Biology subjects

Ocorr, K.

Publications and source records attributed to Ocorr, K..

11 recordsLinked to original sources

bioRxiv2026

Deficiency in MICOS component Chchd3 Compromises Drosophila Heart Function via mitophagy, ROS and ER Stress

A pair of paralogs, Chchd3 and Chchd6, two components of mitochondrial contact site and cristae organizing system (MICOS), have been identified to be candidate pathogenetic genes in congenital heart disease (CHD). Previous research found that knockdown (KD) of the single Chchd3/6 (Chchd3) gene and other MICOS components in Drosophila impaired heart function, likely due to a deficit in mitochondrial organization, ATP production, actomyosin levels, and thus severely diminished contractility. However, the underlying mechanisms of how MICOS deficiency leads to these defects are not clear. Here, we performed genetic manipulations in the Drosophila heart to probe for possible interactions between MICOS-compromised mitochondria and other organelles and processes. We found that moderate reduction in Pink1/parkin-mediated mitophagy synergistically aggravated cardiac Chchd3 KD phenotypes, indicating a major interaction. Further, Chchd3 KD increased the level of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Interestingly, KD of catalase (CAT) also elevated cardiac ROS levels, but surprisingly did not compromise contractility either by itself or in combination with Chchd3 KD to aggravate the cardiac phenotype. However, CAT overexpression (OE) in Chchd3 KD hearts restored contractility, but only partially, even though elevated ROS due to Chchd3 KD was fully normalized. Similarly, counteracting ER stress by overexpressing Xbp1 (or spliced mouse Xbp1) also partially rescued the heart function defects induced by Chchd3 KD. Overall, these data indicate a critical role of mitophagy and ER/oxidative stress in cardiac homeostasis involving Chchd3, which suggests that deficiency of MICOS function contributes to heart dysfunction via multiple stress responsive pathways.

genetics↗

bioRxiv2024

Rigosertib reverses hypertrophic cardiomyopathy in RAF1-associated Noonan syndrome

BackgroundRASopathies constitute a group of rare genetic disorders caused by mutations in genes that reside along the canonical Ras/MAPK signaling pathway, affecting cell growth and differentiation. These syndromes, which include conditions like Noonan syndrome (NS), are characterized by developmental delays, distinctive facial dysmorphia, and a variety of cardiac defects, notably hypertrophic cardiomyopathy (HCM). Despite their prevalence and impact, therapeutic options for RASopathies remain limited. Rigosertib, a novel dual Ras/MAPK and PI3K/AKT pathway inhibitor, is currently in clinical trials for treatment of melanoma and recessive dystrophic epidermolysis bullosa. Here, we identify rigosertib as a candidate therapy for RAF1-associated HCM. Methods and ResultsOur Drosophila screen of clinically relevant drugs and compounds identified rigosertib as broadly effective across a panel of transgenic RASopathy fly transgenic models, indicating that rigosertib may be effective against multiple disease isoforms. Analysis of a Drosophila model targeting a RAF1L613V transgene to the heart found that rigosertib reduced aspects of cardiac hypertrophy. Rigosertib treatment prevented or regressed cellular hypertrophy in human induced pluripotent stem cell-(iPSC-) derived cardiomyocytes homozygous for the NS-associated RAF1S257L allele. We extended these findings to a mammalian model, using Raf1L613V/+ KI mice to explore the therapeutic implications of rigosertib on RAF1-driven HCM. Longitudinal six-week treatment with rigosertib in these mice resulted in significant improvement in left ventricular chamber dimension and posterior wall thickness, total heart mass, size of individual cardiomyocytes (CMs), as well as reversal of cardiac hypertrophy. Rigosertib treatment also led to normalized fetal gene expression and inhibition of ERK and AKT pathway activities in primary CMs isolated from Raf1L613V/+ mice. Cardiac function, as assessed by echocardiography, showed significant improvement in ejection fraction and fractional shortening, with molecular studies confirming downregulation of hypertrophic markers and signaling pathways. Together with the Drosophila data, these mammalian results support the potential and use for rigosertib to reverse pathological hypertrophy in NS through targeted pathway inhibition in patients. Moreover, in addition to its effects in the heart, rigosertib treatment in mice also significantly improved other NS-associated syndromic features, including increasing bone growth and correcting craniofacial abnormalities. ConclusionsTaken together, our findings suggest rigosertib effectively normalizes and reverses RASopathy-associated HCM as well as other NS-associated syndromic features, supporting its potential for development as a promising treatment for RAF1-associated HCM and, potentially, other RASopathies-dependent pathologies. This study not only highlights the therapeutic potential of rigosertib but also demonstrates the utility of an integrated approach using Drosophila, iPSC and mammalian models to elucidate drug effects across complex biological systems.

