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 (MAPK) 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 using 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 MAPK kinase 6 (MKK6), a direct inducer of p38, were generated to investigate if 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 MKK6-p38 developed interstitial and perivascular fibrosis in the heart, lung and kidney due to enhanced myofibroblast numbers. Mechanistic experiments show that p38 transduces cytokine and mechanical signals into myofibroblast differentiation through the transcription factor serum-response factor (SRF) and the signaling effector calcineurin. Conclusions These findings suggest that signals from diverse modes of injury converge on p38α MAPK 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.

The differentiation of fibroblasts into myofibroblasts mediates tissue wound healing and fibrotic remodelling, although the molecular programme underlying this process remains poorly understood. Here we perform a genome-wide screen for genes that control myofibroblast transformation, and identify the RNA-binding protein muscleblind-like1 (MBNL1). MBNL1 overexpression promotes transformation of fibroblasts into myofibroblasts, whereas loss of Mbnl1 abrogates transformation and impairs the fibrotic phase of wound healing in mouse models of myocardial infarction and dermal injury. Mechanistically, MBNL1 directly binds to and regulates a network of differentiation-specific and cytoskeletal/matrix-assembly transcripts to promote myofibroblast differentiation. One of these transcripts is the nodal transcriptional regulator serum response factor (SRF), whereas another is calcineurin Aβ. CRISPR-Cas9-mediated gene-editing of the MBNL1-binding site within the Srf 3′UTR impairs myofibroblast differentiation, whereas in vivo deletion of Srf in fibroblasts impairs wound healing and fibrosis. These data establish a new RNA-dependent paradigm for myofibroblast formation through MBNL1.

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Fibrosis is the excess collagen deposition that occurs with nearly every form of heart disease. A myriad of different stimuli including mechanical strain, cytokines and neuroendocrine effectors like TGFβ and Angiotensin II induce fibrotic matrix deposition. Current paradigms suggest that the differentiation of resident fibroblasts into myofibroblasts underlies the heart’s fibrotic response through yet unknown molecular mechanisms. Recently the p38 mitogen activated protein kinase (MAPK) signaling axis was implicated as a nodal pathway regulating myofibroblast differentiation in vitro , but never examined in vivo . To tactically address the role of p38 in the heart’s fibrotic response tamoxifen-inducible Cre recombinase knock-in mice (Tcf21 MCM ) were used to excise p38α from cardiac fibroblasts of conditional p38α knockout mice (p38 F/F ) subjected to ischemic injury. Deletion of p38 in fibroblasts reduced scar area by more than 50%, which we ascribe to the significantly reduced number of myofibroblasts in these hearts. Moreover, p38 F/F - Tcf21 MCM mice had markedly improved diastolic function relative to control littermates with intact p38 activity. Conversely, mice with fibroblast-specific expression of a constitutively active MAPK kinase 6 transgene (MKK6 Tg), which directly initiates p38α activity, developed interstitial and perivascular fibrosis at baseline. These mice had severe diastolic dysfunction in comparison to non-transgenic littermates with myofibroblasts present throughout heart. To investigate whether p38 MAPK regulates the fibrotic response in other tissues, p38α was deleted from activated dermal fibroblasts in mice using a tamoxifen-inducible periostin Cre recombinase (Postn MCM ) that were subjected to subcutaneous circular wounds. Mice lacking p38 signaling in activated fibroblasts had significantly delayed wound closure relative to p38 replete littermates, whereas mice expressing the activated MKK6 transgene in activated fibroblasts had accelerated wound closure and scarring suggesting that p38α activity in fibroblasts is universally vital to the fibrotic phase of wound healing. Collectively, these data suggest that p38 inhibition is a viable candidate for anti-fibrotic therapies.