Transcriptional Dysregulation Underlies Both Monogenic Arrhythmia Syndrome and Common Modifiers of Cardiac Repolarization

Bersell, Kevin; Yang, Tao; Mosley, Jonathan D.; Glazer, Andrew M.; Hale, Andrew T.; Kryshtal, Dmytro O.; Kim, Kyungsoo; Steimle, Jeffrey D.; Brown, J.; Salem, Joe‐Elie; Campbell, Courtney C; Hong, Charles C.; Wells, Quinn S.; Johnson, Amanda N; Short, Laura; Blair, Marcia; Behr, Elijah R.; Petropoulou, Evmorfia; Jamshidi, Yalda; Benson, Mark D.; Keyes, Michelle J.; Ngo, Debby; Vasan, Ramachandran S.; Yang, Qiongqiong; Gerszten, Robert E.; Shaffer, Christian M.; Parikh, Shan; Sheng, Quanhu; Kannankeril, Prince J.; Moskowitz, Ivan P.; York, John D.; Wang, Thomas J.; Knollmann, Björn C.; Roden, Dan M.

doi:10.1161/circulationaha.122.062193

Cited by 10 publications

(8 citation statements)

References 107 publications

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“… 4 , 6 Moreover, elevated cytoplasmic Na + commonly observed during IR may also act as a preconditioning stimulus in PI3Kα‐deficient hearts, rendering them resistant to IR injury. Because late Na + current is known to be arrhythmogenic and detrimental to long‐term Ca 2+ homeostasis, 14 , 34 , 35 the current results suggest a more ambivalent role of the current: protective in the settings of the acute ischemia–reperfusion model, but physiologically detrimental in the long term. 14 , 34 , 35 …”

Section: Discussionmentioning

confidence: 77%

Loss of PI3Kα Mediates Protection From Myocardial Ischemia–Reperfusion Injury Linked to Preserved Mitochondrial Function

Zhabyeyev

McLean

Bassiouni

et al. 2023

JAHA

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Background Identifying new therapeutic targets for preventing the myocardial ischemia–reperfusion injury would have profound implications in cardiovascular medicine. Myocardial ischemia–reperfusion injury remains a major clinical burden in patients with coronary artery disease. Methods and Results We studied several key mechanistic pathways known to mediate cardioprotection in myocardial ischemia–reperfusion in 2 independent genetic models with reduced cardiac phosphoinositide 3‐kinase‐α (PI3Kα) activity. P3Kα‐deficient genetic models (PI3KαDN and PI3Kα‐Mer‐Cre‐Mer) showed profound resistance to myocardial ischemia–reperfusion injury. In an ex vivo reperfusion protocol, PI3Kα‐deficient hearts had an 80% recovery of function compared with ≈10% recovery in the wild‐type. Using an in vivo reperfusion protocol, PI3Kα‐deficient hearts showed a 40% reduction in infarct size compared with wild‐type hearts. Lack of PI3Kα increased late Na + current, generating an influx of Na + , facilitating the lowering of mitochondrial Ca 2+ , thereby maintaining mitochondrial membrane potential and oxidative phosphorylation. Consistent with these functional differences, mitochondrial structure in PI3Kα‐deficient hearts was preserved following ischemia–reperfusion injury. Computer modeling predicted that PIP3, the product of PI3Kα action, can interact with the murine and human Na V 1.5 channels binding to the hydrophobic pocket below the selectivity filter and occluding the channel. Conclusions Loss of PI3Kα protects from global ischemic–reperfusion injury linked to improved mitochondrial structure and function associated with increased late Na + current. Our results strongly support enhancement of mitochondrial function as a therapeutic strategy to minimize ischemia–reperfusion injury.

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Section: Discussionmentioning

confidence: 77%

Loss of PI3Kα Mediates Protection From Myocardial Ischemia–Reperfusion Injury Linked to Preserved Mitochondrial Function

Zhabyeyev

McLean

Bassiouni

et al. 2023

JAHA

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“…Stem cell experiments were carried out in the ‘C2’ line previously characterized by our laboratory 40 . At 60-80% confluency, stem cells began differentiation to cardiomyocytes using a previously described chemical method 41,53 . RNA analyses and patch clamp studies were performed at days 35-40 of differentiation.…”

Section: Methodsmentioning

confidence: 99%

ParSE-seq: A Calibrated Multiplexed Assay to Facilitate the Clinical Classification of Putative Splice-altering Variants

