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CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of ?₂AR in isogenic hPSC-derived cardiomyocytes.

ABSTRACT: During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (?₂AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions. To overcome these limitations, we used CRISPR/Cas9 gene editing technology in human pluripotent stem cells (hPSCs) to create a unique suite of four isogenic homozygous variants at amino acid positions 16(G/R) and 27(G/Q), which reside in the N terminus of the ?₂AR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level ?₂AR signaling dominated over ?₁AR . Examining changes in beat rates and responses to isoprenaline, Gi coupling, cyclic AMP (cAMP) production, downregulation, and desensitization indicated that responses were often heightened for the GE variant, implying differential dominance of both polymorphic location and amino acid substitution. This finding was corroborated, since GE showed hypersensitivity to doxorubicin-induced cardiotoxicity relative to GQ and RQ variants. Thus, understanding the effect of ?₂AR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact.

SUBMITTER: Kondrashov A

PROVIDER: S-EPMC7733025 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of β2AR in isogenic hPSC-derived cardiomyocytes.

Kondrashov Alexander A Mohd Yusof Nurul A N NAN Hasan Alveera A Goulding Joëlle J Kodagoda Thusharika T Hoang Duc M DM Vo Nguyen T N NTN Melarangi Tony T Dolatshad Nazanin N Gorelik Julia J Hill Stephen J SJ Harding Sian E SE Denning Chris C

Molecular therapy. Methods & clinical development 20201027

During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (β2AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanisti ...[more]

PMID: 33335946

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Project description:BackgroundDesminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P. We created the first CRISPR-Cas9 engineered rat model for a muscle disease by mirroring the R350P mutation in humans.MethodsUsing CRISPR-Cas9 technology, Des c.1045-1046 (AGG > CCG) was introduced into exon 6 of the rat genome causing p.R349P. The genotype of each animal was confirmed via quantitative PCR. Six male rats with a mutation in desmin (n = 6) between the age of 120-150 days and an equal number of wild type littermates (n = 6) were used for experiments. Maximal plantar flexion force was measured in vivo and combined with the collection of muscle weights, immunoblotting, and histological analysis. In addition to the baseline phenotyping, we performed a synergist ablation study in the same animals.ResultsWe found a difference in the number of central nuclei between desmin mutants (1 ± 0.4%) and wild type littermates (0.2 ± 0.1%; P < 0.05). While muscle weights did not differ, we found the levels of many structural proteins to be altered in mutant animals. Dystrophin and syntrophin were increased 54% and 45% in desmin mutants, respectively (P < 0.05). Dysferlin and Annexin A2, proteins associated with membrane repair, were increased two-fold and 32%, respectively, in mutants (P < 0.05). Synergist ablation caused similar increases in muscle weight between mutant and wild type animals, but changes in fibre diameter revealed that fibre hypertrophy in desmin mutants was hampered compared with wild type animals (P < 0.05).ConclusionsWe created a novel animal model for desminopathy that will be a useful tool in furthering our understanding of the disease. While mutant animals at an age corresponding to a preclinical age in humans show no macroscopic differences, microscopic and molecular changes are already present. Future studies should aim to further decipher those biological changes that precede the clinical progression of disease and test therapeutic approaches to delay disease progression.

Dataset Information

CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of ?₂AR in isogenic hPSC-derived cardiomyocytes.

Publications

CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of β<sub>2</sub>AR in isogenic hPSC-derived cardiomyocytes.

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CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of ?2AR in isogenic hPSC-derived cardiomyocytes.

Publications

CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of β<sub>2</sub>AR in isogenic hPSC-derived cardiomyocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of ?₂AR in isogenic hPSC-derived cardiomyocytes.