PI(4,5)P 2 role in Transverse-tubule membrane formation and muscle function.

Transverse (T)-tubules - vast, tubulated domains of the muscle plasma membrane - are critical to maintain healthy skeletal and heart contractions. How the intricate T-tubule membranes are formed is not well understood, with challenges to systematically interrogate in muscle. We established the use of intact Drosophila larval body wall muscles as an ideal system to discover mechanisms that sculpt and maintain the T-tubule membrane network. A muscle-targeted genetic screen identified specific phosphoinositide lipid regulators necessary for T-tubule organization and muscle function. We show that a PI4KIIIα - Skittles/PIP5K pathway is needed for T-tubule localized PI(4)P to PI(4,5)P 2 synthesis, T-tubule organization, calcium regulation, and muscle and heart rate functions. Muscles deficient for PI4KIIIα or Amphiphysin , the homolog of human BIN1 , similarly exhibited specific loss of transversal T-tubule membranes and dyad junctions, yet retained longitudinal membranes and the associated dyads. Our results highlight the power of live muscle studies, uncovering distinct mechanisms and functions for sub-compartments of the T-tubule network relevant to human myopathy. Summary: T-tubules - vast, tubulated domains of the muscle plasma membrane - are critical to maintain skeletal and heart contractions. Fujita et al . establish genetic screens and assays in intact Drosophila muscles that uncover PI(4,5)P 2 regulation critical for T-tubule maintenance and function. Key Findings: PI4KIIIα is required for muscle T-tubule formation and larval mobility. A PI4KIIIα-Sktl pathway promotes PI(4)P and PI(4,5)P 2 function at T-tubules. PI4KIIIα is necessary for calcium dynamics and transversal but not longitudinal dyads. Disruption of PI(4,5)P 2 function in fly heart leads to fragmented T-tubules and abnormal heart rate.

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 R egulated T ranscription C o-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 Ca 2+ -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.

A single DPE core promoter motif contributes to in vivo transcriptional regulation and affects cardiac function.

Transcription is initiated at the core promoter, which confers specific functions depending on the unique combination of core promoter elements. The downstream core promoter element (DPE) is found in many genes related to heart and mesodermal development. However, the function of these core promoter elements has thus far been studied primarily in isolated, in vitro or reporter gene settings. tinman (tin) encodes a key transcription factor that regulates the formation of the dorsal musculature and heart. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we show that a substitution mutation of the functional tin DPE motif within the natural context of the core promoter results in a massive perturbation of Tinman's regulatory network orchestrating dorsal musculature and heart formation. Mutation of endogenous tin DPE reduced the expression of tin and distinct target genes, resulting in significantly reduced viability and an overall decrease in adult heart function. We demonstrate the feasibility and importance of characterizing DNA sequence elements in vivo in their natural context, and accentuate the critical impact a single DPE motif has during Drosophila embryogenesis and functional heart formation.

Mitochondrial MICOS complex genes, implicated in hypoplastic left heart syndrome, maintain cardiac contractility and actomyosin integrity.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes from these patients for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (goliath, E3 ubiquitin ligase), or SPTBN1 (ß-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.

Multiplatform modeling of atrial fibrillation identifies phospholamban as a central regulator of cardiac rhythm.

Atrial fibrillation (AF) is a common and genetically inheritable form of cardiac arrhythmia; however, it is currently not known how these genetic predispositions contribute to the initiation and/or maintenance of AF-associated phenotypes. One major barrier to progress is the lack of experimental systems to investigate the effects of gene function on rhythm parameters in models with human atrial and whole-organ relevance. Here, we assembled a multi-model platform enabling high-throughput characterization of the effects of gene function on action potential duration and rhythm parameters using human induced pluripotent stem cell-derived atrial-like cardiomyocytes and a Drosophila heart model, and validation of the findings using computational models of human adult atrial myocytes and tissue. As proof of concept, we screened 20 AF-associated genes and identified phospholamban loss of function as a top conserved hit that shortens action potential duration and increases the incidence of arrhythmia phenotypes upon stress. Mechanistically, our study reveals that phospholamban regulates rhythm homeostasis by functionally interacting with L-type Ca2+ channels and NCX. In summary, our study illustrates how a multi-model system approach paves the way for the discovery and molecular delineation of gene regulatory networks controlling atrial rhythm with application to AF.

Deacetylase-dependent and -independent role of HDAC3 in cardiomyopathy.

