Fibroblast growth factor receptor 1 signaling in adult cardiomyocytes increases contractility and results in a hypertrophic cardiomyopathy

Fibroblast growth factors (FGFs) and their receptors are highly conserved signaling molecules that have been implicated in postnatal cardiac remodeling. However, it is not known whether cardiomyocyte-expressed FGF receptors are necessary or sufficient for ventricular remodeling in the adult heart. To determine whether cardiomyocytes were competent to respond to an activated FGF receptor, and to determine if this signal would result in the development of hypertrophy, we engineered a doxycycline (DOX)-inducible, cardiomyocyte-specific, constitutively active FGF receptor mouse model (αMHC-rtTA, TRE-caFgfr1-myc). Echocardiographic and hemodynamic analysis indicated that acute expression of caFGFR1 rapidly and directly increased cardiac contractility, while chronic expression resulted in significant hypertrophy with preservation of systolic function. Subsequent histologic analysis showed increased cardiomyocyte cross-sectional area and regions of myocyte disarray and fibrosis, classic features of hypertrophic cardiomyopathy (HCM). Analysis of downstream pathways revealed a lack of clear activation of classical FGF-mediated signaling pathways, but did demonstrate a reduction in Serca2 expression and troponin I phosphorylation. Isolated ventricular myocytes showed enhanced contractility and reduced relaxation, an effect that was partially reversed by inhibition of actin-myosin interactions. We conclude that adult cardiomyocytes are competent to transduce FGF signaling and that FGF signaling is sufficient to promote increased cardiomyocyte contractility in vitro and in vivo through enhanced intrinsic actin-myosin interactions. Long-term, FGFR overexpression results in HCM with a dynamic outflow tract obstruction, and may serve as a unique model of HCM

In vivo induction of caFGFR1 in adult cardiomyocytes leads to a hypercontractile phenotype with the development of a dynamic obstruction in the proximal left ventricle.

<p>(A) Representative pressure-volume (PV) loops at baseline (1) and following increased afterload (2) in DTG mice and littermate controls induced with DOX chow for 24 hours. The slope of the end systolic pressure-volume relationship (ESPVR, dotted line) is significantly elevated in DTG mice (n=3 for DTG and control). (B) Representative pressure (top) and dP/dt (bottom) tracings suggesting the presence of a dynamic obstruction in DTG mice induced for 42 days (n_DTG=3, n_control=2). (C) Representative color Doppler images (blue = blood outflow, red = blood inflow) and (D) spectral Doppler tracings illustrating flow convergence (yellow arrow) and a high velocity late-peaking jet originating from the point of dynamic mid-cavity obstruction in DTG mice. (E) Quantitation of LV peak outflow velocity (LV POV) at the point of flow convergence. Error bars = standard deviation. *p<0.05, **p<0.01, +p<0.001.</p

Ventricular myocytes from double transgenic mice have impaired relaxation and enhanced contraction.

<p>(A) Representative signal averaged sarcomere length measurements during contraction recorded from acutely-induced DTG (solid line) and control cardiomyocytes (dashed line). (B) DTG myocytes had significantly shorter sarcomere lengths at both systole and diastole, (C) with an overall increase in fractional shortening (n=4 mice, 17 myocytes total for both control and DTG). (D) Addition of BDM caused significant relaxation of both DTG and control myocytes, and (E) resulted in a greater change in sarcomere length in DTG myocytes (n=3 mice each, 19 control myocytes, 23 DTG myocytes). Error bars = standard deviation. **p<0.01, +p<0.001 DTG vs. controls; # p<0.05 vs. respective baseline measurements.</p

Transgene induction for 42 days, followed by DOX removal for 42 days leads to partial phenotype reversal.

<p>(A,B) LV internal diastolic diameter (LVIDd, A) and LV mass index (LVMI, B) returned to near control levels after removal of DOX for 42 days (42 off). (C) LV diastolic posterior wall thickness (LVPWd) was significantly reduced in DTG mice (n=6) following DOX removal (42 off), but remained significantly elevated compared to controls (n=4). (D) Despite decreasing significantly following removal of DOX, peak outflow velocity (LV POV) from the proximal ventricle was still significantly elevated compared to controls. (E) Cardiomyocyte cross-sectional area (CM CSA) was smaller than that seen following six weeks of caFGFR1 induction (42 on), but cells were still significantly larger than controls following DOX removal (42 off). (F) Western blot analysis showing a significant decrease but persistent expression of caFGFR1 following removal of DOX (DTG 42d ON 42d OFF), compared to transgene expression after three weeks of induction (DTG 21d; protein samples from six weeks of induction were unavailable). (G-I) QRT-PCR analysis demonstrating continued overexpression of Fgfr1 in DTG hearts following 42 days off DOX (gray bar), despite a large reduction from the level of expression observed following 42 days on DOX (black bar, G). Expression of downstream mediators of FGF signaling (H) and markers of pathological LV remodeling (I) are decreased to control levels 42 days following DOX removal. Error bars = standard deviation. *p<0.05, **p<0.01, +p<0.001 compared to controls. #p<0.05 compared to DTG mice 42 days on DOX.</p

