Increased afterload induces pathological cardiac hypertrophy: a new in vitro model

Increased afterload results in ‘pathological’ cardiac hypertrophy, the most important risk factor for the development of heart failure. Current in vitro models fall short in deciphering the mechanisms of hypertrophy induced by afterload enhancement. The aim of this study was to develop an experimental model that allows investigating the impact of afterload enhancement (AE) on work-performing heart muscles in vitro. Fibrin-based engineered heart tissue (EHT) was cast between two hollow elastic silicone posts in a 24-well cell culture format. After 2 weeks, the posts were reinforced with metal braces, which markedly increased afterload of the spontaneously beating EHTs. Serum-free, triiodothyronine-, and hydrocortisone-supplemented medium conditions were established to prevent undefined serum effects. Control EHTs were handled identically without reinforcement. Endothelin-1 (ET-1)- or phenylephrine (PE)-stimulated EHTs served as positive control for hypertrophy. Cardiomyocytes in EHTs enlarged by 28.4 % under AE and to a similar extent by ET-1- or PE-stimulation (40.6 or 23.6 %), as determined by dystrophin staining. Cardiomyocyte hypertrophy was accompanied by activation of the fetal gene program, increased glucose consumption, and increased mRNA levels and extracellular deposition of collagen-1. Importantly, afterload-enhanced EHTs exhibited reduced contractile force and impaired diastolic relaxation directly after release of the metal braces. These deleterious effects of afterload enhancement were preventable by endothelin-A, but not endothelin-B receptor blockade. Sustained afterload enhancement of EHTs alone is sufficient to induce pathological cardiac remodeling with reduced contractile function and increased glucose consumption. The model will be useful to investigate novel therapeutic approaches in a simple and fast manner. Electronic supplementary material The online version of this article (doi:10.1007/s00395-012-0307-z) contains supplementary material, which is available to authorized users.


SDS-Page/Western-Blot analysis
The middle sections of EHTs (Fig. 1e) were homogenized in lysis buffer (Tris 30 mM at pH 8.8, 3 % SDS, 10 % glycerol, EDTA 5 mM, sodium fluoride 30 mM, and aprotinin 2 mg/L) in a mechanical sample disruption system with stainless steel beads (Qiagen Tissue Lyser). For immunoblotting, aliquots of denatured protein were subjected to SDS-PAGE on a 10 % polyacrylamide gel, and separated proteins were electrophoretically transferred onto nitrocellulose membranes. Nonspecific binding was blocked by incubation with 5 % nonfat dry milk for 2 h at room temperature.

Whole transcriptome analysis
RNA was isolated (see above) from 6 EHTs per group (control, AE, ET-1, PE). According to manufacturer's protocol 250 ng RNA was subjected to first and second strand synthesis, followed by [4] an in vitro transcription (IVT) amplification that incorporated dU-biotin (Illumina TotalPrep Amplification Kit, Ambion). Subsequently cRNA was purified and hybridized on RatRef-12 Expression BeadChips containing 21,910 probes (Illumina BD-27-302). Arrays were washed, blocked, and streptavidin-Cy3 stained for reading on a BeadArray Reader (Illumina). Gene expression levels and sample clustering by correlation were calculated after quantile normalization using GenomeStudio version 1.6.0 software (Illumina). Differentially expressed genes were identified using the Illumina custom error model, multiple testing correction was performed using Benjamini and Hochberg False Discovery Rate. Data-mining was performed with DAVID Bioinformatics Resource 6.7 (NIAID, NIH) according to a recently published protocol [3]. Its functional annotation chart module was utilized to investigate whether KEGG pathways (Kyoto Encyclopedia of Genes and Genomes) were overrepresented in our differentially expressed genes [4].
Up-or downregulated genes in the rat afterload-enhanced EHTs were compared with published gene lists of transverse aortic constriction in mice. Rat gene symbols were converted to mouse gene symbols using the Mouse Genome Database (MGD) from the Mouse Genome Informatics website, The Jackson Laboratory, Bar Harbor, Maine (www.informatics.jax.org; 08/0212) [1]. The mouse TAC I data set was taken from the supplemental data of an article from Toischer et al. [6]. The mouse TAC II data set from Lee et al. [5] was obtained via the Gene Expression Omnibus of the NCBI (accession: GSE 29446). Whenever applicable lists were cured with the DAVID Bioinformatics Resource 6.7.

Glucose consumption
The glucose content of the EHT cell culture medium was measured with a blood gas analyzer (ABL90 FLEX, Radiometer GmbH). Glucose consumption (in 24 hours) of beating EHTs was calculated by subtracting the measured glucose concentration after 24 hours from the known glucose concentration in fresh medium and subsequent multiplying by the medium volume (1.5 mL). [5]

Macroscopic dimensions of EHTs
The CTMV software (Pforzheim, Germany) captured pictures of each analyzed EHT automatically.
Diameters of EHTs were measured from these images at the narrowest point in diastole with the ImageJ 1.44p software (National Institute of Health). Resting lengths of EHTs in diastole were measured identically.

Electron microscopy
EHTs were incubated in 2,3-butanedione monoxime (BDM, Sigma-Aldrich B0753) at 30 mM for 10 minutes to stop cardiac muscle contraction and allow uniform relaxation. After overnight fixation in 0.36 % glutardialdehyde EHTs were postfixed with 1 % osmium tetroxide for 2 h. Dehydration of samples was followed by embedding in epon. Ultrathin sections of 50 nm were prepared, contrasted with uranyl acetate and lead nitrate and examined under a LEO 912AB transmission electron microscope with Omega energy filter (formerly from Zeiss).

Hormone measurements and determination of enzyme activity
Free and total triiodothyronine (T 3 ) concentrations as well as lactate dehydrogenase (LDH) and creatine kinase (CK) enzyme activity were measured in appropriate medium without phenol red. All analyses were performed with routine tests in a Clinical Chemistry Department.

Statistics
Results are presented as mean±SEM. All statistical tests were performed in GraphPad Prism version 5.02. In detail, 1-way ANOVA and Dunnett's multiple comparison post test (to compare to controls) or 1-way ANOVA and Bonferroni's multiple comparison post test (to compare all groups) was used for more than 2 groups, or Student's unpaired t test for 2 groups. Repeated measures 1-way ANOVA followed by Dunnett's multiple comparison post test (to compare to controls) was performed for matched measurements (Online Fig. V). Non-Gaussian data was analyzed by Kruskal-Wallis test and Dunn's multiple comparison post test. P<0.05 or less was considered statistically significant. P-values [6] are displayed graphically as follows: * p<0.05, ** p<0.01, *** p<0.001, ns = not significant.
Quantitative PCR data analyses were carried out using the ΔΔ-Ct method. Online Table I Composition of the reconstitution mix to generate EHTs. Reconstitution mix was prepared on ice and blended carefully. For each EHT 100 µL of it was mixed briefly with 3 µL thrombin (100 U/mL, Sigma-Aldrich T7513) and pipetted into the agarose slot, already containing the tubes of the silicone rack