High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

<div><p>Fibroblast growth factor 2 (FGF-2) is a multifunctional protein synthesized as high (Hi-) and low (Lo-) molecular weight isoforms. Studies using rodent models showed that Hi- and Lo-FGF-2 exert distinct biological activities: after myocardial infarction, rat Lo-FGF-2, but not Hi-FGF-2, promoted sustained cardioprotection and angiogenesis, while Hi-FGF-2, but not Lo-FGF-2, promoted myocardial hypertrophy and reduced contractile function. Because there is no information regarding Hi-FGF-2 in human myocardium, we undertook to investigate expression, regulation, secretion and potential tissue remodeling-associated activities of human cardiac (atrial) Hi-FGF-2. Human patient-derived atrial tissue extracts, as well as pericardial fluid, contained Hi-FGF-2 isoforms, comprising, respectively, 53%(±20 SD) and 68% (±25 SD) of total FGF-2, assessed by western blotting. Human atrial tissue-derived primary myofibroblasts (hMFs) expressed and secreted predominantly Hi-FGF-2, at about 80% of total. Angiotensin II (Ang II) up-regulated Hi-FGF-2 in hMFs, via activation of both type 1 and type 2 Ang II receptors; the ERK pathway; and matrix metalloprotease-2. Treatment of hMFs with neutralizing antibodies selective for human Hi-FGF-2 (neu-Ab^Hi-FGF-2) reduced accumulation of proteins associated with fibroblast-to-myofibroblast conversion and fibrosis, including α-smooth muscle actin, extra-domain A fibronectin, and procollagen. Stimulation of hMFs with recombinant human Hi-FGF-2 was significantly more potent than Lo-FGF-2 in upregulating inflammation-associated proteins such as pro-interleukin-1β and plasminogen-activator-inhibitor-1. Culture media conditioned by hMFs promoted cardiomyocyte hypertrophy, an effect that was prevented by neu-Ab^Hi-FGF-2<i>in vitro</i>. In conclusion, we have documented that Hi-FGF-2 represents a substantial fraction of FGF-2 in human cardiac (atrial) tissue and in pericardial fluid, and have shown that human Hi-FGF-2, unlike Lo-FGF-2, promotes deleterious (pro-fibrotic, pro-inflammatory, and pro-hypertrophic) responses <i>in vitro.</i> Selective targeting of Hi-FGF-2 production may, therefore, reduce pathological remodelling in the human heart.</p></div

Detection of Hi-FGF-2 in human atrial myofibroblasts.

<p><b>Panel (A)</b> shows two sets of western blots analyzing FGF-2 isoforms. The first set, (i), is a composite of two blots (separated by a broken line) and analyzes FGF-2 isoforms in hMF lysates (20 µg/lane), from atrial myofibroblast primary cultures obtained from 10 patients (patients 11–20), and correspondingly labeled as C11–20. The second set, (ii), also a composite of two blots separated by a broken line, analyzes FGF-2 isoforms in atrial tissue lysates from patients 11–20, and labelled T11–20 (50 µg/lane). The hMF blots or tissue blots were also probed for, respectively, β-tubulin (β-tub), or Troponin-T (TnT), as indicated. Following densitometry of the hMF blots, the % contribution of each FGF-2 isoform to the total FGF-2 signal was determined for each individual lane, and cumulative results (mean±SD) are included in graph form (n = 10). <b>In Panel (B)</b>, a western blot shows FGF-2 signals from 0.5 and 0.2 ng/lane of recombinant histidine tagged Lo-FGF-2 (FGF-2<i>^His</i>), atrial tissue lysates (T11, T15 and T17, loaded at 50 µg/lane), side by side with FGF-2 signals from lysates obtained from corresponding primary hMF cultures (C11, C15 and C17, loaded at 10 µg/lane). The graph shows comparisons between tissue and cell lysates for their relative total FGF-2 content, assessed by densitometry as optical density (O.D.) units (n = 3). Measurements corresponding to cell FGF-2 were multiplied by 5, to correct for the 5-fold difference in total protein loading. In both panels, comparisons between groups are indicated by brackets, where P>0.05 is marked as ns, while P<0.001, 0.01, are marked as ***, or **, respectively. <b>Panels C and D</b> show immunofluorescence images of hMFs stained for, (C), Hi-FGF-2 (green), as well as, (D), alpha smooth muscle actin (red) and nuclei (blue). White arrows point to cytosolic Hi-FGF-2; pink and pale- pink arrows arrows point to nuclear and nucleolar Hi-FGF-2, respectively. Grey sizing bars correspond to 20 µm.</p

