KLF12 Aggravates Angiotensin II‐Induced Cardiac Remodeling in Male Mice by Transcriptionally Inhibiting SMAD7

Figures

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  Figure 1. KLF12 is upregulated in failing heart. A, Cardiac KLF12 expression detected by Western blot in human (Con, n=6; DCM, n=6). B, Relative KLF12 mRNA level (n=6). C, Relative Klf12 mRNA level in mice (n=6). D, Cardiac KLF12 expression detected by Western blot in mice (n=6). E, Representative images of KLF12 (green), POSTN (red) in 12‐week‐Ang II‐infused heart samples. Nuclei were stained with DAPI (blue). Hearts were subjected to immunofluorescence staining to detect the cellular location of KLF12 (scale bar=100 μm, n=6). F, Relative Klf12 mRNA level (n=6). G, Cardiac KLF12 expression detected by Western blot in CFs (n=6). H, Schematic representation of KLF12 expression cassette in the lentivirus vectors engineered to overexpress exogenous KLF12 or negative control in CFs expression mir‐1a‐3p and mir‐133a‐3p. I, Protocol diagram for the study design. J, Representative Western blot images and statistical results (n=6). All data are expressed as the mean±SD, and analyzed using 1‐way ANOVA followed by Tukey post hoc test. *P<0.05 vs the matched group. AMCF indicates angiotensin II‐infused adult mouse cardio fibroblast; AMCM, adult mouse cardiac myocytes; Ang II, angiotensin II; CF,…

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  Figure 2. Fibroblast‐specific overexpression of KLF12 aggravates cardiac remodeling in mice. A, The volcano map of RNA‐sequencing data set. B, Representative images of Masson and quantitative results of fibrotic area (scale bar=100 μm, n=6). C, Representative image of the whole mount of the mouse heart (scale bar=3 mm, n=6). D, Heart weight‐to‐tibia length in mice (n=6). E, Heart weight‐to‐body weight () in mice (n=6). F and G, Representative images of WGA and quantitative results of cell area (scale bar=100 μm, n=6). H, Relative α‐Mhc mRNA levels in murine hearts (n=6). All data are expressed as the mean±SD, and analyzed using 1‐way ANOVA followed by Tukey post hoc test. *P<0.05 vs the matched group. Ang II indicates angiotensin II; HW/BW, heart weight‐to‐body weight; HW/TL, heart weight‐to‐tibia length; KLF12, Krüppel‐like factor 12; and WGA, wheat germ agglutinin.

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  Figure 3. Fibroblast‐specific overexpression of KLF12 aggravates cardiac dysfunction. A, Cardiac function was presented as EF (n=6). B, Representative M‐mode echocardiography was recorded (scale bar=10 mm, n=6). C and D, Cardiac function was analyzed by transthoracic echocardiography and presented as LVIDd and IVSd (n=6). E, Protocol diagram for the study design. F, The ratio of HW/TL (n=6). G and H, Representative images of WGA and quantitative results of cell area (scale bar=100 μm, n=6). I and J, Representative images of Masson and quantitative results of fibrotic area (scale bar=100 μm, n=6). K and L, Cardiac function was analyzed by transthoracic echocardiography and presented as EF and LVIDd (n=6). All data are expressed as the mean±SD, and analyzed using 1‐way ANOVA followed by Tukey post hoc test. *P<0.05 vs the matched group. Ang II indicates angiotensin II; EF, ejection fraction; HW/TL, heart weight‐to‐tibia length; IVSd, interventricular septum thickness; KLF12, Krüppel‐like factor 12; LVIDd, left ventricular end‐diastolic size; TAC, transverse aortic constriction; and WGA, wheat germ agglutinin.

