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. 2017 Aug 8;136(6):549-561.
doi: 10.1161/CIRCULATIONAHA.116.026238. Epub 2017 Mar 29.

Fibroblast-Specific Genetic Manipulation of p38 Mitogen-Activated Protein Kinase In Vivo Reveals Its Central Regulatory Role in Fibrosis

Jeffery D Molkentin  1 , Darrian Bugg  2 , Natasha Ghearing  2 , Lisa E Dorn  2 , Peter Kim  2 , Michelle A Sargent  2 , Jagadambika Gunaje  2 , Kinya Otsu  2 , Jennifer Davis  1
Affiliations

Fibroblast-Specific Genetic Manipulation of p38 Mitogen-Activated Protein Kinase In Vivo Reveals Its Central Regulatory Role in Fibrosis

Jeffery D Molkentin et al. Circulation. .

Abstract

Background: In the heart, acute injury induces a fibrotic healing response that generates collagen-rich scarring that is at first protective but if inappropriately sustained can worsen heart disease. The fibrotic process is initiated by cytokines, neuroendocrine effectors, and mechanical strain that promote resident fibroblast differentiation into contractile and extracellular matrix-producing myofibroblasts. The mitogen-activated protein kinase p38α (Mapk14 gene) is known to influence the cardiac injury response, but its direct role in orchestrating programmed fibroblast differentiation and fibrosis in vivo is unknown.

Methods: A conditional Mapk14 allele was used to delete the p38α encoding gene specifically in cardiac fibroblasts or myofibroblasts with 2 different tamoxifen-inducible Cre recombinase-expressing gene-targeted mouse lines. Mice were subjected to ischemic injury or chronic neurohumoral stimulation and monitored for survival, cardiac function, and fibrotic remodeling. Antithetically, mice with fibroblast-specific transgenic overexpression of activated mitogen-activated protein kinase kinase 6, a direct inducer of p38, were generated to investigate whether this pathway can directly drive myofibroblast formation and the cardiac fibrotic response.

Results: In mice, loss of Mapk14 blocked cardiac fibroblast differentiation into myofibroblasts and ensuing fibrosis in response to ischemic injury or chronic neurohumoral stimulation. A similar inhibition of myofibroblast formation and healing was also observed in a dermal wounding model with deletion of Mapk14. Transgenic mice with fibroblast-specific activation of mitogen-activated protein kinase kinase 6-p38 developed interstitial and perivascular fibrosis in the heart, lung, and kidney as a result of enhanced myofibroblast numbers. Mechanistic experiments show that p38 transduces cytokine and mechanical signals into myofibroblast differentiation through the transcription factor serum response factor and the signaling effector calcineurin.

Conclusions: These findings suggest that signals from diverse modes of injury converge on p38α mitogen-activated protein kinase within the fibroblast to program the fibrotic response and myofibroblast formation in vivo, suggesting a novel therapeutic approach with p38 inhibitors for future clinical application.

Keywords: fibroblasts; fibrosis; mice, transgenic; mitogen-activated protein kinase kinases; myocardial infarction.

