Another-regulin regulates cardiomyocyte calcium handling via integration of neuroendocrine signaling with SERCA2a activity

Figures

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  Fig. 1.. ALN KO mice have normal cardiac structure and function. (A) To generate ALN KO mice, CRISPR gRNAs were used to target exons 1 and 2 of the mouse ALN gene resulting in a 220 bp deletion and 1 bp insertion. (B) ALN protein expression analyzed in heart homogenates from WT and ALN KO mice by Western blotting. (C) Representative images of histological sections stained with H&E from 12-month-old mice with the indicated genotypes. Scale bar is 1 mm. (D) Heart weight to body weight (HW/BW) ratios of 12-month-old WT and ALN KO mice. Data are expressed as mean ± SD and statistical comparison between groups was performed using an unpaired student’s t-test. (E-I) Serial echocardiography (ECHO) was performed to measure left ventricular (LV) function and structure in mice with the indicated genotypes. (E) Representative para-sternal short-axis view (left) and M-mode tracings (right) from 12-month-old mice. M-mode images were used to quantify ejection fraction (F), fractional shortening (G), and LV posterior wall thickness during diastole (H) and systole (I). Data are expressed as mean ± SD and number of mice analyzed are indicated within each graph. Statistical analysis was performed by 2-way…

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  Fig. 2.. ALN KO cardiomyocytes display an intermediate phenotype in Ca2+ handling compared to WT and PLN KO cardiomyocytes. (A) Representative fractional shortening tracings measured by sarcomere length during pacing-induced cardiomyocyte contraction in cells from 8-week-old mice with the indicated genotypes. (B) Representative Ca2+ transient traces recorded in Fura-2 loaded cardiomyocytes from mice with the indicated genotypes. (C, D) Ca2+ transients were used to quantify mean peak transient amplitude (C) and cytosolic Ca2+ decay rates (D) in cardiomyocytes from mice with the indicated genotypes. Transient decay rates (Tau) were measured by fitting a single exponential to the decay phase of the Ca2+ transient. (E) Quantification of cardiomyocyte fractional shortening measured by sarcomere length for the indicated genotypes. Data points in panels A-E represent individual cardiomyocytes from n = 6 mice per genotype. Statistical analysis was performed by One-way ANOVA with Bonferroni post-hoc testing. Abbreviations: WT, wildtype; PLN, phospholamban; ALN/PLN dKO, ALN/PLN double-knockout.

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  Fig. 3.. The microprotein ALN shares sequence homology with PLN and is phosphorylated on Serine 19. (A). Sequence alignment of ALN and PLN highlighting their conserved transmembrane domain (blue). Identical residues within the PLN/ALN SERCA2a binding domain are underlined. Validated PLN phosphorylation and candidate phosphorylation sites of ALN are indicated in yellow (PLN) and green (ALN). The ALN phosphorylation site at S19 is indicated with an asterisk. (B) AlphaFold prediction of ALN and PLN structures with phosphorylation sites indicated. AlphaFold model confidence is depicted with the following colors: dark blue, very high; light blue, high; yellow, low. (C) Western blot analysis of protein lysates from AD293 cells expressing HA-ALN-WT separated by Phos-tag (top) or conventional (bottom) SDS-PAGE. Phos-tag SDS-PAGE separates HA-ALN into a phosphorylated species (pALN, top) and a non-phosphorylated species (ALN, bottom). (D) Western blot analysis of protein lysates from AD293 cells transfected with WT HA-ALN or versions of ALN with candidate phosphorylation mutated to alanine separated by Phos-tag SDS-PAGE. The loss of the high molecular weight species of ALN in the ALN-S19A lane…

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  Fig. 4.. ALN S19 phosphorylation regulates its interaction with SERCA2a. (A) Co-immunoprecipitation experiments performed on protein extracts from AD293 cells expressing WT or phospho-mutant versions of HA-tagged ALN or PLN with Myc-tagged SERCA2a. Pulldowns were performed with an antibody for Myc (SERCA2a). Immunoblots are representative of 3 independent experiments. (B) Average FRET from Cer-SERCA2a to YFP-ALN variants. (C) The dependence of FRET on expression level (WT, gray; S19A, red; S19D, green). FRET increased with protein concentration toward a maximum (FRETmax). (D) FRETmax values for SERCA2a-ALN (WT, S19A, S19D) obtained from hyperbolic fitting of data (as in panel C). (E) Fluorescent lifetime decays for mCyRFP-SERCA2a donor alone (Cntrl) or in the presence of acceptor mMaroon1-ALN (WT, S19A, S19D). In the absence of ALN, mCyRFP-SERCA2a was characterized by a long lifetime. Decays were obtained from cells. (F) Comparison of amplitude-weighted average fluorescence lifetimes. (G) Quantification of a short lifetime component τ2 obtained from fitting of the decay data with a multiexponential model. (H) Quantification of short lifetime component τ3. S19D-expressing cells…

