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FHL2 expression and variants in hypertrophic cardiomyopathy.

Friedrich FW, Reischmann S, Schwalm A, Unger A, Ramanujam D, Münch J, Müller OJ, Hengstenberg C, Galve E, Charron P, Linke WA, Engelhardt S, Patten M, Richard P, van der Velden J, Eschenhagen T, Isnard R, Carrier L - Basic Res. Cardiol. (2014)

Bottom Line: HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins.We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT).Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM.

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Effect of phenylephrine or endothelin-1 on hypertrophic parameters after transduction of cardiac myocytes with FHL2 wild-type or variants. a, b Cardiac myocytes were isolated from neonatal rats, transduced with AAV6 (MOI 100,000) encoding FLAG-tagged FHL2 WT or mutants (R177Q, T171M, V187L) and then subjected to phenylephrine (PE; gray bars), endothelin-1 (ET1, black bars) or DMSO (white bars) for 48 h (n = 3, experiments performed in triplicates). Cell area was determined using an automated microscopic edge detection algorithm. c–i Cardiac myocytes were isolated from neonatal rats cardiac myocytes, transduced with AAV6 (MOI 30,000) encoding FLAG-tagged FHL2 WT or mutants (R177Q, T171M, V187L) and then subjected to either 50 µM phenylephrine (black bars) or H2O (white bars) for 48 h (n = 5). c Representative Western blots stained with antibodies directed against endogenous rat FHL2 (only stained with antibody against endogenous FHL2), exogenous FLAG-tagged human FHL2, a membrane showing endogenous FHL2 (lower band) and exogenous FHL2 (stronger FLAG signal above endogenous FHL2), or calsequestrin (protein loading control). Molecular weight marker (MW) indicates 37 and 55 kDa. d Quantification of protein levels of endo- and exogenous FHL2, normalized to calsequestrin, and related to FHL2 NT and WT, respectively (n = 5–6 wells per group). Levels of mRNA of (e) total (with primers detecting rat and human) FHL2,fNppa, gNppb, hActa1, and iRcan1.4. Values are related to non-transduced (NT) cardiac myocytes in basal conditions. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. non-transduced (NT) cells in the same condition, two-way ANOVA followed by Bonferroni’s comparison post test. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. basal conditions, unpaired Student’s t test, for protein quantification one-way ANOVA followed by Dunnett’s post test *p < 0.05 vs. WT. Scale bars 100 µm
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Fig3: Effect of phenylephrine or endothelin-1 on hypertrophic parameters after transduction of cardiac myocytes with FHL2 wild-type or variants. a, b Cardiac myocytes were isolated from neonatal rats, transduced with AAV6 (MOI 100,000) encoding FLAG-tagged FHL2 WT or mutants (R177Q, T171M, V187L) and then subjected to phenylephrine (PE; gray bars), endothelin-1 (ET1, black bars) or DMSO (white bars) for 48 h (n = 3, experiments performed in triplicates). Cell area was determined using an automated microscopic edge detection algorithm. c–i Cardiac myocytes were isolated from neonatal rats cardiac myocytes, transduced with AAV6 (MOI 30,000) encoding FLAG-tagged FHL2 WT or mutants (R177Q, T171M, V187L) and then subjected to either 50 µM phenylephrine (black bars) or H2O (white bars) for 48 h (n = 5). c Representative Western blots stained with antibodies directed against endogenous rat FHL2 (only stained with antibody against endogenous FHL2), exogenous FLAG-tagged human FHL2, a membrane showing endogenous FHL2 (lower band) and exogenous FHL2 (stronger FLAG signal above endogenous FHL2), or calsequestrin (protein loading control). Molecular weight marker (MW) indicates 37 and 55 kDa. d Quantification of protein levels of endo- and exogenous FHL2, normalized to calsequestrin, and related to FHL2 NT and WT, respectively (n = 5–6 wells per group). Levels of mRNA of (e) total (with primers detecting rat and human) FHL2,fNppa, gNppb, hActa1, and iRcan1.4. Values are related to non-transduced (NT) cardiac myocytes in basal conditions. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. non-transduced (NT) cells in the same condition, two-way ANOVA followed by Bonferroni’s comparison post test. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. basal conditions, unpaired Student’s t test, for protein quantification one-way ANOVA followed by Dunnett’s post test *p < 0.05 vs. WT. Scale bars 100 µm