molecular biology↗

bioRxiv2023

The nutrient sensor CRTC & Sarcalumenin / Thinman represent a new pathway in cardiac hypertrophy

Obesity and type 2 diabetes are at epidemic levels and a significant proportion of these patients are diagnosed with left ventricular hypertrophy. CREB Regulated Transcription Co-activator (CRTC) is a key regulator of metabolism in mammalian hepatocytes, where it is activated by calcineurin (CaN) to increase expression of gluconeogenic genes. CaN is known its role in pathological cardiac hypertrophy, however, a role for CRTC in the heart has not been identified. In Drosophila, CRTC null mutants have little body fat and exhibit severe cardiac restriction, myofibrillar disorganization, cardiac fibrosis and tachycardia, all hallmarks of heart disease. Cardiac-specific knockdown of CRTC, or its coactivator CREBb, mimicked the reduced body fat and heart defects of CRTC null mutants. Comparative gene expression in CRTC loss- or gain-of-function fly hearts revealed contra-regulation of genes involved in glucose, fatty acid, and amino acid metabolism, suggesting that CRTC also acts as a metabolic switch in the heart. Among the contra-regulated genes with conserved CREB binding sites, we identified the fly ortholog of Sarcalumenin, which is a Ca2+-binding protein in the sarcoplasmic reticulum. Cardiac knockdown recapitulated the loss of CRTC cardiac restriction and fibrotic phenotypes, suggesting it is a downstream effector of CRTC we named thinman (tmn). Importantly, cardiac overexpression of either CaN or CRTC in flies caused hypertrophy that was reversed in a CRTC mutant background, suggesting CRTC mediates hypertrophy downstream of CaN, perhaps as an alternative to NFAT. CRTC novel role in the heart is likely conserved in vertebrates as knockdown in zebrafish also caused cardiac restriction, as in fl ies. These data suggest that CRTC is involved in myocardial cell maintenance and that CaN-CRTC- Sarcalumenin/tmn signaling represents a novel and conserved pathway underlying cardiac hypertrophy.

cell biology↗

bioRxiv2022

The deacetylase dependent and independent role of HDAC3 in cardiomyopathy

BackgroundCardiomyopathy is a common disease of cardiac muscle that negatively affects cardiac function. HDAC3 commonly functions as co-repressor by removing acetyl moieties from histone tails. However, a deacetylase-independent role of HDAC3 has also been described. Cardiac deletion of HDAC3 causes reduced cardiac contractility accompanied by lipid accumulation. The molecular function of HDAC3 in cardiomyopathy remains unknown. We have used the powerful genetic tools in Drosophila to investigate the enzymatic and non-enzymatic roles of HDAC3 in cardiomyopathy. Methods and ResultsUsing the Drosophila heart model, we showed that cardiac-specific HDAC3 knockdown leads to prolonged systoles and reduced cardiac contractility. Immunohistochemistry revealed structural abnormalities characterized by myofiber disruption in HDAC3 knock down hearts. Cardiac-specific HDAC3 knockdown showed increased levels of whole body triglycerides and increased fibrosis. The introduction of deacetylase-dead HDAC3 mutant in HDAC3 KD background showed comparable results with wild type HDAC3 in aspects of contractility and Pericardin deposition. However, deacetylase-dead HDAC3 mutants failed to improve triglyceride accumulation. ConclusionsOur data indicate that HDAC3 plays a deacetylase-independent role in maintaining cardiac contractility and preventing Pericardin deposition as well as a deacetylase-dependent role to maintain triglyceride homestasis.

physiology↗

bioRxiv2022

Multiplatform Modeling of Atrial Fibrillation Identifies Phospholamban as Central Regulator of Cardiac Rhythm