O’Neill,

Yang,

Laudeman

et al. 2023

Preprint

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BackgroundInterpreting the clinical significance of putative splice-altering variants outside 2-base pair canonical splice sites remains difficult without functional studies.MethodsWe developed Parallel Splice Effect Sequencing (ParSE-seq), a multiplexed minigene-based assay, to test variant effects on RNA splicing quantified by high-throughput sequencing. We studied variants in SCN5A, an arrhythmia-associated gene which encodes the major cardiac voltage-gated sodium channel. We used the computational tool SpliceAI to prioritize exonic and intronic candidate splice variants, and ClinVar to select benign and pathogenic control variants. We generated a pool of 284 barcoded minigene plasmids, transfected them into Human Embryonic Kidney (HEK293) cells and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), sequenced the resulting pools of splicing products, and calibrated the assay to the American College of Medical Genetics and Genomics scheme. Variants were interpreted using the calibrated functional data, and experimental data were compared to SpliceAI predictions. We further studied some splice-altering missense variants by cDNA-based automated patch clamping (APC) in HEK cells and assessed splicing and sodium channel function in CRISPR-edited iPSC-CMs.ResultsParSE-seq revealed the splicing effect of 224SCN5Avariants in iPSC-CMs and 244 variants in HEK293 cells. The scores between the cell types were highly correlated (R2=0.84). In iPSCs, the assay had concordant scores for 21/22 benign/likely benign and 24/25 pathogenic/likely pathogenic control variants from ClinVar. 43/112 exonic variants and 35/70 intronic variants with determinate scores disrupted splicing. 11 of 42 variants of uncertain significance were reclassified, and 29 of 34 variants with conflicting interpretations were reclassified using the functional data. SpliceAI computational predictions correlated well with experimental data (AUC = 0.96). We identified 20 uniqueSCN5Amissense variants that disrupted splicing, and 2 clinically observed splice-altering missense variants of uncertain significance had normal function when tested with the cDNA-based APC assay. A splice-altering intronic variant detected by ParSE-seq, c.1891-5C>G, also disrupted splicing and sodium current when introduced into iPSC-CMs at the endogenous locus by CRISPR editing.ConclusionsParSE-seq is a calibrated, multiplexed, high-throughput assay to facilitate the classification of candidate splice-altering variants.

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“…This work highlights the role of the TBX5 mutation in BrS and the reversibility of the phenotype in hiPSC-CMs. The study also showed that the same pathological phenotype could be replicated in hiPSC-CMs from healthy controls with the introduction of the same mutation [ 81 ]. In another paper, De La Roche and coauthors modified healthy hiPSC lines by introducing the point mutation A735V to the SCN5A gene, which had been reported by four separate clinical centres around Europe, America, and Japan.…”

Section: Brugada Syndrome Experimental Modelsmentioning

confidence: 98%

Automated Patch-Clamp and Induced Pluripotent Stem Cell-Derived Cardiomyocytes: A Synergistic Approach in the Study of Brugada Syndrome

Melgari

Calamaio

Frosio

et al. 2023

IJMS

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The development of high-throughput automated patch-clamp technology is a recent breakthrough in the field of Brugada syndrome research. Brugada syndrome is a heart disorder marked by abnormal electrocardiographic readings and an elevated risk of sudden cardiac death due to arrhythmias. Various experimental models, developed either in animals, cell lines, human tissue or computational simulation, play a crucial role in advancing our understanding of this condition, and developing effective treatments. In the perspective of the pathophysiological role of ion channels and their pharmacology, automated patch-clamp involves a robotic system that enables the simultaneous recording of electrical activity from multiple single cells at once, greatly improving the speed and efficiency of data collection. By combining this approach with the use of patient-derived cardiomyocytes, researchers are gaining a more comprehensive view of the underlying mechanisms of heart disease. This has led to the development of more effective treatments for those affected by cardiovascular conditions.

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Transcriptional Dysregulation Underlies Both Monogenic Arrhythmia Syndrome and Common Modifiers of Cardiac Repolarization

Cited by 10 publications

References 107 publications

Loss of PI3Kα Mediates Protection From Myocardial Ischemia–Reperfusion Injury Linked to Preserved Mitochondrial Function

Loss of PI3Kα Mediates Protection From Myocardial Ischemia–Reperfusion Injury Linked to Preserved Mitochondrial Function

ParSE-seq: A Calibrated Multiplexed Assay to Facilitate the Clinical Classification of Putative Splice-altering Variants

Automated Patch-Clamp and Induced Pluripotent Stem Cell-Derived Cardiomyocytes: A Synergistic Approach in the Study of Brugada Syndrome

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