Research ArticleCardiac Physiology

Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart

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Science Signaling  21 Jul 2015:
Vol. 8, Issue 386, pp. ra72
DOI: 10.1126/scisignal.aaa5876
  • Fig. 1 B56α-deficient mice display increased PP2A activity and abnormal heart rate regulation.

    (A) Wild-type (WT) (n = 6) and B56α+/− hearts (n = 6) had no difference in total protein phosphatase activity [P = N.S. (not significant)]. B56α−/− hearts (n = 6) showed increased protein phosphatase abundance compared to WT hearts (*P < 0.05 for B56α−/− compared to WT). A.U., arbitrary unit. (B) PP2A-specific phosphatase activity was significantly increased in B56α+/− and B56α−/− hearts compared to WT hearts (n = 6 hearts per genotype; *P < 0.05 compared to WT; #P < 0.05 of B56α+/− to B56α−/−). (C) PP2A-specific phosphatase activity was significantly higher in B56α−/− than in WT cerebellar lysates (*P < 0.05 compared to WT; n = 4 mice per genotype). (D) At baseline, B56α+/− mice display decreased heart rate compared with WT mice (n = 4 mice per genotype; *P < 0.05). bpm, beats per minute. (E and F) Representative 5-s ECG recordings showing bradycardia and heart rate variability in WT mice (E) compared with B56α+/− (F) littermates. (G) Heart rate distribution of WT and B56α+/− mice demonstrates the leftward shift to lower heart rates in B56α+/− mice when plotted as % of total incidences at defined heart rate intervals (bars represent all data points from n = 4 mice per genotype; *P < 0.05). (H) The average SD from each 5-s RR interval at the indicated heart rates, plotted to demonstrate the increased heart rate variability in B56α+/− compared to WT mice (n = 4 mice per genotype; *P < 0.05). (I) Quantification of mean heart rate for conscious WT and B56α−/− mice (n = 4 mice per genotype; *P < 0.05). (J) Heart rate distribution for WT and B56α−/− mice demonstrates a leftward shift to lower heart rates in B56α−/− mice when plotted as % of total incidences at defined heart rate intervals (bars represent all data points from n = 4 mice per genotype; *P < 0.05). (K) The average SD from each 5-s averaged RR interval at the indicated heart rates, plotted to demonstrate the increased heart rate variability in B56α−/− compared to WT mice (n = 4 mice per genotype; *P < 0.05). (L) WT and B56α+/− mice displayed similar peak heart rates after isoproterenol (Iso) injection (n = 3 mice per genotype; P = N.S.). (M) Heart rate recovery after isoproterenol injection was longer in B56α+/− mice than in WT mice (n = 3 mice per genotype; *P < 0.05). (N and O) After acetylcholine receptor activation with carbachol (CCH), B56α+/− mice had a more pronounced reduction in heart rate and increased period for heart rate recovery compared to WT mice (n = 3 mice per genotype; *P < 0.05 compared to WT).

  • Fig. 2 Myocytes from B56α-deficient mice display aberrant Ca2+ regulation.

    (A to D) Representative line-scan images and corresponding Ca2+ transients normalized to the basal fluorescence (F0) of 1 Hz–stimulated ventricular myocytes, in the absence or presence of Iso. (E) Spark frequency was decreased in B56α+/− ventricular myocytes at baseline compared to WT myocytes (*P < 0.05). (F) Iso increased Ca2+ wave frequency for both WT and B56α+/− ventricular myocytes (*P < 0.05). Ca2+ wave frequency was decreased in B56α+/− + Iso compared to WT + Iso ventricular myocytes (#P < 0.05). (G) Mean SR Ca2+ load in WT and B56α+/− ventricular myocytes at baseline ± Iso (*P < 0.05 B56α+/− compared to B56α+/− + Iso; #P < 0.05 WT + Iso compared with B56α+/− + Iso). (H) Ca2+ transient amplitudes in WT and B56α+/− ventricular myocytes at baseline ± Iso (*P < 0.05 from respective baselines; #P < 0.05 WT + Iso compared to B56α+/− + Iso). (I to L) Representative line-scan images and corresponding Ca2+ transients normalized to F0 of 1 Hz–stimulated atrial myocytes, in the absence or presence of Iso. (M) Iso increased Ca2+ wave frequency for both WT and B56α+/− atrial myocytes (*P < 0.05). Ca2+ wave frequency was decreased in B56α+/− + Iso compared to WT + Iso atrial myocytes (#P < 0.05). (N) Ca2+ transient amplitudes in WT and B56α+/− atrial myocytes at baseline ± Iso (*P < 0.05 from baseline; #P < 0.05 WT + Iso compared to B56α+/− + Iso). For (A) to (M), myocytes were isolated from n = 4 mice per genotype. A minimum of 20 myocytes were analyzed for each independent myocyte preparation.

