Loss of Y-chromosomal genes in macrophages induces paracrine cardiac fibroblast activation

Lukas Zanders (Frankfurt am Main)1, M. Shumliakivska (Frankfurt am Main)2, F. Fleck (Frankfurt am Main)1, W. Abplanalp (Frankfurt am Main)2, A. M. Zeiher (Frankfurt am Main)3, S. Dimmeler (Frankfurt am Main)1

1Goethe Universität Frankfurt am Main Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland; 2Goethe Universität Frankfurt am Main Institute of Cardiovascular Regeneration and Department of Cardiology Frankfurt am Main, Deutschland; 3Goethe Universität Frankfurt am Main Institute of Cardiovascular Regeneration Frankfurt am Main, Deutschland

 

Background: Sex differences in the incidence, presentations and prognosis are an understudied hallmark of cardiovascular disease. Mosaic loss of the Y chromosome (mLOY) in blood leukocytes is the most frequent chromosomal aberration in men, affecting more than half of males over the age of 90. mLOY is associated with increased all-cause- and cardiovascular mortality. Patients with mLOY undergoing TAVR show an increased mortality. Mice transplanted with myeloid LOY cells exerted a fibrotic myocardial phenotype upon left ventricular pressure overload. It is currently unclear, if one or multiple genes cause the effects of mLOY PBMCs on the cardiovascular system and through which – potentially therapeutically targetable – mechanisms these effects are exerted.

Aim: To identify Y-chromosomal genes involved in cardiovascular disease and elucidate their cellular and molecular mechanisms.

Methods: We used single cell RNA sequencing (scRNA-Seq) datasets from both THP-1 monocytes and human PBMCs to investigate the expression levels of Y-chromosomal genes in monocytes. Using siRNA pools, we silenced the expression of the top expressed genes in THP-1 M0 macrophages and collected conditioned media from these cells. Primary human fibroblasts and neonatal rat cardiomyocytes were treated with these conditioned media. In fibroblasts, collagen 1A1 protein expression was assessed by immunohistochemistry and myocyte hypertrophy was measured in cardiomyocytes (Fig. 1B). Conditioned medium from DNMT3A-silenced THP-1 macrophages was used as a positive control.

Results: We found 9 genes in the male-specific region of the Y chromosome that were highly expressed in scRNA-Seq datasets of THP-1 and patient monocytes (Fig. 1A)7 of those were protein-coding and 2 were non-coding genes. Silencing efficacy was sufficient as shown by RT-qPCR analyses (Fig. 1C). Treatment of cardiac fibroblasts with conditioned media from macrophages silenced for ZFY(1.44-fold, p = 0.03), TTTY14 (1.30-fold, p = 0.03), LINC00278 (1.8-fold, p = 0.002) as well as the positive control DNMT3A (1.9-fold, p = 0.01) induced collagen 1A1 protein expression (Fig. 1D). Treatment of neonatal rat cardiomyocytes with conditioned medium for 72 hours did not induce cardiomyocyte hypertrophy.

Conclusion: Silencing of ZFYTTTY14 or the lincRNA LINC00278 in macrophages induces paracrine activation of cardiac fibroblasts. These data suggest a differential effect of mLOY on different myocardial cell types and might guide the way to a better understanding of the role of mLOY in cardiovascular disease.

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