FHL2 expression and variants in hypertrophic cardiomyopathy

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. Electronic supplementary material The online version of this article (doi:10.1007/s00395-014-0451-8) contains supplementary material, which is available to authorized users.


Plasmid constructs
As starting material for all further cloning, FHL2 cDNA was PCR-amplified with human healthy donor heart cDNA as template. For the production of adeno-associated virus (AAV) serotype 6 (AAV6) with FLAG-tagged FHL2 cDNA a linker-PCR with specific primers containing 5'-CACC-BamHI-Kozak-ATG-FLAG2x and 3'-BsaBI sites in addition to the homologues sequence of FHL2 were used (Supplemental Table 3). After successful amplification and gel electrophoresis, the corresponding PCR fragment was gel-eluted and ligated into the pEF5/FRT/V5-D-TOPO expression kit (Invitrogen). Mutations identified in the present study were introduced into wild-type (WT) FHL2 cDNA via PCR mutagenesis (primers are given in Supplemental Table 4). All cloning steps were verified by sequencing.

Production of adeno-associated virus
The FLAG-tagged FHL2 variants were subcloned into the pdsAAV-CMV-MLC260 vector under the control of the CMV-enhanced myosin light chain-2v promoter for AAV6 production as described previously [1,2]. AAV6 titers ranged from 1.69 to 3.68x10 12 virus genomes per ml (Vg/ml).

Gene expression analysis
RNA from EHTs and NRCMs was isolated using the SV Total RNA Isolation kit. Shortly, NRCMs and EHTs were PBS-washed three times and then further processed using the SV Total RNA Isolation kit according to the manufacturer's instructions. 175 μl of RNA lysis buffer reagent were added to each well/EHT, followed either by scraping the cells or by mechanical tissue lysing of the EHTs with stainless steel beads (Qiagen Tissue Lyser, 2 min, 30 Hz). RNA concentrations and purities were measured by spectrophotometry (NanodropTM ND-1000; Thermo Fisher Scientific Inc.). For NRCMs, 50 ng RNA were transcribed into cDNA using the SuperScript® III™ Reverse Transcriptase kit (Life Technologies) and for EHTs 100 ng RNA were transcribed into cDNA using the SuperScript® VILO™ Reverse Transcriptase kit (Life Technologies) as described previously [3].
For native FHL2 expression analysis, RNA was isolated from powdered human and mouse ventricular samples using the SV Total RNA Isolation kit (Promega) and 75 ng (human) or 200 ng (mouse) transcribed into cDNA using the SuperScript® III Reverse Transcriptase kit (Life Technologies). C t values were normalized to G alpha s (G s ) for mouse and to tumor protein translationally-controlled 1 (TPT1) for human samples.
For Western blot analysis, EHTs were PBS-washed three times and then further processed by mechanical tissue lysing with stainless steel beads (Qiagen Tissue Lyser, 2 min, 30 Hz) in M-Per Mammalian Protein Extraction reagent (Thermo Scientific). Proteins were extracted from powdered human and mouse ventricular samples and Western blotting for human, mouse and EHT proteins was performed as described before [1]. Protein loading was normalized by calsequestrin staining (Dianova, 1:2500), and subsequently membranes were stained with a monoclonal antibody directed against FHL2 (MBL, 1:1000 or 2500) and/or the FLAG epitope (Sigma-Aldrich, 1:5000). Since the FLAG-tag inhibited the antibody detecting the endogenous FHL2, we first incubated with the antibody detecting the native FHL2 and then with the antibody detecting the FLAG-tag. Due to differences in binding affinity of the antibodies no quantification of overexpression was possible.