Biology subjects

Kushner, J.

Publications and source records attributed to Kushner, J..

3 recordsLinked to original sources

bioRxiv2025

FGF13 regulates cardiomyocyte impulse propagation via Cx43 trafficking independent of voltage-gated sodium channels

BackgroundFibroblast growth factor homologous factor (FHF) variants associate with arrhythmias. Although FHFs are best characterized as regulators of voltage gated sodium channel (VGSC) gating, recent studies suggest broader, non-VGSC-related functions, including regulation of Cx43 gap junctions and/or hemichannels, mechanisms that have generally been understudied or disregarded. MethodsWe assessed cardiac conduction and cardiomyocyte action potentials in mice with constitutive cardiac-specific Fgf13 ablation (cFgf13KO) while targeting Cx43 gap junctions and hemichannels pharmacologically. Using immunostaining and biochemistry, we characterized FGF13 regulation of Cx43 abundance and subcellular distribution. With proximity labeling proteomics, we investigated novel candidate mechanisms underlying FGF13 regulation of Cx43. ResultsFGF13 ablation prolonged the QRS and QT intervals on the surface electrocardiogram. Carbenoxolone, a Cx43 gap junction uncoupler, markedly prolonged the QRS duration leading to conduction system block in cFgf13KO but not in WT mice. Optical mapping revealed markedly decreased conduction velocity (CV) during ventricular pacing. Microscopy revealed markedly perturbed trafficking of Cx43, reduced localization in the intercalated disc, and suggested decreased membrane Cx43 but increased Cx43 hemichannels in cardiomyocytes from cFgf13KOmice. Resting membrane potential (RMP) was depolarized and APD50 was prolonged in cFgf13KOcardiomyocytes. Both were restored towards wildtype (WT) values with Gap19 (a Cx43 hemichannel inhibitor), expression of FGF13, or expression of a mutant FGF13 incapable of binding to VGSCs, emphasizing VGSC-independent regulation by FGF13. To assess the functional impact of RMP depolarization, hearts were subjected to hypokalemia, which had no effect in WT hearts but fully rescued CV in cFgf13KO hearts. Proteomic analyses revealed candidate roles for FGF13 in the regulation of vesicular-mediated transport. Biochemistry and immunocytochemistry showed that FGF13 ablation destabilized microtubules and reduced the expression of tubulins and MAP4, the major cardiac microtubule regulator. ConclusionsFGF13 regulates microtubule-dependent trafficking and targeting of Cx43, thereby impacting cardiac impulse propagation via VGSC-independent mechanisms.

physiology↗

bioRxiv2025

The nucleus activates mechano-responsiveness via FHOD-associated LINC complexes

The nucleus is the defining organelle of eukaryotic cells. It is usually considered a target organelle for cellular inputs. Here, we find that the nucleus is not simply a "passive" responder, but an active organelle directing the mechanical properties of the actin cytoskeleton it engages. Biochemically, interaction of FHOD formins with nesprin-2 of the nuclear LINC complex activates their actin bundling activity making them more potent than known bundlers like fascin or -actinin. In cells, FHOD-associated LINC complexes enhance the mechanical resistance of nuclear-engaged actin cables in polarizing fibroblasts and sarcomeres in developing cardiomyocytes. Hypertrophic cardiomyopathy-associated variants of FHOD3 are defective in these processes. In mice, the FHOD3 R637P disease-causing allele results in embryonic lethality when homozygous and in stress-induced cardiac hypertrophy when heterozygous. These results show that the nucleus actively directs its mechanical environment and that disruption of this capability in heart leads to cardiac hypertrophy.

cell biology↗

bioRxiv2025

Probing the CaV1.2 interactome in heart failure identifies a positive modulator of inotropy and lusitropy

BackgroundMaladaptive changes in the function, expression and localization of proteins involved in calcium-handling worsen the impaired contractility of systolic heart failure (HF). Standard proteomics techniques require cell lysis and so are unable to characterize changes specific to the critical sub-cellular domain bounded by the T-tubule and the sarcoplasmic reticulum, known as the cardiac dyad. Traditional approaches are also less likely to capture low-affinity protein-protein interactions on lipid membranes. To improve our understanding of heart failure pathophysiology, we applied proximity proteomics to the cardiac dyad of mice with ischemic cardiomyopathy. MethodsUsing two lines of transgenic mice expressing fusion proteins of the engineered ascorbate peroxidase APEX2 with subunits of the cardiac voltage gated calcium channel, CaV1.2, we labeled the dyad proteome of live, intact myocytes from healthy mice (N=6) and mice with coronary artery ligation HF (N=5) by peroxidase-catalyzed biotinylation. Quantitative mass spectrometry with isobaric tandem mass tags (TMT) was used to assess alterations in the local dyad proteome in myocytes from mice with chronic, remodeled HF. We subsequently generated a mouse with inducible cardiac overexpression of Galectin-1 to examine the effects of this protein on cardiac function and more specifically on the calcium-handling properties of mature myocytes, using cellular electrophysiology, calcium imaging, and echocardiography. ResultsFrom mice with HF, we found significant enrichment of 43 proteins defined by their abundance and proximity to transgenic CaV1.2 1C channels, and a significant reduction in 22 proteins, out a of a total of 2326 proteins quantified. We also significantly enriched 286 proteins, and saw a reduction in 13 proteins, defined by proximity and abundance to CaV1.2 {beta}2B-subunits, out of a total 2236 proteins identified. Pathway analysis revealed HF is associated with increased abundance of components of the 26s proteasome and microtubules in the dyad, as well as the dimerizing, carbohydrate binding protein Galectin-1. Cardiac specific overexpression of Galectin-1 in healthy mice increases activation of CaV1.2 and the Ryanodine receptor and accelerates myocyte relaxation through phosphorylation of Phospholamban. ConclusionsUsing proximity proteomics to examine the effects of HF in vivo, we find increased localization of Galectin-1 to the cardiac dyad, and that overexpression of Galectin-1 accelerates calcium kinetics in the heart.

cell biology↗