antibody that recognizes both endogenous mouse and exogenous human B56α (A–D) or with a rabbit polyclonal antibody raised against human PP2A_C (E and F). Arrows indicate the sarcomeric localization of overexpressed B56α. Sections were also stained with Masson's trichrome to detect connective tissue (G and H).

FIGURE 3.

Detection of B56α and PP2A_C in cardiomyocytes by immunofluorescence. A, photomicrographs showing isolated cardiomyocytes from WT (panels a, c, d, f, g, and i) and TG (panels b, e, h, and j) mouse hearts. B, photomicrographs showing detailed analysis of TG cardiomyocytes by confocal microscopy (panels k–m). In detail, green fluorescence staining of B56α (panels a, b, and k), red fluorescence staining of PP2A_C (panels d, e, and l), control without primary antibodies (panels c and f), DAPI nuclei staining (panels g and h), and the overlay (panels i, j, and m). Note the increased signal of B56α and PP2A_C in TG (panels b and e, respectively) compared with WT cardiomyocytes (panels a and d). Confocal analysis revealed a sarcomeric pattern in the distribution of B56α (panel k) and an enrichment of B56α in nuclear membranes (white arrowheads). Scale bar, 50 μm (A) and 10 μm (B).

FIGURE 4.

Expression and activity of PP2A in myocardial fractions. The expression of PP2A subunits was assessed in different myocellular compartments of WT and TG hearts (A). Cyt., cytosol; MF, myofilaments; Mem., membranes. The preparation of crude myocardial fractions was achieved by use of a differential centrifugation protocol. After SDS-PAGE, the separated proteins were transferred to nitrocellulose membranes. Thereafter, immunoblotting was performed to detect the regulatory (B56α), the catalytic (Cα), or the scaffolding (Aα) subunit of PP2A, as well as specific marker proteins, indicating the purity of myocardial fractions. Shown is the quantification of the protein expression of PP2A subunits in fractions prepared of mouse ventricles (B). PP activity was measured in cytosolic, myofilament, and membrane fractions in the absence and presence of 3 nm okadaic acid to differentiate between PP2A and PP1 activity (C). *, p < 0.05 versus WT; +, p < 0.05 versus PP1.

FIGURE 5.

Recording of contractile function on isolated hearts. Isolated hearts of WT and B56α-overexpressing (TG) mice were perfused in the Langendorff mode. A–D, contractile parameters were recorded under basal conditions and after β-adrenergic stimulation. A, maximum rate of LV pressure development; B, maximum amplitude of LV pressure; C, maximum rate of LV pressure decline; D, mean aortic flow. E and F, basal (E) and ISO-stimulated (F) contractility was also studied on isolated mouse hearts at an afterload of 50 mm Hg using the work-performing mode. *, p < 0.05 versus WT; +, p < 0.05 versus basal.

FIGURE 6.

Measurement of Ca²⁺ transients and sarcomere length in isolated cardiomyocytes. Intracellular Ca²⁺ transients and cardiomyocyte contraction were measured simultaneously at 0.5-Hz stimulation frequency in isolated cardiomyocytes of WT and TG mice under basal conditions and after application of ISO. A–D, shown are the summarized data of [Ca]_i peak amplitude (A), sarcomere length shortening (B), the time to 50% [Ca]_i decay (C), and the time of 50% relaxation (D). E, the normalized data of basal [Ca]_i peak amplitude and SL shortening were correlated. The slope of this relation can be used as an indicator of myofilament Ca²⁺ sensitivity. *, p < 0.05 versus WT; +, p < 0.05 versus basal.

FIGURE 7.

Analysis of l-type Ca²⁺ channel currents in isolated cardiomyocytes. The peak current-voltage relation was obtained from WT and TG cardiomyocytes under basal conditions (A) and after administration of ISO (C). L-type Ca²⁺ channels were activated by 400-ms depolarizing pulses after a prepulse to −40 mV to the indicated test potentials (−80 mV holding potential). Currents were related to individual cell capacitances. Shown is the conductance-voltage relation in the absence (B) and presence (D) of 1 μm ISO. The values were normalized to the maxima and fitted to a Boltzmann function. *, p < 0.05 versus WT, RM-analysis of variance with Holm-Sidak post hoc test.

FIGURE 8.

Expression of regulatory proteins in crude heart homogenates. Mouse heart tissue from WT and B56α-overexpressing (TG) mice was homogenized, and solubilized proteins were separated on polyacrylamide gels. After the transfer to nitrocellulose membranes, blots were consecutively probed with specific antibodies directed against either membrane-associated proteins (A) or myofilament regulatory proteins (B). Homogenates were also prepared from Langendorff-perfused hearts treated with ISO to obtain a maximum β-adrenergic stimulation. Samples were analyzed by SDS-PAGE, and blots were consecutively probed with phospho-specific antibodies as described under “Experimental Procedures.” Shown are representative immunoblots (C) and the quantitative analysis of phosphorylated regulatory membrane-associated (sarcolemma or SR) and myofilament proteins under basal (bars labeled B) and ISO-stimulated conditions (D). Caffeine-induced Ca²⁺ release was determined in isolated cardiomyocytes that were loaded with Fluo-4. Shown are F₁/F₀ peak amplitudes and decay kinetics (E). Cav, L-type Ca²⁺ channel; SER, sarco(endo)plasmic reticulum Ca²⁺ ATPase; RyR, ryanodine receptor; CSQ, calsequestrin; ANK, ankyrin B; TRN, triadin 1. *, p < 0.05 versus WT.