Biology subjects

Blackwell, D. J.

Publications and source records attributed to Blackwell, D. J..

8 recordsLinked to original sources

bioRxiv2026

RyR2-Mediated Ca2+ Leak Drives Progressive Arrhythmogenic Cardiomyopathy

Background Abnormal sarcoplasmic reticulum (SR) Ca2+ release through ryanodine receptor 2 (RyR2) is a well-established mechanism of ventricular arrhythmias. Whether persistent RyR2 dysfunction can also initiate the structural remodeling characteristic of arrhythmogenic cardiomyopathy (ACM) remains unknown. We tested the hypothesis that chronic RyR2-mediated Ca2+ leak is sufficient to couple electrical instability to progressive cardiomyopathy. Methods We generated a rabbit carrying the severe gain-of-function RyR2-V2475F mutation and characterized disease progression using longitudinal cardiac phenotyping, continuous electrocardiographic monitoring, cellular electrophysiology and Ca2+ imaging, histopathology, biochemical analyses, and single-nucleus RNA sequencing (snRNA-seq). Therapeutic potential was evaluated by continuous treatment with the selective RyR2 inhibitor ent-verticilide for two months. Results RyR2-V2475F rabbits developed spontaneous ventricular tachyarrhythmias, sudden cardiac death, and progressive right ventricular (RV)-dominant fibrofatty cardiomyopathy recapitulating major features of human ACM. Malignant ventricular arrhythmias preceded overt structural remodeling and were associated at the cellular level with early spontaneous Ca2+ release and early afterdepolarizations. Early disease was characterized by remodeling of excitation-contraction coupling with preserved SR Ca2+ load and contractile function despite RyR2 dysfunction; upon disease progression, these compensatory changes were accompanied by SR Ca2+ depletion, impaired RV contractility and reduced left ventricular filling. Single-nucleus transcriptomics identified early fibroblast expansion and predicted cardiomyocyte-centered paracrine signaling before advanced fibrofatty infiltration. Cardiomyocytes also exhibited an altered lipid-metabolic transcriptome and intracellular lipid accumulation, accompanied by enhanced mitochondrial Ca2+ uptake. Selective RyR2 inhibition markedly reduced ventricular tachycardia burden and preserved RV contraction and LV filling during disease progression. Conclusions Severe RyR2 gain-of-function can lead to both malignant ventricular arrhythmias and progressive arrhythmogenic cardiomyopathy in a large-animal model, establishing persistent intracellular Ca2+ dysregulation as a mechanistic link between electrical and structural heart disease. The ability of selective RyR2 inhibition to suppress arrhythmias and preserve cardiac function identifies RyR2 as a potentially actionable therapeutic target in arrhythmogenic cardiomyopathy.

physiology↗

bioRxiv2026

Calmodulin mutation N54I causes autosomal dominant CPVT in mice

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by stress- or catecholamine-induced ventricular arrhythmias in the absence of overt structural heart disease. Mutations in RYR2 and CASQ2 account for most genetically defined cases, although pathogenic variants in the three genes encoding calmodulin (CALM1-3) have also been linked to CPVT. Because all three CALM genes encode an identical calmodulin protein, pathogenic calmodulin variants are expected to be expressed in only a small fraction of total cellular calmodulin, raising the question of whether this limited abundance is sufficient to produce an arrhythmogenic phenotype in vivo. We generated a heterozygous mouse model carrying the human disease-associated N54I-equivalent mutation, N54I, in Calm1. Mutant calmodulin accounted for 13.7% of total cardiac calmodulin, consistent with expression from one of six Calm alleles. Under basal conditions, N54I/+ mice exhibited normal growth, survival, cardiac morphology, and surface electrocardiogram parameters. However, cardiomyocytes isolated from N54I/+ mice had increased rates of RyR2-mediated spontaneous calcium release. Following catecholaminergic challenge with isoproterenol and caffeine, N54I/+ mice exhibited significantly more premature ventricular contractions and arrhythmias than wild-type littermates. Exercise challenge in conscious mice similarly provoked ventricular ectopy and ventricular tachycardia. In addition, N54I/+ mice exhibited abnormalities of atrial and sinoatrial electrical activity, including premature atrial contractions, ectopic P waves, atrioventricular conduction slowing, and beat-to-beat variability. These findings demonstrate that expression of the N54I calmodulin variant from a single Calm1 allele is sufficient to produce a CPVT phenotype in vivo. This model provides experimental evidence linking a human disease-associated calmodulin variant to arrhythmogenesis and demonstrates the functional dominance of mutant calmodulin in the heart.

