Lack of MTTP Activity in Pluripotent Stem Cell-Derived Hepatocytes and Cardiomyocytes Abolishes apoB Secretion and Increases Cell Stress

Abstract

Abetalipoproteinemia (ABL) is an inherited disorder of lipoprotein metabolism resulting from mutations in microsomal triglyceride transfer protein (MTTP). In addition to expression in the liver and intestine, MTTP is expressed in cardiomyocytes, and cardiomyopathy has been reported in several ABL cases. Using induced pluripotent stem cells (iPSCs) generated from an ABL patient homozygous for a missense mutation (MTTP^R46G), we show that human hepatocytes and cardiomyocytes exhibit defects associated with ABL disease, including loss of apolipoprotein B (apoB) secretion and intracellular accumulation of lipids. MTTP^R46G iPSC-derived cardiomyocytes failed to secrete apoB, accumulated intracellular lipids, and displayed increased cell death, suggesting intrinsic defects in lipid metabolism due to loss of MTTP function. Importantly, these phenotypes were reversed after the correction of the MTTP^R46G mutation by CRISPR/Cas9 gene editing. Together, these data reveal clear cellular defects in iPSC-derived hepatocytes and cardiomyocytes lacking MTTP activity, including a cardiomyocyte-specific regulated stress response to elevated lipids.

Keywords: abetaliproteinemia; apoB; cardiac stress; iPSC-derived hepatocytes and cardiomyocytes; induced pluripotent stem cells; lipid accumulation.

Figure 1. Abolished apoB secretion in mutant MTTP^R46G differentiated hepatocytes

(A and B) Quantification of messenger RNA MTTP and MTTP protein by real-time PCR and Western blot, respectively. (C) Microsomal triglyceride transfer activity was measured. (D and E) Quantification of messenger RNA and protein of apoB 100 by real-time PCR and Western blot, respectively. (F) ApoB protein secreted into media after a 16 hour incubation was measured by ELISA. (G) Newly synthesized cellular apoB was analyzed by immunoprecipitation and SDS-PAGE after a 20 minute label with [³⁵S]methionine/cysteine in the presence or absence of ALLN. (H) Quantification of newly synthesized apoB protein with normalization to albumin after 20 min pulse with [³⁵S]methionine/cysteine. (I) Pulse-chase of newly synthesized apoB for 30min, 60min, and 120min. Graph shows the percentage relative to initial amount of apoB at the end of the label. ±S.D. *P<0.05 **P<0.01. Values are means for three independent experiments.

Figure 2. Accumulated lipid droplets in mutant MTTP^R46G differentiated hepatocytes

(A) Representative images of lipid droplets stained with Oil Red O. Yellow arrowheads indicate red positive lipid stains. Hematoxylin stains the nuclei blue. (B and C) Cellular triglyceride (TG) and total cholesterol (TC) content was determined by enzymatic assays and normalized to total cellular protein as measured by BCA assay. (D and E) Cells were incubated with [³H]-OA(5 uCi/ml)+0.8mM OA for 4 hours. Cellular and medium TG were extracted, separated by TLC, and quantified by liquid scintillation counting. TG counts are normalized to protein contents determined by Lowry protein assay. ± S.D. (n=4) *P<0.05 Values are means for three independent experiments. Scale bar: 400 μm

Figure 3. Correction of C136G in MTTP rescues the ABL phenotype

(A) Schematic strategy for correction of C136G in MTTP by CRISPR/Cas9. (B) iPSC from ABL patient was transfected with plasmids containing guide RNA and Cas9. Genomic DNA was extracted from GFP⁺ colonies and subjected to PCR amplification. Subsequent DpnII digestion was applied to identify the positively targeted clones. (C) The corrected iPSC lines were tested for expression of pluripotency markers by real-time PCR. (D) Expression of hepatic genes was analyzed by real-time PCR in hepatocytes derived from the corrected iPSC lines. (E–G) Amount of newly synthesized apoB in the cell or secreted in the medium was measured at the end of a 2-hour label with [³⁵S]methionine/cysteine. F is a Western blot for apoB in the media. (H) Cellular lipid accumulation by Oil Red O staining following rescue of the C136G MTTP mutation by CRISPR/Cas9. Scale bar: 400 μm ± S.D. *P<0.05. **P<0.01 Values are means for three independent experiments.

Figure 4. Abolished apoB and elevated lipid storage upon fatty acids treatment in cardiomyocytes was rescued by correction of MTTP^R46G

(A and B) ApoB transcript and medium apoB protein upon fatty acids treatment were quantified by real-time PCR and ELISA, respectively. Cells were incubated with Oleic Acid (OA: 1mM) or Palmitic Acid (PA: 0.5mM) for 48 hours. (C) Representative images of neutral lipid staining in cardiomyocytes. Cardiac Troponin T stains cardiac muscle fiber proteins as green. Nile Red stains lipid droplet as red. DAPI stains nuclei as blue. (D) Mean Fluorescence Intensity of Nile Red was analyzed by ImageJ. Fold relative to control is shown. (E and F) Newly synthesized cellular TG content is quantified in cardiomyocytes (E) and in the culture media (F) following a 24 hour label with [¹⁴C] oleic acid and lipid extraction. TG counts are normalized to protein contents determined by Lowry protein assay ± S.D. *P<0.05, Values are means for three independent experiments. Scale bar: 25 μm

Figure 5. MTTP^R46G cardiomyocytes are hypersensitive to metabolic stresses

(A) Representative images showing sunitinib (15 μM) and PA (0.5mM) induced apoptosis as visualized by TUNEL positive cells (green). DAPI stains nuclei blue. (B) Percentage of sunitinib induced TUNEL positivity was quantified by cell counting using ImageJ (>1000 nuclei were counted per genotype). (C) Representative images showing expression of cleaved Caspase 3 in sunitinib and PA treated cardiomyocytes. Troponin T (green), Cleaved caspase 3 (red), and DAPI (blue). (D) Mean fluorescence density for cleaved Caspase 3 was quantified using ImageJ. Fold change relative to control is shown. (E) Quantification of TUNEL positivity in response to Hypoxia/Reoxygenation and PA. (F) Representative images showing expression of cleaved Caspase 3 in hypoxia/reoxygenation and PA treated cardiomyocytes. Troponin T (white), Cleaved caspase 3 (green), Nile Red (red), and DAPI (blue). Graph to the right shows mean fluorescence density for cleaved Caspase 3 was quantified using ImageJ. Fold change relative to control is shown. *P<0.05. Values are means for three independent experiments ± S.D. Scale bar: A=150 μm, C=40 μm.

Figure 6. Increased expression of stress response genes in MTTP^R46G cardiomyocytes

(A) Expression of stress associated genes, such as Caspase3, Caspase9, Bak, ANP, BNP, Hsp32, and HSP70-2 in response to sunitinib (SU) and PA by real-time PCR. (B) Expression of stress genes including Caspase3, p53, ANP, and BNP, in response to H/R and PA. ± S.D. *P<0.05. Values are means for three independent experiments.