Approximately 3–5% of inherited hypertrophic cardiomyopathies result from lysosome storage disorders [1
]. One of these cardiomyopathies is Fabry disease (FD), which is caused by alpha-galactosidase A (GLA) deficiency leading to accumulation of globotriaosylceramide (Gb3) in several tissues. FD is particularly manifested in renal and cardiovascular dysfunctions [2
]. A number of in vivo and in vitro studies revealed that the loss of GLA results in left ventricular hypertrophy and develops into heart failure, myocardial infarction and life-threatening arrhythmias due to Gb3 deposition. The levels of Gb3 and its deacylated derivative, globotriaosylsphingosine (lyso-Gb3) in plasma and tissues are used as diagnostic biomarkers of FD, and are applied for the screening/monitoring of FD patients by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [3
]. However, the limited sample volume and variable levels of lyso-Gb3 in different matrices makes the quantitation very challenging [7
]. Furthermore, the detailed mechanism of how Gb3 accumulation results in hypertrophic cardiomyopathy still needs to be elucidated. In order to develop new therapeutic strategies for FD-associated vasculopathy, it is essential to understand the underlying pathogenesis mechanisms, as well as to discover the potential prognostic biomarkers.
Previously, we generated CRISPR/Cas9-edited GLA-null human HEK293 cells; however, this cell line may not be an appropriate model with which to study pathological events occurring in cardiomyocytes [8
]. The pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs), offer a great potential for modelling human diseases as they can be differentiated into the tissue affected by the pathology. Although several FD-specific iPSC lines have been obtained from patients carrying different GLA
gene mutations, which claimed to be useful for FD cardiomyopathy research [9
], one major limitation of such approach is the influence of variable genetic background, which can be significant even for monogenic, dominant and highly penetrant disease in FD [11
]. Currently, CRISPR/Cas9 emerges as a powerful genome editing technique, providing the opportunity to efficiently delete genes to establish isogenic cells [13
]. Therefore, our strategy in this study was to generate GLA
knockout in human pluripotent stem cells by CRISPR/Cas9-mediated gene editing, and compare them with the parental cells of the same genetic background to study the mechanisms of FD-associated cardiomyopathy.
Recently, it became increasingly clear that lysosomal storage disorders have an impact on autophagic dysfunction [16
]. Dysregulated ceramide metabolism can trigger cytotoxic signaling cascades, including apoptosis and necroptosis, missorting and accumulation of these sphingolipids in the membrane subdomains may destabilize lipid bilayer and cause their permeabilization [17
]. Exosomes are small vesicles secreted upon fusion of multivesicular endolysosomal compartments with the plasma membrane and are derived from the intraluminal vesicles (ILVs) of those organelles. Exosomes may participate in the control of cellular homeostasis by promoting the release of intracellular harmful components, including proteins, lipids or nucleic acids. Emerging evidence from the studies of normal development, as well as multiple disease studies, is beginning to reveal a coordinated exosome–autophagy response that functions to maintain homeostasis through lysosomal degradation and release of cellular cargo [18
]. However, little effort has been made to investigate the impact of autophagic dysfunction in FD on biogenesis and secretion of exosomes.
In the current study, we applied CRISPR/Cas9-mediated genomic editing to deplete GLA expression in ESC-derived cardiomyocytes to recapitulate FD cardiac hypertrophy in vitro and performed proteomic analysis by LC-MS/MS. We identified that Rab GTPase signaling-related vesicle secretion is the factor that may initiate or exacerbate the development of FD-associated cardiomyopathy. Such information will be extremely important for potential application in the prevention of and in interventions for the adverse effects of the cardiac hypertrophy in FD patients.