physiology↗

bioRxiv2026

Dynamic-Structure Redesign of Calmodulin Reveals Mechanistic Constraints on Ryr2 Regulation

2.Calmodulin (CaM) is a highly conserved Ca2+ sensor that regulates hundreds of cellular targets through Ca2+ -dependent conformational dynamics. Despite its central role in Ca2+ signaling and disease, its evolutionary conservation and structural flexibility have suggested that CaM is resistant to rational redesign. Here, using the cardiac Ca2+ release channel Ryanodine receptor 2 (RyR2) as a model system, we tested whether incorporating conformational dynamics into computational protein design enables functional reengineering of CaM. We first applied a static structure-based redesign to increase CaM-RyR2 affinity. Although the resulting variant bound more tightly to both the RyR2 peptide and the intact channel in vitro, it distorted peptide geometry and worsened Ca2+ leak in cardiomyocytes ex vivo. Guided by molecular dynamics simulations, we then developed a dynamic-structure redesign strategy that preserves conformational integrity while strengthening binding. The resulting CaM variant exhibited increased RyR2 affinity and reduced pathological Ca2+ leak in a disease-relevant model. These findings show that improved binding affinity alone is insufficient to enhance physiological regulation and that successful CaM redesign requires preservation of conformational dynamics. More broadly, they demonstrate that integrating conformational dynamics into protein redesign can enable functionally predictive engineering of flexible regulatory protein-protein interactions.

biophysics↗

bioRxiv2023

ent-Verticilide B1 inhibits type 2 ryanodine receptor channels and is antiarrhythmic in Casq2-/- mice

Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in single RyR2 channel assays, [3H]ryanodine binding assays, and in Casq2-/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single-channels and [3H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca2+ release in Casq2-/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 min and half-life of 45 min after intraperitoneal administration of 3 mg/kg in mice. Both 3 mg/kg and 30 mg/kg ent-B1 significantly reduced catecholamine-induced ventricular arrhythmia in Casq2-/- mice. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. Significance statementThe cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

pharmacology and toxicology↗

bioRxiv2023

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here we used fluorescence lifetime (FLT)-measurements of Forster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multi-exponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent-verticilide, yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.

biophysics↗

bioRxiv2023

High-throughput functional mapping of variants in an arrhythmia gene, KCNE1, reveals novel biology

BackgroundKCNE1 encodes a 129-residue cardiac potassium channel (IKs) subunit. KCNE1 variants are associated with long QT syndrome and atrial fibrillation. However, most variants have insufficient evidence of clinical consequences and thus limited clinical utility. ResultsHere, we demonstrate the power of variant effect mapping, which couples saturation mutagenesis with high-throughput sequencing, to ascertain the function of thousands of protein coding KCNE1 variants. We comprehensively assayed KCNE1 variant cell surface expression (2,554/2,709 possible single amino acid variants) and function (2,539 variants). We identified 470 loss-of-surface expression and 588 loss-of-function variants. Out of the 588 loss-of-function variants, only 155 had low cell surface expression. The latter half of the protein is dispensable for protein trafficking but essential for channel function. 22 of the 30 KCNE1 residues (73%) highly intolerant of variation were in predicted close contact with binding partners KCNQ1 or calmodulin. Our data were highly concordant with gold standard electrophysiological data ({rho} = -0.65), population and patient cohorts (32/38 concordant variants), and computational metrics ({rho} = -0.55). Our data provide moderate-strength evidence for the ACMG/AMP functional criteria for benign and pathogenic variants. ConclusionsComprehensive variant effect maps of KCNE1 can both provide insight into IKs channel biology and help reclassify variants of uncertain significance.

genomics↗

bioRxiv2022

RyR2 inhibition with dantrolene is antiarrhythmic, antifibrotic, and improves cardiac function in chronic ischemic heart disease

