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. 2019 Mar;124(5):737-746.
doi: 10.1161/CIRCRESAHA.118.314350.

Protein Phosphatase 2A Regulates Cardiac Na+ Channels

Mona El Refaey  1   2 , Hassan Musa  1   2 , Nathaniel P Murphy  1   2 , Ellen R Lubbers  1   2 , Michel Skaf  1   2 , Mei Han  1   2 , Omer Cavus  1   2 , Sara N Koenig  1   2 , Michael J Wallace  1   2 , Daniel Gratz  1   3 , Elisa Bradley  1   4 , Katherina M Alsina  5   6   7 , Xander H T Wehrens  8 , Thomas J Hund  1   4   3 , Peter J Mohler  1   4   2
Affiliations

Protein Phosphatase 2A Regulates Cardiac Na+ Channels

Mona El Refaey et al. Circ Res. 2019 Mar.

Abstract

Rationale: Voltage-gated Na+ channel ( INa) function is critical for normal cardiac excitability. However, the Na+ channel late component ( INa,L) is directly associated with potentially fatal forms of congenital and acquired human arrhythmia. CaMKII (Ca2+/calmodulin-dependent kinase II) enhances INa,L in response to increased adrenergic tone. However, the pathways that negatively regulate the CaMKII/Nav1.5 axis are unknown and essential for the design of new therapies to regulate the pathogenic INa,L.

Objective: To define phosphatase pathways that regulate INa,L in vivo.

Methods and results: A mouse model lacking a key regulatory subunit (B56α) of the PP (protein phosphatase) 2A holoenzyme displayed aberrant action potentials after adrenergic stimulation. Unbiased computational modeling of B56α KO (knockout) mouse myocyte action potentials revealed an unexpected role of PP2A in INa,L regulation that was confirmed by direct INa,L recordings from B56α KO myocytes. Further, B56α KO myocytes display decreased sensitivity to isoproterenol-induced induction of arrhythmogenic INa,L, and reduced CaMKII-dependent phosphorylation of Nav1.5. At the molecular level, PP2A/B56α complex was found to localize and coimmunoprecipitate with the primary cardiac Nav channel, Nav1.5.

Conclusions: PP2A regulates Nav1.5 activity in mouse cardiomyocytes. This regulation is critical for pathogenic Nav1.5 late current and requires PP2A-B56α. Our study supports B56α as a novel target for the treatment of arrhythmia.

Keywords: ankyrins; arrhythmias, cardiac; calcium-calmodulin-dependent protein kinase type 2; phosphorylation; physiology.

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Conflict of interest statement

DISCLOSURES

XHTW is a founding partner of Elex Biotech, a start-up company that developed drug molecules that target ryanodine receptors for the treatment of cardiac arrhythmia disorders. Other authors have no conflicts.