2. Materials and Methods
2.1. CRISPR/Cas9 Plasmid Construction and Transfection
The CRISPR/Cas9 with T2A-eGFP co-expression vector pSpCas9(BB)–2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid). The exon 1 of GLA
was selected for guiding RNA design and the sequence (5′-AGGAACCCAGAACTACATCT-3′) was cloned into PX458 (abbreviated as GLA-Cas9-GFP) as previously described [8
]. The GLA
-specific targeting plasmid was transfected into hESC line (WA09) by electroporation using Nucleofector System (Lonza, Basel, Switzerland) following the manufacturer’s protocol. Briefly, hESC cells were cultured to 80–90% confluence, then harvested and washed with PBS without Ca2+
. Approximately 4 × 105
cells were resuspended in the pre-mixture solution with 2 μg GLA-Cas9-GFP plasmid, and the optimized protocol (program B016) was used for electroporation. The cells were plated on Matrigel-coated 6-well plate in mTeSR1 medium containing 10 μM Y27632. 48 h later, the proportion of cells expressing EGFP was enriched by flow cytometry using FACSCalibur (BD Biosciences, San Jose, CA, USA). Three days later, cells were detached with TrypLE (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), separated into single cells and seeded with a density of 1 cell/well of a 96-well dish.
2.2. Analysis of CRISPR/Cas9-Induced Mutations in GLA Gene
To identify the presence of indels in GLA gene, the genomic DNA was extracted and used for PCR amplification of the target site with the primer pair 5′-CACACACCAACCTCTAACGATACC-3′ (forward) and 5′-CCAGGAAAGGTCACACAGAGAAAG-3′ (reverse). PCR products were TA-cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). Subsequently, DNA from the clones #19 and #27 was sequenced using T7 forward and Sp6 Reverse primer. Vector NTI software was used to align the results of sequencing and determine the indel spectra in GLA target site.
2.3. hESC Culture and Differentiation to Cardiomyocytes
The hESCs were cultured on tissue culture dishes coated with Geltrex (Life Technologies, Thermo Fisher Scientific) in mTeSR1 culture medium (STEMCELL Technologies, Vancouver, BC, Canada) with daily media changes. The cells were passaged every 3–4 days using Accutase (STEMCELL Technologies). The undifferentiated phenotype of the hESCs was checked daily using a light microscope. In order to differentiate hESCs to cardiomyocytes, they were dissociated by Versene (Life Technologies, Thermo Fisher Scientific), resuspended in mTeSR1 + 5 μM Y27632 and seeded onto Geltrex-coated plates at a density of 3 × 105 cells/cm2 and grown for the next four days with daily medium change. Following that, the cells were treated with 6 µM CHIR99021 (Selleckchem, Houston, TX, USA) in insulin-free RPMI/B27 medium (Life Technologies) for 24 h. The medium was replaced with basal medium for another 2 days. At day 3, the culture medium was subsequently replaced with 5 µM IWP-2 (Tocris Bioscience, Minneapolis, MN, USA) in insulin-free RPMI/B27 for 48 h. On day 7, the culture medium was changed to RPMI/B27 containing insulin (Life Technologies, Thermo Fisher Scientific), and the culture medium was refreshed every 3 days thereafter.
2.4. Alkaline Phosphatase Staining
Cells were washed in PBS twice, fixed with 80% alcohol for at least 2 h at 4 °C, followed by soaking in ddH2O for 2–3 min, and 100 mM Tris-HCl (pH 8.2–8.5) for 5 min. Alkaline phosphatase substrate working solution (Vector Laboratories, Burlingame, CA, USA) was added for 1 h and stained colonies were visualized under light microscope.
2.5. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated with TRIzol reagent (Invitrogen, Thermo Fisher Scientific) and quantified by spectrophotometry at 260 nm. 3 μg of total RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific) at 55 °C for 1 h into complementary DNA, which was then used as the template for subsequent PCR reactions. The PCR reactions were run with the following cycling conditions: 94 °C for 5 min, followed by 25 or 30 cycles at 94 °C (denaturation) for 30 s, 58–62 °C for 30 s (annealing), 72 °C for 45 s (synthesis). The primer sequences are shown in Supplementary Tables S1 and S2
. Amplified RT-PCR products were analyzed on 2% agarose gels and visualized using ethidium bromide staining and SPOT camera system (Diagnostic Instruments, Sterling Height, MI, USA).
