Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disease that causes sudden death in young adults and athletes. ARVC is characterized by extensive electrical involvement, ...though structural defects are equally important as fibro‐fatty replacement of myocardium leads to ventricular dysfunction and failure. ARVC is termed a “disease of the desmosome” as 40% of mutations in ARVC patients are found in the cardiac desmosomal components, with plakophilin‐2 (PKP2) being the most frequently mutated desmosomal gene in ARVC. Studies of PKP2 ARVC populations suggest that a majority of mutations impact PKP2 protein levels via diverse mechanisms (nonsense mutation, insertion/deletion mutation, splice site mutation). There are currently no effective treatments or cures for ARVC, thus, strategies elevating PKP2 protein levels would represent a clinically relevant avenue to address a large portion of ARVC populations. We hypothesize that PKP2 protein dose is a critical driver of ARVC, and that early delivery of PKP2 via adeno‐associated virus (AAV) can prevent disease development. Through CRISPR‐Cas9 we generated a novel knock‐in mouse model harboring a human equivalent PKP2 mutation (IVS10‐1 G>C) that impacts PKP2 RNA splicing, PKP2 protein levels, and is sufficient to recapitulate all classic ARVC disease features. PKP2 homozygous mutant (PKP2 Hom) mice are viable at birth yet display adult hallmarks of ARVC including ventricular arrhythmias, right and left ventricular dysfunction, and fibro‐fatty replacement of myocardium leading to sudden death starting at 4 weeks of age. PKP2 Hom mice display reduced PKP2 protein levels, which results in a disruption of desmosomal and gap junction proteins by the onset of disease features. Using a cardiotropic AAV9, early delivery at postnatal day 2 of PKP2 (AAV9‐PKP2) resulted in restoration of PKP2 protein to wild type levels by 4 weeks of age in PKP2 Hom mice. This caused a significant improvement in cell‐cell junction protein levels. Cardiac function (both left and right ventricles) and electrophysiology were significantly improved at 4 weeks of age as well. Histological analysis further showed improved morphology in PKP2 Hom mice treated with AAV9‐PKP2, as well as significantly less fibrosis throughout ventricular myocardium. Kaplan‐Meier survival curves highlighted a dramatic improvement in survival in PKP2 Hom mice treated with AAV9‐PKP2, suggesting early administration of AAV9‐PKP2 resulted in a beneficial and durable impact on cardiac function. We provide a novel mouse model that incorporates PKP2 patient genetics and serves as an ideal platform to evaluate therapies for ARVC. Early administration of PKP2 via AAV9 represents an effective and clinically relevant approach to prevent ARVC disease development.
Abstract only Introduction: Arrhythmogenic cardiomyopathy (ACM) is a rare, incurable inherited cardiac disease resulting in mortality through sudden death and heart failure. ACM cases are linked to ...mutations in desmosomal genes, with the most frequent related to plakophilin-2 (PKP2), resulting in haploinsufficiency. LX2020 is an adeno-associated viral (AAV) based gene therapy intended to restore human PKP2 (hPKP2) protein expression. However, its efficacy in disease settings and safety has not been established. Hypothesis: LX2020 mediated hPKP2 expression is sufficient to scaffold the desmosomal complex to prevent cardiomyocyte loss and circumvent structural damage to improve cardiac electrical and mechanical function as well as promote survival, while being safe and well tolerated. Methods: LX2020 was intravenously administered to PKP2 mutant mice with severe ACM and non-human primates (NHPs) at various doses and was evaluated 12 weeks post-dose for efficacy and safety, respectively. Endpoints in mice involved effect of LX2020 on various cardiac disease-relevant phenotypes and survival, while safety, tolerability, biodistribution and expression was assessed in NHPs. Results: In ACM disease settings, LX2020 showed dose-dependent increase in hPKP2 expression that was sufficient to restore desmosomal proteins and cell-cell junctional complexes inhibiting further cardiac structural damage resulting in improved electrical and mechanical dysfunction, ultimately leading to improved survival. In NHPs, LX2020 showed dose-dependent biodistribution in various organs including the heart, with increases in cardiac hPKP2 mRNA and protein expression that were safe and well tolerated. Notably, LX2020 showed cardiac hPKP2 expression in mice and NHPs, which reached above or close to healthy human PKP2 protein levels, respectively, indicating that LX2020 could lead to therapeutic levels of PKP2 expression. Conclusions: Collectively, these data support the efficacy of LX2020 in restoring cardiac hPKP2 expression and rescuing the progressively advancing severe ACM diseae, while being safe and well tolerated. LX2020 offers a promising gene therapy that provides maximal therapeutic benefit with minimal safety risk for PKP2-ACM populations.
