P752Pericyte/mural cells of ischemic human hearts show impairment of mechanotransduction, attenuating YAP signaling

Abstract

Abstract Background The Hippo pathway, that is upregulated in human heart failure, can restrain cardiac regeneration and impair arteriogenesis, preventing the nuclear translocation of the transcriptional coactivator YAP. Actin dynamics and mechanotransduction pathways are critical regulators of YAP subcellular localization. Aim To investigate if cardiac pathology alters mechanotransduction pathways, modulating the nucleocytoplasmic shuttling of YAP. Methods and results PDGFRb+ NG2+Tbx18+cardiac pericytes/mural cells (PM) cultured from normal atria (PMnorm, n=6) and from ischemic failing hearts (PMfail, n=6) were compared. From a transcriptomic standpoint, biological processes and molecular functions associated with cell adhesion and actin cytoskeleton were differentially expressed by PMfail vs PMnorm. The morphological analysis of the two cell types showed that PMfail were significantly larger (2,455±160μm2 vs 3518±334μm2, p=0.04) and less polarized (aspect ratio: 3±0.9 vs 2±0.1, p=0.002) with respect to PMnorm. Consistently, PMfail showed a significantly smaller number of Paxillin+ focal adhesions/cell area and impaired migratory properties. Furthermore, PMfail showed contact inhibition of cell proliferation at significantly lower cell density with respect to PMnorm, paralleled by a significantly lower fraction of cells expressing YAP in their nuclei at a density of 500 cells/mm2 (28.1±12.7% vs 87.0±23.6%, p=0.029). Given the link between mechanotransduction, response to substrate stiffness and extracellular matrix composition, we evaluated the responses of PM plated on a soft (16kPa), intermediate (231kPa) and very hard (in order to GPa) polyacrylamide gels coated with two different fibronectin (FN) concentrations (1 and 25μg/mL). Atomic force microscopy measure of cell stiffness, showed that, while PMnorm significantly increase their intracellular stiffness (804±311Pa vs 1,320±394Pa, p=0.014) as a function of extracellular stiffness (soft vs very hard), PMfail do not. These results were paralleled by YAP and Myocardin Related Transcription Factor-A (MRTF-A) shuttling, that are translocated in the nuclei of PMnorm in response to substrate stiffness and fibronectin concentration significantly more than in the nuclei of PMfail cells. Analysis of CTGF, CYR61 and ANKRD1 gene expression corroborated these data. Finally, we demonstrated that serum deprivation, inhibitors of both actin polymerization (latrunculin-A), and Rho-associated Kinase (ROCK, Y23763) significantly inhibited the nuclear translocation of both YAP and MRTF-A and cell proliferation. Conversely, the MEK1/2 inhibitor I-1040 exerted opposite effects. Conclusion PM isolated from ischemic hearts are characterized by altered mechanotransduction properties that impair a correct nuclear translocation of the cotranscriptional regulators YAP and MRTF-A in response to environmental cues. Pharmacologic modulation of regulators of this pathway may revert the pathologic phenotype.