Cardiomyopathy is a common disease of cardiac muscle that negatively affects cardiac function. HDAC3 commonly functions as corepressor 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, but the molecular function of HDAC3 in cardiomyopathy remains unknown. We have used powerful genetic tools in Drosophila to investigate the enzymatic and nonenzymatic roles of HDAC3 in cardiomyopathy. Using the Drosophila heart model, we showed that cardiac-specific HDAC3 knockdown (KD) leads to prolonged systoles and reduced cardiac contractility. Immunohistochemistry revealed structural abnormalities characterized by myofiber disruption in HDAC3 KD hearts. Cardiac-specific HDAC3 KD 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. Our 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 homeostasis.

Age-dependent Lamin changes induce cardiac dysfunction via dysregulation of cardiac transcriptional programs.

As we age, structural changes contribute to progressive decline in organ function, which in the heart act through poorly characterized mechanisms. Taking advantage of the short lifespan and conserved cardiac proteome of the fruit fly, we found that cardiomyocytes exhibit progressive loss of Lamin C (mammalian Lamin A/C homologue) with age, coincident with decreasing nuclear size and increasing nuclear stiffness. Premature genetic reduction of Lamin C phenocopies aging's effects on the nucleus, and subsequently decreases heart contractility and sarcomere organization. Surprisingly, Lamin C reduction downregulates myogenic transcription factors and cytoskeletal regulators, possibly via reduced chromatin accessibility. Subsequently, we find a role for cardiac transcription factors in regulating adult heart contractility and show that maintenance of Lamin C, and cardiac transcription factor expression, prevents age-dependent cardiac decline. Our findings are conserved in aged non-human primates and mice, demonstrating that age-dependent nuclear remodeling is a major mechanism contributing to cardiac dysfunction.

Genetic architecture of natural variation of cardiac performance from flies to humans.

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.

Nascent polypeptide-Associated Complex and Signal Recognition Particle have cardiac-specific roles in heart development and remodeling.

Establishing a catalog of Congenital Heart Disease (CHD) genes and identifying functional networks would improve our understanding of its oligogenic underpinnings. Our studies identified protein biogenesis cofactors Nascent polypeptide-Associated Complex (NAC) and Signal-Recognition-Particle (SRP) as disease candidates and novel regulators of cardiac differentiation and morphogenesis. Knockdown (KD) of the alpha- (Nacα) or beta-subunit (bicaudal, bic) of NAC in the developing Drosophila heart disrupted cardiac developmental remodeling resulting in a fly with no heart. Heart loss was rescued by combined KD of Nacα with the posterior patterning Hox gene Abd-B. Consistent with a central role for this interaction in cardiogenesis, KD of Nacα in cardiac progenitors derived from human iPSCs impaired cardiac differentiation while co-KD with human HOXC12 and HOXD12 rescued this phenotype. Our data suggest that Nacα KD preprograms cardioblasts in the embryo for abortive remodeling later during metamorphosis, as Nacα KD during translation-intensive larval growth or pupal remodeling only causes moderate heart defects. KD of SRP subunits in the developing fly heart produced phenotypes that targeted specific segments and cell types, again suggesting cardiac-specific and spatially regulated activities. Together, we demonstrated directed function for NAC and SRP in heart development, and that regulation of NAC function depends on Hox genes.

Depletion of cardiac cardiolipin synthase alters systolic and diastolic function.

Cardiolipin (CL) is a major cardiac mitochondrial phospholipid maintaining regular mitochondrial morphology and function in cardiomyocytes. Cardiac CL production includes its biosynthesis and a CL remodeling process. Here we studied the impact of CL biosynthesis and the enzyme cardiolipin synthase (CLS) on cardiac function. CLS and cardiac CL species were significantly downregulated in cardiomyocytes following catecholamine-induced cardiac damage in mice, accompanied by increased oxygen consumption rates, signs of oxidative stress, and mitochondrial uncoupling. RNAi-mediated cardiomyocyte-specific knockdown of CLS in Drosophila melanogaster resulted in marked cardiac dilatation, severe impairment of systolic performance, and slower diastolic filling velocity assessed by fluorescence-based heart imaging. Finally, we showed that CL72:8 is significantly decreased in cardiac samples from patients with heart failure with reduced ejection fraction (HFrEF). In summary, we identified CLS as a regulator of cardiac function. Considering the cardiac depletion of CL species in HFrEF, pharmacological targeting of CLS may be a promising therapeutic approach.

Atrial Fibrillation Genomics: Discovery and Translation.