Many different approaches have been made to explain the nature of dark matter (DM), but it remains and unsolved mystery of our universe. In this work we examine a type II two-Higgs-doublet model ...extended by a complex singlet (2HDMS), where the pseudo-scalar component of the singlet acts as a natural DM candidate. The DM candidate is stabilized by a Z'2 symmetry, which is broken spontaneously by the singlet acquiring a vacuum expectation value (vev). This vev in turn causes the scalar component of the singlet to mix with the scalar components of the two doublets, which results in three scalar Higgs particles. Additionally we aim to include an excess around 95 GeV, which was observed at CSM and LEP and can be explained by one of the three scalar Higgs particles. After introducing the model, we apply experimental and theoretical constraints and find a viable benchmark point. We then look into the DM phenomenology as well as collider phenomenology.
The Two Higgs Doublet model extended with a complex scalar singlet (2HDMS) is a well-motivated Beyond Standard Model candidate addressing several open problems of nature. In this work, we focus on ...the dark matter (DM) phenomenology of the complex scalar singlet where the real part of the complex scalar obtains a vacuum expectation value. The model is characterized by an enlarged Higgs spectrum comprising six physical Higgs bosons and a pseudoscalar DM candidate. We address the impact of accommodating the 95 GeV excess on the 2HDMS parameter space and DM observables after including all theoretical and experimental constraints. Finally, we look into the prospects of this scenario at HL-LHC and future lepton colliders for a representative benchmark.
Abstract only
Introduction:
Arrhythmogenic right ventricular cardiomyopathy (ARVC) increases risk of sudden cardiac death (SCD) and can progress to heart failure. Clinical studies have established ...40-50% prevalence of mutations to desmosomal genes, with plakophilin-2 (PKP2) being the most frequent. To date, no effective treatment exists for ARVC. LX2020 is a novel AAV gene therapy that restores cardiac expression of human PKP2. A proof-of-concept study with LX2020 was conducted in a severe novel PKP2 mutant mouse model that exhibits all classic phenotypes of ARVC.
Hypothesis:
LX2020 mediated PKP2 expression is sufficient to scaffold the desmosomal cell-cell complex to promote survival as well as preserving cardiac electrical and mechanical function.
Methods:
A total of N=16 PKP2 Hom mice were randomized for vehicle/formula control (VC), low dose (LD) and high dose (HD). LX2020 was administered at postnatal day 2 and mice were analyzed 4-weeks post administration. Endpoints included survival, EKG, cardiac MRI and desmosomal levels of PKP2, desmoplakin (DSP), desmoglein-2 (DSG2), plakoglobin (JUP), along with fascia-adherens (N-Cad), and gap junction (CX43), using western blot analyses.
Results:
LX2020 showed dose dependent improvements in ARVC related deficits in PKP2 mice, including survival. HD resulted in 100% survival of PKP2 mutant mice, while LD resulted in 60% survival, when compared to 50% survival with VC. ECG data revealed suppression of ectopic beats in 100% of PKP2 mutant mice at HD and 75% suppression at LD when compared to 60% with VC. MRI HD improved left and right ejection fraction, as well as reduced right and left end-diastolic and systolic volumes, when compared to VC. LD showed a mild impact on left ventricular end-diastolic and end-systolic volumes like HD. Mechanistically, while LD showed limited increase in cardiac PKP2 expression, HD showed substantial cardiac PKP2 expression. Significantly increased levels of other desmosomal proteins, especially DSP and DSG2, were seen suggesting scaffolding functions to PKP2.
Conclusions:
These data indicate that the restoration of cardiac PKP2 expression can halt or slow ARVC disease progression, making LX2020 a promising gene therapy candidate for treating PKP2 mutation patients.
Regulated protein degradation through the ubiquitin–proteasome and lysosomal/autophagy systems is critical for homeostatic protein turnover in cardiac muscle and for proper cardiac function. The ...discovery of muscle-specific components in these systems has illuminated how aberrations in their levels are pivotal to the development of cardiac stress and disease. New evidence suggests that equal importance in disease development should be given to ubiquitously expressed degradation components. These are compartmentalized within cardiac muscles and, when mislocalized, can be critical in the development of specific cardiac diseases. Here, we discuss how alterations in the compartmentalization of degradation components affect disease states, the tools available to investigate these mechanisms, as well as recent discoveries that highlight the therapeutic value of targeting these pathways in disease.
The mitogen activated protein kinase (MAPK)-extracellular regulated kinase 1/2 (ERK1/2) pathway is a central downstream signaling pathway that is activated in cardiac muscle cells during mechanical ...and agonist-mediated hypertrophy. Studies in genetic mouse models deficient in ERK-associated MAPK components pathway have further reinforced a direct role for this pathway in stress-induced cardiac hypertrophy and disease. However, more recent studies have highlighted that these signaling pathways may exert their regulatory functions in a more compartmentalized manner in cardiac muscle. Emerging data has uncovered specific MAPK scaffolding proteins that tether MAPK/ERK signaling specifically at the sarcomere and plasma membrane in cardiac muscle and show that deficiencies in these scaffolding proteins alter ERK activity and phosphorylation, which are then critical in altering the cardiac myocyte response to stress-induced hypertrophy and disease progression. In this review, we provide insights on ERK-associated scaffolding proteins regulating cardiac myofilament function and their impact on cardiac hypertrophy and disease.
