Faculty Bibliography

BACKGROUND/UNASSIGNED:Alterations in microtubule dynamics have been shown to affect cardiomyocyte membrane stiffness and modulate ion channels, including the cardiac sodium channel. While conditions, such as heart failure and Duchenne muscular dystrophy, are associated with increased detyrosination of microtubules and reduced sodium current, a potential role for microtubule detyrosination in arrhythmogenic cardiomyopathy has not been explored. We here investigated the impact of microtubule detyrosination on membrane stiffness, cardiac sodium channel distribution, and function in mouse and human models of arrhythmogenic cardiomyopathy. METHODS/UNASSIGNED:-c.2013delC, and isogenic control human-induced pluripotent stem cell-derived-cardiomyocytes were incubated for 2 to 4 hours with compounds known to decrease microtubule detyrosination (parthenolide, 10 µmol/L; EpoY, 20 µmol/L) or vehicle (dimethyl sulfoxide). Immunocytochemistry, mechano-scanning ion conductance microscopy, patch-clamp analysis, and stochastic optical reconstruction microscopy were performed. RESULTS/UNASSIGNED:-c.2013delC human-induced pluripotent stem cell-derived-cardiomyocytes displayed increased microtubule detyrosination and reduced sodium current compared with isogenic control human-induced pluripotent stem cell-derived-cardiomyocytes, which were both prevented by parthenolide and EpoY. CONCLUSIONS/UNASSIGNED:Increased microtubule detyrosination secondary to loss of PKP2 impacts cardiomyocyte (dys)function beyond the desmosome, contributing to both electrical and mechanical alterations in the setting of arrhythmogenic cardiomyopathy. Our findings identify microtubule detyrosination as a novel therapeutic target in pathophysiological conditions, such as arrhythmogenic cardiomyopathy, aimed at improving both contractile and electrical function.

BACKGROUND/UNASSIGNED:-deficiency in epicardium-derived cells (EPDCs) contributes to fibro-inflammatory signaling and ACM pathogenesis. METHODS/UNASSIGNED:in cardiomyocytes (Pkp2-cKO), in EPDC (Pkp2-eKO), or in both cardiomyocyte and EPDC (Pkp2-ceKO) via the tissue-specific expression of tamoxifen-inducible Cre recombinase. Nonmyocyte populations were isolated 21 days posttamoxifen injection for single-cell RNA-sequencing. Immunohistochemistry, flow cytometry, quantitative reverse transcription polymerase chain reaction, and echocardiography were used to interrogate cardiac physiology and cellular composition. RESULTS/UNASSIGNED:deletion in cardiomyocytes induced a moderate fibro-inflammatory EPDC phenotype, while deletion in EPDC did not elicit a pathological phenotype, suggesting cardiomyocyte involvement is necessary for ACM pathogenesis. Proinflammatory fibroblasts acquired the senescence-associated secretory phenotype, correlating with elevated senescence associated-βgal staining in the right ventricle. Gene expression, flow cytometry, and histological data also revealed an exaggerated inflammatory response in Pkp2-ceKO mice, which progresses from right to left ventricular predominance. Importantly, macrophages and B cells accumulate in both Pkp2-cKO and Pkp2-ceKO mice compared with controls. Although B-cell depletion delays the early inflammatory and fibrosis response, it did not alter end-stage cardiac physiology. CONCLUSIONS/UNASSIGNED:

BACKGROUND:are the most common cause of familial arrhythmogenic right ventricular cardiomyopathy. This study tests whether plakophilin-2 (PKP2) deficiency only in cardiomyocytes is sufficient to provoke premature aging and proinflammatory senescence in nonmyocyte, cardiac resident cells. METHODS:We studied mice with cardiomyocyte-specific, tamoxifen-activated loss of PKP2 (cardiomyocyte-specific conditional knockout of plakophilin-2) using conventional and multiplex imaging, cytokine arrays, epigenetic clocks, spatial transcriptomics, expansion and structured illumination microscopy, and correlative data analysis. We examined nonmyocytes and cardiomyocytes for premature aging and senescence. RESULTS:We observed senescence-associated heterochromatin foci in nonmyocytes, predominantly in cells positive for α-smooth muscle actin staining. Cytokines in media of nonmyocyte cells were consistent with senescence-associated secretory phenotype. Epigenetic clocks identified premature aging. Multiplex immunohistochemistry showed nonmyocyte cells in niches, intermingled with cardiomyocytes. Spatial transcriptomics showed overrepresentation of senescence-associated secretory phenotype-related transcripts, predominantly in myocyte-rich areas of the left ventricle. Senescence-associated heterochromatin foci and increased epigenetic age were not found in cardiomyocytes from cardiomyocyte-specific conditional knockout of plakophilin-2 hearts, although we observed structural features associated with premature aging. Cross-reference analysis showed correlation between the cardiomyocyte-specific conditional knockout of plakophilin-2 cardiac proteome and that of mice 5 or 6 times their chronological age, as well as transcriptional signatures of neurodegenerative diseases. CONCLUSIONS:Loss of PKP2 expression only in adult cardiac myocytes is sufficient to induce proinflammatory senescence in nonmyocytes, and overall premature cardiac aging. This is the first study to intersect cellular senescence and premature aging with desmosomal arrhythmogenic cardiomyopathies. We speculate that cell-agnostic molecular signatures, biomarkers, and pharmacology of senescence and of neurodegenerative diseases may be relevant to diagnose or treat PKP2 arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND:Pathogenic variants in plakophilin-2 (PKP2) cause arrhythmogenic cardiomyopathy (ACM) with intracellular calcium dysregulation as a major component of its arrhythmia phenotype. Recent adeno-associated viral (AAV) based Pkp2 (AAV-PKP2) gene therapy has shown promising results in a few different PKP2-associated ACM models. Fibroblast growth factor 21 (FGF21) has multiple cardioprotective effects and has recently emerged as a promising therapeutic agent for cardiovascular disease. OBJECTIVES/OBJECTIVE:To assess the efficacy and impact on calcium regulation of a novel AAV8-based FGF21 gene therapy on adult cardiac-specific, tamoxifen-activated PKP2 knockout (PKP2-cKO) mice. METHODS:Experiments were performed using a PKP2-cKO murine model. AAV8-FGF21 was delivered to adult mice by a single tail vein injection 7 days before tamoxifen-activated PKP2-cKO. Cardiac functions were monitored by echocardiography and electrocardiography. Intracellular calcium transients were investigated in acute isolated adult mouse cardiomyocytes and calcium fluorescent signals were acquired with the IonOptix system. RESULTS:Loss of PKP2 expression caused cardiac mechanical dysfunction and pro-arrhythmic phenotype in adult mouse models. AAV-mediated delivery of FGF21 mitigated the progression of biventricular structural changes, decreased the occurrence of adrenergic arrhythmias, and rescued intracellular calcium imbalance in the setting of PKP2 haploinsufficiency. In contrast, acute in vitro FGF21 treatment for 1 hour had no effect on intracellular calcium transients. CONCLUSIONS:These beneficial effects of AAV8-FGF21 on the PKP2-ACM phenotype suggest a therapeutic landscape for various targeted cardiomyopathies.

