FHF (fibroblast growth factor homologous factor) 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 (connexin 43) gap junctions and hemichannels, mechanisms that have generally been understudied or disregarded. We assessed cardiac conduction and cardiomyocyte action potentials in mice with constitutive cFgf13KO while targeting Cx43 gap junctions and hemichannels pharmacologically. We characterized FGF (fibroblast growth factor) 13 regulation of Cx43 abundance and subcellular distribution. With proximity labeling proteomics, we investigated novel candidate mechanisms underlying FGF13 regulation of Cx43. FGF13 ablation prolonged the QRS and QT intervals. Carbenoxolone, a Cx43 gap junction uncoupler, markedly prolonged the QRS duration, leading to conduction system block in cFgf13KO but not in wild-type mice. Optical mapping revealed markedly decreased conduction velocity during ventricular pacing. Microscopy revealed perturbed trafficking of Cx43, reduced localization in the intercalated disc, and suggested decreased membrane Cx43 but increased Cx43 hemichannels in cardiomyocytes from cFgf13KO mice. Resting membrane potential was depolarized, and action potential duration at 50% repolarization was prolonged in cFgf13KO cardiomyocytes. Both were restored toward wild-type 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 resting membrane potential depolarization, hearts were subjected to hypokalemia, which had no effect in wild-type hearts but fully rescued conduction velocity in cFgf13KO hearts. Proteomic analyses revealed candidate roles for FGF13 in the regulation of vesicular-mediated transport. FGF13 ablation destabilized microtubules and reduced the expression of tubulins and MAP4, the major cardiac microtubule regulator. FGF13 regulates microtubule-dependent trafficking and targeting of Cx43 and impacts cardiac impulse propagation via VGSC-independent mechanisms.

Cardiac hypertrophy, defined as a stress-induced increase in heart mass/size, is a major risk factor for adverse cardiovascular events, including heart failure and arrhythmia. Within this general definition, the orientation of cell and organ growth varies considerably depending on stress type and duration, with important implications for cardiac function, yet little is known regarding the mechanisms that regulate hypertrophic orientation. Here, we evaluated the role of the cytoskeletal protein βIV-spectrin and associated prohypertrophic STAT3 (signal transducer and activator of transcription 3) to direct the orientation of hypertrophic growth. Transgenic mouse models with altered STAT3 signaling through modified interaction with its scaffolding partner βIV-spectrin, or phospho-regulation of STAT3 directly, were evaluated at baseline, and after transaortic constriction, or aortocaval fistula. Unbiased screening of gene expression from these structurally divergent states was evaluated for pathways responsible for directing myocyte length/width. These pathways were tested in vitro using primary mouse myocytes and in vivo to tune growth patterns for therapeutic intervention. Loss of βIV-spectrin or direct STAT3 activation promoted a preferential increase in myocyte length over width, resulting in dilation of the left ventricular chamber (eccentric hypertrophy) and decreased systolic function. Conversely, preservation of βIV-spectrin favored an increase in myocyte width without left ventricular dilation (concentric hypertrophy) and preserved systolic function in response to transaortic constriction or aortocaval fistula. Differential expression of genes associated with microtubules, including the trafficking kinesin motor, KIF20A (kinesin family member 20A), were identified in concentric versus eccentric hypertrophic states. In vitro assays revealed a relationship between βIV-spectrin/STAT3 signaling, KIF20A expression, microtubule density, and spatial distribution of mRNA for the sarcomeric gene actc1. Finally, intervention with pharmacological STAT3 inhibition after chronic 6-week transaortic constriction successfully recovered concentric growth with improved systolic function. These data identify a novel and pivotal role for βIV-spectrin/STAT3 to modify microtubule properties and sarcomeric transcript distribution to direct myocyte geometry in response to chronic stress. These studies further illustrate the unique separation of hypertrophic growth and orientation as distinct pathways in cardiac remodeling.