Human Hi-FGF-2 exerts pro-hypertrophic effect.

<p><b>Panel A.</b> Neonatal rat cardiomyocyte cell surface area (normalized, assigning a value of 1 in control, untreated samples) is shown in response to stimulation with Endothelin 1 (ET-1), serving as a positive control, and a recombinant human Hi-FGF-2 preparation (10 ng protein/ml), n = 320 myocytes/group. CM denotes conditioned medium obtained from unstimulated hMFs while CM* denotes conditioned medium from Ang II-stimulated hMFs. ET-1, recombinant human Hi-FGF-2, as well as CM* (but not CM, or Ang II added at 100 nM) increased myocyte cell surface area significantly. <b>Panel B</b>. Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*or CM* supplemented with neutralizing antibodies to total FGF-2 (neu-Ab^FGF-2), as indicated; n = 480 cells/group. Neutralization of total FGF-2 eliminated the ability of CM* to increase myocytes cell surface area compared to CM. <b>Panel C.</b> Protein synthesis (³H-Leucine incorporation) of cardiomyocytes incubated with CM, CM*, and CM* supplemented with 20 µg/ml neutralizing anti-Hi-FGF-2 antibodies (CM* +neu-Ab^Hi-FGF-2). Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase protein synthesis of cardiomyocytes compared to CM; n = 5 plates/group. <b>D</b>. Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*, and CM* +neu-Ab ^Hi-FGF-2. Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase surface area of cardiomyocytes compared to CM; n = 480/group. Please note that for the experiments shown in B,C,D panels the conditioned media in the first two groups (CM, CM*) were supplemented with non-specific rabbit IgG, at 20 µg/ml. <b>E</b>. Representative images of cardiomyocytes stained for anti-N-cadherin (green), alpha-actinin (red) and nuclei (blue), and incubated with CM, CM*, and CM* +neu-Ab (FGF-2). Sizing bar in (iii) coresponds to 100 µM. In all graphs, brackets show comparison between groups, where *, **, ***, ns correspond to P<0.05, <0.01, <0.001, or P>0.05.</p

Detection of Hi-FGF-2 in human atrial tissue.

<p><b>Panel (A)</b> shows representative western blot images of human atrial extracts (hA1, hA2, hA3, 50 µg/lane) probed for FGF-2 with an antibody detecting all FGF-2 isoforms. Expected migration of all human FGF-2 isoforms (34, 24, 22–22.5, and 18 kDa), corresponding to Hi- or Lo-FGF-2, is indicated by arrows; please note that the 34 kDa isoform is not detectable in tissue lysates. Western blots were also probed for cardiac troponin T (TnT) to verify equivalent loading of lanes. Samples hA1, hA2 were analyzed in small (8.3×5.5 cm²) 15% polyacrylamide gels, while hA3 was analyzed in a large (16×11.5 cm²) 15% polyacrylamide gel. The included graph shows percentage of each isoform over total FGF-2, where, n = 45; comparisons between groups are indicated by brackets, where *** and ** denote P<0.001, and P<0.01, respectively. <b>Panels (B) and (C)</b> show images from patient-derived serial atrial sections, subjected to (B) incubation with purified anti-Hi-FGF-2 antibodies followed by immunohistochemical visualization of antigen-antibody complexes (brown color) as well as nuclear staining (hematoxylin, blue), and (C) similar procedures as in B but without the anti-Hi-FGF-2 antibodies. Incubation with anti-Hi-FGF-2 antibody elicits extensive immunostaining, in what appears to be nuclear as well as cytosolic sites in cardiomyocytes; staining of non-cardiomyocytes located at the epicardium is indicated by arrows. <b>Panels (D) and (E)</b> are close-up images from human atrial tissue sections stained as in (B), showing cellular and subcellular distribution of Hi-FGF-2. <b>Panels (G) and (F)</b> show human atrial sections subjected to double-immunofluorescence staining for Hi-FGF-2 (green), and either vimentin (G, red), or desmin (F, red). In all images, yellow or pink arrows point, respectively, to nuclear or cytosolic sites within cardiomyocytes. Blue arrows in (D) point to small connective tissue cells, likely fibroblasts. Green arrows in (E) point to endothelial cells, lining a vessel. White arrows in (G) and (F) point to non-myocytes, found at or near the epicardial region. These cells are positive for vimentin, but not desmin. In (F), co-staining with desmin confirms presence of Hi-FGF-2 in atrial cardiomyocytes. Sizing bars in (B) or (D,E,F,G) correspond to 250 or 100 µM, respectively. Insets within panels G and F are shown in larger magnification in Fig S2.</p