BACKGROUND:Plakophilin-2 (PKP2) is a component of the desmosome. Pathogenic variants can lead to arrhythmogenic cardiomyopathy (PKP2-ACM). In PKP2-ACM patients, exercise and catecholamine surges negatively impact arrhythmia incidence and severity. OBJECTIVE:To characterize remodeling of the sympathetic input and adrenergic response in hearts of PKP2-deficient mice (PKP2cKO) subjected to endurance exercise. METHODS:transient dynamics. Separately, we evaluated distribution of sympathetic terminals in PKP2cKO trained hearts vs controls. RESULTS:Exercise led to increased abundance of sarcolemma β1-ARs in control, and decreased abundance in PKP2cKO-myocytes. OCT3 knockdown drastically reduced the response of trained PKP2cKO-myocytes to norepinephrine but not isoproterenol, indicating preserved response to native catecholamines by intracellular (dyad-associated) receptors in the setting of a reduced sarcolemma pool. In tissue, we observed reduced abundance of sympathetic terminals, and heterogeneous distribution across the myocardium. CONCLUSION/CONCLUSIONS:Endurance exercise in PKP2-deficient myocytes leads to reduced pool of functional β1-ARs in the sarcolemma and yet availability of intracellular receptors, which can activate selected (and heterogeneous) routes of intracellular signaling cascades. We speculate that remodeling of nerve terminals affects sympathetic input distribution and hence, regional modulation of excitability and conduction. These changes can facilitate cell-generated triggered activity and heterogeneity of the underlying substrate, setting the stage for life-threatening arrhythmias.

BACKGROUND:Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy associated with a high risk of ventricular arrhythmia (VA). Several animal models have been used to postulate a therapeutic role of the inhibition of the ryanodine 2 receptor via the use of flecainide for this disease. Clinical data describing its use are scarce, however, especially in patients without implantable cardioverter-defibrillators or with left ventricular (LV) involvement. OBJECTIVES/OBJECTIVE:This study sought to report safety and effectiveness long-term, multicenter data on the impact of flecainide therapy on arrhythmic outcomes in patients with a definite diagnosis of ARVC. METHODS:Patients with definite ARVC receiving flecainide at 12 academic institutions were enrolled in the study. Baseline was defined as the time of flecainide initiation. Premature ventricular complex burdens, nonsustained ventricular tachycardia (NSVT) rates, and sustained VA yearly/rates were collected and compared while on and off flecainide. Side effects and flecainide discontinuation were tracked. Analyses were performed in the overall cohort as well as stratifying for genotype (gene positive vs negative; plakohpillin-2 [PKP-2] vs non PKP-2) and for LV involvement. RESULTS:; LV ejection fraction 55.9 ± 7.3%; right ventricular ejection fraction 44.5 ± 10.5% at baseline) were enrolled, with 66 patients (34.6%) showing LV involvement. The median dose of flecainide was 200 mg/d [150-200 mg/d], with 166 patients (86.9%) also taking a beta-blocker. The median follow-up time on flecainide was 4.2 years [1.9-6.3 years]. Flecainide was well tolerated, with a low (7.9%) discontinuation rate. After flecainide initiation, a significant reduction in the 24-hour premature ventricular complex burden and in the rate of nonsustained ventricular tachycardia was observed (2,190 vs 418; P < 0.001; 35.1% vs 21.5%; P = 0.003). For patients with prior VA events, a significant reduction in the amount of VA episodes/y (1.1 [0.4-1.6] episodes/y vs 0 [0-0.3] episodes/y; P < 0.001) was observed. These safety and effectiveness findings were consistent across genotype subgroups, as well as in patients with and without LV involvement. CONCLUSIONS:Flecainide use had a favorable safety profile and was associated with an observed to a significant reduction in arrhythmic burden in patients with ARVC, irrespective of the underlying genotype or LV involvement.