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia in humans, present in > 33 million people worldwide. Although AF is often developed secondary to cardiovascular diseases, endocrine disorders, or lifestyle factors, recent GWAS studies have identified >200 genetic variants that substantially contribute to AF risk. However, it is currently not known how these genetic predispositions contribute to the initiation and/or maintenance of AF-associated phenotypes. In this context, one major barrier to progress is the lack of experimental systems enabling to rapidly explore the function of large cohort of genes on rhythm parameters in models with human atrial relevance. To address these modeling challenges, we have developed a new multi-model platform enabling 1) high-throughput characterization of the role of AF-associated genes on action potential duration and rhythm parameters at the cellular level, using human iPSC-derived atrial-like cardiomyocytes (ACMs), and at the whole organ level, using the Drosophila heart model, and 2) validation of the physiological relevance of our experimental results using computational models of heterogenous human adult atrial myocytes (HAMs) and tissue. As proof of concept, we screened a cohort of 20 AF-associated genes and identified Phospholamban (PLN) loss of function as a top conserved hit that significantly shortens action potential duration in ACMs, HAMs and fly cardiomyocytes. Remarkably, while PLN knock-down (KD) was not sufficient to induce arrhythmia phenotypes, addition of environmental stressors (i.e fibroblasts, {beta}-adrenergic stimulation) to the model systems, led to the robust generation of irregular beat to beat intervals, delayed after depolarizations, and triggered action potentials, as compared to controls. Finally, to delineate the mechanism underlying PLN KD-dependent arrhythmia, we used a logistic regression approach in HAM populations, and predicted that PLN functionally interacts with both NCX (loss of function) and L-type calcium channels (gain of function) to mediate these arrhythmic phenotypes. Consistent with our predictions, co-KD of PLN and NCX in ACMs and flies, led to increased arrhythmic events, while treatment of ACMs with L-type calcium channel inhibitor, verapamil, reverted these phenotypes. In summary, these results collectively demonstrate that our integrated multi-model system approach was successful in identifying and characterizing conserved roles (i.e regulation of Ca2+ homeostasis) for AF-associated genes and phenotypes, and thus paves the way for the discovery and molecular delineation of new gene regulatory networks controlling atrial rhythm with application to AF.

pathology↗

bioRxiv2021

Genetic architecture of natural variations of cardiac performance in flies

Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. We investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP). Genome Wide Associations Studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that we identified were used to map potential regulatory non-coding regions, which in turn were employed to predict Transcription Factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart specific knockdown. Additionally, we showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, we showed that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, we documented correlations between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 and EGR2 in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans.

genetics↗

bioRxiv2020

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk

BackgroundKCNMA1 encodes the -subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited and KCNMA1 has not been investigated as an AF candidate gene. Methods and ResultsKCNMA1 sequencing in 118 patients with familial AF identified a novel complex variant in one kindred. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced cardiac arrhythmias. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. ConclusionsOur data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish and fly and suggest that KCa1.1 loss of function may predispose to AF.

genetics↗

bioRxiv2020

Zebrafish follistatin-like 1b regulates cardiac contraction during early development

Extracellular glycoprotein Follistatin-like protein 1 (FSTL1) has been reported to be involved in multiple signaling pathways and biological processes during development and disease. In addition, some mouse studies have suggested an inhibitory role of FSTL1 in BMP signaling, in certain context. Yet, whether FSTL1 has biological functions in early heart development are largely unknown. In our functional studies, CRISPR/Cas9 genome editing was used to create fstl1a and fstl1b insertion/deletion mutations in zebrafish and our results suggest that fstl1b is important in regulating heart development during early embryogenesis. in situ hybridization revealed that fstl1b transcripts are expressed in the developing zebrafish heart, and fstl1b homozygous (-/-) mutant exhibited pericardial edema and showed significantly reduced contractility in the ventricle, with incomplete penetrance. We found that zebrafish fstl1b (-/-) mutant hearts formed a collapsed ventricle with strong reductions in the sarcomere structure protein alpha-actinin, although the number of cardiomyocytes was comparable to wild type control siblings. Further, normal levels of fstl1b seems to prevent the expansion of Bmp signaling into ventricular myocytes, which is consistent with the previously established model of Bmp-dependent cardiac contraction. Together these results reveal the zebrafish ortholog fstl1b regulates sarcomere structure and cardiac contractile function. In vertebrate developing heart, it might function as a key component in a novel pathway that constrains Bmp signaling from ventricular myocytes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2019

Patient-specific functional genomics and disease modeling suggest a role for LRP2 in Hypoplastic Left Heart Syndrome

Congenital heart diseases (CHD), such as hypoplastic left heart syndrome (HLHS), are considered to have complex genetic underpinnings that are poorly understood. Here, an integrated multi-disciplinary approach was applied to identify novel genes and underlying mechanisms associated with HLHS. A family-based strategy was employed that coupled whole genome sequencing (WGS) with RNA sequencing of patient-derived induced pluripotent stem cells (iPSCs) from a sporadic HLHS proband-parent trio to identify, prioritize and functionally evaluate candidate genes in model systems. Consistent with the hypoplastic phenotype, the probands iPSCs had reduced proliferation capacity. Filtering WGS for rare de novo, recessive, and loss-of-function variants revealed 10 candidate genes with recessive variants and altered expression compared to the parents iPSCs. siRNA/RNAi-mediated knockdown in generic human iPSC-derived cardiac progenitors and in the in vivo Drosophila heart model revealed that LDL receptor related protein LRP2 and apolipoprotein APOB are required for robust hiPSC-derived cardiomyocyte proliferation and normal hear structure and function, possibly involving an oligogenic mechanism via growth-promoting WNT and SHH signaling. LRP2 was further validated as a CHD gene in a zebrafish heart model and rare variant burden testing in an HLHS cohort. Collectively, this cross-functional genetic approach to complex congenital heart disease revealed LRP2 dysfunction as a likely novel genetic driver of HLHS, and hereby established a scalable approach to decipher the oligogenic underpinnings of maladaptive left heart development. One sentence summaryWhole genome sequencing and a multi-model system candidate gene validation - human iPSC-derived cardiomyocytes and Drosophila and zebrafish hearts - identified lipoprotein LRP2 as a new potential driver in congenital heart disease and suggests a deficit in proliferation as a hallmark of hypoplastic left heart syndrome.

