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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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Evaluation of chronic phenylephrine stimulation on contractile parameters of rat-engineered heart tissue transduced with FHL2 wild-type or variants. Rat EHTs were transduced at day 0 with AAV6 (MOI 1,000) encoding FLAG-tagged FHL2 wild type (WT) or mutants (R177Q, T171M, V187L). On day 13, serum content was reduced to 0 %, on day 14 EHTs were treated with 20 µM phenylephrine (black bars) or without (white bars) for 7 days. Measurements of a force of contraction, b contraction velocity, and c relaxation velocity at day 21 are shown. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT in the same condition, two-way ANOVA followed by Bonferroni’s multiple comparison post test. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. H2O, unpaired Student’s t test. Number of EHTs is indicated in the bars
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Fig4: Evaluation of chronic phenylephrine stimulation on contractile parameters of rat-engineered heart tissue transduced with FHL2 wild-type or variants. Rat EHTs were transduced at day 0 with AAV6 (MOI 1,000) encoding FLAG-tagged FHL2 wild type (WT) or mutants (R177Q, T171M, V187L). On day 13, serum content was reduced to 0 %, on day 14 EHTs were treated with 20 µM phenylephrine (black bars) or without (white bars) for 7 days. Measurements of a force of contraction, b contraction velocity, and c relaxation velocity at day 21 are shown. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT in the same condition, two-way ANOVA followed by Bonferroni’s multiple comparison post test. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. H2O, unpaired Student’s t test. Number of EHTs is indicated in the bars

Mentions: Since expression of FHL2 variants did not induce an exaggerated hypertrophy after gene transfer in cardiac myocytes, we sought to evaluate whether they would affect contraction parameters of EHTs. EHTs were derived from neonatal rat heart cells, transduced or not with AAV6 encoding FHL2 WT or mutants. After 14 days of culture and transduction, EHTs were subjected to PE or control medium for 7 days. Force of contraction did not differ significantly between groups under basal conditions (no PE), although a tendency to higher force was observed in EHTs expressing T171M and V187L mutants (and p < 0.05 vs WT using Student’s t test; Fig. 4a). Furthermore, T171M EHTs exhibited higher velocity of both contraction and relaxation than WT EHTs (and p < 0.01 vs WT using Student’s t test; Fig. 4b–c). V187L EHTs showed a trend to higher contraction velocity (and p < 0.05 vs WT using Student’s t test; Fig. 4b). EHTs transduced with R177Q did not differ to WT EHTs. Chronic PE stimulation reduced all parameters in WT-, R177Q- and V187L-transduced EHTs, but had a blunted effect in T171M-transduced EHTs (Fig. 4a–c). As observed in NRCMs, mRNA levels for Nppa, Nppb, Acta1 and Rcan1.4 did not differ between groups under basal conditions (Online Fig. 2). Chronic PE stimulation induced accumulation of Nppa and Nppb mRNAs in all groups, but had no major effect on Rcan1.4 mRNA level (Online Fig. 2).Fig. 4


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)

Evaluation of chronic phenylephrine stimulation on contractile parameters of rat-engineered heart tissue transduced with FHL2 wild-type or variants. Rat EHTs were transduced at day 0 with AAV6 (MOI 1,000) encoding FLAG-tagged FHL2 wild type (WT) or mutants (R177Q, T171M, V187L). On day 13, serum content was reduced to 0 %, on day 14 EHTs were treated with 20 µM phenylephrine (black bars) or without (white bars) for 7 days. Measurements of a force of contraction, b contraction velocity, and c relaxation velocity at day 21 are shown. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT in the same condition, two-way ANOVA followed by Bonferroni’s multiple comparison post test. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. H2O, unpaired Student’s t test. Number of EHTs is indicated in the bars
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Evaluation of chronic phenylephrine stimulation on contractile parameters of rat-engineered heart tissue transduced with FHL2 wild-type or variants. Rat EHTs were transduced at day 0 with AAV6 (MOI 1,000) encoding FLAG-tagged FHL2 wild type (WT) or mutants (R177Q, T171M, V187L). On day 13, serum content was reduced to 0 %, on day 14 EHTs were treated with 20 µM phenylephrine (black bars) or without (white bars) for 7 days. Measurements of a force of contraction, b contraction velocity, and c relaxation velocity at day 21 are shown. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT in the same condition, two-way ANOVA followed by Bonferroni’s multiple comparison post test. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. H2O, unpaired Student’s t test. Number of EHTs is indicated in the bars
Mentions: Since expression of FHL2 variants did not induce an exaggerated hypertrophy after gene transfer in cardiac myocytes, we sought to evaluate whether they would affect contraction parameters of EHTs. EHTs were derived from neonatal rat heart cells, transduced or not with AAV6 encoding FHL2 WT or mutants. After 14 days of culture and transduction, EHTs were subjected to PE or control medium for 7 days. Force of contraction did not differ significantly between groups under basal conditions (no PE), although a tendency to higher force was observed in EHTs expressing T171M and V187L mutants (and p < 0.05 vs WT using Student’s t test; Fig. 4a). Furthermore, T171M EHTs exhibited higher velocity of both contraction and relaxation than WT EHTs (and p < 0.01 vs WT using Student’s t test; Fig. 4b–c). V187L EHTs showed a trend to higher contraction velocity (and p < 0.05 vs WT using Student’s t test; Fig. 4b). EHTs transduced with R177Q did not differ to WT EHTs. Chronic PE stimulation reduced all parameters in WT-, R177Q- and V187L-transduced EHTs, but had a blunted effect in T171M-transduced EHTs (Fig. 4a–c). As observed in NRCMs, mRNA levels for Nppa, Nppb, Acta1 and Rcan1.4 did not differ between groups under basal conditions (Online Fig. 2). Chronic PE stimulation induced accumulation of Nppa and Nppb mRNAs in all groups, but had no major effect on Rcan1.4 mRNA level (Online Fig. 2).Fig. 4

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