AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP-170

Abstract

AMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated disks.

Keywords: AMPK; CLIP-170; intercalated disk; microtubule.

Figure 1. Phosphorylation levels of AMPK significantly increased at the intercalated disks in adult mouse heart together with its substrate CLIP‐170

1. A, B

   Immunoblot analysis of the phosphorylation level of CLIP‐170, AMPK, and ACC in heart (A) and liver (B) along with the developmental stages. α‐Tubulin was used as a loading control.

2. C–E

   Quantitative analysis of Western blotting shown in (A). Multiple tissues from each stage were collected and mixed to minimize variation. The data represent three independent sample collections for the heart and liver. *P < 0.05, **P < 0.01 vs E15.5. One‐way ANOVA followed by Dunnett post hoc test.

3. F–H

   Quantitative analysis of Western blotting shown in (B). Multiple tissues from each stage were collected and mixed to minimize variation. The data represent three independent sample collections for the heart and liver. **P < 0.01 vs E15.5. One‐way ANOVA followed by Dunnett post hoc test.

4. I

   Immunostained images of adult mouse heart tissue. These were stained with a phosphorylated AMPK antibody (I, upper), a phosphorylated CLIP‐170 antibody (I, lower), and a plakoglobin antibody (middle). The representative images from more than five analyzed tissues were shown.

Data information: Scale bar, 20 μm (I).

Figure EV1. AMPK and its upstream kinase, LKB1, localized at the intercalated disks in adult mouse heart

1. Immunostained images of adult mouse heart tissue. These were stained with an AMPKβ₂ antibody (upper, left), an LKB1 antibody (bottom, left), and a plakoglobin antibody (center). The representative images from more than three analyzed tissues were shown.

2. Cardiomyocytes transfected with adenovirus vector encoding AMPKβ₂‐mCherry were observed by a fluorescent (mCherry; red) and bright‐field microscope. White arrows indicate that AMPKβ₂ localized at the cell–cell junction site of the cardiomyocytes. The data represent two independent experiments.

3. Quantitative analysis of FRET signal (YFP/CFP) from organelle‐specific ABKAR. Number of cells analyzed: Cyto‐ABKAR in HeLa (n = 117), Cyto‐ABKAR in cardiomyocytes (n = 112), PM‐ABKAR in HeLa (n = 27), PM‐ABKAR in cardiomyocytes (n = 94). Data means ± SD. All measurements were normalized to the average of Cyto‐ABKAR in HeLa cells. Statistical analysis was done by ANOVA followed by post hoc analysis (Turkey). CM: cardiomyocytes. **P < 0.01.

4. Organelle‐specific expression of Cyto‐ or PM‐ABKAR was confirmed by fluorescent microscope.

5. Immunostained images of adult cardiomyocytes obtained by Langendorff method. PPase⁺; lambda phosphatase treatment.

6. Quantitative analysis of intensity of pAMPK or CLIP‐170 in (E). Box and whisker plots showed the 25^th percentile (bottom line of each box), median (middle line of each box), 75^th percentile (top line of each box), maximum and minimum (each whisker). Number of cells analyzed, pAMPK; Control, n = 29, PPase⁺, n = 28, pCLIP‐170; Control, n = 29, PPase⁺, n = 26. **P < 0.01 vs Control.

Data information: Scale bar, 20 μm (A, B), 10 μm (D), 20 μm (E).

Figure 2. The localization of AMPK was regulated by contraction of the cardiomyocytes

1. A, B

   Immunostained images of neonatal rat cardiomyocytes 2 h after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK‐461(MYK‐461, middle row) and 4 h after washing out of MYK‐461 (Wash out, bottom row). These cells were stained with a phosphorylated AMPK antibody, an AMPKβ₂ antibody, and a N‐cadherin antibody.

2. C, D

   Immunostained images of neonatal rat cardiomyocytes 2 h after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK‐461(MYK‐461, bottom row) and stained with a connexin 43 antibody, a plakoglobin antibody, an LKB‐1 antibody, and an N‐cadherin antibody. The representative images from four independent experiments were shown.

3. E

   Quantification of pAMPK, AMPKβ₂, Connexin 43, or LKB1 that was localized with a cell–cell junction marker (N‐cadherin or Plakoglobin). Box and whisker plots showed the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Numbers in the brackets indicate the cells that were analyzed for quantification. Differences among multiple groups were compared by one‐way ANOVA, followed by a post hoc comparison using the Tukey method. Two‐tailed Student's t‐test was used to analyze differences between two groups. *P < 0.05, **P < 0.01. n.s., not significant.

