Fig 2: JMJD1C represses AMPK activation in cardiomyocytes. (A,B) The AMPK signaling pathway is repressed in hypertrophic hearts in humans and mice. **p < 0.01 vs. control or saline. n = 4 in each group. (C) Jmjd1c knockdown activates AMPK signaling. NRCMs were infected with adenovirus expressing shJmjd1c or shCtrl for 24 h, followed by Ang II treatment for an additional 24 h. **p < 0.01 vs. Ad-shCtrl. n = 3 in each group. (D) JMJD1C overexpression represses AMPK activation. NRCMs were infected with adenovirus expressing Jmjd1c for 24 h, followed by Ang II treatment for an additional 24 h. **p < 0.01 vs. Ad-Ctrl. n = 3 in each group.

Fig 3: AMPK participates in the role of JMJD1C during cardiomyocyte hypertrophy. (A) Prkaa1 knockdown in cardiomyocytes. The cells were treated with adenovirus expressing shPrkaa1 or with a control virus for 48 h (B,C). Prkaa1 knockdown blocks the influence of Jmjd1c knockdown on Ang II-induced increase in cardiomyocytes. The cells with/without Prkaa1 or Jmjd1c knockdown were subjected to hypertrophy induction with Ang II for 48 h. Then cardiomyocyte size (B) and gene expression (C) were analyzed. **p < 0.01 vs. shCtrl. ##p < 0.01 vs. shCtrl (D). Metformin activates AMPK. The indicated cardiomyocytes were treated with metformin (1 mM) for 24 h. **p < 0.01 vs. Ad-Ctrl; ##p < 0.01 vs. Ad-Jmjd1c. n = 3 in each group (E,F). Metformin-mediated activation of AMPK blocks the influence of JMJD1C overexpression on cardiomyocyte hypertrophy induced by Ang II stimuli. NRCMs were infected with/without adenovirus carrying Jmjd1c for 24 h; then the cells were treated with Ang II (1 µM) metformin (1 mM) for an additional 48 h. Cardiomyocyte size (E) and gene expression (F) were analyzed **p < 0.01. (G) STO-609 blocks the influence of Jmjd1c knockdown on the Ang II-induced increase in cardiomyocytes. **p < 0.01. (H) Camkk2 knockdown blocks the influence of Jmjd1c knockdown on Ang II-induced increase in cardiomyocytes. **p < 0.01. All results are from three independent experiments.

Fig 4: JMJD1C regulates AMPK in a CAMKK2-dependent manner. (A,B) JMJD1C does not affect activation of LKB1 or levels of its activation partners STRAD and MO25 in NRCMs. The cells were infected with adenovirus expressing Jmjd1c, shJmjd1c, or the control constructs for 24 h, followed by Ang II treatment for an additional 24 h. n = 3 in each group. (C) Effects of JMJD1C on CAMKK2 expression. NRCMs were treated as in (A). **p < 0.01 vs. Ad-Ctrl or shCtrl. n = 3 in each group. (D,E) Effects of JMJD1C on CAMKK2 mRNA expression. NRCMs were treated as in (A). **p < 0.01 vs. Ad-Ctrl or shCtrl. (F) Chromatin immunoprecipitation (ChIP) assay showing the binding of JMJD1C, JMJD2A, and JMJD3 on the FHL1, ß-MHC, and CAMKK2 promoters. IgG was used for negative control, and the enrichment was normalized to the input. ***p < 0.001 vs. IgG. (G) ChIP assay showing that JMJD1C overexpression reduced H3K9me1 enrichment at the CAMKK2 promoter in cardiomyocytes. ***p < 0.001 (H). The inhibition of CAMKK2 blocks the effects of JMJD1C on AMPK activation in NRCMs. The cells were treated with adenovirus expressing shJmjd1c or shCtrl; then the cells were treated with Ang II and the CAMKK2 inhibitor STO-609 (20 µM) for an additional 24 h. All results are from three independent experiments. **p < 0.01 vs. Ad-shCtrl+PBS; ##p < 0.01 vs. Ad-shCtrl+STO-609. n = 3 in each group.

Fig 5: JMJD1C promotes cardiomyocyte hypertrophy. (A) Jmjd1c knockdown in neonatal rat cardiomyocytes (NRCMs). The cells were infected with adenovirus expressing shJmjd1c or shCtrl for 48 h. ***p < 0.001 vs. shCtrl. (B,C) Jmjd1c knockdown reduces the Ang II-mediated increase in the size of cardiomyocytes (B) and the expression of fetal genes (C). Cardiomyocytes infected with adenovirus expressing shJmjd1c or shCtrl were treated with Ang II (1 µM) for 48 h to induce hypertrophy. Then a-actinin staining was performed and cardiomyocyte size was analyzed. **p < 0.01 vs. shCtrl+PBS, ##p < 0.01 vs. shCtrl+Ang II. (D) Exogenous Jmjd1c overexpression in NRCMs. The cells were infected with adenovirus overexpressing Jmjd1c for 48 h. ***p < 0.001 vs. Ad-Ctrl. (E,F) Exogenous Jmjd1c overexpression enhances the increase in cardiomyocyte size (E) and hypertrophic fetal gene expression (F) induced by Ang II. NRCMs with/without Jmjd1c overexpression were treated with Ang II for 48 h to induce hypertrophy. Then cardiomyocyte size was analyzed. **p < 0.01 vs. Ad-Ctrl+PBS, ##p < 0.01 vs. Ad-Ctrl+Ang II. All results are from three independent experiments.