PURPOSE OF REVIEW: Our understanding of the fundamental cellular and molecular factors leading to atrial fibrillation (AF) remains stagnant despite significant advancement in ablation and device technologies. Diagnosis and prevention strategies fall behind that of treatment, but expanding knowledge in AF genetics holds the potential to drive progress. We aim to review how an understanding of the genetic contributions to AF can guide an approach to individualized risk stratification and novel avenues in drug discovery. RECENT FINDINGS: Rare familial forms of AF identified monogenic contributions to the development of AF. Genome-wide association studies (GWAS) further identified single-nucleotide polymorphisms (SNPs) suggesting polygenic and multiplex nature of this common disease. Polygenic risk scores accounting for the multitude of associated SNPs that each confer mildly elevated risk have been developed to translate genetic information into clinical practice, though shortcomings remain. Additionally, novel laboratory methods have been empowered by recent genetic findings to enhance drug discovery efforts. AF is increasingly recognized as a disease with a significant genetic component. With expanding sequencing technologies and accessibility, polygenic risk scores can help identify high risk individuals. Advancement in digital health tools, artificial intelligence and machine learning based on standard electrocardiograms, and genomic driven drug discovery may be integrated to deliver a sophisticated level of precision medicine in this modern era of emphasis on prevention. Randomized, prospective studies to demonstrate clinical benefits of these available tools are needed to validate this approach.

Fat-body brummer lipase determines survival and cardiac function during starvation in Drosophila melanogaster.

The cross talk between adipose tissue and the heart has an increasing importance for cardiac function under physiological and pathological conditions. This study characterizes the role of fat body lipolysis for cardiac function in Drosophila melanogaster. Perturbation of the function of the key lipolytic enzyme, brummer (bmm), an ortholog of the mammalian ATGL (adipose triglyceride lipase) exclusively in the fly's fat body, protected the heart against starvation-induced dysfunction. We further provide evidence that this protection is caused by the preservation of glycerolipid stores, resulting in a starvation-resistant maintenance of energy supply and adequate cardiac ATP synthesis. Finally, we suggest that alterations of lipolysis are tightly coupled to lipogenic processes, participating in the preservation of lipid energy substrates during starvation. Thus, we identified the inhibition of adipose tissue lipolysis and subsequent energy preservation as a protective mechanism against cardiac dysfunction during catabolic stress.

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

BACKGROUND: KCNMA1 encodes the α-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: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. RESULTS: A complex KCNMA1 variant was identified in 1 kindred with AF. 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 fibrillatory cardiac contractions. 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. CONCLUSIONS: Our 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.

Prolonged Exposure to Microgravity Reduces Cardiac Contractility and Initiates Remodeling in Drosophila.

Understanding the effects of microgravity on human organs is crucial to exploration of low-earth orbit, the moon, and beyond. Drosophila can be sent to space in large numbers to examine the effects of microgravity on heart structure and function, which is fundamentally conserved from flies to humans. Flies reared in microgravity exhibit cardiac constriction with myofibrillar remodeling and diminished output. RNA sequencing (RNA-seq) in isolated hearts revealed reduced expression of sarcomeric/extracellular matrix (ECM) genes and dramatically increased proteasomal gene expression, consistent with the observed compromised, smaller hearts and suggesting abnormal proteostasis. This was examined further on a second flight in which we found dramatically elevated proteasome aggregates co-localizing with increased amyloid and polyQ deposits. Remarkably, in long-QT causing sei/hERG mutants, proteasomal gene expression at 1g, although less than the wild-type expression, was nevertheless increased in microgravity. Therefore, cardiac remodeling and proteostatic stress may be a fundamental response of heart muscle to microgravity.

Patient-specific genomics and cross-species functional analysis implicate LRP2 in hypoplastic left heart syndrome.

Congenital heart diseases (CHDs), including hypoplastic left heart syndrome (HLHS), are genetically complex and poorly understood. Here, a multidisciplinary platform was established to functionally evaluate novel CHD gene candidates, based on whole-genome and iPSC RNA sequencing of a HLHS family-trio. Filtering for rare variants and altered expression in proband iPSCs prioritized 10 candidates. siRNA/RNAi-mediated knockdown in healthy human iPSC-derived cardiomyocytes (hiPSC-CM) and in developing Drosophila and zebrafish hearts revealed that LDL receptor-related protein LRP2 is required for cardiomyocyte proliferation and differentiation. Consistent with hypoplastic heart defects, compared to patents the proband's iPSC-CMs exhibited reduced proliferation. Interestingly, rare, predicted-damaging LRP2 variants were enriched in a HLHS cohort; however, understanding their contribution to HLHS requires further investigation. Collectively, we have established a multi-species high-throughput platform to rapidly evaluate candidate genes and their interactions during heart development, which are crucial first steps toward deciphering oligogenic underpinnings of CHDs, including hypoplastic left hearts.

