A perinuclear calcium compartment regulates cardiac myocyte hypertrophy

Figures

• 

  Fig. 1.. Identification of an mAKAPβ-dependent perinuclear Ca2+ compartment regulating local calcineurin activity. A, Compartment-specific biosensors: D3cpv Cameleon contains mutant calmodulin (mCam) and Ca2+/calmodulin-binding domain of myosin light chain kinase (mMLCK) inserted between ECFP and cpVenus fluorescent proteins, such that Ca2+-dependent mCaM binding of the mMLCK peptide increases FRET signal [29]. CaNAR2 contains between Cerulean 3 and YPet fluorescent proteins the N-terminal regulatory domain of NFATc2, that is highly phosphorylated in unstimulated cells and that upon CaN dephosphorylation changes conformation increasing FRET signal [26]. The intensiometric Ca2+ sensor GCaMP6s contains a calmodulin-binding M13 peptide, circularly permuted green fluorescent protein (cpGFP), and a variant mCam, that upon Ca2+-dependent M13-mCam binding increases cpGFP fluorescence [25]. Nesprin-1α (blue bar) has spectrin-like repeat (SR) domains, that mediate mAKAPβ binding, and a C-terminal transmembrane Klarsicht, ANC-1, Syne Homology (KASH) domain, that results in nuclear envelope localization [7]. B, Neonatal rat ventricular myocytes infected with adenovirus expressing Cameleon,…

• 

  Fig. 2.. Local Ca2+ ion and ryanodine receptor activity are required for Ca2+ −CaN signaling in the perinuclear mAKAPβ – nesprin-1α compartment. A, Recombinant mCherry-tagged nesprin-1α fusion proteins for the study of perinuclear Ca2+ signaling. Carp parvalbumin-β contains 2 EF-hand domains that bind Ca2+ with high affinity (Kd ≈ 10−8 d mol/L) [30]. B–D, FRET imaging of Iso-stimulated (1 μmol/L) neonatal rat ventricular myocytes co-infected with adenovirus expressing Cameleon-nesprin (B), Cameleon (C), or CaNAR2-nesprin (D) and either mCherry-parv-nesprin or mCherry-nesprin control. E–F, FRET imaging of Iso-stimulated (1 μmol/L) adult rat ventricular myocytes co-infected with adenovirus expressing CaNAR2-nesprin (E) or CaNAR2 (F) and either mCherry-parv-nesprin or mCherry-nesprin control. G-H, Protein complexes were immunoprecipitated from neonatal myocyte extracts using antibodies for nesprin-1α and mAKAPβ and detected by Western Blot. n = 3. I–K, Imaging of neonatal myocytes expressing Cameleon-nesprin and treated with Iso (1 μmol/L), ryanodine (5 nmol/L or 5 μmol/L) or xestospongin C (3 μmol/L) as indicated. Representative tracings and peak amplitudes for FRET ratio (R normalized to…

• 

  Fig. 3.. Perinuclear Ca2+ in the mAKAPβ – nesprin-1α compartment ion is required for NFAT nuclear translocation and myocyte hypertrophy, but not myocyte contractility. A-B, Neonatal rat ventricular myocytes, infected with adenovirus expressing GFP-tagged NFATc1 and either mCherry-nesprin or mCherry-Parv-nesprin, were stimulated with Iso (1 μmol/L) for 24 h, before assay of NFAT localization by fluorescence microscopy. Fraction of cells with majority NFAT nuclear localization was determined. Bar - 10 μm. C—D, Neonatal myocytes expressing either mCherry-nesprin or mCherry-Parv-nesprin were stimulated for 48 h with Iso (1 μmol/L) before staining with atrial natriuretic factor antibody (ANF, red). Fraction of cells with prominent ANF staining was determined. E–F, Adult rat ventricular myocytes were infected with adenovirus expressing either mCherry-nesprin (Ctrl) or mCherry-Parv-nesprin (PDE) and stimulated for 48 h with Iso (1 μmol/L) before staining for α-actinin (green) and nuclei (DAPI, blue) and fluorescent imaging. Myocyte length and width were measured. Bar - 25 μm. In A-F each data point represents the mean value of >22 cells that were measured for each individual biological…

