Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

Abstract

Background: Recent interest in understanding cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow the abscission of daughter cardiomyocytes.

Methods: Here, we use a proteomics screen to identify adducin, an actin capping protein previously not studied in cardiomyocytes, as a regulator of sarcomere disassembly. We generated many adeno-associated viruses and cardiomyocyte-specific genetic gain-of-function models to examine the role of adducin in neonatal and adult cardiomyocytes in vitro and in vivo.

Results: We identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phospho-isoforms of α-adducin in vitro and in vivo identified Thr445/Thr480 phosphorylation of a short isoform of α-adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin.

Conclusions: These results highlight an important mechanism for coordinating cytoskeletal morphological changes during cardiomyocyte mitosis.

Keywords: Irak4; adducin; alpha-actinin; cardiac proliferation; sarcomere disassembly.

Figure 1.. Identification of Adducin as a key protein associated with sarcomere disassembly and cardiomyocyte proliferation.

A. Schematic representation of sarcomere disassembly morphology. B. Co-immunoprecipitation coupled with mass spectrometry workflow, utilizing Tnnt2 pulldown to identify associated proteins from total heart extracts 3 days post-MI at P1 (P1MI) and P7 (P7MI). C. Table summarizing key proteins associated with Tnnt2, as identified from Co-IP/MS. Notations: *Peptide Spectrum Matches (PSM) indicates the number of spectra assigned to peptides that contributed to the inference of the protein; **MIC Sin represents the normalized spectral index statistic for each protein, calculated from the intensity of fragment ions in each spectrum assigned to a protein; ***Ratio between groups derived from MIC Sin values. D. Co-IP of Add1 from P1MI and P7MI, probed for Tnnt2 and α-Actinin. E. (Left) Representative WB; (Right) Quantitative comparison of Add1 and Add3 protein expression at three days following sham surgery or MI at P1 and P7. The pH3-S10 is included as a biomarker to indicate the occurrence of MI. F. (Left) Representative WB; (Right) Quantification of endogenous expression of Add1 and Add3 at different postnatal ages. Gapdh serves as a loading control in E and F. G. Percentage of cardiomyocytes expressing Adducins in neonatal hearts. H. Immunostaining for selected cytoskeletal proteins (Add1, Add3, Spectrin, Filamin) in (Left) proliferative and (Right) non-proliferative neonatal cardiomyocytes. Arrows indicate proliferative cardiomyocytes. Arrowheads in Filamin staining refers to the nucleus location. Scale bar: 10 μm (H). Data are presented as mean±sd. Statistical analyses: Two-way ANOVA with Tukey's post-hoc test (E), Kruskal-Wallis with uncorrected Dunn's test (F); * p < 0.05, ** p < 0.01.

Figure 2.. Overexpression of Add1 isoform 2 disassembles cardiomyocyte sarcomeres.

A. (Top) Schematic detailing the exon-intron structure of the Add1 gene. The red rectangle highlights exon15, the alternative splicing target. (Bottom) Depicts alternative splicing of exon15 in mRNA: inclusion results in Add1_i2 with 636 amino acids, while exclusion produces Add1_i1 with 732 amino acids. The corresponding protein schematics are displayed alongside. B. (Left) Representative WB; (Right) Quantitative comparison of Add1_i2 endogenous expression across different ages, with Gapdh as an internal control. C. Representative images of NRVMs transduced with AAV6 viral particles for Add1_i1, Add1_i2, and GFP control, at day 3. Cells were immunostained with α-Actinin (Actn2, red) to visualize sarcomere structure. Green fluorescence indicates Add1_i1, Add1_i2, or GFP expression. High-magnification views of selected areas are shown to the right, depicting varying states of sarcomere assembly: (i) and (iii) partially disassembled sarcomeres; (ii) and (iv) fully disassembled sarcomeres; (v) assembled sarcomeres. Dotted lines mark cardiomyocyte borders. D. Percentage of sarcomere disassembly in response to Adducin overexpression in C. E. Generation strategy for Add1_i2 transgenic mice. F. H&E staining of WT and Add1_i2 transgenic hearts. G. HW/BW comparison in control and Add1_i2 transgenic mice. H. (Top) Images showing Add1_i2 expression patterns in transgenic mice at P14 and P28. (Bottom) Insets show enlarged views of individual cardiomyocytes. I. Proportion of cardiomyocytes expressing Add1_i2 in transgenic mice. J. (Top) Images representing sarcomere patterns in Add1_i2 transgenic mice at P14 and P28. (Bottom) Insets show enlarged views of individual cardiomyocytes. Dotted lines indicate the outer and inner borders of sarcomeres. K. Comparisons of sarcomere disassembly scored with age-matched WT littermates indicate significant sarcomere thinning and central clearance in Add1_i2 transgenic mice at P14. Scale bar 10 μm (C, H, J); 1 mm (F). Data are presented as mean±sd. Statistical analyses: Kruskal-Wallis with uncorrected Dunn's test (B), Mann-Whitney U test (two-tailed) (I),two-way ANOVA with Tukey's post-hoc test (D, K; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p<0.0001.