Figures

Figure 1.
Figure 1.. B56α KO mice display aberrant cardiomyocyte excitability.
(A-B) Representative action potential (APs, 1.0 Hz pacing) and summary of APD at 50%, 75% and 95% repolarization for 0.5 and 1.0 Hz pacing in WT and B56α KO myocytes. (C-D) AP amplitudes (APA) and maximum upstroke velocity (dv/dtmax) in WT and B56α KO myocytes. Results are shown for 0.5 and 1.0 Hz pacing frequencies (for B-D, WT, N=3; n= 9 and B56α KO, N=3; n=8; *p<0.05).
Figure 2.
Figure 2.. B56α KO ventricular myocytes display decreased sensitivity to isoproterenol-induced APD prolongation.
(A-D) Representative APs (1.0 Hz pacing) and summary of APD at 50%, 75% and 95% repolarization at 0.5 and 1.0 Hz pacing in WT and B56α KO myocytes ±100nM Iso. (E-H) Action potential amplitudes (APA) and maximum upstroke velocity (dv/dtmax) in WT and B56α KO myocytes ±Iso. Results are shown for 0.5 and 1.0 Hz pacing frequencies (WT, N=3; n=9 and B56α KO, N=3; n=8 *p<0.05).
Figure 3.
Figure 3.. Identification of Nav1.5 as PP2A target.
Computer simulations to predict the mechanism underlying changes in cell excitability in the setting of B56α-deficiency. (A) Regression coefficients showing relative impact of changes in ion channel conductance/transport rates on action potential duration at 90% repolarization (APD) in the Hund-Rudy dynamic cell model. Abbreviations are as follows: inwardly rectifying K current (gK1), rapid delayed rectifier K+ current (gKr), slow delayed rectifier K+ current (gKs), L-type Ca2+ current (gCa(L)), fast inward Na+ current (gNa), subspace and bulk Na+/Ca2+ exchanger (gNaCa(ss) and gNaCa(bulk), respectively), Na+/K+ ATPase (gNak), late Na+ current (gNa,L), Ca2+ release from SR (grel), transient outward K+ current (gto), Ca2+ translocation from network to junctional SR (gtr) and Ca2+ uptake into SR (gup). (B) APD90 (expressed relative to WT) in experiment (B56α KO), and in the following computational models: 1) model with parameter values identified in the regression analysis with smallest sum-of-squared error compared to experiment (Mutant), and 2) model with parameter values selected as average of 10 best solutions (Mutant10). (C) Corresponding model parameters (expressed relative to WT) for Mutant and Mutant10 computational models.
Figure 4.
Figure 4.. B56α KO myocytes display decreased INa,L.
(A-B) Representative recordings of whole cell INa from WT and B56α KO ventricular myocytes. (C) Current-voltage relationship, (D) voltage-dependent activation and voltage-dependent inactivation curves, and (E) time-dependent recovery of INa in WT and B56α KO ventricular myocytes. No significant difference was observed in peak INa at experimental voltages ranging from −60 to −15mV (p=N.S.), in V½ as determined by Boltzmann fits of the steady-state voltage-dependent inactivation (p=N.S.) and time-dependent recovery (p=N.S.; N=5,5, n=22,21 combined genders). (F) Representative INa,L traces from WT and B56α KO ventricular myocytes. (G) Quantification of INa,L from WT and B56α KO ventricular myocytes at baseline (WT, N=4; n=10 and B56α KO, N=4; n=9; *p<0.05). (H) No difference in cell capacitance was observed between WT and B56α KO myocytes (p=N.S.; shown for INa,L and INa experiments).
Figure 5.
Figure 5.. PP2A/B56α complex associates with Nav1.5 regulatory complex.
(A-E) Co-immunoprecipitation experiments were performed from detergent-soluble lysates of adult mouse hearts using beads conjugated to Nav1.5 Ig or control Ig. Bound protein was eluted and immunoblotted with ankyrin-G (A), CaMKIIδ (B), PP2A-C (C), PP2A-B56α (D) or PP2A-A (E). Data are representative of experiments repeated three times. (F) Co-immunoprecipitation experiments were performed from detergent-soluble lysates of adult mouse hearts using beads conjugated to PP2A-C Ig or control Ig. Bound protein was immunoblotted with ankyrin-G. (G) Co-immunoprecipitation experiments were performed from human left ventricular lysates (non-failing hearts) using beads conjugated to Nav1.5Ig or control Ig. Bound protein was immunoblotted with PP2A-C. Experiments were repeated three times. (H-K) Pull-down experiments were performed using detergent-soluble extracts of WT mouse hearts using glutathione S-transferase (GST) or GST-AnkG. Bound protein was analyzed using antibodies specific for PP2A-B56α (H), PP2A-C (I), PP2A-A (J) or PP2A-B56γ (L). Data are representative of experiments repeated three times.
Figure 6.
Figure 6.. B56α KO mice display decreased Nav1.5 Ser571 phosphorylation.
(A-G) Immunoblots and quantitative analysis of PP2A-B56α (WT, N=5; B56α KO, N=7; p<0.05), PP2A-C (WT, N=7; B56α KO, N=7; p=N.S.), CaMKIIδ (WT, N=7; B56α KO, N=7; p=N.S.) Nav1.5 (WT, N=11; B56α KO, N=11; p=N.S.), Nav1.5 pSer571 (WT, N=11; B56α KO, N=10; p<0.05) and GAPDH expression in WT and B56α KO mouse hearts following isoproterenol treatment. B56α KO mice showed a significant decrease in both B56α expression and Nav1.5 Ser571 phosphorylation, as well as the ratio of Nav1.5 Ser571 phosphorylation to Nav1.5 expression (WT, N=11; B56α KO, N=10; p<0.05) following isoproterenol treatment. For Nav1.5pS571/total Nav1.5ratio, data is expressed based on normalized protein expression versus GAPDH as shown in E and F. We observed no difference in GAPDH expression between genotypes (WT, N=11; B56α KO, N=10; p=N.S.).
Figure 7.
Figure 7.. B56α KO myocytes are insensitive to isoproterenol-induced increases of INa,L.
(A-B) Representative voltage-gated Na+ current (INa) traces from WT and B56α KO ventricular myocytes ± isoproterenol (100nM). (C-D) Summary data (mean ± SEM) for peak INa (at −30 mV), and INa,L in response to 100nM Iso (*p<0.05). (E) Cell capacitance was not different between groups (p=N.S., WT, WT Iso N=5, 4; n=10, 9 and B56α KO, B56α KO Iso N=4,4; n= 9,7).
Figure 8.
Figure 8.. Intercalated disc Nav1.5 macromolecular signaling complex.
Via direct interaction of Nav1.5 with ankyrin-G and βIV spectrin, CaMKIIδ resides in close proximity with its target molecule, Nav1.5. Our new findings implicate ankyrin-G in targeting the PP2A complex via B56α to the Nav1.5 intercalated disc complex to balance CaMKII-dependent phosphorylation. Together, CaMKIIδ and PP2A regulate INa,L in heart.
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