2.6. Immunofluorescence Staining
First, the cells were rinsed in PBS and fixed with 1% (v
) paraformaldehyde for 10 min followed by treatment with 70% ethanol (v
) for 10 min at room temperature. The cells were permeabilized with 0.1% NP-40 (Sigma-Aldrich, St. Louis, MO, USA) for 20 min, then washed twice with PBS. To block cells, blocking solution (0.3% BSA and 5% serum in PBS) was applied for 30 min. Cells were incubated with primary antibodies in the blocking solution overnight at 4 °C, washed three times in PBS, then stained with secondary antibodies at 1:200 in PBS for 1 h at room temperature. Cells were washed three times in PBS, and nuclei were stained with Hoechst 33,342 (Life Technologies, Thermo Fisher Scientific) at 1:5000 in PBS for 5 min at room temperature. Prior to imaging, cells mounted with SlowFade Gold Antifade Mountant (Millipore, Sigma, Burlington, MA, USA). Images were obtained using fluorescent microscopy and a digital camera. Antibody for characterization of pluripotency is listed in Supplementary Table S3
2.7. Measurement of Cardiomyocyte Size
The size of iPSC-derived cardiomyocytes was evaluated by measuring the cell area. Twenty days after cardiac induction, the spontaneously beating embryoid bodies were dissociated into single cells using Accutase™ (STEMCELL Technologies). These cells were then plated onto gelatin-coated dishes for further experiments and analysis. Subsequently, the cellular images of cTnT-positive cells were recorded at days 30, 40 and 60 post-induction of differentiation using FV10i confocal microscope (Olympus, Tokyo, Japan). The pixel area of cTnT-positive cells was measured and analyzed using ImageJ software package (NIH). About total two hundred cells were analyzed in five independent experiments.
2.8. Western Blotting
Cells were lysed in RIPA lysis buffer (0.5M Tris-HCl, pH 7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA, protease inhibitor), and the protein lysates were subjected to SDS-PAGE followed by electroblotting onto a PVDF membrane. Membranes were probed with the following primary antibodies: α-galactosidase A, TSG101 (GeneTex, Irvine, CA, USA), CD63 (Santa Cruz Biotechnology, Dallas, TX, USA), Calnexin (Abcam, Cambridge, MA, USA) GDRID2, VPS36, VTI1A (Proteintech Group, Wuhan, Hubei, P.R.C), LC3 (Novus Biologicals, Centennial, CO, USA), Rab11 and GAPDH (Cell Signaling Technology, Denver, MA, USA). The bands were visualized by chemiluminescence detection reagents.
2.9. Lipid Extraction
The cells were grown to confluency on 150 mm cell culture dishes and harvested by scraping into 700 μL PBS. The cell suspension was transferred into 16 mm × 100 mm glass tubes, and 1 mL chloroform and 2.4 mL methanol were added. After water bath sonication, protein was precipitated by centrifuging at 2400× g for 30 min. The supernatant was transferred to a new glass tube and 4.5 mL chloroform and 1.2 mL 0.9% NaCl were added. The sample was centrifuged at 900× g for 5 min. The upper aqueous phase was discarded, and the lower organic phase was washed twice with 2 mL methanol and 0.8 mL 0.9% NaCl. The lower phase was extracted using a 1 mL glass syringe (Hamilton, Reno, NV, USA) and transferred to a new glass tube. Lipids were dried under a stream of nitrogen.