  • Fig. 3 B56α+/− hearts display reduced RyR2 phosphorylation.

    (A) Quantification of myocyte proteins from WT and B56α+/− heart lysates. Total RyR2 abundance was unchanged, and B56α+/− mice showed reduced phosphorylation (p) of RyR2 at Ser2808 and Ser2814 (*P < 0.05; abundance was normalized to total RyR2). The total abundance of PLB, SERCA2, PKA catalytic (cat) subunit, CaMKIIδ, or protein phosphatase 1 (PP1), and the phosphorylation of PLB at Ser16 or Thr17 (normalized to total PLB) did not differ. n = 4 mouse hearts per genotype; *P < 0.05. (B) Representative immunoblots of WT and B56α+/− lysates.

  • Fig. 4 Ectopic B56α expression decreases PP2A and parasympathetic activity.

    (A) Four weeks after injection of AAV9 B56α–IRES mCherry into mice, ventricular myocytes were isolated to confirm mCherry expression. (B) Example images of control WT myocytes, as well as AAV9 mCherry (mCh) and AAV9 mCherry B56α-transduced atrial and ventricular cardiomyocytes. Data are representative of three independent experiments per genotype. Scale bars, 20 μm. (C) Representative immunoblot of B56α, mCherry, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in three independent experiments of WT and B56α–overexpressing (OE) hearts. (D) Similar abundance of B56α in atria and ventricle of mouse hearts transduced with AAV9 mCherry B56α (n = 3 hearts per genotype; P = N.S.). (E) Relative PP2A activity in WT and B56α OE hearts (n = 4 hearts per genotype; *P < 0.05 compared to WT). (F) Conscious B56α-OE mice had higher resting heart rates than did WT mice (n = 3 mice per experimental group; *P < 0.05). (G) Conscious B56α OE mice had higher peak heart rates after isoproterenol injection than WT, B56α+/−, or control mCherry-transduced mice (n = 3 mice per experimental group; *P < 0.05). (H and I) Heart rates and heart rate recovery [t1/2 (half-time)] of WT mice transduced with mCherry or mCherry B56α were compared to WT and B56α+/− mice (reported in Fig. 1) after injection of carbachol to activate acetylcholine receptors. B56α OE mice displayed a blunted heart rate response compared with WT, B56α+/−, and mCherry-transduced mice (n = 3 mice per experimental group; *P < 0.05). Heart rate recovery was more rapid in B56α OE mice than in WT mice, B56α+/− mice, or control mice transduced with AAV9 mCherry (n = 3 mice per experimental group; *P < 0.05). B56α expression in B56α+/− mice (gray symbols) restored carbachol heart rate response to levels similar to WT mice (n = 3 mice per experimental group; P = N.S. between WT and B56α+/− + B56α OE). (J and K) Representative immunoblots and quantification of WT and B56α OE mouse cardiac lysates illustrating increased phosphorylation of RyR2 at Ser2808 and Ser2814 (n = 3 hearts per experimental group; *P < 0.05; phosphorylation was normalized to total RyR2). The total abundance of RyR2, GAPDH, or PP2A/C did not differ between experimental groups (n = 3 hearts per experimental group; P = N.S.).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/386/ra72/DC1

    Fig. S1. B56α+/− mice display reduced B56α abundance without changes in the abundance of PP2A core subunits.