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Figures

Figure 1
Figure 1
p38 transduces chemical and mechanical stimuli to initiate myofibroblast differentiation. A, Western blot analysis showing highly efficient deletion of p38 protein from Mapk14fl/fl MEFs 4 days after AdCre infection versus Adβgal control infection. GAPDH was used as a loading control. B, Immunofluorescent-based quantification of the percent of MEFs with αSMA+ stress fibers in Mapk14fl/fl MEFs versus Mapk14fl/fl MEFs with AdCre infection to delete p38α protein expression. MEFs were also infected with the additional adenoviruses shown along the x-axis or treated with TGFβ to alter signaling in these cells. Error bars are SEM, N≥530, *P<0.05 vs Mapk14fl/fl. C, Representative images and D, quantification of contracted collagen gel matrices seeded with Mapk14fl/fl MEFs virally transduced with Adβgal or AdCre and treated with TGFβ or AngII. The white arrows show the direction of contraction, while the dashed white outline shows the full uncontracted collagen gel. Average contraction ± SEM, N=3, *P<0.05 vs Mapk14fl/fl. The entire area of the picture is 28 mm across in panel C. E, Immunofluorescent images and F, quantification of the number of Mapk14fl/fl MEFs with αSMA+ stress fibers (green) after 48 hours of cyclic stretching (20% strain) at 0.5 and 1.0 Hz. Scale bar = 50 μm. Data shows the average number of MEFs with αSMA+ stress fibers normalized to the total number of nuclei ± SEM, N≥80, *P<0.05.
Figure 2
Figure 2
Mapk14 deletion in resident cardiac fibroblasts minimizes the fibrotic response to ischemic injury. A, Top panel: schematic showing the Tcf21 locus containing a tamoxifen-regulated MerCreMer (MCM) cDNA and a Mapk14-loxP targeted allele contained within mice so that with tamoxifen administration Cre-dependent recombination can inactivate the Mapk14 gene. Lower panel: schematic of the tamoxifen dosing scheme used for these experiments with age listed in days (d). B, Western blot analysis for p38α protein from purified cardiac fibroblasts isolated from hearts of Mapk14fl/fl Tcf21MCM mice after tamoxifen induction. GAPDH was used as a loading control. C, Kaplan-Meier plot of survival of the indicated genotypes of mice over 10 days after IR injury. D, Quantification and E, Masson's trichrome stained histological images from the hearts of the indicated mice 28 days after IR injury. Blue staining indicates fibrosis. Scale bar = 150 μm. Average fibrotic area ± SEM, N=3 for sham and N≥8, *P<0.05 vs Mapk14fl/fl and Tcf21MCM. F, Immunofluorescent staining and quantification of myofibroblast numbers (white arrows) in the cardiac infarction border zone 28 days after injury. Myofibroblasts are αSMA (red) positive and negative for isolectin-B4 (green). Scale bar = 50 μm. Data represent the average number of αSMA+/isolectin-B4- cells/section ± SEM, 5 sections were measured per heart, N=3 for sham and N≥8 mice per group. G, Quantification of ventricular weight (VW) normalized to body weight (BW) 28 days after ischemic injury, Average VW/BW ± SEM, *P<0.05 vs Mapk14fl/fl and Tcf21MCM. Echocardiography of H, left ventricle inner diameter (LVID) and I, fractional shortening (FS%) from Mapk14fl/fl-Tcf21MCM, Mapk14fl/fl and Tcf21MCM mice 28 days post IR injury. Data are averages ± SEM, *P<0.05 vs Mapk14fl/fl and Tcf21MCM. The legend above panels D and E represent all of the data in the figure. Number of mice analyzed is shown within the bars of panels D,G-I
Figure 3
Figure 3
Mapk14 deletion in myofibroblasts reduces the fibrotic response to ischemia injury. A, Top panel: schematic showing the Postn locus containing a tamoxifen-regulated MerCreMer (MCM) cDNA and a Mapk14-LoxP targeted allele contained within mice so that with tamoxifen administration Cre-dependent recombination can inactivate the Mapk14 gene. Lower panel: schematic of the tamoxifen dosing scheme listed in days (d). B, Western blot analysis of p38α protein expression in eGFP sorted and purified fibroblasts from the hearts of the indicated mice after MI injury. GAPDH was used as a loading control. C, Kaplan-Meier plot of survival in the indicated genotypes of mice over 10 days after MI injury. D, Quantification and E, Masson's trichrome stained histological images from the hearts of the indicated mice 28 days after IR injury. Blue staining indicates fibrosis. Scale bar = 150 μm. Average fibrotic area ± SEM, N≥5, *P<0.05 vs Mapk14fl/fl and PostnMCM. F, Immunofluorescent staining and quantification of myofibroblast numbers (white arrows) in the cardiac infarction border zone 28 days after IR injury. Myofibroblasts are αSMA (red) positive and negative for the endothelial marker isolectin-B4 (green). Scale bar = 50 μm. Data represent the average number of αSMA+/isolectin-B4- cells/section ± SEM, 5 sections were measured per heart, N≥5 mice per group. G, Quantification of ventricular weight (VW) normalized to body weight (BW) 28 days after IR injury, Average VW/BW ± SEM, *P<0.05 vs Mapk14fl/fl and PostnMCM. Echocardiography of H, left ventricle inner diameter (LVID) and I, fractional shortening (FS%) from Mapk14fl/fl PostnMCM, Mapk14fl/fl and PostnMCM mice 28 days post IR injury. Data are averages ± SEM, *P<0.05 vs Mapk14fl/fl and PostnMCM. The legend next to panel I represent all of the data in the figure. Number of mice analyzed is shown within the bars of panels D,G-I