Figure 3.. Role of Add1_i1 phosphorylation on sarcomere disassembly.

A. Endogenous phospho-Adducin patterns in neonatal mouse hearts: phospho-sites (T445/T480, S12/S355, S714/S724, S408/S436/S481) co-stained with sarcomeric proteins Tnnt2 or α-Actinin. Arrows and arrowheads indicate colocalization with disassembled and assembled sarcomeres, respectively. B. NRVM showing Add1_pT445 expression, stained with α-Actinin (red) and pT445 (green), noting its translocation from the nucleus to the cytoplasm during mitosis. C. (Left) Representative WB; (Right) Quantitative comparison of Add1_pT445 endogenous expression in relative to Gapdh. Tnnt2 is used as an age indicator. D. Representative images of NRVM transduced with AAV6 viral particles for phospho- or non-phospho mimic T445/T480. (Top left) Adducin mutants are expressed with 3 tandem FLAG epitopes at the N-terminus. Cells were immunostained with α-Actinin (red) to visualize sarcomere structure. Adducin mutants are detected by FLAG antibody (green). High magnification images of representative region (insets) were shown on the right. Dotted lines were drawn around the cell border. E. Quantitative analysis of disassembled sarcomeres induced by Adducin overexpression in D. F. Generation strategy for cardiac-specific Add1_i1 phospho transgenic mice. G. H&E staining of control and cardiac-specific Add1_i1 phospho transgenic. H. HW/BW in control and phospho-transgenic mice. I. Images showing phospho Add1_i2 expression patterns in transgenic mice at P14 and P28, with (Bottom) high magnification views of selective phospho-Adducin mimic overexpression cardiomyocytes. J. Proportion of cardiomyocytes expressing pAdd1 from I. K. Sarcomere patterns in phospho Add1_i1 transgenic mice at P14 and P28, with insets showing high magnification images. Dotted lines are drawn around the outer and inner edges of cardiomyocytes. L. Quantitative analysis of sarcomere disassembly. Scale bar 10 μm (A, B, D, I, K); 1 mm (G). Data are presented as mean±sd. Statistical analyses: Kruskal-Wallis with uncorrected Dunn's test (C) or Mann-Whitney U test (two-tailed) (J) or two-way ANOVA with Tukey's post-hoc test (E, L); *p < 0.05, **p < 0.01, ***p < 0.001 and ****p<0.0001.

Figure 4.. Co-expression of non-phosphorylated Add1_i2 with Add3 induces partially sarcomere disassembly.

A. HEK293T were transfected with 3xFLAG-Add1 variants, either with or without 3xTY1-Add3, and treated with cycloheximide (CHX) to halt protein translation; cell collection at 0, 24, 48 hours post-treatment. (Left) Representative WB; (Right) Assessment of relative protein levels of Add1_i1 and Add1_i2 (normalized FLAG to Gapdh), in the absence or presence of Add3. B. Generation strategy for cardiac-specific Add1_i2/Add3 dTG mice. Both expression constructs were driven by an α-MHC promoter. C. qPCR analysis of Add1_i2 mRNA in 2-month-old low expression dTG(L) hearts, normalized to WT. D. (Left) H&E staining and (Right) HW/BW comparison in 2-month-old dTG(L) mice. E. (Left) Echocardiography and (Right) left ventricular systolic function in 2-month-old dTG(L) mice. F. Sarcomere patterns in 2-month-old WT and dTG(L) hearts and high-magnification images of specific regions are provided. The dotted lines indicate cardiomyocyte borders, with green insets highlighting partially disassembled sarcomeres. Arrows indicate corresponding Adducin expression. G. qPCR analysis of Add1_i2 mRNA in 2-month-old low expression dTG(H) hearts, normalized to WT. H. (Left) H&E staining and (Right) HW/BW comparison in 2-month-old dTG(H) mice. I. (Left) Echocardiography and (Right) left ventricular systolic function in 1-month-old dTG(H) mice. J. (Top) Sarcomere pattern in 1-month-old dTG(H) hearts with robust disassembly. (Bottom) High-magnification images of specific regions show the regions with a lack of disassembly. K. (Left) Evidence of cardiomyocyte fragmentation and (Right) High magnification of the inset indicate colocalization of Add1 and Tnnt2 in fragmented sarcomere (arrows). Scale bar 10 μm (F, J, K); 1 mm (D, H). Data are presented as mean±sd. Statistical analyses: repeated measures two-way ANOVA with Tukey's post-hoc test (A) or Mann-Whitney U test (two-tailed) (C, E, G, H, I) were used to determine statistical significance; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p<0.0001.

