KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

Figures

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  Fig. 1. KLK11 expression is overexpressed in cardiac hypertrophy in humans. a Expression of mRNA of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (MYH7) in control and hypertrophic heart tissues in humans (n = 5). ***P < 0.001 by unpaired Student’s t test. HCM, hypertrophic cardiomyopathy. b Expression of mRNA of kallikrein 11 (KLK11) in control and hypertrophic heart tissues in humans (n = 5). ***P < 0.001 by unpaired Student’s t test. c Expression of protein of KLK11 in control and hypertrophic hearts (n = 5). ***P < 0.001 by unpaired Student’s t test

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  Fig. 2. KLK11 expression is upregulated in murine hypertrophic hearts. a Fraction shortening and the ejection fraction of control and hypertrophic hearts in mice (n = 5). Cardiac hypertrophy in C57BL/6 mice was induced by TAC surgery for four weeks. **P < 0.01 by unpaired Student’s t test. b Heart weight of control and hypertrophic hearts in mice (n = 5). Cardiac hypertrophy in C57BL/6 mice was induced by TAC surgery for four weeks. HW, heart weight; BW, body weight; TL, tibia length. **P < 0.01 by unpaired Student’s t test. c Expression of mRNA of ANP, BNP, and MYH7 in control and hypertrophic heart tissues in mice (n = 5). Cardiac hypertrophy in C57BL/6 mice was induced by TAC surgery for four weeks. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; MYH7, β-myosin heavy chain; **P < 0.01 vs Sham by unpaired Student’s t test. d Expression of mRNA of KLK11 in control and hypertrophic heart tissues in mice (n = 5). Cardiac hypertrophy in C57BL/6 mice was induced by TAC surgery for four weeks. **P < 0.001 by unpaired Student’s t test. e Expression of protein of KLK11 in control and hypertrophic heart tissues in mice (n = 3). Cardiac hypertrophy in C57BL/6 mice was induced by…

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  Fig. 3. KLK11 regulates cardiac hypertrophy in vitro. a siRNA knockdown of KLK11 in cardiomyocytes (n = 3). Mouse cardiomyocytes were transfected with siCtrl or siKLK11 for 48 h. mRNA and protein levels of KLK11 were analyzed. ***P < 0.001 by unpaired Student’s t test. b KLK11 knockdown reduces Ang II-induced hypertrophic growth of cardiomyocytes (n = 3). Mouse cardiomyocytes were transfected with siCtrl or siKLK11 for 24 h, followed by Ang II (1 μM) treatment for an additional 48 h. Representative images for a-actinin staining and quantitative results are shown. **P < 0.01 by one-way ANOVA with Tukey post-hoc test. c KLK11 knockdown reduces Ang II-induced overexpression of hypertrophic genes in cardiomyocytes (n = 3). Mouse cardiomyocytes were transfected with siCtrl or siKLK11 for 24 h, followed by Ang II (1 μM) treatment for an additional 48 h. **P < 0.01 by one-way ANOVA with Tukey post-hoc test. d Adenovirus-mediated overexpression of KLK11 in cardiomyocytes (n = 3). Mouse cardiomyocytes were infected with AdCtrl or AdKLK11 for 48 h. mRNA and protein levels of KLK11 were analyzed. ***P < 0.001 by unpaired Student’s t test. e KLK11 overexpression promoted Ang II-induced hypertrophic…

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  Fig. 4. KLK11 regulates cardiac hypertrophy in vivo. a KLK11 was knocked down in murine hearts with AAV9-mediated shRNA. n = 5 in each group. One week–old male C57BL/6 mice received a single intravenous injection of saline or AAV9 vector via the jugular vein. 12 weeks later, the mice received a TAC or sham surgery. The knockdown efficiency was confirmed by quantitative real-time PCR.***P < 0.001 by unpaired Student’s t test. AAV9, adeno-associated virus 9. b KLK11 knockdown rescues TAC-induced decline in fraction shortening and ejection fraction. One week–old male C57BL/6 mice received a single intravenous injection of saline or AAV9 vector via the jugular vein. Eight weeks later, the mice received a TAC or sham surgery. n = 8 in sham groups, n = 10 in TAC groups. **P < 0.01 by one-way ANOVA with Tukey post-hoc test. c KLK11 knockdown represses TAC-induced increase in heart weight to body weight ratio (HW/BW) and heart weight to tibia length ratio (HW/TL). One week–old male C57BL/6 mice received a single intravenous injection of saline or AAV9 vector via the jugular vein. Eight weeks later, the mice received a TAC or sham surgery. n = 8 in sham groups, n = 10 in TAC groups. **P < 0.01 by…

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  Fig. 5. KLK11 promotes protein synthesis. a KLK11 knockdown represses Ang II-induced protein synthesis in cardiomyocytes (n = 3). Mouse cardiomyocytes were transfected with siRNA for 24 h, followed by protein synthesis analysis by monitoring [3H]-leucine incorporation in the presence of Ang II (1 μM). ***P < 0.001 by one-way ANOVA with Tukey post-hoc test. b KLK11 overexpression promotes Ang II-induced protein synthesis in cardiomyocytes (n = 3). Mouse cardiomyocytes were infected with adenovirus for 24 h, followed by protein synthesis analysis by monitoring [3H]-leucine incorporation in the presence of Ang II (1 μM).. ***P < 0.001 by one-way ANOVA with Tukey post-hoc test. c KLK11 knockdown represses the phosphorylation of S6K1 and 4EBP1 in cardiomyocytes (n = 3). Mouse cardiomyocytes were transfected with siRNA for 24 h, followed by Ang II (1 μM) treatment for an additional 24 h. Representative western blots and quantitative results are shown. **P < 0.001 by one-way ANOVA with Tukey post-hoc test. d KLK11 overexpression promotes phosphorylation of S6K1 and 4EBP1 in mouse cardiomyocytes (n = 3). Mouse cardiomyocytes were infected with adenovirus for 24 h, followed by Ang II (1 μM)…

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  Fig. 6. KLK11 regulates mTOR signaling to target cardiac hypertrophy. a KLK11 knockdown represses the phosphorylation of Akt-mTOR (n = 3). Mouse cardiomyocytes were transfected with siRNA for 24 h, followed by Ang II (1 μM) treatment for an additional 24 h. Representative western blots and quantitative results are shown. **P < 0.01 by one-way ANOVA with Tukey post-hoc test. b KLK11 overexpression promotes phosphorylation of Akt-mTOR (n = 3). Mouse cardiomyocytes were infected with adenovirus for 24 h, followed by Ang II (1 μM) treatment for an additional 24 h. Representative western blots and quantitative results are shown. **P < 0.01 by one-way ANOVA with Tukey post-hoc test. c KLK11 knockdown represses the phosphorylation of Akt-mTOR in vivo (n = 3). One week–old male C57BL/6 mice received a single intravenous injection of saline or AAV9 vector via the jugular vein. Eight weeks later, the mice received a TAC or sham surgery. Heart tissues from hypertrophic hearts with/without KLK11 knockdown were analyzed. Representative western blots and quantitative results are shown. **P < 0.01 by one-way ANOVA with Tukey post-hoc test. d Rapamycin represses KLK11-mediated phosphorylation of S6K1…