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  Figure 4. KLF12 is required for cardiac fibroblast‐to‐myofibroblast conversion. A, Relative α‐Sma, Col1, and Postn mRNA levels in CFs (n=6). B and C, Immunofluorescence staining of α‐SMA (green) and quantitative results (scale bar=100 μm, n=6). D, Representative images of 5‐ethynyl‐2′‐deoxyuridine (red). Nuclei were stained with DAPI (blue). (scale bar=100 μm, n=6). E, The proliferative rate of CFs (n=6). F, The treatment schedule of CFs‐derived ConM on NRCMs. G, Representative images of α‐actinin (green). Nuclei were stained with DAPI (blue). (scale bar=100 μm, n=6). H, The quantitative results of cell area (n=6). I, Representative images of Ki67 (red) and Vimentin (green) in heart samples. Nuclei were stained with DAPI (blue). Hearts were subjected to immunofluorescence staining to detect the fibroblast proliferation in heart tissue (scale bar=100 μm, n=6). All data are expressed as the mean±SD, and analyzed using 1‐way ANOVA followed by Tukey post hoc test. *P<0.05 vs the matched group. α‐SMA indicates alpha smooth muscle actin; AMCF, angiotensin II‐infused adult mouse cardio fibroblast; Ang II, angiotensin II; CF, cardio fibroblast; ConM, conditioned medium; KLF12, Krüppel‐like factor…

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  Figure 5. KLF12 exacerbates cardiac remodeling via transcriptionally inhibiting Smad7. A through D, Representative Western blot images and statistical results (n=6). E, Relative Smad6 mRNA levels (n=6). F, Relative Smad7 mRNA levels (n=6). G and I, Representative Western blot images and statistical results (n=6). J, A conserved UPRE was identified in the Smad7 promoter across different species. K, Smad7 promoter activity was measured by a luciferase assay (n=6). L, Smad7 promoter activity was measured by a luciferase assay (n=6). M, Independent ChIP‐PCR was performed with Klf12‐overexpressing CFs (n=6). O, Relative Smad7 mRNA levels (n=6). P, Representative Western blot images and statistical results (n=6). All data are expressed as the mean±SD, and analyzed using 1‐way ANOVA followed by Tukey post hoc test. *P<0.05 vs the matched group. Ang II indicates angiotensin II; ChIP‐PCR, chromatin immunoprecipitation‐polymerase chain reaction; KLF12, Krüppel‐like factor 12; NS, not significant; TGF‐β, transforming growth factor beta; UPRE, unfolded protein response element; and WT, wild type.

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  Figure 6. Smad7 effectively reverses the conversion of cardiac fibroblasts to myofibroblasts induced by KLF12. A, Relative α‐Sma mRNA levels (n=6). B, The quantitative results of α‐SMA intensity (n=6). C, Relative Col1 mRNA levels (n=6). D, Relative Postn mRNA levels (n=6). E, The proliferative rate of CFs (n=6). F, Representative images of 5‐ethynyl‐2′‐deoxyuridine (red). Nuclei were stained with DAPI (blue). (scale bar=100 μm, n=6). G through J, Relative Smad7, α‐Sma, Col1, and Postn mRNA levels (n=6). K, Relative α‐Sma mRNA levels (n=6). L, The quantitative results of α‐SMA intensity (n=6). M and N, Relative Col1 and Postn mRNA levels (n=6). All data are expressed as the mean±SD, and analyzed using 1‐way ANOVA followed by Tukey post hoc test. *P<0.05 vs the matched group, # P<0.05 siSmad7 vs siRNA, $ P<0.05 siSmad7# vs siRNA. α‐SMA indicates alpha smooth muscle actin; Ang II, angiotensin II; CF, cardio fibroblast; and KLF12, Krüppel‐like factor 12.

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  Figure 7. Overexpression Smad7 attenuates Ang II‐induced cardiac remodeling and improved cardiac function in with KLF12 overexpression. A, The ratio of HW/TL (n=6). B and C, Representative images of WGA and quantitative results of cell area (scale bar=100 μm, n=6). D, Relative Anp, Bnp, and β‐Mhc mRNA levels (n=6). E and F, Representative images of Masson and quantitative results of fibrotic area (scale bar=100 μm, n=6). G, Relative Col1, α‐Sma, and Ctgf mRNA levels (n=6). H and I, Cardiac function was analyzed by transthoracic echocardiography and presented as EF and LVIDd (n=6). All data are expressed as the mean±SD, and analyzed using 1‐way ANOVA followed by Tukey post hoc test. *P<0.05 versus the matched group. Ang II indicates angiotensin II; Con, control; EF, ejection fraction; HW/TL, heart weight‐to‐tibia length; KLF12, Krüppel‐like factor 12; LVIDd, left ventricular end‐diastolic size; and WGA, wheat germ agglutinin.