Inducible constitutively-active FGFR1 genetic system shows minimal baseline activity and rapid activation of transgene expression.

<p>(A) Schematic of a tetracycline response element (TRE)-<i>Fgfr31c</i>(R248C)<i>-</i>c-myc transgene. (B) Western blot analysis demonstrates minimal baseline expression (DTG 0d), rapid induction (DTG 1d), and consistent expression of caFGFR1 following feeding of DOX chow for various lengths of time (DTG 7d, 21d, 168d). ß-tubulin expression demonstrates equal protein loading. (C-E) Uninduced DTG mice show no evidence of hypertrophy on gross measurement of biventricular weight/body weight ratio (HW/BW, B; n_DTG=7, n_control=7), histological measurement of mean cardiomyocyte cross sectional area (CM CSA, C; n_DTG=5, n_control=5), or echocardiographic measurement of diastolic left ventricular posterior wall thickness (LVPWd, D; n_DTG=47, n_control=23). (F) Minimal expression of caFGFR1 is sufficient to induce a small, but significant, increase in outflow velocity from the proximal LV (n_DTG=72, n_control=43). (G) QRT-PCR analysis (n_DTG=4, n_control=4) shows no significant changes in markers of pathological cardiac remodeling. (H) QRT-PCR analysis (DTG: n_0d=4, n_1d=7, n_7d=5, n_21d=6, n_168d=4) shows a significant elevation in Fgfr1 mRNA in uninduced DTG animals (0d) that dramatically increases within one day of transgene induction (1d). (I) No significant changes were seen in downstream transcription factors Etv5 or Etv4 at baseline (0d), but significant induction was observed after one day of DOX induction (1d). Fold change is versus corresponding time-matched controls (not shown; n_0d=4, n_1d=6, n_7d=4, n_21d=5, n_168d=4). Error bars = standard deviation. *p<0.05, ** p<0.01, + p<0.001. </p

Induction of caFGFR1 in adult cardiomyocytes results in the development of concentric hypertrophy with preservation of systolic function over time.

<p>(A) LV diastolic posterior wall thickness (LVPWd, A) was significantly elevated by one day of induction, while LV mass index (LVMI) was significantly elevated (B) and LV internal diastolic diameter (LVIDd) was significantly decreased (C) within seven days of DOX induction. (D) Systolic function was maintained throughout 6 months of transgene induction. Error bars = standard deviation. *p<0.05, **p<0.01, +p<0.001. 0d: n_DTG=47, n_control=23; 1d: n_DTG=8, n_control=4; 7d: n_DTG=17, n_control=7; 42d: n_DTG=14, n_control=12; 84d: n_DTG=4, n_control=4; 126d: n_DTG=4, n_control=4; 168d: n_DTG=4, n_control=4. (E) Representative short axis echocardiographic images depicting the progression of hypertrophy in DTG animals from baseline to 168 days of induction. </p

DTG mice have significantly decreased troponin I phosphorylation and Serca2 expression.

<p>(A) Representative Western blots depicting no changes in the expression of phosphorylated protein kinase A (P-PKA) and decreased expression of phosphorylated troponin I (P-TnI) and Serca2. ß-tubulin was used as a loading control. Independent blots are separated by a thick black line. (B) Densitometric quantitation of p-TnI/t-TnI and Serca2, demonstrating a significant decrease in both by seven days of caFGFR1 induction. Fold change is versus corresponding time-matched controls (not shown). DTG (shown): n_0d=5, n_1d=8, n_7d=8, n_21d=6, n_168d=2; controls (not shown): n_0d=2, n_1d=5, n_7d=4, n_21d=3, n_168d=2. Error bars = standard deviation. *p<0.05, **p<0.01.</p