Mentions: It has been shown that FHL2 overexpression reduced PE-induced hypertrophy in cardiac myocytes [44], suggesting an antihypertrophic role of FHL2. Therefore, we sought to evaluate whether FHL2 mutants would act differently. NRCMs were transduced with AAV6 encoding FLAG-tagged FHL2 WT or mutants, and treated with PE or ET1 for 48 h. Cell area was determined using an automated microscopic edge detection algorithm and mRNA levels of FHL2 and markers of hypertrophy were quantified by RT-qPCR (Fig. 3). Under basal conditions, cell area did not differ between the groups (Fig. 3a, b), although total FHL2 (exogenous human and endogenous rat) mRNA levels were ~two- to fourfold higher in FHL2-transduced NRCMs (Fig. 3e). To evaluate an overexpression on protein level, we used an antibody detecting the endogenous, native FHL2 directed against the N-terminal regions and an antibody only recognizing the exogenous FLAG-tagged FHL2. Staining with the FHL2 antibody revealed only one band in all samples (Fig. 3c). This suggests that the FHL2 antibody only recognized the endogenous FHL2 protein, likely because of the presence of the FLAG tag at the N-terminal end, which could hinder the binding of the FHL2 antibody. The endogenous FHL2 level did not differ significantly between the groups (Fig. 3d). We then stained the membranes with the antibody detecting the FLAG tag. Exogenous FLAG-tagged FHL2 was overexpressed in all groups (except for NT) with some slight difference (Fig. 3c, d). Due to differences in binding affinities of the antibodies no quantification of overexpression (endogenous + exogenous FHL2) was possible. Furthermore, mRNA levels of atrial natriuretic peptide (Nppa), brain natriuretic peptide (Nppb) and the NFAT-target gene regulator of calcineurin (Rcan1.4) did not differ between all groups, whereas α-skeletal actin (Acta1) mRNA levels were ~90 % lower in FHL2-transduced than non-transduced NRCMs (Fig. 3f–i). Both PE and ET1 increased cardiac myocyte area in all groups, but to a lower extent in FHL2-transduced than non-transduced NRCMs (Fig. 3b). This was associated with a lower activation of Acta1 gene expression in FHL2-transduced NRCMs. Conversely, the PE-induced increased mRNA levels of Nppa, Nppb and Rcan1.4 did not differ between the groups, except for a partial inhibition on Rcan1.4 in T171M-transduced NRCMs (Fig. 3f–i). The amount of overexpressed FHL2 was also lower in PE-treated NRCMs. These data suggest that FHL2 WT has an antihypertrophic effect and FHL2 mutants do not lose this feature.Fig. 3


FHL2 expression and variants in hypertrophic cardiomyopathy.

Friedrich FW, Reischmann S, Schwalm A, Unger A, Ramanujam D, Münch J, Müller OJ, Hengstenberg C, Galve E, Charron P, Linke WA, Engelhardt S, Patten M, Richard P, van der Velden J, Eschenhagen T, Isnard R, Carrier L - Basic Res. Cardiol. (2014)

