An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility

Abstract

Background: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell-derived cardiomyocytes.

Methods: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward.

Results: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding.

Conclusion: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.

Trial registration: ClinicalTrials.gov NCT02417311.

Keywords: cardiac hypertrophy; epigenetics; tissue engineering.

Conflict of interest statement

Drs Eschenhagen and Hirt are cofounders of EHT Technologies GmbH, Hamburg. The other authors report no conflicts.

Figures

Figure 1.

Contraction kinetics of DNA methyltransferase(DNMT) 3A knockoutengineered heart tissue (EHT). A, Normalized average contraction peaks of EHT from wild-type and knockout lines at a stimulation frequency of 1.5 Hz. Mean±SEM. B, Relaxation times from peak to 80% relaxation at 1.5 Hz. n=7-12 EHTs per group. C, Ratio of myosin heavy chain (MHC) isoforms in wild-type and DNMT3A−/− EHTs. MYH7 and MYH6 abundance was measured by NanoString analysis, n=3. D, Ratio of atrial (MYL7, MLC2A) and ventricular myosin light chain (MYL2, MLC2V) isoforms, measured by RNA sequencing. n=3. E, Relative expression of atrial-specific marker genes. n=3, False discovery rate- (FDR-) adjusted P value. F, PITX2 promoter methylation, measured by reduced representation bisulfite sequencing. n=3–4. G, Pacing capture of wild-type and knockout EHT at frequencies from 0.7 Hz to 6 Hz, beating frequency of each EHT line plotted against the stimulation frequency. Each dot represents a single EHT measurement. ***P<0.001.

Figure 2.

Contraction behavior in serum-free culture conditions. Force development (A) and RR scatter (B) as a surrogate parameter for beating arrhythmias, with R being the interval between 2 neighboring force peaks, of wild-type and (C–D) knockout engineered heart tissue (EHT) over culture time under both standard serum-containing (green plus) and serum-free culture conditions (red minus). The dotted line marks beginning of serum withdrawal. Mean±SEM, n=51–58 EHTs from 3 batches. Representative contraction peaks of wild-type (E) and knockout EHTs (F) under serum-free conditions.

Figure 3.

Gene expression changes in homozygous DNA methyltransferase(DNMT) 3A knockout engineered heart tissue(EHT). Transcript abundance of (A) low-expression genes and (B) high-expression genes in DNMT3A−/− EHTs. All values normalized to wild-type (dashed line); n=3 per group, **P<0.001, ***P<0.001. C, Pathway mapping of significantly differentially expressed genes in DNMT3A−/− vs wild-type. Dotted line represents significance threshold of P<0.05. Red, cardiac-related; blue, signaling-related; yellow, metabolism-related pathways.

Figure 4.

Morphology of DNA methyltransferase(DNMT) 3A knockout engineered heart tissue(EHT). Histological analysis of (A) wild-type and (B) DNMT3A−/− EHTs. Left, Hematoxylin and eosin staining (H&E); scale bar, 50 µm. Middle, MLC2V staining; scale bar, 50 µm. Right, Lipid staining with Oil Red O; scale bar, 500 µm, red dots mark lipid accumulations. Transmission electron microscopy of wild-type (C) and knockout (D) EHTs showing intact sarcomeric structures but degenerated mitochondria in knockout EHT. Yellow arrowheads, sarcomeres; black arrowheads, mitochondria. Scale bar, 500 nm.

Figure 5.

Upregulation ofperoxisome proliferator-activated receptor gamma (PPARγ) is responsible for lipid accumulation inengineered heart tissue (EHT). A, Normalized PPARγ transcripts in wild-type and DNA methyltransferase (DNMT) 3A−/− EHTs. n=3 EHTs per group, ***P<0.001. B, PPARγ promoter methylation in wild-type and DNMT3A−/− EHTs measured by bisulfite sequencing. Black dot, methylated cytosine; white dot, unmethylated cytosine. C, Hematoxylin and eosin staining (H&E) staining of vehicle (left) and PPARγ-inhibitor GW9662–treated DNMT3A−/− EHTs (right); scale bar, 50 µm. D, H&E staining of DNMT3A−/− EHTs cultured for 4 weeks in serum-containing conditions; scale bar, 50 µm.

Figure 6.

Effect of DNA methyltransferase(DNMT) 3A knockout on engineered heart tissue(EHT) glucose metabolism. Effect of restrictive feeding with lactate or glucose-only medium on frequency (A), force (B), and RR scatter, with R being the interval between 2 neighboring force peaks, (C) of wild-type and knockout EHTs. n=8, *P<0.05, ***P<0.001. D, Relative transcript abundance of glycolytic genes in DNMT3A−/− #2 EHTs after 1 week of serum-free culture. n=3, values normalized to wild-type (dashed line). E and F, Quantification of hypoxia-inducible factor 1α (HIF-1α) protein by Western blot of DNMT3A−/− #1 and isogenic control EHTs cultured either with lipoprotein-deficient serum (LDS), control serum, or dialyzed serum. Loading control = ERK1/2; n=2 to 3 EHTs per group.

Figure 7.

Effect of growth factor treatment of engineered heart tissue(EHT) under serum-free conditions. A, Effect of growth factor treatment (GFs; 20 ng/mL transforming growth factor [TGF]-β1, 10 ng/mL basic fibroblast growth factor [bFGF], 20 ng/mL insulin-like growth factor 1 [IGF-1], 10 ng/mL endothelial growth factor [EGF]) on force under serum-free conditions. n=10–15 EHTs from 2 batches per group. B and C, Quantification of hypoxia-inducible factor 1α (HIF-1α) protein by Western blot of wild-type and DNA methyltransferase (DNMT) 3A−/− EHTs cultured either with serum-free medium or with serum-free medium containing GFs. Loading control = ERK1/2; n=3 EHTs per group. D, Relative transcript abundance of glycolytic genes in DNMT3A−/− EHTs after 1 week of serum-free culture ± GFs. n=3, values normalized to wild-type (dashed line). Effect of GF treatment on glucose consumption (E) and lactate generation (F) of wild-type and DNMT3A−/− EHTs. n=4, values normalized to wild-type (dashed line). ns indicates nonsignificant, *P<0.05, **P<0.01, ***P<0.001.

Figure 8.

Overview key experiments and findings. Dashed boxes indicate key experimental findings; colored boxes indicate conclusions drawn from experimental findings.