Data information: Scale bar, 20 μm (A‐D).

Figure 3. AMPK regulated longitudinal microtubule dynamics through the phosphorylation of CLIP‐170 in cardiomyocytes

1. GFP time‐lapse images of neonatal rat cardiomyocytes expressing EGFP‐CLIP‐170 WT 0 and 15 min after treatment with 20 μM compound C (left side panel) and expressing EGFP‐CLIP‐170 S311A (right side panel). Higher magnification of white square (upper row) showing CLIP‐170 migrated longitudinally toward the cell–cell junctions.

2. Beeswarm plots of comet length of EGFP‐CLIP‐170. The comet length from multiple cells in different fields was analyzed. Number of comets analyzed, Pre: n = 32, Cpd. C: n = 36, CLIP S311A: n = 68. Data means ± SD. Differences among multiple groups were compared by one‐way ANOVA, followed by a post hoc comparison using the Tukey method. **P < 0.01 vs Pre.

3. GFP time‐lapse images of neonatal rat cardiomyocytes expressing EGFP‐CLIP‐170 WT treated with control siRNA (siCL, left) or siRNA targeting both AMPKα₁ and α₂ (siAMPKα₁α₂, right). White dotted lines in the images showed the connected cardiomyocyte not expressing EGFP‐CLIP‐170 WT.

4. Beeswarm plots of comet length of EGFP‐CLIP‐170. The comet length from multiple cells from different fields was analyzed. Number of comets analyzed, siCL: n = 178, siAMPKα₁α₂: n = 184. Data means ± SD. Two‐tailed Student's t‐test was used to analyze differences between two groups. **P < 0.01 vs siCL.

Data information: Scale bar, 10 μm (A, C).

Figure EV2. Knockdown of AMPKα₁α₂

1. (A, B) Knockdown by siRNA was confirmed by real‐time PCR (A) and Western blotting (B). The data were confirmed by three independent experiments. Student's t‐tests, **P < 0.01 vs siControl.

Figure 4. AMPK‐CLIP‐170 signal at the intercalated disk controlled the cell shape in cardiomyocytes

1. GFP time‐lapse images of neonatal rat cardiomyocytes expressing EGFP‐CLIP‐170 WT 0, 2 and 3 h after treatment with 2 μM MYK‐461.

2. Immunostained images of neonatal rat cardiomyocytes 4 h after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK‐461(MYK‐461, bottom row). These cells were stained with an α‐tubulin (green) and a plakoglobin (red) antibody.

3. Box and whisker plots of the cell size of a cardiomyocyte 2 h after treatment with or without 2 μM MYK‐461, showing the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), and maximum and minimum (each whisker). Adenovirus expressing EGFP‐CLIP‐170 (adCLIP) mutant was used (WT, S311A, S311D). Numbers in the graph indicate n number of the cells from 2 independent experiments. Control (−/−): n = 784, MYK‐461: n = 939, CLIP WT: n = 656, CLIP WT + MYK‐461: n = 619, CLIP S311A: n = 405, CLIP S311A + MYK‐461: n = 635, CLIP S311D: n = 613, CLIP S311D + MYK‐461: n = 735. Data means ± SD. Differences among multiple groups were compared by one‐way ANOVA, followed by a post hoc comparison using the Tukey method. **P < 0.01 vs Control, ^†† P < 0.01 vs CLIP WT, n.s., not significant.

Data information: Scale bar, 10 μm (A, B).

Figure EV3. Generation of cardiomyocytes‐specific CLIP‐170 S311A‐overexpressing TG mice and S311D TG mice

1. Immunoblot analysis of the CLIP‐170 in control (Cont) and two lines of S311A TG heart (Tg #3 and #9).

2. Immunoblot analysis of the CLIP‐170 in control (Cont) and S311D TG heart. α‐Tubulin was used as a loading control. Tissues were collected 1 week after tamoxifen induction.

Figure 5. Inducible heart‐specific CLIP‐170 S311A overexpressing transgenic mouse shows cardiac dysfunction

1. Beeswarm plots of the echocardiographic parameter (ejection fraction) of individual Cre control (CLIP‐170 S311A ^+/+ ; α‐MHC‐MerCreMer ^+/−), Control mice (CLIP‐170 S311A^{flox /+} ; α‐MHC‐MerCreMer ^−/−), and CLIP‐170 S311A overexpressing mice (CLIP‐170 S311 A^{flox /+} ; α‐MHC‐MerCreMer ^+/−), before (Pre), 2 weeks (Tx2w), 8 weeks (Tx8w), 26 weeks (Tx26w), and over 1 year (Tx1y) after tamoxifen induction. Number of mice (Cre control, Control, S311A), Pre (6, 2, 3), Tx2w (9, 5, 5), Tx8w (9, 10, 14), Tx12w (7, 10, 14), Tx26w (3, 7, 8), Tx52w (3, 7, 9).