Identification of MYOM2 as a candidate gene in hypertrophic cardiomyopathy and Tetralogy of Fallot, and its functional evaluation in the Drosophila heart.

The causal genetic underpinnings of congenital heart diseases, which are often complex and multigenic, are still far from understood. Moreover, there are also predominantly monogenic heart defects, such as cardiomyopathies, with known disease genes for the majority of cases. In this study, we identified mutations in myomesin 2 (MYOM2) in patients with Tetralogy of Fallot (TOF), the most common cyanotic heart malformation, as well as in patients with hypertrophic cardiomyopathy (HCM), who do not exhibit any mutations in the known disease genes. MYOM2 is a major component of the myofibrillar M-band of the sarcomere, and a hub gene within interactions of sarcomere genes. We show that patient-derived cardiomyocytes exhibit myofibrillar disarray and reduced passive force with increasing sarcomere lengths. Moreover, our comprehensive functional analyses in the Drosophila animal model reveal that the so far uncharacterized fly gene CG14964 [herein referred to as Drosophila myomesin and myosin binding protein (dMnM)] may be an ortholog of MYOM2, as well as other myosin binding proteins. Its partial loss of function or moderate cardiac knockdown results in cardiac dilation, whereas more severely reduced function causes a constricted phenotype and an increase in sarcomere myosin protein. Moreover, compound heterozygous combinations of CG14964 and the sarcomere gene Mhc (MYH6/7) exhibited synergistic genetic interactions. In summary, our results suggest that MYOM2 not only plays a critical role in maintaining robust heart function but may also be a candidate gene for heart diseases such as HCM and TOF, as it is clearly involved in the development of the heart.This article has an associated First Person interview with Emilie Auxerre-Plantié and Tanja Nielsen, joint first authors of the paper.

Systemic and heart autonomous effects of sphingosine Δ4 desaturase deficiency in lipotoxic cardiac pathophysiology.

Lipotoxic cardiomyopathy (LCM) is characterized by cardiac steatosis, including the accumulation of fatty acids, triglycerides and ceramides. Model systems have shown the inhibition of ceramide biosynthesis to antagonize obesity and improve insulin sensitivity. Sphingosine Δ4 desaturase (encoded by ifc in Drosophila melanogaster) enzymatically converts dihydroceramide into ceramide. Here, we examine ifc mutants to study the effects of desaturase deficiency on cardiac function in Drosophila Interestingly, ifc mutants exhibited classic hallmarks of LCM: cardiac chamber dilation, contractile defects and loss of fractional shortening. This outcome was phenocopied in global ifc RNAi-mediated knockdown flies. Surprisingly, cardiac-specific ifc knockdown flies exhibited cardiac chamber restriction with no contractile defects, suggesting heart autonomous and systemic roles for ifc activity in cardiac function. Next, we demonstrated that ifc mutants exhibit suppressed Sphingosine kinase 1 (Sk1) expression. Ectopic overexpression of Sk1 was sufficient to prevent cardiac chamber dilation and loss of fractional shortening in ifc mutants. Partial rescue was also observed with cardiac- and fat-body-specific Sk1 overexpression. Finally, we showed that cardiac-specific expression of Drosophila inhibitor of apoptosis (dIAP) also prevented cardiac dysfunction in ifc mutants, suggesting a role for caspase activity in the observed cardiac pathology. Collectively, we show that spatial regulation of sphingosine Δ4 desaturase activity differentially affects cardiac function in heart autonomous and systemic mechanisms through tissue interplay.

Overexpression of Kif1A in the Developing Drosophila Heart Causes Valvar and Contractility Defects: Implications for Human Congenital Heart Disease.

Left-sided congenital heart defects (CHDs) are among the most common forms of congenital heart disease, but a disease-causing gene has only been identified in a minority of cases. Here, we identified a candidate gene for CHDs, KIF1A, that was associated with a chromosomal balanced translocation t(2;8)(q37;p11) in a patient with left-sided heart and aortic valve defects. The breakpoint was in the 5' untranslated region of the KIF1A gene at 2q37, which suggested that the break affected the levels of Kif1A gene expression. Transgenic fly lines overexpressing Kif1A specifically in the heart muscle (or all muscles) caused diminished cardiac contractility, myofibrillar disorganization, and heart valve defects, whereas cardiac knockdown had no effect on heart structure or function. Overexpression of Kif1A also caused increased collagen IV deposition in the fibrous network that normally surrounds the fly heart. Kif1A overexpression in C2C12 myoblasts resulted in specific displacement of the F-actin fibers, probably through a direct interaction with G-actin. These results point to a Kif1A-mediated disruption of F-actin organization as a potential mechanism for the pathogenesis in at least some human CHDs.