Aging is a major, yet unmodifiable, cardiovascular risk factor and is associated with vascular alterations, increased cardiac fibrosis, and inflammation, all of which contribute to impaired cardiac function. However, the microenvironment inciting age-related alterations within the multicellular architecture of the cardiac tissue is unknown. We investigated local microenvironments in aged mice hearts by applying an integrative approach combining single-nucleus RNA sequencing and spatial transcriptomics of 3- and 18-month-old mice. We defined distinct cardiac niches and studied changes in their cellular composition and functional characteristics. We treated mice with broad-spectrum senolytics dasatinib and quercetin, and endothelial-specific senolytic fisetin and studied their effects on senescence and macrophage populations. Integration of spatial transcriptomics data across 3- and 18-month-old hearts allowed the identification of 11 cardiac niches, which were characterized by distinct cellular composition and functional signatures. Aging did not alter the overall proportions of cardiac niches but led to distinct regional changes, particularly in the left ventricle. While cardiomyocyte-enriched niches showed disrupted circadian clock gene expression, vascular niches showed major changes in proinflammatory and profibrotic signatures and altered cellular composition. We particularly identified larger vessel-associated cellular niches as key hotspots for activated fibroblasts and bone marrow-derived Lyve1- (lymphatic vessel endothelial hyaluronan receptor 1) and resident Lyve1+ macrophages in aged hearts, with interactions of both cell types through the C3:C3ar1 (complement C3 and Complement C3a receptor 1) axis. These niches were also enriched in senescent cells exhibiting high expression of immune evasion mechanisms that may impair senescent cell clearance. Removal of senescent cells by senolytics reduced the presence of Lyve1- macrophages. Our findings indicate that the perivascular microenvironment is particularly susceptible to age-related changes and serves as a primary site for inflammation-driven aging, so-called inflammaging. This study provides new insights into how aging reshapes cardiac cellular architecture, highlighting vessel-associated niches as potential therapeutic targets for age-related cardiac dysfunction.

Mitochondrial calcium (Ca2+) is a key regulator of cardiac energetics by stimulating the tricarboxylic acid cycle during elevated workload. Atrial fibrillation (AF) is associated with a reduction in cytosolic Ca2+ transient amplitude, but its effect on mitochondrial Ca2+ handling and cellular redox state has not been explored. Cardiac myocytes isolated from patient-derived right atrial biopsies were subjected to workload transitions using patch-clamp stimulation and β-adrenergic stimulation (isoproterenol). In conjunction, nicotinamide adenine dinucleotide (phosphate)/flavin adenine dinucleotide (NAD[P]H/FAD) autofluorescence, cytosolic and mitochondrial [Ca2+] were monitored using epifluorescence microscopy. Sarcoplasmic reticulum and mitochondria were imaged using electron microscopy and tomography and stimulated emission depletion microscopy. The effects of the mitochondrial Ca2+ uptake enhancer ezetimibe on proarrhythmic activity in atrial myocytes and on AF burden in patients were investigated. Mitochondrial Ca2+ accumulation during increased workload was blunted in AF, and was associated with impaired regeneration of nicotinamide adenine dinucleotide and flavin adenine dinucleotide. Nanoscale imaging revealed spatial disorganization of sarcoplasmic reticulum and mitochondria, associated with microtubule destabilization. This was confirmed in human induced pluripotent stem cell-derived cardiac myocytes, where treatment with the microtubule destabilizer nocodazole displaced mitochondria and increased proarrhythmic Ca2+ sparks, which were rescued by MitoTEMPO. Ezetimibe also reduced the occurrence of arrhythmogenic Ca2+ release events both in AF myocytes and nocodazole-treated human induced pluripotent stem cell-derived cardiac myocytes. Retrospective patient analysis also revealed a reduced AF burden in patients on ezetimibe treatment. Mitochondrial Ca2+ uptake and accumulation are impaired in atrial myocytes from patients with AF. The disturbed spatial association between sarcoplasmic reticulum and mitochondria driven by destabilized microtubules may underlie impaired Ca2+ transfer in AF. Enhancing mitochondrial Ca2+ uptake potentially protects against arrhythmogenic events.