Abstract only Human genetic studies and mouse models have classically linked mutations/deficiencies in components of the desmosomal cell-cell junction to arrhythmogenic right ventricular ...cardiomyopathy (ARVC). However, growing evidence points to the importance of the desmosome in cardiac diseases beyond ARVC, which include electrical diseases that have no impact on cardiac structure/morphology. The mechanisms of how defects in cardiac desmosomal protein homeostasis drive these distinct forms of cardiac disease remain elusive to the field. To uncover mechanisms that underlie the distinct pathophysiology (structural versus non-structural) encompassed by desmosomal mutations/loss, we performed an unbiased yeast-two-hybrid screen using an adult human heart cDNA library and the desmosomal protein, desmoplakin (DSP) to uncover new regulators of cardiac desmosomal protein homeostasis. We identified synaptosomal-associated protein 29 (SNAP29), as a novel DSP-interacting protein in the adult human heart. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy; however, its role at the desmosome and in the heart is undefined. We show that SNAP29 is a subcomponent of the cardiac desmosome, as it co-localizes with DSP in the adult heart and DSP-deficient hearts harbor loss of SNAP29. Cardiomyocyte-specific SNAP29 knockout (SNAP29-cKO) mice displayed baseline and pacing-induced ventricular arrhythmias in an age-dependent manner in the absence of cardiac structural and functional deficits. We show that a loss of a subset of desmosomal proteins and connexin43 as well as upregulation of selective autophagy-mediated degradation underlie SNAP29 deficient cardiomyocyte arrhythmias. In line with this, acute blockade of autophagy was sufficient to rescue desmosomal and connexin43 protein levels as well as arrhythmias in SNAP29 deficient cardiomyocytes. In conclusion, SNAP29 insulates a subset of desmosomal and gap junction proteins from selective autophagy-mediated degradation to restrict cardiac arrhythmias. Thus, loss of SNAP29-desmosome-gap junction interactome may predispose the heart to desmosomal based diseases of an electrical nature.
Abstract only
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disease that causes sudden death in young adults and athletes. ARVC is termed a “disease of the desmosome” as ...40% of mutations in ARVC patients are found in the cardiac desmosomal components, with plakophilin‐2 (PKP2) being the most frequently mutated desmosomal gene in ARVC. Evidence from humans suggests that altered RNA splicing may be a critical mechanism through which PKP2 patient genetics drive ARVC. Studies have suggested that approximately one third of all human disease‐causing mutations are a result of defects in RNA splicing; highlighting the importance of testing the relevance of this mechanism in human ARVC. However, there are no models and limited mechanistic insights into how mutations in RNA splicing impact ARVC disease pathogenesis. We hypothesize that RNA splicing mutations in PKP2 are sufficient to recapitulate classic disease features associated with human ARVC and can provide valuable mechanistic insight into how altered RNA splicing impacts disease. Through CRISPR‐Cas9 we generated a novel knock‐in mouse model harboring a human equivalent PKP2 mutation (IVS10‐1 G>C) that impacts RNA splicing and is sufficient to recapitulate all classic ARVC disease features. PKP2 homozygous mutant (PKP2 Hom) mice are viable at birth yet display adult hallmarks of ARVC including ventricular arrhythmias, right and left ventricular dysfunction, and fibro‐fatty replacement of myocardium leading to sudden death. RNA and sequencing analyses of exons spanning the PKP2 mutation site reveals a larger PKP2 transcript that retains a 54 base pair portion of the intronic sequence in PKP2 Hom hearts. However, RNA analysis of exons outside the PKP2 mutation site reveals PKP2 RNA transcripts at similar levels to wild type PKP2, suggesting that total RNA levels are not impacted by the mutation. Instead, we show that PKP2 Hom hearts express a higher molecular weight mutant PKP2 protein in the absence of endogenous PKP2. Analysis of PKP2 Hom neonatal hearts and cardiomyocytes reveal early desmosomal protein loss coupled with a susceptibility to baseline arrhythmias in the absence of overt cardiac disease features at this stage (e.g., no changes in heart weight/body weight ratios, no upregulation of cardiac stress and fibrotic markers). We provide a novel mouse model that highlights the sufficiency and molecular consequences of a PKP2 RNA splicing mutation that triggers all of the classic early and adult onset disease features associated with human ARVC.
Support or Funding Information
National Science Foundation (Graduate Research Fellowship Program), National Institutes of Health (NIH R01, HL142251‐A1)
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