BackgroundVentricular tachycardia (VT) is responsible for sudden death in chronic ischemic heart disease (CIHD) patients. The cardiac ryanodine receptor (RyR2) releases Ca2+ from the sarcoplasmic reticulum (SR) and links electrical excitation to contraction. RyR2 hyperactivity has been widely documented in CIHD and may contribute to VT risk and progressive LV remodeling. ObjectiveTo test the hypothesis that targeting RyR2 hyperactivity plays a mechanistic role in VT inducibility and progressive heart failure in CIHD that can be prevented by the RyR2 inhibitor dantrolene. MethodsCIHD was induced in C57BL/6J mice by left coronary artery ligation. Four weeks later, mice were randomized to either acute or chronic (6 weeks via osmotic mini-pump) treatment with dantrolene or vehicle. VT inducibility was assessed by programmed stimulation in vivo and in isolated hearts. Electrical substrate remodeling was assessed by optical mapping. Ca2+ sparks and spontaneous Ca2+ releases were measured in isolated cardiomyocytes. Cardiac remodeling was assessed by histology and qRT-PCR. Cardiac function and contractility were assessed by echocardiography. ResultsCompared to vehicle, acute dantrolene treatment reduced VT inducibility and improved LV contractility in vivo. Optical mapping in isolated hearts demonstrated reentrant VT prevention by dantrolene, which normalized the shortened refractory period (VERP) and prolonged action potential duration (APD), preventing APD alternans. In single CIHD cardiomyocytes, dantrolene normalized RyR2 hyperactivity and prevented spontaneous SR Ca2+ release. Chronic dantrolene treatment reduced peripheral muscle strength but had no adverse effects on body weight or mortality. Chronic dantrolene not only reduced VT inducibility but also reduced peri-infarct fibrosis and prevented the progression of LV dysfunction in CIHD mice. ConclusionRyR2 hyperactivity plays a mechanistic role for VT risk, infarct remodeling, and contractile dysfunction in CIHD mice. Our data provide proof of concept for the anti-arrhythmic and anti-fibrotic efficacy of dantrolene in CIHD. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIThe mouse CIHD model is a more clinically relevant model in which treatment is started late after infarction, when heart failure is already established. C_LIO_LIAcute and chronic dantrolene treatment suppresses VT inducibility by restoring myocyte APD, terminating APD alternans and normalizing VERP. C_LIO_LIChronic dantrolene treatment prevents pathological remodeling and peri-infarct fibrosis, the substrate for reentry VT. Cardiac function is improved with chronic dantrolene therapy. C_LI Clinical ImplicationsO_LITreatment with dantrolene, which is already approved for clinical use, is a promising therapy in patients with ischemic heart disease, in whom other antiarrhythmic drugs are contraindicated. C_LIO_LIDantrolene inhibition of RyR2 not only suppresses VT but also improves cardiac function in chronic ischemic heart disease. C_LI

pharmacology and toxicology↗

bioRxiv2020

High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KCNH2

BackgroundKCHN2 encodes the KV11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that decrease IKr can cause Type 2 Long QT syndrome, usually due to mistrafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would help clinicians identify and treat individuals at risk of a major cardiac event. The volume of reported non-synonymous KCNH2 variants preclude the use of conventional electrophysiologic methods for functional study. ObjectiveTo report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in KCNH2. MethodsWe develop a method to quantitate KCNH2 variant trafficking on a pilot region of 11 residues in the S5 helix, and generate trafficking scores for 220/231 missense variants in this region. ResultsFor 5/5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We additionally compare our results with planar patch electrophysiology and find that loss-of-trafficking variants do not produce IKr, but that some variants which traffic normally may still be functionally compromised. ConclusionsHere, we describe a new method for detecting trafficking-deficient variants in KCNH2 in a multiplexed assay. This new method accurately generates trafficking data for variants in KCNH2 and can be readily extended to all residues in Kv11.1 and to other cell surface proteins. CLINICAL IMPLICATIONSHundreds of KCNH2 variants have been observed to date, and thousands more will be found as clinical and population sequencing efforts become increasingly widespread. The major mechanism of KV11.1 loss of function is misfolding and failure to traffic to the cell surface. Deep mutational scanning of KCNH2 trafficking is a scalable, high-throughput method that can help identify new loss of function variants and decipher the large number of KCNH2 variants being found in the population.

genetics↗