Figure 5.. Co-expression of phosphor mimic Add1_i2 with Add3 promotes sarcomere disassembly.

A. (Top) Schematic strategy for generating cardiac-specific phospho-mimic Add1_i2/Add3 double transgenic mice (p-dTG), with T445 and T480 Glu substitutions in Add1_i2 to mimic phosphorylation, driven by an α-MHC promoter. (Bottom) H&E staining of 2-month-old control and p-dTG hearts. B. HW/BW in WT and p-dTG mcie in P21 and 2-month-old mice. C. (Left) Echocardiography and (Right) left ventricular systolic function in P21 and 2-month-old mice p-dTG mice. D. qPCR results for Add1_i2 in 2-month-old p-dTG. E. Sarcomere structure in p-dTG hearts at P7 (i), P14 (ii), P21 (iii) and 2-month-old (iv). High-magnification images of selected regions show sarcomere absence in areas with high Adducin expression (arrows). Dotted lines outline cardiomyocyte borders. F. Identification of 7 kinase candidates potentially phosphorylating (Left) T445 and (Right) T480 using 245 Ser/Thr peptide kinase assays, with corrected kinase activity values depicted. G. Verification of Nek1, Dcam1, and Irak4 kinase activity in phosphorylating T445 and T480 using Serine/Threonine Kinase dose-response assays. Each kinase was tested at three concentrations in triplicates. H. Endogenous expression pattern of (Left) Irak4 and (Right) Nek1 in the regenerating neonatal heart, with positive signals indicated (arrows). I. (Top) Schematic of Irak4 expression in AAV6 constructs. Irak4 (green) overexpression in NRVMs causes sarcomere disassembly (stained with α-actinin, red) disassembly (i) and translocation of Add1_pT445 (red) from nucleus to cytoplasm (ii). Arrowheads indicate Adducin is expressed in the nuclei in Irak4 negative cells. J. (Top left) Schematic of Irak4 expression in AAV9 constructs. (Top right) Experimental design for neonatal AAV injection. (Bottom) Results post AAV9-Irak4-GFP injection in CD1 pups at P1, showing intense sarcomere disassembly in cardiomyocytes compared to littermate controls at P15. Scale bar 10 μm (E, H, I, J); 1 mm (A). Data are presented as mean±sd. Statistical analyses: Two-way ANOVA with Bonferroni's multiple comparisons test (B, C), Mann-Whitney U test (two-tailed) (D); *p < 0.05, **p < 0.01, ***p < 0.001 and ****p<0.0001.

Figure 6.. Analysis of Co-IP/MS with purified Adducin complex.

A. The table enumerates key proteins associated with Add1, as identified through Co-IP/MS, with a similar strategy as 1C. B. Schematic of the purification process of short and long Adducin complexes. C. Schematic of Co-IP/MS by purified adducin complex pulldown D. Bar graph for Canonical Pathways enriched in each sample. FDR: false discovery rate. E. Network plot of cytoskeleton proteins from the pull-down assay interact with Adducin long or Adducin short isoforms at different regenerative time points (P1 and P21). Different color labels indicate common and unique proteins among sample groups (Red: all sample groups, Yellow: common to all groups, Green: common to two or three groups, Blue: unique to each group). F. PLA result shows the direct interaction between Add1_pT445 and α-actinin in P4 heart tissue. Red dots (arrows) represent fluorescent signals of close interactions. Interaction between α-actinin and Tnnt2 serves as a positive control. G. Immuno-EM image details the subcellular location of Add1_pT445 in cardiomyocytes with disassembled sarcomeres in neonatal P1MI hearts. Two red boxes are magnified in the right 2 panels showing Add1_pT445 at (i) z-disks, and (ii) at z-disks associated with the plasma membrane in cardiomyocytes with disassembled sarcomeres. Red arrows indicate the location of Adducin. Scale bar 1 μm (G); 10 μm (F).