    Fig. S2. Wild-type and B56α+/− mice have similar QT intervals.

    Fig. S3. Wild-type and B56α+/− mice have similar peak heart rates after exercise.

    Fig. S4. B56α+/− mice display an aberrant response to adrenergic stimulation.

    Fig. S5. B56α−/− mice display phenotypes associated with increased parasympathetic activity.

    Fig. S6. B56α+/− mice display exaggerated cholinergic response to carbachol administration.

    Fig. S7. B56α+/− and B56α−/− mice display normal ECG responses after inhibition of muscarinic acetylcholine receptors.

    Fig. S8. B56α-deficient mice display reduced heart rate after inhibition of sympathetic and parasympathetic signaling.

    Fig. S9. B56α−/− hearts display reduced phosphorylation of RyR2.

    Fig. S10. B56α associates with PP2A/C and RyR2 but is not required for the interaction between RyR2 and PP2A/C.

    Fig. S11. B56α+/− and B56α−/− atria display reduced phosphorylation of RyR2.

    Fig. S12. Myofilament proteins are phosphorylated to a similar extent in wild-type and B56α+/− mice.

    Fig. S13. The PP2A core enzyme is differentially localized in B56α+/− and B56α−/− myocytes.

    Fig. S14. B56α abundance is increased in ankyrin-B–deficient hearts.

  • Supplementary Materials for:

    Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart

    Sean C. Little, Jerry Curran, Michael A. Makara, Crystal F. Kline, Hsiang-Ting Ho, Zhaobin Xu, Xiangqiong Wu, Iuliia Polina, Hassan Musa, Allison M. Meadows, Cynthia A. Carnes, Brandon J. Biesiadecki, Jonathan P. Davis, Noah Weisleder, Sandor Györke, Xander H. Wehrens, Thomas J. Hund, Peter J. Mohler*

    *Corresponding author. E-mail: peter.mohler{at}osumc.edu

    This PDF file includes:

    • Fig. S1. B56α+/− mice display reduced B56α abundance without changes in the abundance of PP2A core subunits.
    • Fig. S2. Wild-type and B56α+/− mice have similar QT intervals.
    • Fig. S3. Wild-type and B56α+/− mice have similar peak heart rates after exercise.
    • Fig. S4. B56α+/− mice display an aberrant response to adrenergic stimulation.
    • Fig. S5. B56α−/− mice display phenotypes associated with increased parasympathetic activity.
    • Fig. S6. B56α+/− mice display exaggerated cholinergic response to carbachol administration.
    • Fig. S7. B56α+/− and B56α−/− mice display normal ECG responses after inhibition of muscarinic acetylcholine receptors.
    • Fig. S8. B56α-deficient mice display reduced heart rate after inhibition of sympathetic and parasympathetic signaling.
    • Fig. S9. B56α−/− hearts display reduced phosphorylation of RyR2.
    • Fig. S10. B56α associates with PP2A/C and RyR2 but is not required for the interaction between RyR2 and PP2A/C.
    • Fig. S11. B56α+/− and B56α−/− atria display reduced phosphorylation of RyR2.
    • Fig. S12. Myofilament proteins are phosphorylated to a similar extent in wild-type and B56α+/− mice.
    • Fig. S13. The PP2A core enzyme is differentially localized in B56α+/− and B56α−/− myocytes.
    • Fig. S14. B56α abundance is increased in ankyrin-B–deficient hearts.

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    Citation: S. C. Little, J. Curran, M. A. Makara, C. F. Kline, H.-T. Ho, Z. Xu, X. Wu, I. Polina, H. Musa, A. M. Meadows, C. A. Carnes, B. J. Biesiadecki, J. P. Davis, N. Weisleder, S. Györke, X. H. Wehrens, T. J. Hund, P. J. Mohler, Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart. Sci. Signal. 8, ra72 (2015).

    © 2015 American Association for the Advancement of Science

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