The homeostatic chemokine CCL21 (C-C motif chemokine ligand 21) is abnormally elevated in coronary artery disease. Plasma CCL21 levels have been found to be independently associated with adverse outcomes after acute coronary syndrome. However, the specific effects of CCL21 on coronary artery disease-associated platelet activation and thrombosis remain poorly understood. We examined the effects of CCL21 on platelet activation, spreading, clot retraction, in vitro shear stress-induced thrombus formation, in vivo arterial thrombus formation, middle cerebral artery occlusion-induced brain injury, and myocardial ischemia-reperfusion injury. We also investigated the underlying mechanisms and the therapeutic impacts of a CCL21 antibody on platelet activation and in vivo thrombosis in atherosclerosis. CCL21 potentiated agonist-induced platelet activation, including aggregation, dense granule release, P-selectin exposure, integrin αIIbβ3 activation, spreading, and clot retraction. Furthermore, CCL21 enhanced in vivo thrombosis, whole blood thrombus formation, and middle cerebral artery occlusion-induced brain injury. Mechanistically, CCL21 binds to platelet CCR7 (C-C motif chemokine receptor 7), a G-protein-coupled receptor previously unreported in platelets, activating Gi (inhibitory G protein) and G13 signaling pathways to enhance platelet activation. A CCL21 antibody attenuated platelet activation and inhibited in vivo thrombosis in patients with coronary artery disease and atherosclerotic ApoE-/- mice. In addition, this antibody mitigated microvascular thrombosis, safeguarding the hearts of atherosclerotic ApoE-/- mice from severe ischemia-reperfusion injury. CCL21 enhances platelet activation and atherothrombosis by binding to platelet CCR7 and thus activating downstream Gi and G13 signaling pathways. A CCL21 antibody can counteract these effects in the context of coronary artery disease, supporting its potential as a preventive therapy for thrombotic complications.

Peripheral artery disease is a severe ischemic vascular pathology without effective pharmacological approaches and improving angiogenesis to recover blood perfusion is a promising therapeutic strategy. Endothelial cells are the primary cell type contributing to angiogenesis in response to ischemia. However, the molecular mechanisms regulating ischemia-induced angiogenesis remain elusive. We used a discovery-driven approach to identify elevated SRSF1 (serine/arginine splicing factor 1) expression in endothelial cells after ischemia. We used loss- and gain-of-function approaches to explore the role of SRSF1 in angiogenesis both in vivo and in vitro. A mouse model of hindlimb ischemia was used to evaluate ischemia-induced angiogenesis. We also investigated the mechanisms through transcriptome, enhanced crosslinking and immunoprecipitation sequencing, RNA pull-down, and chromatin immunoprecipitation-quantitative polymerase chain reaction analysis. Proteomic analyses identified endogenous SRSF1 accumulated in endothelial cells of the ischemic muscle and responded to hypoxia. Mice deficient in endothelial SRSF1 exhibited impaired blood flow recovery and impaired vasculature formation after hindlimb ischemia. Importantly, overexpression of SRSF1 enhanced blood flow recovery and angiogenesis after hindlimb ischemia. SRSF1 overexpression enhanced the angiogenic functions of human endothelial cells, promoting tube formation, sprouting capability, and cell migration, while SRSF1 knockdown suppressed these functions. Mechanistically, SRSF1 modulated the alternative splicing of ATF3 (activating transcription factor 3) by directly binding to ATF3 premRNA, and SRSF1 overexpression elevated full-length ATF3 transcript at the expense of truncated ATF3Δzip2 transcript. ATF3 then bound directly to the KLF2 (Krüppel-like factor 2) promoter, suppressed KLF2 expression and downstream S1PR1 (sphingosine-1-phosphate receptor 1) signaling. Through upregulation of full-length ATF3 and downregulating KLF2-S1PR1 signaling, SRSF1 promoted endothelial tube formation and angiogenesis. In addition, alprostadil, the prostaglandin E1 analog, could activate the SRSF1 signaling to improve endothelial angiogenesis in vitro and in vivo. Our findings identified SRSF1 as a novel regulator of ischemia-induced angiogenesis that enhances endothelial angiogenic functions by regulating the ATF3-KLF2-S1PR1 pathway. These results suggest that modulation of endothelial SRSF1 may represent a promising therapeutic approach for treating ischemic vascular diseases.