Metastable Atrial State Underlies the Primary Genetic Substrate for MYL4 Mutation-Associated Atrial Fibrillation

Abstract

Background: Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with heart failure, stroke, and increased mortality. The myocardial substrate for AF is poorly understood because of limited access to primary human tissue and mechanistic questions around existing in vitro or in vivo models. Methods: Using an MYH6:mCherry knock-in reporter line, we developed a protocol to generate and highly purify human pluripotent stem cell–derived cardiomyocytes displaying physiological and molecular characteristics of atrial cells. We modeled human MYL4 mutants, one of the few definitive genetic causes of AF. To explore non–cell-autonomous components of AF substrate, we also created a zebrafish Myl4 knockout model, which exhibited molecular, cellular, and physiologic abnormalities that parallel those in humans bearing the cognate mutations. Results: There was evidence of increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models, as well as abnormal expression and localization of cytoskeletal proteins, and loss of intracellular nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide + hydrogen. To identify potentially druggable proximate mechanisms, we performed a chemical suppressor screen integrating multiple human cellular and zebrafish in vivo endpoints. This screen identified Cx43 (connexin 43) hemichannel blockade as a robust suppressor of the abnormal phenotypes in both models of MYL4 (myosin light chain 4)–related atrial cardiomyopathy. Immunofluorescence and coimmunoprecipitation studies revealed an interaction between MYL4 and Cx43 with altered localization of Cx43 hemichannels to the lateral membrane in MYL4 mutants, as well as in atrial biopsies from unselected forms of human AF. The membrane fraction from MYL4-/- human embryonic stem cell derived atrial cells demonstrated increased phospho-Cx43, which was further accentuated by retinoic acid treatment and by the presence of risk alleles at the Pitx2 locus. PKC (protein kinase C) was induced by retinoic acid, and PKC inhibition also rescued the abnormal phenotypes in the atrial cardiomyopathy models. Conclusions: These data establish a mechanistic link between the transcriptional, metabolic and electrical pathways previously implicated in AF substrate and suggest novel avenues for the prevention or therapy of this common arrhythmia.

Androgen Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men

“…Cell culture and differentiation. H9 human embryonic stem cells were maintained and differentiated as described previously 10,12 . Briefly, human embryonic stem cells (hESCs) were maintained in mTeSR medium.…”

“…The bulk RNA sequencing dataset analyzed here was previously reported 12 . Briefly, human pluripotent stem cell (H9 with knock-in MYH6:mCherry reporter)-derived cardiomyocytes were prepared as described previously 10,12 . Reporter tagged cardiomyocytes were isolated from the negative fraction of the culture by FACS sorting.…”

“…To identify candidate drugs capable of modulating ACE2 protein levels, we took advantage of our previously established methods to generate cardiac cells from human embryonic stem cells at large scale [10][11][12] and performed a high content screen with a library of 1443 FDA-approved drugs and a subsequent in silico screen with the ZINC15 library of over 9 million drug-like compounds. We discovered that drugs most effective in reducing ACE2 protein levels converge on androgen receptor (AR) signaling inhibition as a common mechanism of action.…”

Long Non-Coding RNAs in Atrial Fibrillation: Pluripotent Stem Cell-Derived Cardiomyocytes as a Model System

“…Recent advances in atrial lineage differentiation of pluripotent stem cells has allowed the culture of pure and functional atrial myocytes to model AF [ 151 , 152 , 153 , 154 , 155 , 156 ]. Zheng et al [ 153 ] showed that exogenous activation of retinoic acid (RA) signaling in differentiating human embryonic stem cells (hESCs) promoted an atrial phenotype as assessed by action potential characteristics and Ca 2+ handling.…”

Gap19, a Cx43 Hemichannel Inhibitor, Acts as a Gating Modifier That Decreases Main State Opening While Increasing Substate Gating

“…Making use of Gap19, we demonstrated protection against cardiac ischemia/reperfusion injury and cardiomyocyte cell death [ 6 , 14 ]. Gap19 is increasingly used to probe for Cx43 HC involvement in various cardiac, brain, and other disease models [ 7 , 8 , 15 , 16 , 17 , 18 ], making a more detailed analysis indicated and timely.…”