genetics↗

bioRxiv2019

Extra-cellular matrix induced by steroids through a G-protein coupled receptor in a Drosophila model of renal fibrosis

Aldosterone is produced by the mammalian adrenal cortex to modulate blood pressure and fluid balance, however excessive, prolonged aldosterone production promotes fibrosis and kidney failure. How aldosterone triggers disease may involve actions that are independent of its canonical mineralocorticoid receptor. Here we present a Drosophila model of renal pathology caused by excess extra-cellular matrix formation, stimulated by exogenous aldosterone and insect ecdysone steroids. Chronic administration of aldosterone or ecdysone induces expression and accumulation of collagen-like pericardin at adult nephrocytes - podocyte-like cells that filter circulating hemolymph. Excess pericardin deposition disrupts nephrocyte (glomerular) filtration and causes proteinuria in Drosophila, hallmarks of mammalian kidney failure. Steroid-induced pericardin arises from cardiomyocytes associated with nephrocytes, reflecting an analogous role of mammalian myofibroblasts in fibrotic disease. Remarkably, the canonical ecdysteroid nuclear hormone receptor, ecdysone receptor EcR, is not required for aldosterone or ecdysone to stimulate pericardin production or associated renal pathology. Instead, these hormones require a cardiomyocyte-associated G-protein coupled receptor, dopamine-EcR (dopEcR), a membrane-associated receptor previously characterized in the fly brain as affecting behavior. This Drosophila renal disease model reveals a novel signaling pathway through which steroids may potentially modulate human fibrosis through proposed orthologs of dopEcR.\n\nSignificance StatementAldosterone regulates salt and fluid homeostasis, yet excess aldosterone contributes to renal fibrosis. Aldosterone acts through a nuclear hormone receptor, but an elusive, G-protein coupled receptor (GPCR) is thought to also mediate the hormones pathology. Here we introduce a Drosophila model of renal fibrosis. Flies treated with human aldosterone produce excess extra-cellular matrix and that causes kidney pathology. Flies treated with the insect steroid ecdysone produce similar pathology, and from this analogous response we identify an alternative receptor through which steroids mediate renal fibrosis -- the GPCR dopamine-Ecdysone Receptor (dopEcR). dopEcR functions in heart muscle cells associated with nephrocytes, analogous to the role of myofibroblasts in human fibrosis. This finding opens avenues to identify mammalian GPCR homologs of dopEcR through which aldosterone mediates renal fibrosis.

pathology↗

bioRxiv2017

Heart-Specific Activin Signaling Promotes Cardiomyopathy And Organismal Aging Through Autophagy Inhibition

Age-dependent loss of cardiac tissue homeostasis largely impacts heart performance and contributes significantly to cardiovascular diseases later in life. Cellular quality control machinery, such as autophagy/lysosome system, plays a crucial role in maintaining cardiac health and preventing age-induced cardiomyopathy and heart failure. However, how aging alters the autophagy/lysosome system to impact cardiac function remains largely unknown. Here using Drosophila heart as a model system, we show that activin signaling, a member of TGF-beta superfamily, negatively regulates cardiac autophagy and cardiac health during aging. We found that cardiac-specific knockdown of Daw, an activin-like protein in Drosophila, increased cardiac autophagy and prevented age-related cardiac dysfunction, including arrhythmia and bradycardia (slow heart rate). Inhibition of autophagy blocked Daw knockdown-mediated cardioprotection. Consistently, cardiac-specific expression of constitutively activated activin type I receptor Babo disrupted cardiac function at young ages. Intriguingly, the key autophagy regulator, mechanistic target of rapamycin complex 1 (mTORC1), was not involved in activin-mediated autophagy. Instead, activin signaling genetically interacted with Rictor, the key subunit of mTORC2, to regulate autophagy and cardiac aging. Knockdown of Daw increased the mRNA expression of Rictor and the phosphorylation of AKT in fly hearts. Finally, cardiac-specific silencing of Daw not only improved cardiac health, but also prolonged lifespan. Thus, our findings highlight an emerging role of activin signaling and mTORC2 in the regulation of autophagy and cardiac aging.

genetics↗