2.10. Quantification of Gb3 by Thin Layer Chromatography (TLC)
First, 100 nmole of total phospholipid was applied to a silica high performance TLC plate (Sigma-Aldrich, St. Louis, MO, USA). The plate was first developed in a solvent system consisting of chloroform/methanol (98:2), and air-dried. The plate was then developed in a solvent system consisting of chloroform/methanol/acetic acid/water (61/31/5/3) and air-dried. Plates were submerged in 8% cupric sulfate pentahydrate in water/methanol/H3PO4 (60:32:8), and charred for 10 min at 150 °C, or were sprayed with 1% orcinol in 11% H2SO4 and charred at 130 °C for 5 min. Plates were scanned and densinometry measured using ImageJ software. Lipids were quantified by running Gb3 standards on a plate (Matreya LLC, State College, PA, USA).
2.11. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis
LC-MS/MS analysis was performed using Orbitrap Mass Analyzer (Thermo Fisher Scientific), according to the manufacturer’s protocol. Briefly, each sample of digested peptides was reconstituted in 20 μL of 0.1% formic acid. Peptides were first separated by the nanoflow HPLC on Agilent 1100 (Agilent Technologies, Santa Clara, CA, USA) using C18 column (Agilent Technologies) with a flow rate of 0.4 μL/min, and were ionized after passing through the nanospray tip (New Objective, Woburn, MA, USA). LC gradient for the LC-MS/MS system ramped from 2–40% ACN in 120 min, and the system was set up for automated data-dependent acquisition, with a mode of 200–2000 m/z full scan for the maximum three most intense peaks from each Orbitrap MS scan. Peptides with +2 or +3 charge were further subjected to CID. Spectra were obtained in raw data files with Xcalibur (version 2.0 SR2). Protein identification was accomplished by TurboSEQUEST (Thermo Fisher Scientific) using the UniProt database. A protein was confirmed once three peptides with Xcorr >2.5 were matched in sequencing.
2.12. Transmission Electron Microscopy
The morphology of differentiated cardiomyocytes was characterized using JEM-2000 EX II transmission electron microscope (JEOL, Tokyo, Japan). The cardiomyocytes were covered with 400 mesh carbon-coated copper TEM grid. After 15 min, the grid was tapped with filter paper to remove the excess water followed by staining with 1% phosphotungstic acid (Sigma-Aldrich) for 20 min. The samples were allowed to air-dry for 24 h and then observed under TEM.
2.13. Array-Based Comparative Genomic Hybridization (CGH-Array
Genomic DNA was isolated and intermittently sonicated using a Digital Sonifier 450 sonicator probe (Branson Ultrasonics, Danbury, CT, USA). DNA samples were amplified using the GenomePlex WGA kit (Thermo Fisher Scientific). Genomic DNA ULS Labeling Kit (Agilent) was used to label the amplified DNA with either Cy3 or Cy5. As recommended by Agilent, 2.0–2.5 μg of amplified DNA was used as the input starting material for each labeling reaction. Scanning and image analysis were conducted according to Agilent Oligonucleotide Array-based CGH for Genomic DNA analysis Protocol (version 4.0). Microarrays were scanned using an Agilent G2565BA DNA Microarray Scanner (Agilent). Agilent Feature Extraction software (v9.1.3) was used to extract data from raw microarray image files. Agilent CGH Analytics software (v3.4) was used to visualize, detect and analyze the aberration patterns from CGH microarray profiles.
2.14. Exosome Isolation and Characterization
Exosomes were isolated from cell culture supernatants using Total Exosome Isolation Reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. Culture media samples were centrifuged at 2000× g for 30 min to remove cells and debris. The supernatant was transferred to sterile tubes and an exosome precipitation solution was added at a 2:1 ratio. Samples were mixed and left overnight at 4 °C. Samples were then centrifuged at 10,000× g for 60 min and supernatant carefully removed. The precipitated exosome pellets were re-suspended with PBS and either used immediately or stored at −80 °C until required. Immunoaffinity capture assay was used to characterize the purity of exosomes using CD63 antibody-conjugated dynabeads. Exosomes were incubated with antibody-conjugated dynabeads overnight and washed by PBS containing 0.1% BSA two times. Further, CD63 PE-conjugated antibody was used to stain the exosome-bound dynabeads. All samples were examined by flow cytometry using FACSCanto System (BD Biosciences) and FACSDIVA software was used to analyze the population of exosomes.
