Biology subjects

Saffitz, J.

Publications and source records attributed to Saffitz, J..

4 recordsLinked to original sources

bioRxiv2026

Gating Mechanism of the Human Connexin 45 Gap Junction Channel

Gap junction channels formed by the 21-member human connexin family enable direct intercellular exchange of ions and small signaling metabolites, coordinating electrical coupling across cardiac, neural and epithelial tissues. Connexin 45 (Cx45), encoded by GJC1, mediates impulse conduction in the atrioventricular node, His bundle, and Purkinje fibers, where disease-linked mutations cause progressive atrioventricular block and familial atrial fibrillation, yet no experimental structure has been reported, and its regulatory mechanism remains undefined. Here, we determine the structural basis of Cx45 gating and Ca2+ regulation using cryo-electron microscopy, mutational analysis, and molecular dynamics simulations. Cryo-EM structures of the apo (2.76 [A]), Ca2+-bound (2.65 [A]), and E41A mutant (3.55 [A]) channel reveal a neck constriction formed by Y45, establishing a steric gate distinct from other connexins. Ca2+ associates with E41, stabilizing the neck via electrostatic remodeling without global conformational change. Together, these data define a dual steric-electrostatic mechanism for Cx45 regulation and provide a structural framework for isoform-specific connexin gating relevant to cardiac physiology and conduction disease.

biochemistry↗

bioRxiv2024

Interleukin-1β Drives Disease Progression in Arrhythmogenic Cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is a genetic form of heart failure that affects 1 in 5000 people globally and is caused by mutations in cardiac desmosomal proteins including PKP2, DSP, and DSG2. Individuals with ACM suffer from ventricular arrhythmias, sudden cardiac death, and heart failure. There are few effective treatments and heart transplantation remains the best option for many affected individuals. Here we performed single nucleus RNA sequencing (snRNAseq) and spatial transcriptomics on myocardial samples from patients with ACM and control donors. We identified disease-associated spatial niches characterized by co-existence of fibrotic and inflammatory cell types and failing cardiac myocytes. The inflammatory-fibrotic niche co-localized to areas of cardiac myocyte loss and was comprised of FAP (fibroblast activation protein) and POSTN (periostin) expressing fibroblasts and macrophages expressing NLRP3 (NLR family pyrin domain containing 3) and NFB activated genes. Using homozygous Desmoglein-2 mutant (Dsg2mut/mut) mice, we identified analogous populations of Postn expressing fibroblasts and inflammatory macrophage populations that co-localized within diseased areas. Detailed single cell RNA sequencing analysis of inflammatory macrophage subsets that were increased in ACM samples revealed high levels of interleukin-1{beta} (Il1b) expression. To delineate the possible benefit of targeting IL-1{beta} in ACM, we treated Dsg2mut/mut mice with an anti-IL-1{beta} neutralizing antibody and observed attenuated fibrosis, reduced levels of inflammatory cytokines and chemokines, preserved cardiac function, and diminished conduction slowing and automaticity, key mechanisms of arrhythmogenesis. These results suggest that currently approved therapeutics that target IL-1{beta} or IL-1 signaling may improve outcomes for patients with ACM.

immunology↗

bioRxiv2024

Computational Modeling of Effects of PKP2 Gene Therapy on Ventricular Conduction Properties in Arrhythmogenic Cardiomyopathy

BackgroundPatients with arrhythmogenic cardiomyopathy (ACM) due to pathogenic variants in PKP2, the gene for the desmosomal protein plakophilin-2, are being enrolled in gene therapy trials designed to replace the defective allele via adeno-associated viral (AAV) transduction of cardiac myocytes. Evidence from experimental systems and patients indicates that ventricular myocytes in PKP2 ACM have greatly reduced electrical coupling at gap junctions and reduced Na+ current density. In previous AAV gene therapy trials, <50% of ventricular myocytes have generally been transduced. MethodsWe used established computational models of ventricular cell electrophysiology to define the effects of varying levels of successful gene therapy on conduction in patients with PKP2 ACM. Conduction velocity and development of conduction block were analyzed in tissue constructs composed of cells with levels of electrical coupling and Na+ current density observed in experimental studies. ResultsWe observed a non-linear relationship between conduction velocity and the proportion of transduced cells. Conduction velocity increased only modestly when up to 40% of myocytes were transduced. Conduction block did not occur in tissue constructs with moderate levels of uncoupling (0.10 or 0.15 of normal) as this degree of coupling was sufficient to allow electrotonic current to pass through diseased cells. Thus, low levels of transduction, likely to occur in phase 1 clinical trials, do not appear to pose a major safety concern. However, our models did not incorporate potential effects of fibrosis and immune signaling, both of which will presumably be present in PKP2 ACM patients undergoing gene therapy. ConclusionsThe extent of successful ventricular myocyte transduction anticipated to be achieved in PKP2 AAV gene therapy trials will likely not restore conduction velocity to levels sufficient to decrease risk of reentrant arrhythmias. What is Known- Patients with arrhythmogenic cardiomyopathy due to pathogenic variants in PKP2 (the gene for the desmosomal protein plakophilin-2) are now being enrolled in gene therapy trials. - Experimental and clinical observations indicate that patients with arrhythmogenic cardiomyopathy have slow ventricular conduction with a propensity to conduction block due to source-sink mismatch. - <50% of ventricular myocytes are usually transduced after adeno-associated viral gene therapy. What the Study Adds- At anticipated levels of successful transduction of ventricular myocytes, little change in conduction velocity will be achieved in patients with arrhythmogenic cardiomyopathy due to variants in PKP2. - Higher levels of transduction could produce conditions that increase risk of conduction block, especially in the presence of areas of non-conducting fibrofatty scar tissue.

physiology↗

bioRxiv2024

Inhibition of Soluble Epoxide Hydrolase Reduces Inflammation and Myocardial Injury in Arrhythmogenic Cardiomyopathy

Previous studies have implicated persistent innate immune signaling in the pathogenesis of arrhythmogenic cardiomyopathy (ACM), a familial non-ischemic heart muscle disease characterized by life-threatening arrhythmias and progressive myocardial injury. Here, we provide new evidence implicating inflammatory lipid autocoids in ACM. We show that specialized pro-resolving lipid mediators are reduced in hearts of Dsg2mut/mut mice, a well characterized mouse model of ACM. We also found that ACM disease features can be reversed in rat ventricular myocytes expressing mutant JUP by the pro-resolving epoxy fatty acid (EpFA) 14,15-eicosatrienoic acid (14-15-EET), whereas 14,15-EE-5(Z)E which antagonizes actions of the putative 14,15-EET receptor, intensified nuclear accumulation of the desmosomal protein plakoglobin. Soluble epoxide hydrolase (sEH), an enzyme that rapidly converts pro-resolving EpFAs into polar, far less active or even pro-inflammatory diols, is highly expressed in cardiac myocytes in Dsg2mut/mut mice. Inhibition of sEH prevented progression of myocardial injury in Dsg2mut/mut mice and led to recovery of contractile function. This was associated with reduced myocardial expression of genes involved in the innate immune response and fewer pro- inflammatory macrophages expressing CCR2, which mediate myocardial injury in Dsg2mut/mut mice. These results suggest that pro-inflammatory eicosanoids contribute to the pathogenesis of ACM and, further, that inhibition of sEH may be an effective, mechanism-based therapy for ACM patients.

cell biology↗