Both AT-1R and AT-2R mediate the Ang II-induced ERK activation in hMFs.

<p>Panel A shows western blot of activated (phosphorylated) pERK, and total ERK, in hMFs stimulated for 30 minutes with with Ang II (lanes 1,2,3), Ang II + PD123319 (lanes 4,5,6), Ang II + Losartan (lanes 7,8,9), and Ang II +PD123319 +Losartan (lanes 10,11,12), in the absence (-) or presence (+) of neutralizing anti-FGF-2 antibodies (neu-Ab^FGF-2), as indicated. Please note that the western blot for pERK in the groups incubated with neu-Ab^FGF-2 is not directly comparable to the western blot for pERK in the groups incubated in the absence of neu-Ab^FGF-2 (different exposures). Panel B shows pERK/ERK ratios in the groups shown in panel A. Brackets show statistically significant differences between groups, where *, **, ***, correspond to P<0.05, 0.01, and 0.001, respectively.</p

Calreticulin Induces Dilated Cardiomyopathy

<div><p>Background</p><p>Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.</p><p>Methodology/Principal Findings</p><p>We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.</p><p>Conclusions/Significance</p><p>We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy <i>in vivo</i> leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.</p></div

Angiotensin II promotes upregulation of cell-associated human Hi-FGF-2 via AT-1R and AT-2R.

<p><b>Panel A</b>: western blot, and corresponding cumulative data, showing the effect of Ang II on Hi-FGF-2 accumulation by hMFs, in the absence or presence of either losartan (AT-1R inhibitor) or PD123319 (AT-2R inhibitor). Lanes 1–3; 4–6; 7–9; 10–12 correspond to lysates from, respectively, untreated (Control)-;Ang II-stimulated-; Ang II stimulated in the presence of losartan; and Ang II-stimulated in the presence of PD123319- hMFs. Ang II promotes Hi-FGF-2 upregulation which is significantly decreased by either losartan or PD123319. <b>Panel B</b>: western blot and cumulative densitometry data showing the effect of Ang II on Hi-FGF-2 accumulation in the absence or presence of simultaneous inhibition of both AT-1R and AT-2R. Lanes 1–3; 4–6; 7–9 correspond to lysates from, respectively, untreated (Control)-;Ang II-stimulated-; Ang II stimulated in the presence of both losartan and PD123319- hMFs. Relative levels of Hi-FGF-2 in the presence of both AT-1R and AT-2R inhibitors are not significantly different to those of unstimulated cells. <b>Panels C and D</b>. Western blots showing expression, respectively, of AT-1R or AT-2R by hMFs, and relative levels of these receptors after 24 h stimulation with Ang II. After 24 hour stimulation, levels of AT-1R, but not AT-R2, decrease compared to unstimulated cells. Signal for β-actin is also shown in A-D, serving as loading control. <b>E</b>. Densitometry data showing the effect of Ang II receptor inhibitors on baseline Hi-FGF-2 accumulation by hMFs in the abcence of stimulation by added Ang II. Incubation of unstimulated hMFs with losartan (but not PD123319) significantly decreased baseline Hi-FGF-2 levels. Sample size n = 3 (all graphs); *, **, *** indicates P<0.05, <0.01, <0.001, respectively; and ns denotes non-significance difference at P>0.05.</p

Selective neutralization of extracellular human Hi-FGF-2 attenuates expression of pro-fibrotic proteins.