2.15. Quantification of Isolated Exosomes
Acetylcholinesterase activity assay was performed using EXOCET Exosome Quantitation Kit (System Biosciences, Palo Alto, CA, USA). Isolated exosomes were re-suspended with PBS and lysed with lysis buffer. Each sample was incubated at 37 °C for 5 min and mixed with reaction buffer in 96-well plates. Mixed samples were incubated for 20 min at room temperature and quantified by spectrophotometry at OD 405 using standard samples containing known numbers of exosomes. The final number of exosomes was converted to micrograms, per the manufacturer’s guideline. To characterize exosomes, isolated exosome preparations were re-suspended with PBS and analyzed using Zetasizer Nano dynamic light scattering system (Malvern Instruments, Malvern, UK).
2.16. Mitochondrial Superoxide Stress Quantification
Mitochondrial superoxide production was quantified using MitoSOX Red mitochondrial superoxide indicator (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, the cells were incubated with 5 µM MitoSOX Red in the culture medium in the dark for 60 min at 37 °C. Stained cells were counterstained as desired and mounted and analyzed by fluorescent microscopy.
2.17. Annexin V Staining
Cells were washed once with PBS and analyzed using FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s protocol.
2.18. GLA Enzyme Activity Assay
Cells were washed twice with 1X PBS and were lysed in 60 μL lysis buffer (27 mM sodium citrate, 46 mM sodium phosphate dibasic, 0.5% Triton X-100). 10 μL of cell lysate was added to 50 μL assay buffer containing 6 mM 4-methylumbelliferyl-α-d-galactopyranoside (Sigma-Aldrich) and 117 mM N-acetyl-D-galactosamine (Sigma-Aldrich) and incubated at 37 °C for 1 hr. The 4-methylumbelliferone (Sigma-Aldrich) dissolved in methanol was used as standard ranging from 0.15 μM to 5000 μM. Thereafter, 70 μL glycine-carbonate solution (pH 10.8) was then added to stop the reaction and fluorescence was detected by microplate reader (em/ex = 365/448 nm). The enzyme activity was normalized by protein concentration of cell lysate.
2.19. Statistical Analysis
The quantifiable data are presented as the means ± standard deviation (SD) and compared with Student’s t-test by GraphPad Prism 6 (GraphPad Prism Software). * p < 0.05, ** p < 0.01, *** p < 0.005 and **** p < 0.001.
The lysosome storage dysfunction (LSD) is the major factor in etiology of inherited hypertrophic cardiomyopathy, including FD; however, the underlying mechanisms of FD-associated cardiomyopathy are not fully understood. In order to expand the therapeutic strategies for FD, the construction of in vitro disease models using primary human cells is essential and unavoidable. Whereas the patients’ cells can be used to directly model the effects of drugs on humans, their availability and capacity for expansion are limited and finite compared to in vitro derived cell lines, especially for vital organs such as heart and brain. These drawbacks restrict the capacity of these models to faithfully simulate human disease. By comparison, ESCs can sidestep these limitations and thus provide a powerful and versatile tool for disease therapy, as well as basic research. Combined with the advancements in genome editing technology though CRISPR/Cas9, it is now possible to model human diseases in a physiologically, pathologically, and genetically relevant manner.
