Varied Responses to a High m.3243A>G Mutation Load and Respiratory Chain Dysfunction in Patient-Derived Cardiomyocytes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patient Cells and Induced Pluripotent Stem Cell Culture
2.2. Differentiation and Characterization of Cardiomyocytes
2.3. Flow Cytometry
2.4. Seahorse Analysis for Cellular Bioenergetics
2.5. Immunocytochemistry
2.6. Calcium Imaging
2.7. ROS Analysis
2.8. DNA Extraction
2.9. Quantitative PCR
2.10. Determination of the m.3243A>G Mutation Load
2.11. Western Blotting
2.12. ATP Concentration
2.13. Lactate Concentration
2.14. Statistical Analysis
3. Results
3.1. Cells with High mtDNA Mutation Load Show Normal Cardiac Differentiation Potential
3.2. Mitochondrial Mutations Do Not Induce Drastic Effects on Mitochondrial Mass or Morphology in iPSC-CMs
3.3. High m.3243A>G Mutation Load Impairs Oxidative Respiration in iPSC-CMs
3.4. Compensatory Glucose Metabolism Is Upregulated in the MIDD Patient’s Cells upon High Mutation Load
3.5. High m.3243A>G Mutation Load Elevates ROS Production in the CMP Patient’s Cells
3.6. High m.3243A>G Mutation Load Induces Altererations in Calcium-Signaling in the CMP Patient’s Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A. Additional Methodological Details
Type | Antibody | Origin | Company (Product Code) | Application (Dilution) |
---|---|---|---|---|
Primary | Cardiac Troponin T | Goat | Abcam (ab64623) | IF (1:500) |
TOM20 | Rabbit | Cell Signaling (42406) | IF (1:200) WB (1:500) | |
Porin | Rabbit | Abcam (ab15895) | WB (1:1000) | |
Total OXPHOS Human WB antibody | Mouse | Abcam (ab110411) | WB (1:1000) | |
Myosin Heavy Chain | Mouse | Millipore (05-716) | IF (1:400) | |
α-Tubulin | Mouse | Abcam (ab40742) | WB (1:2000) | |
Secondary | Anti-mouse, Alexa fluor 488 | Donkey | Invitrogen (A21202) | IF (1:300) |
Anti-goat, Alexa fluor 568 | Donkey | Invitrogen (A11057) | IF (1:300) | |
Anti-goat, Alexa fluor 488 | Donkey | Invitrogen (A11055) | IF (1:300) | |
Anti-rabbit, Alexa fluor 594 | Donkey | Invitrogen (A21207) | IF (1:300) | |
Anti-mouse, biotinylated | Horse | Vector Laboratories (BA-2000) | WB (1:3000) | |
Anti-rabbit, biotinylated | Goat | Vector Laboratories (BA-1000) | WB (1:5000) |
Gene | Sequence 5′->3′ | Reference |
---|---|---|
APP forward | GCCTGCCTGATCCTCCAAAT | |
APP reverse | AGGGTAGCGGATGATTCAGCC | |
CYTB forward | TGTGTGCTCTCCCAGGTCTA | |
CYTB reverse | CAGTTCTGGATGGTCACTGG | |
ANP forward | GATCTGCCCTCCTAAAAAGCAA | |
ANP reverse | ATCTCCGCAGGCTCCGA | |
ATP2A2 forward | GCGGGCAATCTACAACAACA | |
ATP2A2 reverse | GAGCAGCTGAACAGGAATCAAA | |
RYR2 forward | AACTACTGTCCAAGCAGAGGAAAAG | |
RYR2 reverse | CCGATGCCTTGGCAGATTAT | |
SLC2A1 forward | GTCTGGCATCAACGCTGTCTT | |
SLC2A1 reverse | CTGCTCGCTCCACCACAAAC | |
SLC2A4 forward | CGACCAGCATCTTCGAGACAG | |
SLC2A4 reverse | TCCACCAACAACACCGAGACC | |
IRS1 forward | AACTGGACATCACAGCAGAATGAA | |
IRS1 reverse | CAAACTGAAATGGATGCATCGTACC | |
IRS2 forward | GCCACCATCGTGAAAGAGTGA | |
IRS2 reverse | TTGCCTTGTTGGTGCCTCAT | |
LDHA forward | AGCCCGATTCCGTTACCT | [44] |
LDHA reverse | CACCAGCAACATTCATTCCA | [44] |
GAPDH forward | AATGAAGGGGTCATTGATGG | |
GAPDH reverse | AAGGTGAAGGTCGGAGTCAA | |
TUBA3 forward | CATTGCCAATCTGGACACC | |
TUBA3 reverse | GCAACAACCTCTCCTCTTCG | |
HSF1 forward | TGAGAATGAGGCTCTGTGG | |
HSF1 reverse | CTGAATGAGCTTGTTGACGA | |
HSPB7 forward | AGCGAAGGTGTTCATGACG | |
HSPB7 reverse | CCTATGAGTTTGCGGTGGAC | |
NFE2L2 forward | GGTTGCCCACATTCCCAAATC | [45] |
NFE2L2 reverse | CAAGTGACTGAAACGTAGCCG | [45] |
HMOX1 forward | TTCAAGCAGCTCTACCGCTC | [45] |
HMOX1 reverse | GAACGCAGTCTTGGCCTCTT | [45] |
NQO1 forward | TGCAGCGGCTTTGAAGAAGAAAG | [46] |
NQO1 reverse | TCGGCAGGATACTGAAAGTTCGC | [46] |
GCLC forward | ATGGAGGTGCAATTAACAGAC | [46] |
GCLC reverse | ACTGCATTGCCACCTTTGCA | [46] |
GCLM forward | GCTGTATCAGTGGGCACAG | [46] |
GCLM reverse | CGCTTGAATGTCAGGAATGC | [46] |
Forward primer | 5′-CCACCCAAGAACAGGGTTTG-3’ |
Reverse primer | 5′-GGAATTGAACCTCTGACTGTAAAGTTT-3’ |
3243A probe | VIC-AGATGGCAGAGCCCGGTA-MGB |
3243G probe | FAM-AGATGGCAGGGCCCGGTA-MGB |
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Ryytty, S.; Modi, S.R.; Naumenko, N.; Shakirzyanova, A.; Rahman, M.O.; Vaara, M.; Suomalainen, A.; Tavi, P.; Hämäläinen, R.H. Varied Responses to a High m.3243A>G Mutation Load and Respiratory Chain Dysfunction in Patient-Derived Cardiomyocytes. Cells 2022, 11, 2593. https://doi.org/10.3390/cells11162593
Ryytty S, Modi SR, Naumenko N, Shakirzyanova A, Rahman MO, Vaara M, Suomalainen A, Tavi P, Hämäläinen RH. Varied Responses to a High m.3243A>G Mutation Load and Respiratory Chain Dysfunction in Patient-Derived Cardiomyocytes. Cells. 2022; 11(16):2593. https://doi.org/10.3390/cells11162593
Chicago/Turabian StyleRyytty, Sanna, Shalem R. Modi, Nikolay Naumenko, Anastasia Shakirzyanova, Muhammad Obaidur Rahman, Miia Vaara, Anu Suomalainen, Pasi Tavi, and Riikka H. Hämäläinen. 2022. "Varied Responses to a High m.3243A>G Mutation Load and Respiratory Chain Dysfunction in Patient-Derived Cardiomyocytes" Cells 11, no. 16: 2593. https://doi.org/10.3390/cells11162593