Serine biosynthesis as a novel therapeutic target for dilated cardiomyopathy

Abstract

Aims: Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro.

Methods and results: Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions.

Conclusions: A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.

Keywords: Cardiomyocytes; Clinical-trial-in-a-dish; Dilated cardiomyopathy; Drug screening; Induced pluripotent stem cells; Phenotypic screens; Precision medicine.

Structured Graphical Abstract

Activation of serine biosynthesis pathway with a dual kinase inhibitor treatment rescues DCM contraction deficit. A kinase inhibitor screening was conducted in iPSC-derived cardiomyocytes, and the resulting hits identified were combined into one single treatment, PPi, that improved the contractile response of the cells. Mechanistically, PPi activated the serine biosynthesis pathway, translating in turn into a more efficient mitochondrial respiration and energy production. Finally, PPi rescued DCM phenotype in multiple gene mutations associated to DCM.

Figure 1

Generation and validation of the dilated cardiomyopathy in vitro model using induced pluripotent stem cell-cardiomyocytes. (A) Genome editing approaches targeting the TNNT2 c.547C > T mutation. (B) Sanger sequencing of the three isogenic lines: patient heterozygous mutant (TNNT2^HET), genome-edited homozygous mutant (TNNT2^HOM), and gene-corrected line (TNNT2^CORR). (C) High-throughput contractility analysis using vector motion mapping. (D) Representative contractility traces of isogenic induced pluripotent stem cell-cardiomyocytes. (E) Contraction amplitude analyses. Mean ± standard deviation, n = 17–44, three differentiation batches per genotype. (F) Schematics of the 3D-engineered heart tissue contractility analysis. (G) Representative force profile of the isogenic 3D-engineered heart tissues. (H) Quantitation of force generation by the 3D-engineered heart tissues. Mean ± standard deviation. n = 12–24 from four differentiation batches.

Figure 2

High-throughput phenotypic drug screening in induced pluripotent stem cell-cardiomyocytes. (A) Schematic of high-throughput kinase inhibitor screen in dilated cardiomyopathy TNNT2^HOM induced pluripotent stem cell-cardiomyocytes. Cells were plated in 384-well plates and treated with a chemical kinase inhibitor library (160 compounds), and contractility was measured with automated kinetic imaging. Hits were further validated in micropatterned single-cell induced pluripotent stem cell-cardiomyocytes and 3D-engineered heart tissues. (B) Kinase inhibitor screen scatter plot: peak amplitude is plotted on the y-axis against 160 corresponding kinase inhibitors on the x-axis. The dashed lines represent 2.5 SDs from the vehicle control mean (solid line). The two hit compounds identified (Gö 6976 and SB 203580) are indicated; and assay controls (OM and Myk-461) are also shown. The screen was performed in duplicate plates. (C) Relative contraction amplitude and (D) relaxation time of TNNT2^HOM induced pluripotent stem cell-cardiomyocytes treated for 72 h with the two hit compounds, Gö 6976, SB 203580, alone, or in combination (PPi). For comparison, TNNT2^HOM induced pluripotent stem cell-cardiomyocytes treated with OM, and untreated TNNT2^CORR induced pluripotent stem cell-cardiomyocytes are shown. Mean ± standard deviation from three independent differentiation batches. (E and F) Representative vector motion maps of micropatterned TNNT2^HOM induced pluripotent stem cell-cardiomyocytes treated with vehicle control (Control) or PPi for 72 h (scale bar = 20 μm) (n = 14 and 17, respectively). Two independent differentiation batches. (G) Total forces (ΣF) of micropatterned single TNNT2^HOM induced pluripotent stem cell-cardiomyocytes. (H) Representative 3D-engineered heart tissues contractile force traces and (I) 3D-engineered heart tissues contraction force, relative to baseline (ΔForce) of each engineered heart tissue. Box-and-whisker plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum from three independent differentiation batches.

