Einfluss von GATA Transkriptionsfaktoren auf die durch mechanische Belastung induzierte myokardiale Fibroblasten Aktivierung, Migration und die Ausschüttung protektiver parakriner Faktoren bei Herzerkrankungen
Zusammenfassung der Projektergebnisse
In this study, we analyzed the role of the transcription factors GATA-4 and GATA-6 in cardiac fibroblasts during pressure overload induced cardiac remodeling. While single deletion of Gata4 or Gata6 in activated cardiac fibroblasts did not exert any phenotypic effect on cardiac function, we found a more decreased systolic function and signs of aggravated heart failure in mice with combined deletion of Gata4 and Gata6 in heart fibroblasts. Histological analysis revealed an impaired angiogenic response in the hearts of mice lacking Gata4 and Gata6 in cardiac fibroblasts, while single deletion of Gata4 or Gata6 had no effect on capillary density. These results support the in vivo relevance of intercellular signaling mechanisms upon cardiac injury. Previous investigations on the role of GATA factors have found that single deletions of Gata4 or Gata6 can be partially or completely compensated by each other in several tissues and disease states 14, 30-32. In line with these results, single deletion of Gata4 or Gata6 in activated cardiac fibroblasts in our study had no detectable effect on the structural composition and function of the heart, suggesting a functional redundancy of GATA-4 and GATA-6 in cardiac fibroblasts. While especially GATA-4 has been extensively investigated in cardiomyocytes, where it acts as a central regulator during cardiac development, maladaptive hypertrophy and even provides regenerative properties, the fibroblast specific functions of GATA-4 and GATA-6 are to our knowledge largely unknown 13, 33-35. We found that among the GATA family of transcription factors, Gata4 and Gata6 show by far the highest RNA expression level in fibroblasts from adult mouse hearts. Previous work had demonstrated that GATA-4 and GATA-6 are even enriched in cardiac fibroblasts versus whole heart tissue, while they are not at all expressed in skin fibroblasts 9. In fact, Gata4 and Gata6 together with Tbx20 and Hand2 were proposed to be part of a cardiogenic gene program in these cells. Although this suggested a heart specific role of this gene program in fibroblasts, its function in the myocardial stress response remained unknown.Considering the major contribution of activated fibroblast to the deposition of extracellular matrix components after pressure overload, we first investigated TAC induced fibrosis. However, neither the total extent nor the composition of cardiac connective tissue was affected in heart tissue with double deletion of Gata4/6 in activated fibroblasts compared to controls. These results raised the question about additional, so far unknown functions of cardiac fibroblasts that could directly affect cardiac function during pathological overload. To enable continuous, heavy mechanical work, the heart strongly depends on oxidative metabolism and sustained energy supply that requires a dense network of blood vessels in close proximity to the working cardiomyocytes. While it is well established that hypertrophying cardiomyocytes stimulate capillary growth, at least in part via increased HIF-1α and GATA-4 dependent signaling, an intercellular crosstalk between fibroblasts and myocardial endothelial cells is less investigated 15. In this study, we show that Gata4/6 expression in cardiac fibroblasts promotes myocardial angiogenesis and thereby helps to maintain cardiac systolic function. Although we did not formally demonstrate a causal relation here, previous work had shown that reduction of cardiac angiogenesis impairs systolic heart function, while increased angiogenesis mediates the opposite 15, 36-38. We identified two signaling pathways that mediate anti-angiogenic signaling from cardiac fibroblasts to endothelial cells upon reduction of fibroblast GATA-4/-6 levels: First, we found a dramatic increase in mRNA expression levels of Angpt4 (angiopoietin-4) in cardiac fibroblasts upon Gata4/6 deletion. While previous studies reported both, pro- or anti-angiogenic functions of angiopoietin-4 depending on dose and study design, we show here that recombinant angiopoietin-4 clearly inhibits endothelial cell migration and tube formation and that siRNA mediated antagonism of the increased Angpt4 RNA levels in cardiac fibroblasts restored angiogenic function in co-cultured HUVECs 39, 40. Second, the Thbs1 RNA levels as well as the expression of its receptor Cd36 were upregulated in Gata4/6-depleted cardiac fibroblasts. While the glycoprotein thrombospondin-1 is well established as an anti-angiogenic secreted factor especially from activated platelets, the influence of increased Cd36 expression levels in cardiac fibroblasts on neighboring endothelial cells remains somewhat unclear. Although we reproducibly found that counteractive downregulation of Cd36 in fibroblasts with lack of Gata4 and Gata6 prevented the anti-angiogenic intercellular effect, further research will be needed to fully understand the underlying mechanisms. Because we detected strongly elevated CD36 deposition in the extracellular matrix of Gata4/6fl-Per-Cre mice, one might speculate that it serves as scaffold protein to increase local thrombospondin levels. A recent study from Vidal et al. found a similar anti-angiogenic gene set in single-nuclei sequencing from aged cardiac fibroblasts, which among other factors, also described the glycoprotein CD36 as one of the receptors mediating intercellular fibroblast endothelial crosstalk 41. Besides the capability to mediate anti-angiogenic properties upon thrombospondin binding, one of the first identified functions of CD36 was the strong interaction with collagen 23. Another possible function might therefore be that CD36 on the surface of fibroblasts is activated by collagen to induce a paracrine gene-program, which in turn inhibits capillary growth. Indeed, Cd36 expressing fibroblasts counteract breast cancer cell growth in an organoid co-culture model by triggering the expression and release of unknown paracrine factors by fibroblasts 42. However, as an in depth investigation of CD36 in cardiac fibroblasts exceeded the scope of this study, further work will be necessary to explore the mechanism of CD36 dependent effects in cardiac fibroblasts. In this study, we focused on the transcription factors GATA-4 and GATA-6 to elucidate the role of the cardiac gene program in myocardial fibroblasts during pathological overload. Because compound deletion of Gata4 and Gata6 in cardiac fibroblasts reduced the adaptive angiogenic response during pressure overload and entailed cardiac dysfunction, we suggest that the cardiac gene-program in fibroblasts acts to maintain a myocardium specific interstitial milieu and high capillarization of the heart. The fibroblast-endothelial interface might therefore play a crucial role in the cardiac stress response and heart failure development and will require further research to enable targeted therapies in the future.
Projektbezogene Publikationen (Auswahl)
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(2020) Fibroblast GATA-4 and GATA-6 promote myocardial adaptation to pressure overload by enhancing cardiac angiogenesis, Basic Research in Cardiology
Gesine M. Dittrich, Natali Froese, Xue Wang, Hannah Kroeger, Honghui Wang, Malgorzata Szaroszyk, Mona Malek-Mohammadi, Julio Cordero, Merve Keles, Mortimer Korf-Klingebiel, Kai C. Wollert, Robert Geffers, Manuel Mayr, Simon J. Conway, Gergana Dobreva, Johann Bauersachs and Joerg Heineke
