Ricular cardiomycardiomyocyte-mediated CF activation. We identified neonatal rat ventricular cardiomyocytes (NRCM) in culture by the expression of cardiomyocyte CBP/p300 Activator Biological Activity marker -actinin (Figure ocytes (NRCM) in culture by the expression of cardiomyocyte marker -actinin (Figure 4A). 4A). Subsequent, we knocked down Nur77 in NRCM (Figure 4B), stimulated the NRCMs with Subsequent, we knocked down Nur77 in NRCM (Figure 4B), stimulated the NRCMs with ISO and ISO and subsequently transferred the conditioned medium to CFs (Figure 4C). Strikingly, subsequently transferred the conditioned medium to CFs (Figure 4C). Strikingly, we found we identified that the expression of MyoFB-associated and ECM genes acta2, postn, col1a1 and that the expression of MyoFB-associated and ECM genes acta2, postn, col1a1 and fn1 was sigfn1 was substantially greater in CFs stimulated with siNur77-NRCM conditioned medium nificantly greater in CFs stimulated with siNur77-NRCM conditioned medium (Figure 4D). (Figure 4D). This difference seemed to originate from Nur77-silencing in NRCMs because This difference seemed to originate from Nur77-silencing in NRCMs considering the fact that ISO stimulation ISO stimulation did not bring about an additive effect on MyoFB gene expression, except for didn’t bring about an additive impact on MyoFB gene expression, except for col1a1 expression. col1a1 expression. Also, CFs proliferated substantially extra when stimulated with On top of that, CFs proliferated considerably more when stimulated with siNur77 NRCM siNur77 NRCM conditioned medium compared to medium from siCon NRCMs (Figure conditioned medium in comparison to medium from siCon NRCMs (Figure 4E). Together, these results4E). With each other, these benefits show that Nur77 reduces the expression of pro-fibrotic parashow that Nur77 reduces the expression of pro-fibrotic paracrine components in NRCMs crine elements in regulates an intricate balance regulates an intricate balance of CF-to-Myand reveal that Nur77NRCMs and reveal that Nur77of CF-to-MyoFB differentiation, with oFB opposing roles with seemingly opposing roles by cardiomyocytes and CFs in fibrosis. seemingly differentiation, by cardiomyocytes and CFs in fibrosis.Int. J. Mol. Sci. 2021, 22, x FOR PEER Evaluation Int. J. Mol. Sci. 2021, 22,7 of 16 7 ofFigure 4. Paracrine things from Nur77-silenced cardiomyocytes market a MyoFB phenotype in Figure 4. Paracrine aspects from Nur77-silenced cardiomyocytes promote a MyoFB phenotype in CF. (A)(A) Primary neonatal rat ventricular cardiomyocytes in culture had been identified by expression CF. Principal neonatal rat ventricular cardiomyocytes in culture had been identified by expression of actinin. Scale bar bar represents 100 (B) Nur77 mRNA expression just after after siRNA-mediated of -actinin. Scale represents one hundred ). m). (B) Nur77 mRNA expressionsiRNA-mediated knockdown of Nur77 in neonatal rat ventricular cardiomyocytes, as measured measured by qPCR. (C) presenknockdown of Nur77 in neonatal rat ventricular cardiomyocytes, as by qPCR. (C) Schematic Schematic presentation of the experimental mRNA(D) mRNA expression markers inmarkers in CF immediately after tation on the experimental setup. (D) setup. expression of MyoFB of MyoFB CF immediately after LPAR1 Inhibitor Source incubation incubation with siCon or siNur77 cardiomyocyte conditioned as assessed by qPCR. by qPCR. (E) with siCon or siNur77 cardiomyocyte conditioned medium, medium, as assessed (E) Proliferation Proliferationincubation with siCon or siNur77 cardiomyocyte conditionedconditioned medium, as of CF right after of CF just after incubation with siCon or s.