<p><b>Panel A</b>. Western blots showing the effect of incubation with either control antibodies (Cont-Ab, 20 µg/ml, lanes 1,2,3), or anti-Hi-FGF-2 antibodies (Neu-Ab^Hi-FGF-2, 20 µg/ml, lanes 4,5,6) on the accumulation of α-SMA, procollagen, SMemb, EDA-Fibronectin (EDA-FN), β-tubulin, and GAPDH, by hMFs, as indicated. <b>Panels B</b>,<b>C</b>,<b>D</b> and <b>E</b> show corresponding quantitative (densitometry) data for α-SMA, procollagen, SMemb, EDA-Fibronectin (EDA-FN), as indicated (±SEM). Incubation with Neu-Ab^Hi-FGF-2 significantly decreased expression of α-SMA, procollagen, SMemb and EDA-Fibronectin, without having any effect on GAPDH or β-tubulin. Brackets show comparisons between groups, where *, **, *** correspond to P<0.05, <0.01, <0.001; n = 3/group.</p

Effect of extracellular-acting FGF-2 isoforms on the accumulation of pro-IL-1β and PAI-1 by hMFs.

<p><b>Panel A</b>, western blot and corresponding cumulative data showing relative pro-IL-1β levels (optical density, O.D. units) in hMF cell lysates from unstimulated cells (lanes 1,2,3) and cells stimulated with 10 ng/ml of a recombinant Hi-FGF-2 preparation (Hi, lanes 4,5,6) or 10 ng/ml of recombinant Lo-FGF-2 (Lo, lanes 7,8,9), as indicated. Both Hi- and Lo-FGF-2 upregulated pro-IL-1β, although the effect of Hi-FGF-2 was significantly more potent. <b>Panel B</b>, western blot and corresponding quantitative data showing relative PAI-1 levels (optical density, O.D. units) in hMF cell lysates from unstimulated cells (lanes 1,2,3) and cells stimulated with Hi-FGF-2 (Hi, lanes 4,5,6) or Lo-FGF-2 (Lo, lanes 7,8,9), as indicated. Hi- but not Lo-FGF-2 upregulated PAI-1 levels. Brackets mark comparisons between groups where *, **, ***, and ns denotes P<0.05, P<0.01, P<0.001, and P>0.05 respectively.</p

ERK and MMP-2 activities mediate the Ang II-induced Hi-FGF-2 upregulation in hMFs.

<p><b>Panel A.</b> Western blot and corresponding cumulative data showing the effect of an ERK inhibitor (U0126), or MMP-2 inhibitor (MMP2i) on the Ang II induced Hi-FGF-2 upregulation. Signal for β-tubulin is also shown, serving as loading control. <b>Panel B.</b> Western blots and corresponding cumulative data showing the effect of Ang II administration on phospho-(P)-ERK and total ERK, after 10–30 minutes and 6–24 hours of stimulation as indicated. The graph shows cumulative data (n = 3) of the ratio between P-ERK/ERK over time (10–30 min, 6–24 hours), in response to Ang II. Minutes and hours are indicated as ‘ and h. <b>Panel (C)</b> Representative zymogram of MMP-2 activity in hMFs, including a positive control band (MMP-2), and corresponding cumulative data, showing relative MMP-2 activity in response to Ang II, over time (10–30 min, 6–24 hours), as indicated. For all graphs, brackets show comparisons between groups; *, **, ***, and ns correspond to P<0.05, P<0.01, P<0.001, and P>0.005, respectively.</p