Persistent Fibrosis in Heart Failure With a Reduced Ejection Fraction Linked to Phenotypic Differences in Human Cardiac Fibroblast Populations

Figure 1. Myocardial passive stiffness and collagen content in myocardial tissues. A and B, Myocardial passive stiffness in control, pre‐LVAD, and post‐LVAD tissue samples. A, Representative examples of passive stretch. B, Summary data. Stress was measured at 15% strain during in vitro passive stretch of human ventricular tissue isolated from myocardium collected from control subjects (n=3), pre‐LVAD patients (n=6), and post‐LVAD patients (n=5). Three LV samples were isolated from each collected myocardial biopsy. Each data point represents an average of 6 consecutive passive stretches of each LV sample. Control tissue had an average stiffness of ≈4 kPa, whereas pre‐LVAD tissue had a significantly higher average stiffness of ≈11 kPa. Post‐LVAD values, ≈10 kPa, continued to be elevated vs. Control, despite hemodynamic unloading by LVAD intervention. *P [lt]0.05 vs Control.: C through E, Representative picrosirius red staining from C. referent control (patient with no history of heart failure). D, Pre‐LVAD (left ventricle collected at LVAD implant). E, Post‐LVAD (LV collected after LVAD removal, (scale bars = 50 microns). Total collagen volume fraction (CVF) quantified for each group, (F)…

Figure 2. Fibroblast numbers increase in pre‐LVAD and post‐LVAD hearts. Myocardial sections stained with vimentin (red), (PECAM, green), and DAPI (nuclei, blue). Cells stained with vimentin only were counted as fibroblasts, PECAM was used to differentiate vimentin+ endothelial cells. A, Control; (B) pre‐LVAD; (C) post‐LVAD (scale bars = 50 microns). (D) Quantification of fibroblast number in control (n=5), pre‐LVAD (n=5), and post‐LVAD (n=3) groups. Both pre‐ and post‐LVAD groups had significantly more fibroblasts than control. *P [lt]0.01 vs control; # P [lt]0.01 vs pre‐LVAD. LVAD indicates left ventricular assist device; and PECAM, platelet endothelial cell adhesion molecule.

Figure 3.. Greater proliferative capacity in pre‐ and post‐LVAD fibroblasts compared with control. A, Fibroblasts were plated at equal density on 2 kPa and 8 kPa and allowed to proliferate for 72 hours. No differences were detected in proliferation on 2 vs 8 kPa for any group, but pre‐LVAD and post‐LVAD cells demonstrated increased cell numbers vs control. Post‐LVAD cells on 2 kPa were significantly different from controls (*P [lt]0.05), n=3 independent cultures. B, through D, Pre and post‐LVAD fibroblasts do not demonstrate overt differences in cell morphology vs control fibroblasts. B, Representative images of control, pre‐LVAD (greens), and post‐LVAD (purples) fibroblasts plated on 2 and 8 kPa substrates. C, Elongation measurements for control (blue bars), pre‐LVAD (green bars), and post‐LVAD (purple bars) fibroblasts on 2 and 8 kPa plates, calculated by fitting an ellipse to the cell outlines then dividing the ellipse major axis length by the minor axis length. D, Cell area calculated by taking the area enclosed by the outlines created by cell profiler in pixels and converting to μm. n=3 independent cultures. LVAD indicates left ventricular assist device.

Figure 4. Fibroblast collagen and α‐SMA production on 2 kPa vs 8 kPa. A, Representative blots for control, pre‐LVAD, and post‐LVAD groups depicting procollagen α1(I) and pC collagen α1(I) in the top panels (≈150 kDa and ≈130 kDa) and in the bottom panels (≈37 kDa). B, Quantification of collagen bands (normalized to β‐Actin) from conditioned media shown as a ratio of total collagen production on 8 kPa to that produced on 2 kPa for control (n=6), pre‐LVAD (n=10), and post‐LVAD (n=6) independent cultures. Control fibroblasts produced ≈2‐fold more collagen on 8 kPa than on 2 kPa, whereas both pre‐ and post‐LVAD fibroblasts did not demonstrate differences in collagen production on 2 kPa vs 8 kPa (ratio ≈1). *P [lt]0.05 vs control. C and D, Fibroblast α‐SMA production on 2 kPa vs 8 kPa. C, Representative blots for control, pre‐LVAD, and post‐LVAD groups depicting α‐SMA in top panels (≈37 kDa) and β‐Actin in bottom panels (≈37 kDa). D, α‐SMA was detected in cell layers and quantified as a ratio of α‐SMA production on 8 kPa to α‐SMA production on 2 kPa after normalization to β‐Actin. Control (n=4), pre‐LVAD (n=9), and post‐LVAD (n=6) independent cultures. Control fibroblasts produced ≈2‐fold to…

Figure 5. Differential gene expression was queried in pre‐LVAD (n=4) and control (n=3) fibroblast cultures on 2 kPa. Expression patterns suggested that the focal adhesion pathway is uncoupled in pre‐LVAD fibroblasts as upstream expression of cell surface receptors (eg, integrin α3 and α5) were decreased in pre‐LVAD cells, whereas downstream mediators of proliferation and survival (eg, MAPK3) were elevated. A, Volcano plot of significant differentially expressed genes (downregulated genes in blue, upregulated genes in red). B, Pathway analysis identified the focal adhesion pathway as one of the most significantly altered signaling motifs in pre‐LVAD fibroblasts on low stiffness, 2 kPa. C, Pathway map suggested an uncoupling with downstream genes (red) showing upregulation despite the downregulation of upstream genes (blue) that might enhance processes such as cell proliferation, motility, and survival. D, Specific genes in the focal adhesion pathway that demonstrated either decreased (blue) or increased (red) expression in pre‐LVAD vs control fibroblasts on 2 kPa by logFC. ECM indicates extracellular matrix; LVAD, left ventricular assist device; MAPK3, map kinase 3; and PDGFB,…

Figure 6. Differential expression analysis between pre‐LVAD (n=4) and control (n=3) fibroblasts on 8 kPa suggested an uncoupling of focal adhesion pathways resulting in increased cell motility, proliferation, and survival in pre‐LVAD cells. A, Volcano plot of significant differentially expressed genes (downregulated genes in blue, upregulated genes in red). B, Pathway analysis identified the focal adhesion pathway as one of the most significantly altered cell signaling motifs in pre‐LVAD fibroblasts vs control on high stiffness. C, Pathway map suggested downstream genes (red) are increased despite the downregulation of upstream genes (blue) at multiple sites in the focal adhesion pathway. D, Specific genes in the focal adhesion pathway that demonstrated either decreased (blue) or increased (red) expression in pre‐LVAD vs control fibroblasts on 8 kPa by logFC. LVAD indicates left ventricular assist device; and MAPK3, map kinase 3.