Figure 4
Figure 4
Mapk14 deletion in myofibroblasts reduces AngII/PE-induced fibrosis and cardiac hypertrophy. A, Quantification of ventricular weight (VW) normalized to body weight (BW) after 2 weeks of AngII/PE infusion by osmotic mini-pump (432 μg/kg/d / 100 mg/kg/d) and tamoxifen induction. Average VW/BW ± SEM, N≥5, *P<0.05 vs Sham, #P<0.05 vs Mapk14fl/fl and PostnMCM. B, Quantification and C, Masson's trichrome-stained transverse heart histological images for fibrosis (blue) in the indicated genotypes of mice after 2 weeks AngII/PE. Scale bar = 1 mm. Average fibrotic area ± SEM, N≥5, *P<0.05 vs Mapk14fl/fl. Echocardiography of D, left ventricle inner diameter (LVID) and E, fractional shortening (FS%). Data are averages ± SEM, N≥5, *P<0.05 vs Sham, # P<0.05 vs Mapk14fl/fl and PostnMCM. The legend next to panel A represent all of the data in the figure.
Figure 5
Figure 5
Constitutive activation of p38 in cardiac fibroblasts enhances fibrotic and hypertrophic remodeling. A, Left panel: schematic of the MKK6 LoxP-dependent and inducible transgene driven by a ubiquitous promoter (CMV β-actin). Right panel: schematic of the tamoxifen dosing scheme used for these experiments with age listed in days (d). B, RT-PCR for the activated MKK6 transgene from cardiac fibroblasts isolated from MKK6-Tg, Tcf21MCM mice and subjected to infection with Adβgal or AdCre. GAPDH expression was used for normalization. C, Immunofluorescent images of fibroblasts isolated from lung and skin of MKK6-Tg mice that were then adenovirally transduced with Adβgal (control) or AdCre and immunostained for αSMA+ stress fibers (green) and nuclei (red). Scale bar = 50 μm. D, Quantification of the area of fibrosis (blue) in transverse histological heart sections with Masson's trichrome staining in the indicated genotypes of mice. Average fibrotic area ± SEM, N≥8, *P<0.05 vs NTG and Tcf21MCM. E, Masson's trichrome-stained histological sections from the indicated tissues of either non-Tg (NTG) or MKK6-Tg mice with the Tcf21MCM allele after 4 months of continuous tamoxifen treatment. Blue staining indicates fibrosis. Scale bar = 25 μm. F, Immunofluorescent staining of hearts from the indicated genotypes of mice after 4 months of continuous tamoxifen treatment. Myofibroblasts are positive for both αSMA (red) and the fibroblast marker vimentin (green) are designated with the white arrows. Scale bar = 100 μm. G, Western blot analysis for the indicated proteins from cardiac fibroblasts purified from Tcf21MCM and MKK6-Tg, Tcf21MCM hearts following 1 month of tamoxifen induction. GAPDH antibody was used as a loading control.
Figure 6
Figure 6
p38 activation in myofibroblasts exacerbates cardiac fibrosis. A, Quantification of ventricular weight (VW) normalized to body weight (BW) 28 days after IR injury in the genotypes of mice shown. Tamoxifen was given 5 days prior to surgery and maintained for the experiment's duration. Data are averages ± SEM, *P<0.05 vs NTG and PostnMCM. B, Quantification of the area of fibrosis (blue) in transverse histological heart sections stained with Masson's trichrome 28 days after IR injury. Data are averages ± SEM, *P<0.05 vs NTG and PostnMCM. C, Echocardiography measured ventricular fractional shortening (FS%) 28 days after IR injury. Data are averages ± SEM. D, Quantification of VW/BW 14 days after AngII/PE infusion. Data are averages ± SEM, *P<0.05 vs NTG; #P<0.05 vs PostnMCM. E, Quantification of the area of fibrosis (blue) in transverse histological heart sections stained with Masson's trichrome 14 days after AngII/PE infusion. Data are averages ± SEM, *P<0.05 vs PostnMCM. F, Echocardiography of ventricular fractional shortening (FS%) 14 days after AngII/PE infusion. Number of mice used is shown in the bars of each panel for the entire figure.
Figure 7
Figure 7
p38 regulates skin wound healing. A, Quantification of in vitro wound closure rates in Mapk14fl/fl and Mapk14fl/fl-AdCre MEF monolayers in serum free culture conditions using live cell imaging. B,C, Quantification of dermal would closure over 7 days measured relative to initial day 0 wound size and corresponding photographs of these punch biopsies from tamoxifen-treated adult NTG, MKK6-Tg, PostnMCM, and Mapk14fl/fl PostnMCM mice at day 0 and 7 of injury. Average closure ± SEM, N≥5, *P<0.05 vs Mapk14fl/fl or PostnMCM. The biopsies in panels C are 6 mm at day 0, which shows sizing of the entire micrograph.
See this image and copyright information in PMC

Comment in

  • p38α: A Profibrotic Signaling Nexus.
    Stratton MS, Koch KA, McKinsey TA. Stratton MS, et al. Circulation. 2017 Aug 8;136(6):562-565. doi: 10.1161/CIRCULATIONAHA.117.028957. Circulation. 2017. PMID: 28784825 Free PMC article. No abstract available.

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