Dataset Information

Phenotypic Drug Screening for Dysferlinopathy Using Patient-Derived Induced Pluripotent Stem Cells.

ABSTRACT: Dysferlinopathy is a progressive muscle disorder that includes limb-girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG-132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient-derived induced pluripotent stem cells. According to the screening, nocodazole was found to effectively increase the level of dysferlin in cells, which, in turn, enhanced membrane resealing following injury by laser irradiation. Moreover, the increase was due to microtubule disorganization and involved autophagy rather than the proteasome degradation pathway. These findings suggest that increasing the amount of misfolded dysferlin using small molecules could represent an effective future clinical treatment for dysferlinopathy. Stem Cells Translational Medicine 2019;8:1017-1029.

SUBMITTER: Kokubu Y

PROVIDER: S-EPMC6766604 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Phenotypic Drug Screening for Dysferlinopathy Using Patient-Derived Induced Pluripotent Stem Cells.

Kokubu Yuko Y Nagino Tomoko T Sasa Katsunori K Oikawa Tatsuo T Miyake Katsuya K Kume Akiko A Fukuda Mikiko M Fuse Hiromitsu H Tozawa Ryuichi R Sakurai Hidetoshi H

Stem cells translational medicine 20190628 10

Dysferlinopathy is a progressive muscle disorder that includes limb-girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG-132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient-derived induced pluripoten ...[more]

PMID: 31250983

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Project description:Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

Project description:Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds.Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome, familial hypertrophic cardiomyopathy, and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations.We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays.

Dataset Information

Phenotypic Drug Screening for Dysferlinopathy Using Patient-Derived Induced Pluripotent Stem Cells.

Publications

Phenotypic Drug Screening for Dysferlinopathy Using Patient-Derived Induced Pluripotent Stem Cells.

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