Silencing of CCR4-NOT complex subunits affects heart structure and function.

The identification of genetic variants that predispose individuals to cardiovascular disease and a better understanding of their targets would be highly advantageous. Genome-wide association studies have identified variants that associate with QT-interval length (a measure of myocardial repolarization). Three of the strongest associating variants (single-nucleotide polymorphisms) are located in the putative promotor region of CNOT1, a gene encoding the central CNOT1 subunit of CCR4-NOT: a multifunctional, conserved complex regulating gene expression and mRNA stability and turnover. We isolated the minimum fragment of the CNOT1 promoter containing all three variants from individuals homozygous for the QT risk alleles and demonstrated that the haplotype associating with longer QT interval caused reduced reporter expression in a cardiac cell line, suggesting that reduced CNOT1 expression might contribute to abnormal QT intervals. Systematic siRNA-mediated knockdown of CCR4-NOT components in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) revealed that silencing CNOT1 and other CCR4-NOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, the cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes in vivo (CNOT1/Not1 and CNOT7/8/Pop2) was either lethal or resulted in dilated cardiomyopathy, reduced contractility or a propensity for arrhythmia. Silencing CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin also affected cardiac chamber size and contractility. Developmental studies suggested that CNOT1/Not1 and CNOT7/8/Pop2 are required during cardiac remodeling from larval to adult stages. To summarize, we have demonstrated how disease-associated genes identified by GWAS can be investigated by combining human cardiomyocyte cell-based and whole-organism in vivo heart models. Our results also suggest a potential link of CNOT1 and CNOT7/8 to QT alterations and further establish a crucial role of the CCR4-NOT complex in heart development and function.This article has an associated First Person interview with the first author of the paper.

TGFB-INHB/activin signaling regulates age-dependent autophagy and cardiac health through inhibition of MTORC2.

Age-related impairment of macroautophagy/autophagy and loss of cardiac tissue homeostasis contribute significantly to cardiovascular diseases later in life. MTOR (mechanistic target of rapamycin kinase) signaling is the most well-known regulator of autophagy, cellular homeostasis, and longevity. The MTOR signaling consists of two structurally and functionally distinct multiprotein complexes, MTORC1 and MTORC2. While MTORC1 is well characterized but the role of MTORC2 in aging and autophagy remains poorly understood. Here we identified TGFB-INHB/activin signaling as a novel upstream regulator of MTORC2 to control autophagy and cardiac health during aging. Using Drosophila heart as a model system, we show that cardiac-specific knockdown of TGFB-INHB/activin-like protein daw induces autophagy and alleviates age-related heart dysfunction, including cardiac arrhythmias and bradycardia. Interestingly, the downregulation of daw activates TORC2 signaling to regulate cardiac autophagy. Activation of TORC2 alone through overexpressing its subunit protein rictor promotes autophagic flux and preserves cardiac function with aging. In contrast, activation of TORC1 does not block autophagy induction in daw knockdown flies. Lastly, either daw knockdown or rictor overexpression in fly hearts prolongs lifespan, suggesting that manipulation of these pathways in the heart has systemic effects on longevity control. Thus, our studies discover the TGFB-INHB/activin-mediated inhibition of TORC2 as a novel mechanism for age-dependent decreases in autophagic activity and cardiac health. Abbreviations: AI: arrhythmia index; BafA1: bafilomycin A1; BMP: bone morphogenetic protein; CQ: chloroquine; CVD: cardiovascular diseases; DI: diastolic interval; ER: endoplasmic reticulum; HP: heart period; HR: heart rate; MTOR: mechanistic target of rapamycin kinase; NGS: normal goat serum; PBST: PBS with 0.1% Triton X-100; PDPK1: 3-phosphoinositide dependent protein kinase 1; RICTOR: RPTOR independent companion of MTOR complex 2; ROI: region of interest; ROUT: robust regression and outlier removal; ROS: reactive oxygen species; R-SMAD: receptor-activated SMAD; SI: systolic interval; SOHA: semi-automatic optical heartbeat analysis; TGFB: transformation growth factor beta; TSC1: TSC complex subunit 1.