Increased FOG-2 in failing myocardium disrupts thyroid hormone-dependent SERCA2 gene transcription

Abstract

Reduced expression of sarcoplasmic reticulum calcium ATPase (SERCA)2 and other genes in the adult cardiac gene program has raised consideration of an impaired responsiveness to thyroid hormone (T3) that develops in the advanced failing heart. Here, we show that human and murine cardiomyopathy hearts have increased expression of friend of GATA (FOG)-2, a cardiac nuclear hormone receptor corepressor protein. Cardiac-specific overexpression of FOG-2 in transgenic mice led to depressed cardiac function, activation of the fetal gene program, congestive heart failure, and early death. SERCA2 transcript and protein levels were reduced in FOG-2 transgenic hearts, and FOG-2 overexpression impaired T3-mediated SERCA2 expression in cultured cardiomyocytes. FOG-2 physically interacts with thyroid hormone receptor-alpha1 and abrogated even high levels of T3-mediated SERCA2 promoter activity. These results demonstrate that SERCA2 is an important target of FOG-2 and that increased FOG-2 expression may contribute to a decline in cardiac function in end-stage heart failure by impaired T3 signaling.

Figure 1. Increased FOG-2 protein in end-stage heart failure

(a) Mean±SEM log-transformed FOG-2 probe intensities in NF (n=6), ICM (n=10), and NICM (n=21) and human heart samples. One-way ANOVA followed by Holm-Sidak analysis, *, p<0.01. (b) Immunoblot analysis of ventricular FOG-2 expression in 12 wk CREB-S133A Tg mice and NTg littermate controls. GAPDH and calsequestrin (CSQ) are protein loading controls.

Figure 2. Generation and characterization of FOG-2 tran sgenic mice

(a) Northern and (b) Immunoblot analysis comparing organ and ventricular FOG-2 expression in FOG-2-med Tg and NTg littermate control mice. 28S is an RNA loading control. (c) Kaplan-Meier survival analysis of FOG-2-med Tg (○, n=75), FOG-2-high Tg (●, n=46) and NTg littermate control (▲, n=44) mice. Differences in survival rates between the Tg and NTg groups were significant by the log rank test (p<0.05). (d) Representative coronal sections of hearts from NTg littermate control and FOG-2-med Tg mice showed no gross hypertrophy of the ventricles. Scale bar, 100 μm. (e) Hemodynamic analysis of FOG-2-med Tg and NTg littermate control mice. The mean±SEM maximum (top panel) and minimum (bottom panel) first derivative of left ventricular pressure tracings (dP/dt) measured at baseline and at indicated time points following stimulation with isoproterenol. Reductions in both maximum and minimum dP/dt in the Tg (●, n=11) compared with NTg (○, n=9) were noted following isoproterenol. Two-way ANOVA with repeated measures followed by Bonferroni analysis, *, p<0.05 versus NTg.

Figure 3. FOG-2 mice ventricles show fetal gene expression pattern including repression of SERCA2

(a) Fold difference of BNP, βMHC and αMHC and SERCA2 mRNA transcripts, each normalized to GAPDH mRNA in 4–6 wk old FOG-2-high Tg mice (n=4) relative to NTg littermate controls (n=4) measured by qRT-PCR. (b) Immunoblot analysis of SERCA2, CSQ and GAPDH protein in ventricular myocardium from FOG-2-high Tg mice. SERCA2 band density normalized to CSQ. (c) Increased SERCA2 protein and reduced FOG-2 protein seen in FOG-2 heterozygote null ventricles. Unpaired t-test, *, p<0.05 versus NTg.

Figure 4. SERCA2 is a downstream target of FOG-2

(a) Immunoblot analysis of neonatal rat ventricular cardiomyocytes lysates following infection with adenoviruses encoding FOG-2 (Ad-FOG2) or LacZ (Ad-LacZ), at the indicated multiplicity of infection (MOI), and stimulation with vehicle (black bars) or T3 (shaded bars). (b) Densitometry analysis of SERCA2 band density normalized to GAPDH. Increased SERCA2 protein seen in T3-treated cells was reduced with increasing Ad-FOG2 (c) Calsequestrin band density normalized to GAPDH did not change with Ad-FOG-2 infection. (d) qRT- PCR of SERCA2 mRNA normalized to GAPDH was significantly reduced by Ad-FOG-2. Two-way ANOVA followed by Bonferroni analysis, *, p<0.05 versus MOI 0/−T3 control; †, p<0.05 versus MOI 0/+T3 control. All data from at least three independent experiments.

Figure 5. FOG-2 physically interacts with TRα1

(a) Co-immunoprecipitation assay of HEK-293 cells transiently co-transfected with FOG-2 and either TRα1 or myc-tagged TRα1. Cell extracts precipitated with FOG-2 antiserum (right), myc antibody (center) and no antibody (left) were analyzed by immunoblot for FOG-2. Interaction between FOG-2 and myc-tagged TRα1 (lane 4) was specific and not a result of non-specific binding of FOG-2 to beads (lanes 1, 2) or binding of the myc-antibody to FOG-2 (lane 3). (b) GST pull-down assays of [³⁵S] methionine-labeled full-length TRα1, TRα1-carboxy terminus only (TRα1 CT) or TRα2-carboxy terminus only (TRα2 CT) with immobilized GST-FOG-2 fusion proteins. Representative results from three experiments are shown. (c) Transient transfection assay of a TREx2-tk-luciferase reporter in cultured cardiomyocytes. FOG-2 abrogation of T3-mediated TRα1 transactivation of a plasmid is not found with the inactive FOG-2-R3A mutant. Two-way ANOVA followed by Holm-Sidak analysis, * p<0.05, versus vector control/−T3 (black bar); †, p<0.05 versus vector control/+T3 control (shaded bar).

Figure 6. FOG-2 significantly abrogates T3-mediated SERCA2 promoter activity

Luciferase reporter assays performed in cultured cardiomyocytes cotransfected with empty vector or TRα1 with empty vector or FOG-2 followed by treatment for 24h with vehicle or T3. Fold activation is expressed as relative luciferase activity normalized to either the vehicle-treated, empty vector control, or the vehicle-treated FOG-2 control when appropriate. Two-way ANOVA followed by Bonferroni was used for statistical analysis. (a) Co-transfection of FOG-2 expression plasmid abrogated TRα1 transactivation of the 0.6kb-SERCA2 luciferase reporter following T3-stimulation. * p<0.05, versus vector control (color-matched bar). (b) Assays using reporter constructs driven by tk alone or with three tandem repeats of TRE1 or TRE2 from the rat SERCA2 proximal promoter. TRα1 (shaded bars) silenced reporter activity in the absence of T3 and transactivated the reporter with T3 stimulation. FOG-2 abrogated T3-mediated TRα1 transactivation. * p<0.05, versus vector control (black bar). (c) Transactivation of 0.6kb-SERCA2 reporter by TRα1 with indicated doses of T3 (nM) was abrogated by FOG-2. * p<0.05, versus vector control (black bar). Data are from at least three independent experiments done in triplicate.