Effect of phenylephrine or endothelin-1 on hypertrophic parameters after transduction of cardiac myocytes with FHL2 wild-type or variants. a, b Cardiac myocytes were isolated from neonatal rats, transduced with AAV6 (MOI 100,000) encoding FLAG-tagged FHL2 WT or mutants (R177Q, T171M, V187L) and then subjected to phenylephrine (PE; gray bars), endothelin-1 (ET1, black bars) or DMSO (white bars) for 48 h (n = 3, experiments performed in triplicates). Cell area was determined using an automated microscopic edge detection algorithm. c–i Cardiac myocytes were isolated from neonatal rats cardiac myocytes, transduced with AAV6 (MOI 30,000) encoding FLAG-tagged FHL2 WT or mutants (R177Q, T171M, V187L) and then subjected to either 50 µM phenylephrine (black bars) or H2O (white bars) for 48 h (n = 5). c Representative Western blots stained with antibodies directed against endogenous rat FHL2 (only stained with antibody against endogenous FHL2), exogenous FLAG-tagged human FHL2, a membrane showing endogenous FHL2 (lower band) and exogenous FHL2 (stronger FLAG signal above endogenous FHL2), or calsequestrin (protein loading control). Molecular weight marker (MW) indicates 37 and 55 kDa. d Quantification of protein levels of endo- and exogenous FHL2, normalized to calsequestrin, and related to FHL2 NT and WT, respectively (n = 5–6 wells per group). Levels of mRNA of (e) total (with primers detecting rat and human) FHL2,fNppa, gNppb, hActa1, and iRcan1.4. Values are related to non-transduced (NT) cardiac myocytes in basal conditions. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. non-transduced (NT) cells in the same condition, two-way ANOVA followed by Bonferroni’s comparison post test. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. basal conditions, unpaired Student’s t test, for protein quantification one-way ANOVA followed by Dunnett’s post test *p < 0.05 vs. WT. Scale bars 100 µm
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Fig3: Effect of phenylephrine or endothelin-1 on hypertrophic parameters after transduction of cardiac myocytes with FHL2 wild-type or variants. a, b Cardiac myocytes were isolated from neonatal rats, transduced with AAV6 (MOI 100,000) encoding FLAG-tagged FHL2 WT or mutants (R177Q, T171M, V187L) and then subjected to phenylephrine (PE; gray bars), endothelin-1 (ET1, black bars) or DMSO (white bars) for 48 h (n = 3, experiments performed in triplicates). Cell area was determined using an automated microscopic edge detection algorithm. c–i Cardiac myocytes were isolated from neonatal rats cardiac myocytes, transduced with AAV6 (MOI 30,000) encoding FLAG-tagged FHL2 WT or mutants (R177Q, T171M, V187L) and then subjected to either 50 µM phenylephrine (black bars) or H2O (white bars) for 48 h (n = 5). c Representative Western blots stained with antibodies directed against endogenous rat FHL2 (only stained with antibody against endogenous FHL2), exogenous FLAG-tagged human FHL2, a membrane showing endogenous FHL2 (lower band) and exogenous FHL2 (stronger FLAG signal above endogenous FHL2), or calsequestrin (protein loading control). Molecular weight marker (MW) indicates 37 and 55 kDa. d Quantification of protein levels of endo- and exogenous FHL2, normalized to calsequestrin, and related to FHL2 NT and WT, respectively (n = 5–6 wells per group). Levels of mRNA of (e) total (with primers detecting rat and human) FHL2,fNppa, gNppb, hActa1, and iRcan1.4. Values are related to non-transduced (NT) cardiac myocytes in basal conditions. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. non-transduced (NT) cells in the same condition, two-way ANOVA followed by Bonferroni’s comparison post test. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. basal conditions, unpaired Student’s t test, for protein quantification one-way ANOVA followed by Dunnett’s post test *p < 0.05 vs. WT. Scale bars 100 µm
Mentions: It has been shown that FHL2 overexpression reduced PE-induced hypertrophy in cardiac myocytes [44], suggesting an antihypertrophic role of FHL2. Therefore, we sought to evaluate whether FHL2 mutants would act differently. NRCMs were transduced with AAV6 encoding FLAG-tagged FHL2 WT or mutants, and treated with PE or ET1 for 48 h. Cell area was determined using an automated microscopic edge detection algorithm and mRNA levels of FHL2 and markers of hypertrophy were quantified by RT-qPCR (Fig. 3). Under basal conditions, cell area did not differ between the groups (Fig. 3a, b), although total FHL2 (exogenous human and endogenous rat) mRNA levels were ~two- to fourfold higher in FHL2-transduced NRCMs (Fig. 3e). To evaluate an overexpression on protein level, we used an antibody detecting the endogenous, native FHL2 directed against the N-terminal regions and an antibody only recognizing the exogenous FLAG-tagged FHL2. Staining with the FHL2 antibody revealed only one band in all samples (Fig. 3c). This suggests that the FHL2 antibody only recognized the endogenous FHL2 protein, likely because of the presence of the FLAG tag at the N-terminal end, which could hinder the binding of the FHL2 antibody. The endogenous FHL2 level did not differ significantly between the groups (Fig. 3d). We then stained the membranes with the antibody detecting the FLAG tag. Exogenous FLAG-tagged FHL2 was overexpressed in all groups (except for NT) with some slight difference (Fig. 3c, d). Due to differences in binding affinities of the antibodies no quantification of overexpression (endogenous + exogenous FHL2) was possible. Furthermore, mRNA levels of atrial natriuretic peptide (Nppa), brain natriuretic peptide (Nppb) and the NFAT-target gene regulator of calcineurin (Rcan1.4) did not differ between all groups, whereas α-skeletal actin (Acta1) mRNA levels were ~90 % lower in FHL2-transduced than non-transduced NRCMs (Fig. 3f–i). Both PE and ET1 increased cardiac myocyte area in all groups, but to a lower extent in FHL2-transduced than non-transduced NRCMs (Fig. 3b). This was associated with a lower activation of Acta1 gene expression in FHL2-transduced NRCMs. Conversely, the PE-induced increased mRNA levels of Nppa, Nppb and Rcan1.4 did not differ between the groups, except for a partial inhibition on Rcan1.4 in T171M-transduced NRCMs (Fig. 3f–i). The amount of overexpressed FHL2 was also lower in PE-treated NRCMs. These data suggest that FHL2 WT has an antihypertrophic effect and FHL2 mutants do not lose this feature.Fig. 3

Bottom Line: HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins.We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT).Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM.

Show MeSH
Related in: MedlinePlus