A Kinase Interacting Protein 1 (AKIP1) promotes cardiomyocyte elongation and physiological cardiac remodelling

Figures

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  Figure 1. Exercise parameters during four-week period of voluntary wheel running, (N = 9–11/group). Shown are, (A) experimental design including voluntary wheel running protocol, (B) daily running distance in kilometres per day (km/day), (C) average daily running distance in kilometres per day (km/day), (D) running time in hours per day (h/day), (E) average running time in hours per day (h/day), and (F) average speed in kilometres per hour (km/h). Mice included were aged 8–12 weeks old. WT = wild type mice, AKIP1-TG = AKIP1 transgenic mice. Graphs represent mean ± standard error of the mean (SEM). Statistical analysis was performed with Student’s t-test, < 0.05 was considered statistically significant. Part of the illustration in panel (A) contains images from Servier Medical Art by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

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  Figure 2. Cardiac hypertrophy parameters and cardiomyocyte size after voluntary wheel running in both WT and AKIP1-TG mice. Shown are, the first three panels as indicators for cardiac hypertrophy, (N = 9–11/group), in (A) left ventricular mass (LV Mass) detected from MRI shown in milligrams (mg) with its typical examples in panel (B) and in (C) the heart weight to tibia length ratio (HW/TL) is shown in milligrams per millimetres (mg/mm). In panel (D), cardiomyocyte cross sectional area in micrometres squared (μm2) is shown, (N = 9–11/group), (E) shows typical examples for cardiomyocyte cross sectional area, scale bars indicate 50 μm. In panel (F), cardiomyocyte width in micrometres (μm), (N = 9–10/group), the latter three panels present initial cardiomyocyte size analysis. Mice included were aged 8–12 weeks old. WT = wild type mice, AKIP1-TG = AKIP1 transgenic mice, Run = Running. Graphs represent mean ± standard error of the mean (SEM). Statistical analysis was performed with Student’s t-test or Mann–Whitney U test, < 0.05 was considered statistically significant. WT Run vs. TG Run: p* < 0.05, p**** < 0.0001.

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  Figure 3. Cardiomyocyte length analysis and its regulation after voluntary wheel running in both WT and AKIP1-TG mice. Shown are (A) cardiomyocyte length in micrometres (μm), (N = 9–10/group), (B) cardiomyocyte length to width ratio (N = 9–10/group), and panel (C) represents typical examples for cardiomyocyte length and width measurements, scale bars indicate 25 μm. In panel (D) mRNA expression level of RSK3 is shown, (N = 8–11/group). Protein level of PP2Ac normalized for α-Tubulin is presented in panel (E), phosphorylation to total SRF (pSRFSer103/tSRF) in panel (F) and representative images of Western blots are presented in panel (G), (N = 5–6/group). Representative Western blot images are taken from the same blot, but not always contiguous; full and original Western blots are included in Supplementary Figures. Mice included were aged 8–12 weeks old. WT = wild type mice, AKIP1-TG = AKIP1 transgenic mice, Run = Running, RSK3 = p90 ribosomal S6 kinase 3, PP2Ac = protein phosphatase 2A catalytic subunit, α-Tubulin = alpha-tubulin, SRF = serum response factor. Graphs represent mean ± standard error of the mean (SEM). Statistical analysis was performed with Student’s t-test or Mann–Whitney U…

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  Figure 4. Fibrosis and angiogenesis levels to determine the presence of additional aspects of cardiac hypertrophy. Shown are, (A) fibrosis levels from Masson staining (N = 9–10/group) with its typical examples in (B), in which scale bars indicate 2 mm in the left panels and 50 μm in the right panels. mRNA levels of collagens are shown in panel (C) (N = 8–9/group). In panel (D) and (E), capillary density (N = 9–10/group) and typical examples are shown, with scale bars indicating 50 μm. Panel (F) shows mRNA levels of markers for angiogenesis (N = 9/group). Mice included were aged 8–12 weeks old. WT = wild type mice, AKIP1-TG = AKIP1 transgenic mice, Run = Running, Col1a1 = collagen type 1 alpha 1 chain, Col3a1 = collagen type 3 alpha 1 chain, HIF1-α = hypoxia inducible factor 1 alpha, VEGFA = vascular endothelial growth factor A. Graphs represent mean ± standard error of the mean (SEM). Statistical analysis was performed with Student’s t-test or Mann–Whitney U test, < 0.05 was considered statistically significant.