• 

  Fig. 4.. mAKAPβ organizes a perinuclear cAMP-PKA signaling compartment. A, AKAR4 consists of a forkhead-associated domain (FHA1) and PKA substrate (LRRATLVD) flanked by Cerulean and cpVenus; PKA substrate phosphorylation induces FHA1 binding, increasing FRET signal [33]. mCherry-PDE-nesprin contains a mutated PDE4D catalytic domain (T349A, K455/456A, S579A, FQF597–599AAA). B, Neonatal rat ventricular myocytes were infected with adenovirus for AKAR4 or AKAR4-nesprin and imaged by confocal microscopy. Bar - 10 μm. Cyan image is shown in grayscale. C, Pseudocolored images showing FRET ratio R (net FRET ÷ background-subtracted donor signal) for representative control shRNA tracings shown in D-G. D-E, FRET measurements for adenovirus-infected neonatal myocytes expressing AKAR4-nesprin (D) or AKAR (E) and either control (Ctrl) or mAKAP shRNA. F-G, FRET measurements for adenovirus-infected neonatal myocytes expressing AKAR4-nesprin (F) or AKAR (G) and either mCherry-PDE-nesprin or mCherry-nesprin control. D-G, Myocytes were stimulated with norepinephrine (NE, 10 nmol/L). Each data point in peak amplitude plots for FRET ratios (R/R0) represents separate tracings (n ≥ 6) obtained using cells from…

• 

  Fig. 5.. Perinuclear cAMP is required for mAKAPβ-associated calcineurin activity. A-B, FRET imaging of neonatal rat ventricular myocytes expressing Cameleon-nesprin (A) or CaNAR2-nesprin (B) were stimulated with isoproterenol (1 μmol/L) and inhibited with H89 PKA inhibitor (1 μmol/L) as indicated. C, FRET imaging of neonatal myocytes expressing CaNAR2-nesprin and either mCherry-PDE-nesprin or mCherry-nesprin control and stimulated with norepinephrine (NE, 10 nmol/L). For each panel, representative tracings for FRET ratio (R/R0) are shown. Each data point in peak amplitude plots for FRET ratios (R/R0) represents separate tracings (n ≥ 6) obtained using cells from 3 independent myocyte preparations. Peak amplitude data were compared by unpaired, two-tailed t-test. *** p ≤ 0.001; **** p ≤ 0.0001.

• 

  Fig. 6.. The phosphorylation of perinuclear ryanodine receptors by PKA is independently regulated by mAKAPβ and local cAMP signaling. Neonatal myocytes infected with adenovirus expressing mAKAP or control shRNA (A,B) or mcherry-PDE-nesprin or control mCherry-nesprin (C,D) were stimulated with isoproterenol (1 μmol/L) as indicated (hr – hours). Protein complexes immunoprecipitated with nesprin-1 (A,B) or mCherry antibody (C,D) were assayed for RyR2 Ser-2808 phosphorylation (A,C), RyR2 Ser-2030 phosphorylation (B,D) and total RyR2 protein by western blotting. Iso induced both Ser-2808 and −2030 RyR2 phosphorylation in both immunoprecipitates and in total extracts (p ≤ 0.0001), although only the phosphorylation of immunoprecipitated RyR2 was dependent upon mAKAP and perinuclear cAMP. n = 4, each using myocytes from an independent preparation. Data were analyzed by matched 2-way ANOVA and Tukey post-hoc testing. **** p ≤ 0.0001.

• 

  Fig. 7.. Perinuclear cAMP at mAKAPβ – nesprin-1α signalosomes is required for NFAT nuclear translocation and myocyte hypertrophy. A-B, Neonatal myocytes were infected with adenovirus expressing GFP-tagged NFATc1 and either mCherry-PDE-nesprin or mCherry-nesprin control followed by stimulation with NE (10 nmol/L) for 24 h before staining with DAPI (blue) and imaging to determine NFAT localization. Representative images of myocytes with predominantly cytosolic and nuclear NFATc1-GFP are shown in A. Bar - 10 μm. C—D, Neonatal myocytes were infected with adenovirus expressing either mCherry-PDE-nesprin or mCherry-nesprin control followed by stimulation with NE (10 nmol/L) for 48 h before staining with ANF antibody and DAPI (blue). Representative images of ANF staining are shown in C. E-G, Adult rat ventricular myocytes were infected with adenovirus expressing either mCherry-nesprin or mCherry-PDE-nesprin and stimulated for 48 h with NE (10 nmol/L) before staining with α-actinin antibodies (green) and DAPI (blue) and fluorescent imaging. Representative images of NE-stimulated myocytes are shown in E. Bar - 25 μm. For all panels, each data point represents the mean value of >50 cells that were…

• 

  Fig. 8.. Model for mAKAPβ signalosomes forming an independent perinuclear Ca2+ and cAMP signaling compartment regulating local calcineurin activity, NFAT nuclear translocation, and myocyte hypertrophy. See text for discussion. Drawing created with BioRender.com.