2. Beeswarm plots of another line of CLIP‐170 S311A overexpressing mice (line 9). Number of mice (Control, S311A), Pre (7, 3), Tx2w (4, 7), Tx8w (6, 4), Tx12w (5, 4), Tx52w (3, 3). Data means ± SD. Statistical significance was determined by two‐way ANOVA followed by post hoc Tukey's multiple comparison test. ^## P < 0.01 vs duration‐matched Cre Control, *P < 0.05, **P < 0.01 vs duration‐matched Control; ^† P < 0.05, ^†† P < 0.01, S311A TG mice 12 vs 52 weeks.

3. Representative long‐axis four‐chamber cardiac magnetic resonance images of CLIP‐170 S311A TG mice and control mice over 1 year after tamoxifen induction. Left column showed systole images, and right column represented diastole images. Three mice from each group were analyzed for cardiac MRI. The representative images were shown.

4. Beeswarm plots showed the ejection fraction from cardiac MRI. LV, left ventricular, RV, right ventricular. Number of mice, (Control, S311A), 1y (3, 3). Data means ± SD. Two‐tailed Student's t‐test was used to analyze differences between two groups. **P < 0.01 vs Control.

5. Beeswarm plots of the echocardiographic parameter (ejection fraction) from CLIP‐170 S311D overexpressing mice and the control. Number of mice (Control, S311D), Tx2w (3, 4), Tx8w (5, 11), Tx26w (4, 11). Data means ± SD. There was no significant difference.

Data information: Scale bar, 5 mm (C).

Figure EV4. Cardiac functional assessment of cardiomyocyte‐specific CLIP‐170 S311A TG mice and S311D TG mice

1. Echocardiographic measurements of S311A TG mice line 3, control and Cre control.

2. Echocardiographic measurements of S311A TG mice line 9 and control.

3. Heart, lung, and body weight of S311A TG mice and its control.

4. Cardiac MRI measurements of S311A line 3 over 1 year after the tamoxifen induction.

5. Echocardiographic measurements of S311D TG mice and control.

Data information: Number of mice are shown in the table. Two‐tailed Student's t‐test was used to analyze differences between two groups. Differences among multiple groups were compared by two‐way ANOVA, followed by a post hoc comparison using the Tukey method.

Figure 6. CLIP‐170 S311A overexpressing transgenic mice showed elongation of the cardiomyocytes with MT accumulation

1. Masson's trichrome staining of the heart of CLIP‐170 S311A overexpressing mice and the control mice from 3 months and 1 year after tamoxifen treatment.

2. Quantitative analysis of cardiac fibrosis. Quantification was made as percentage of fibrosis area (in blue) in the left ventricle. Number of mice, (Control, S311A), 3 months (7, 6), 1 year (5, 6). *P < 0.05 vs Control.

3. Representative immunostained images with an α‐tubulin antibody of CLIP‐170 S311A overexpressing mice heart and control mice heart over 1 year after tamoxifen treatment.

4. Quantitative analysis of intensity of α‐tubulin in (C). Number of mice (Control: n = 7, S311A: n = 8). Average of 5 sections per animal was analyzed. **P < 0.01 vs Control.

5. Representative images of Western blot for an α‐tubulin antibody. GAPDH was used as a loading control.

6. Quantitative analysis of intensity of α‐tubulin in (E). Number of mice (Control: n = 4, S311A: n = 5). *P < 0.05 vs Control.

7. Representative immunostained images of CLIP‐170 S311A overexpressing mice heart and the control mice heart over 1 year after tamoxifen treatment. These were stained with WGA (red) and a plakoglobin antibody (green).

8. Quantitative analysis of (G) cell width, length, and aspect ratio (length/width) were measured. Number of cells analyzed, control, n = 251, S311A, n = 310, **P < 0.01 vs Control.

Data information: Box and whisker plots show the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Two‐tailed Student's t‐test was used to analyze differences between two groups. Differences among multiple groups were compared by one‐way ANOVA, followed by a post hoc comparison using the Tukey method. Scale bar, 100 μm (A), 20 μm (C and G).