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  Fig. 5.. ALN S19 phosphorylation enhances SERCA2a activity. (A) Schematic depicting MyoAAV constructs for cardiomyocyte-specific expression of ALN WT and phosphomutants or MyoAAV-Empty (control). (B) Immunofluorescence (IF) images of heart sections from 8-week-old ALN KO mice infected with the indicated MyoAAVs. Tissue sections were immunostained with antibodies for HA (green) and PLN (red). The merged channel also include WGA (purple) and a nuclear DAPI stain (blue). Scale bars are 100 100 μm. For additional images, see Fig. S4A. (C) HA-ALN protein expression was analyzed by Western blotting of protein extracts from cardiomyocytes isolated from ALN KO mice infected with the indicated MyoAAVs. (D-F) Mean peak Ca2+ transient amplitude (D), cytosolic Ca2+ decay rates (E), and cardiomyocyte fractional shortening (F) quantified from pacing induced Ca2+ transients and sarcomere shortening measurements from Fura-2 loaded cardiomyocytes isolated from 8-week-old ALN KO mice infected with the indicated MyoAAVs. Data points represent individual cardiomyocytes from n = 5 mice. One-way ANOVA with Bonferroni post-hoc testing was used for statistical analysis. (G) Co-immunoprecipitation experiments…

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  Fig. 6.. Gαq/PKC activation increases pALN S19 levels in isolated cardiomyocytes. (A) Isolated cardiomyocytes from 8-week-old WT mice were stimulated with 1nM Iso or 1μM AngII and phosphorylated ALN and PLN levels were analyzed via Western blotting with antibodies for phospho-ALN (pALN-S19) and phospho-PLN (pPLN-S16). Total ALN and PLN (tALN, tPLN) protein levels were also analyzed. Quantification is shown on the right. (B) Schematic of the Gαq pathway with relevant agonists, drugs, and signaling molecules indicated. (C) Adult cardiomyocytes from WT mice were stimulated with Gαq agonists (AngII, 1μM; ET-1, 10nM; PE, 10μM) with or without pre-treatment with a pan-PKC inhibitor (Go6983, 2μM) and protein extracts were analyzed and quantified by Western blotting using antibodies for pALN S19 and total ALN. A Gαs agonist (Iso, 1nM) was used as a negative control. (D) Gαq protein expression analyzed in isolated cardiomyocytes from WT and cardiomyocyte-specific Gαq KO mice by Western blotting. (E) Western blot analysis and quantification of pALN S19 levels in isolated cardiomyocytes from cardiomyocyte-specific Gαq KO mice stimulated with Gαq agonists (AngII, 1μM; ET-1, 10nM; PE, 10μM) or a PKC…

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  Fig. 7.. Phosphorylation of ALN via Gαq stimulation increases cardiomyocyte Ca2+ transient and contractility properties. (A) Schematic of experimental design and methods used for quantification. (B-E) Pacing-induced Ca2+ transients and cardiomyocyte contractility analyzed in Fura-2 loaded cardiomyocytes isolated from 8-week-old mice with the indicated genotypes. Cytosolic Ca2+ decay rates (B, D, Tau) and fractional shortening measured by sarcomere length (C, E) were quantified before and after treatment with Gαs or Gαq agonists. Each datapoint represents a single cardiomyocyte at baseline and after agonist stimulation. 400 nM Iso was used as the Gαs agonist in panels B and C, and 7.5μM AngII was used as the Gαq agonist in panels D and E. Data points represent individual cardiomyocytes from n = 6 mice. 2-way ANOVA with Bonferroni post-hoc testing was used for statistical analysis. Abbreviations: WT, wildtype; ALN/PLN dKO, ALN/PLN double-knockout.

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  Fig. 8.. Working model. Neuroendocrine stimuli activate Gαq-coupled receptors on the cell membrane which initiates the signaling cascade resulting in PKC phosphorylation of ALN, its dissociation from SERCA2a, and increased Ca2+ cycling in cardiomyocytes (Left). In contrast, β-adrenergic agonists activate Gαs-coupled receptors which elevate cAMP and induce PKA to phosphorylate PLN, resulting in SERCA2a disinhibition and increased Ca2+ cycling (Right). Abbreviations: PLC, phospholipase C; DAG, diacyl glycerol; PKC, protein kinase C; AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A.