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  Figure 5. AKIP1 mega clusters localise in the cardiomyocyte nucleus of AKIP1-TG mice. Electron microscopy (EM) mapping to identify various cellular compartments in baseline WT and AKIP1-TG mice. Shown are, in (A) a selection of a nucleus and surrounding cytoplasm of WT (left) and AKIP1-TG (right) mice. Note the atypical clusters inside the nucleus. In (B) electron image and EDX maps of elements as indicated to identify content of nucleus structures observed in AKIP1-TG mice. Note that the aggregates (red ROI and graph) are enriched in nitrogen (arrow) compared to heterochromatin (blued ROI and graph). (C) EM ultra-sections were labelled with anti-AKIP1 antibody. Raw data with zoomable files at high resolution are accessible at www.nanotomy.org. All scale bars indicate 2.5 μm. Mice included were aged 8–12 weeks old. WT = wild type mice, AKIP1-TG = AKIP1 transgenic mice, C = carbon, N = nitrogen, O = oxygen, P = phosphorus, S = sulphur, Os = osmium.

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  Figure 6. Molecular analysis of the physiological growth pathway: Akt-C/EBPß-CITED4 activation. mRNA and protein level analysis of the exercise-induced physiological growth pathway in AKIP1-TG mice. Shown are, (A) ratio of phosphorylated Akt to total Akt (pAktSer473/tAkt) at protein level, (N = 6–7/group), (B) C/EBPß protein levels normalized for GAPDH, (N = 5–6/group), (C) CITED4 protein levels normalized for GAPDH, (N = 8–9/group), (D) typical examples of the described Western blots and (E) CITED4 and DDiT4L mRNA levels, normalized for housekeeping gene 36B4, (N = 8–10/group). Representative Western blot images are taken from the same blot, but not always contiguous; full and original Western blots are included in Supplementary Figures. Mice included were aged 8–12 weeks old. WT = wild type mice, AKIP1-TG = AKIP1 transgenic mice, Run = Running, Akt = protein kinase B, C/EBPß = CCAAT Enhancer Binding Protein Beta, CITED4 = Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 4, DDiT4L = DNA-damage-inducible transcript 4-like. Graphs represent mean ± standard error of the mean (SEM). Statistical analysis was performed with Student’s t-test or Mann–Whitney U test, <…

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  Figure 7. Graphical abstract depicting the molecular signal transduction pathways associated with elongation-mediated physiological cardiac hypertrophy. Increased levels of AKIP1 in combination with an exercise regimen induces activation of the RSK3-PP2Ac-SRF and Akt-C/EBPβ-CITED4 pathway. It remains unknown whether AKIP1 exerts direct or indirect effects, nonetheless increased AKIP1 in cardiomyocytes activates phosphorylation of Akt, which in turn de-represses C/EBPß. The C/EBPß downstream factor CITED4, is subsequently upregulated which results in inhibition of DDiT4L. Consequentially, activation of this pathway in turn leads to established physiological cardiac hypertrophy. Simultaneously, AKIP1 overexpression in concordance with exercise reduces levels of RSK3 and enhances levels of PP2Ac. This synchronized programming dephosphorylates SRF, which entails a molecular switch towards physiological elongation of cardiomyocytes. AKIP1 = A Kinase Interacting Protein 1, Akt = protein kinase B, C/EBPß = CCAAT Enhancer Binding Protein Beta, CITED4 = Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 4, DDiT4L = DNA-damage-inducible transcript 4-like, RSK3 = p90…