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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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Immunofluorescence analysis of rat-engineered heart tissue transduced with FHL2 wild type or variants. EHTs were transduced with AAV6 encoding FLAG-tagged FHL2 wild type (WT) or mutants (R177Q, T171M, V187L) at a MOI of 1,000. After fixation at day 14, EHTs were stained with antibodies directed against the FLAG epitope (green) and cardiac myosin-binding protein C (cMyBP-C, red). Scale bars 10 µm
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Fig5: Immunofluorescence analysis of rat-engineered heart tissue transduced with FHL2 wild type or variants. EHTs were transduced with AAV6 encoding FLAG-tagged FHL2 wild type (WT) or mutants (R177Q, T171M, V187L) at a MOI of 1,000. After fixation at day 14, EHTs were stained with antibodies directed against the FLAG epitope (green) and cardiac myosin-binding protein C (cMyBP-C, red). Scale bars 10 µm

Mentions: Under basal conditions, total FHL2 mRNA levels were ~twofold to threefold higher in FHL2-transduced EHTs than in non-transduced EHTs, suggesting a slight overexpression (Online Fig. 3). As in NRCMs, staining with the FHL2 antibody revealed only one band in all samples (Online Fig. 3b). The endogenous FHL2 level did not differ significantly between the groups (Online Fig. 3b, c). FLAG-tagged FHL2 proteins were detected by Western blot only in FHL2-transduced EHTS, but no major difference was observed between groups (Online Fig. 3b, c). Due to differences in binding affinities of the antibodies no quantification of total (endogenous + exogenous FHL2) was possible. Immunofluorescence analysis revealed a striated pattern for FLAG-FHL2 WT, R177Q, T171M and V187L proteins, mainly in alternation with cMyBP-C doublets, but also within the cMyBP-C doublets, suggesting an additional M-band incorporation (Fig. 5).Fig. 5


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)

Immunofluorescence analysis of rat-engineered heart tissue transduced with FHL2 wild type or variants. EHTs were transduced with AAV6 encoding FLAG-tagged FHL2 wild type (WT) or mutants (R177Q, T171M, V187L) at a MOI of 1,000. After fixation at day 14, EHTs were stained with antibodies directed against the FLAG epitope (green) and cardiac myosin-binding protein C (cMyBP-C, red). Scale bars 10 µm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4215105&req=5

Fig5: Immunofluorescence analysis of rat-engineered heart tissue transduced with FHL2 wild type or variants. EHTs were transduced with AAV6 encoding FLAG-tagged FHL2 wild type (WT) or mutants (R177Q, T171M, V187L) at a MOI of 1,000. After fixation at day 14, EHTs were stained with antibodies directed against the FLAG epitope (green) and cardiac myosin-binding protein C (cMyBP-C, red). Scale bars 10 µm
Mentions: Under basal conditions, total FHL2 mRNA levels were ~twofold to threefold higher in FHL2-transduced EHTs than in non-transduced EHTs, suggesting a slight overexpression (Online Fig. 3). As in NRCMs, staining with the FHL2 antibody revealed only one band in all samples (Online Fig. 3b). The endogenous FHL2 level did not differ significantly between the groups (Online Fig. 3b, c). FLAG-tagged FHL2 proteins were detected by Western blot only in FHL2-transduced EHTS, but no major difference was observed between groups (Online Fig. 3b, c). Due to differences in binding affinities of the antibodies no quantification of total (endogenous + exogenous FHL2) was possible. Immunofluorescence analysis revealed a striated pattern for FLAG-FHL2 WT, R177Q, T171M and V187L proteins, mainly in alternation with cMyBP-C doublets, but also within the cMyBP-C doublets, suggesting an additional M-band incorporation (Fig. 5).Fig. 5

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