Figure 3

PPi activates the de novo serine biosynthesis pathway. (A) Heat map illustrating levels of expression of the top 30 differentially expressed genes in TNNT2^HOM and TNNT2^HET induced pluripotent stem cell-cardiomyocytes after PPi treatment vs. vehicle control (DMSO) (False discovery rate [FDR] < 0.05). (B) Gene Ontology biological processes enrichment analysis of the upregulated transcripts. (C) Graphical representation of enzymes and metabolites of the de novo serine biosynthesis pathway and their integration in cellular metabolism. Genes significantly upregulated in dilated cardiomyopathy-induced pluripotent stem cell-cardiomyocytes upon PPi treatment are indicated throughout the pathway. (D) Schematic showing expected labelling from carbon flow from glucose to serine and glycine when labelled with [¹³C₆]-glucose. (E and F) Abundance of [¹³C₆]-glucose-derived serine and glycine in TNNT2^HET induced pluripotent stem cell-cardiomyocytes cultured with [¹³C₆]-glucose for 72 h post-treatment with vehicle control (Ctrl) or PPi. Vehicle control-treated TNNT2^CORR induced pluripotent stem cell-cardiomyocytes are also shown for comparison. Data represent mean ± standard deviation, n = 9–18 replicates per condition, two independent labelling experiments. (G) Relative contractility of siRNA control- or siPHGDH-transfected TNNT2^HET induced pluripotent stem cell-cardiomyocytes treated with PPi or vehicle control (Ctrl). Box-and-whisker plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. n = 9–12. (H) Relative contractility of siRNA control- or siATF4-transfected TNNT2^HET induced pluripotent stem cell-cardiomyocytes treated with PPi or vehicle control (Ctrl). Box-and-whisker plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. n = 9–12. (I) Relative expression of ATF4 and PHGDH in siRNA control- or siATF4-transfected TNNT2^HET induced pluripotent stem cell-cardiomyocytes treated with PPi or vehicle control (Ctrl). Mean ± standard deviation, n = 6–12. 1-C, one-carbon; 3PG, 3-phosphoglycerate; a-KG, alpha-ketoglutarate; ALDH1L2, aldehyde dehydrogenase 1 family member L2; CHAC1, glutathione-specific gamma-glutamylcyclotransferase 1; Gly, glycine; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; PCK2, phosphoenolpyruvate carboxykinase 2, mitochondrial; PEP, phosphoenolpyruvate; PHGDH, phosphoglycerate dehydrogenase; PSAT1, phosphoserine aminotransferase 1; PSPH, phosphoserine phosphatase; Pyr, pyruvate; Ser, serine; SHMT2, serine hydroxymethyltransferase 2; TCA, tricarboxylic acid.

Figure 4

PPi rescues the mitochondrial dysfunction of TNNT2 mutant induced pluripotent stem cell-cardiomyocytes. (A) Effect of PPi on mitochondrial function in TNNT2 mutant and isogenic control induced pluripotent stem cell-cardiomyocytes. Mitochondrial function was measured by extracellular flux analysis. OCR, cellular oxygen consumption rate. (B and C) Quantitation of mitochondrial functional parameters from (A). Mean ± standard deviation, n = 14–16 replicates per line, three independent differentiation batches. (D) Schematic showing expected labelling of carbon flow from glucose to tricarboxylic acid cycle intermediates when labelled with [¹³C₆]-glucose. (E–G) Abundance of [¹³C₆]-glucose-derived α-ketoglutarate, succinate, and citrate in TNNT2^HET induced pluripotent stem cell-cardiomyocytes cultured in the presence of [¹³C₆]-glucose with vehicle control (Ctrl) or PPi. Vehicle control-treated TNNT2^CORR induced pluripotent stem cell-cardiomyocytes is also shown for comparison. Data represent mean ± standard deviation, n = 9–18 replicates per condition, two independent labelling experiments.

Figure 5

The pseudokinase TRIB3 contributes to the beneficial effects of PPi. (A) Gene set enrichment analysis enrichment plot for mammalian target of rapamycin signalling pathway in TNNT2^HOM induced pluripotent stem cell-cardiomyocytes. (B) Contractility analysis of TNNT2^HOM induced pluripotent stem cell-cardiomyocytes were treated with PPi in the presence of mammalian target of rapamycin inhibitors everolimus and rapamycin; n = 8–16 replicates from three independent differentiation batches. Box-and-whisker plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. (C) Relative contractility of siRNA control- or siTRIB3-transfected TNNT2^HET induced pluripotent stem cell-cardiomyocytes treated with PPi or vehicle control (Ctrl). n = 9–12 replicates from three independent differentiation batches. Box-and-whisker plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. (D) Relative mRNA expression of ATF4, PHGDH, and TRIB3 in siRNA control, siTRIB3- or siATF4- transfected TNNT2^HET induced pluripotent stem cell-cardiomyocytes treated with PPi or vehicle control (Ctrl). Mean ± standard deviation, n = 6–12.

Figure 6

PPi rescues the contractility deficit of dilated cardiomyopathy-induced pluripotent stem cell-cardiomyocytes harbouring pathogenic mutations from diverse gene ontologies. (A) Human induced pluripotent stem cells were derived from five dilated cardiomyopathy patients carrying pathogenic mutations in TTN, PLN, LMNA, TPM1, and LAMA2 genes, and two healthy controls. (B–I) Relative contractility analysis of dilated cardiomyopathy-induced pluripotent stem cell-cardiomyocytes treated with PPi or vehicle control (Control). Box-and-whisker plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum; n = 6–16 replicates per line from three independent differentiation batches.