Mass spectrometry-based proteomics has been recognized as a powerful tool with a potential to uncover detailed changes in protein expression [29
]. To date, most of the proteomics studies performed on FD patients examined FD-affected renal tissue or plasma; however, few studies of protein expression have used FD-affected human heart tissue [30
]. Although it has been revealed that Gb3 accumulation induces endothelial KCa3.1 degradation in Gla
-knockout mice through clathrin and Rab5C, which are the critical components of endosome maturation machinery [32
], the proteomic profiling performed in our study revealed that GLA
knockout led to the downregulation of Rab GTPases, including RAB11 subfamily, which are involved in recycling from an endosomal compartment to the plasma membrane, and was shown to contribute to exosome secretion in neuronal cells [33
], although the molecular mechanism of RAB11 function in exosome secretion has yet to be deciphered, especially regarding its downstream effectors. On the other hand, our proteomic profile also revealed downregulation of Rho GDP-dissociation inhibitor 2 (GDIR2) in GLA-null CMs. This observation is consistent with previously published clinic proteomic profiles, where PBMCs isolated from FD patients were analyzed, and among the downregulated proteins were calnexin, Rho GDP-dissociation inhibitor 1 (GDIR1), Rho GDP-dissociation inhibitor 2 (GDIR2), chloride intracellular channel protein [34
]. Rho GDIs play an important role in regulating Rho GTPases, which are members of the Ras superfamily of GTP binding proteins that participate in the regulation of cytoskeleton and other cellular functions including proliferation, differentiation, and apoptosis [35
]. Rho GDI is ubiquitously expressed and binds to all Rho family proteins, including RhoA [37
]. The small G-protein RhoA regulates the actin cytoskeleton, and its involvement in cell proliferation has also been established. In cardiomyocytes grown in vitro, RhoA induces hypertrophic cell growth and gene expression [38
]. In vivo, however, cardiac-specific overexpression of RhoA leads to development of heart failure [40
]. These correlated evidences suggest that exosome secretion regulated by Rab GTPase/RhoGDI signaling pathway may utilize as a target for the potential therapeutic strategy for FD-associated cardiomyopathy.
FD is characterized by failures of cellular autophagy associated with accumulation of glycogen granules and intracytoplasmic vacuoles that contain autophagic material. Impairment of autophagic flux in FD, which was manifested as defects of autophagosome maturation in renal endothelial cells and mesangial cells [42
]. Autophagy is an evolutionary conserved process of self-degradation of cellular components by autophagosomes, which are delivered to the lysosomal machinery. Several studies have shown that starvation-induced autophagy reduces exosome secretion due to the fusion of multivesicular bodies with autophagic vacuoles [44
]. In contrast, cellular stresses, such as senescence and ER stress, increase exosome secretion [45
]. It is not clear why cells respond to stress by releasing more exosomes, but this could be an alternative way of eliminating waste products. The secreted exosomes may be targeted to and degraded by phagocytes, but they may also have other destinations. Exosomes secreted as waste are likely to affect neighboring cells and possibly induce pathological conditions. Another possibility is that cells may communicate with neighboring cells about intracellular stress by increasing exosome release. Therefore, preventing waste accumulation and rescuing the autophagic ability in FD-affected CMs may be utilized as another therapy approach for FD cardiomyopathy. It has been observed that exosomes derived from CMs harbor a variety of mRNAs, miRNAs and proteins, which may be transferred to the adjacent endothelial cells and modulate their function [47
]. Interestingly, exosomes derived from ESCs/iPSCs were shown to possess regenerative power on CMs by augmenting and modulating endogenous repair mechanisms [48
]. Emerging evidence from the studies of normal development, as well as multiple disease studies, revealed that exosome secretion and autophagy act in a coordinated manner to maintain homeostasis through lysosomal degradation and/or release of cellular cargo [50
]. Therefore, considering the role of exosomes in physiological and pathological conditions, strategies that interfere with the release of exosomes and impair exosome-mediated cell-to-cell communication could potentially be exploited therapeutically in FD cardiomyopathy.
The objective of the present study was to explore the influence of Gb3, a lysosomal glycolipid accumulating in FD-affected cells, on modulation of cellular vesicle cycling and the possible mechanism underlying cardiomyopathy. We demonstrated that disruption of GLA with CRISPR/Cas9 resulted in the complete ablation of GLA protein expression in hESCs. Results from this study may provide mechanistic insights into how Gb3 accumulation modulates vesicles formation, particularly the autophagy flux in CMs. Such information may be extremely important for potential application in prevention and intervention of adverse effects of FD-associated cardiomyopathy.