Genetic and Pathophysiological Basis of Cardiac and Skeletal Muscle Laminopathies.

Nuclear lamins, a type V intermediate filament, are crucial components of the nuclear envelope's inner layer, maintaining nuclear integrity and mediating interactions between the nucleus and cytoplasm. Research on human iPSC-derived cells and animal models has demonstrated the importance of lamins in cardiac and skeletal muscle development and function. Mutations in lamins result in laminopathies, a group of diseases including muscular dystrophies, Hutchison-Gilford progeria syndrome, and cardiomyopathies with conduction defects. These conditions have been linked to disrupted autophagy, mTOR, Nrf2-Keap, and proteostasis signaling pathways, indicating complex interactions between the nucleus and cytoplasm. Despite progress in understanding these pathways, many questions remain about the mechanisms driving lamin-induced pathologies, leading to limited therapeutic options. This review examines the current literature on dysregulated pathways in cardiac and skeletal muscle laminopathies and explores potential therapeutic strategies for these conditions.

Automated assessment of cardiac dynamics in aging and dilated cardiomyopathy Drosophila models using machine learning.

The Drosophila model is pivotal in deciphering the pathophysiological underpinnings of various human ailments, notably aging and cardiovascular diseases. Cutting-edge imaging techniques and physiology yield vast high-resolution videos, demanding advanced analysis methods. Our platform leverages deep learning to segment optical microscopy images of Drosophila hearts, enabling the quantification of cardiac parameters in aging and dilated cardiomyopathy (DCM). Validation using experimental datasets confirms the efficacy of our aging model. We employ two innovative approaches deep-learning video classification and machine-learning based on cardiac parameters to predict fly aging, achieving accuracies of 83.3% (AUC 0.90) and 79.1%, (AUC 0.87) respectively. Moreover, we extend our deep-learning methodology to assess cardiac dysfunction associated with the knock-down of oxoglutarate dehydrogenase (OGDH), revealing its potential in studying DCM. This versatile approach promises accelerated cardiac assays for modeling various human diseases in Drosophila and holds promise for application in animal and human cardiac physiology under diverse conditions.

Diurnal expression of Dgat2 induced by time-restricted feeding maintains cardiac health in the Drosophila model of circadian disruption.

Circadian disruption is associated with an increased risk of cardiometabolic disorders and cardiac diseases. Time-restricted feeding/eating (TRF/TRE), restricting food intake within a consistent window of the day, has shown improvements in heart function from flies and mice to humans. However, whether and how TRF still conveys cardiac benefits in the context of circadian disruption remains unclear. Here, we demonstrate that TRF sustains cardiac performance, myofibrillar organization, and regulates cardiac lipid accumulation in Drosophila when the circadian rhythm is disrupted by constant light. TRF induces oscillations in the expression of genes associated with triglyceride metabolism. In particular, TRF induces diurnal expression of diacylglycerol O-acyltransferase 2 (Dgat2), peaking during the feeding period. Heart-specific manipulation of Dgat2 modulates cardiac function and lipid droplet accumulation. Strikingly, heart-specific overexpression of human Dgat2 at ZT 0-10 significantly improves cardiac performance in flies exposed to constant light. We have demonstrated that TRF effectively attenuates cardiac decline induced by circadian disruption. Moreover, our data suggests that diurnal expression of Dgat2 induced by TRF is beneficial for heart health under circadian disruption. Overall, our findings have underscored the relevance of TRF in preserving heart health under circadian disruptions and provided potential targets, such as Dgat2, and strategies for therapeutic interventions in mitigating cardiac aging, metabolic disorders, and cardiac diseases in humans.

Rapamycin reduces neuronal mutant huntingtin aggregation and ameliorates locomotor performance in Drosophila.

Huntington's disease (HD) is a neurodegenerative disease characterized by movement and cognitive dysfunction. HD is caused by a CAG expansion in exon 1 of the HTT gene that leads to a polyglutamine (PQ) repeat in the huntingtin protein, which aggregates in the brain and periphery. Previously, we used Drosophila models to determine that Htt-PQ aggregation in the heart causes shortened lifespan and cardiac dysfunction that is ameliorated by promoting chaperonin function or reducing oxidative stress. Here, we further study the role of neuronal mutant huntingtin and how it affects peripheral function. We overexpressed normal (Htt-PQ25) or expanded mutant (Htt-PQ72) exon 1 of huntingtin in Drosophila neurons and found that mutant huntingtin caused age-dependent Htt-PQ aggregation in the brain and could cause a loss of synapsin. To determine if this neuronal dysfunction led to peripheral dysfunction, we performed a negative geotaxis assay to measure locomotor performance and found that neuronal mutant huntingtin caused an age-dependent decrease in locomotor performance. Next, we found that rapamycin reduced Htt-PQ aggregation in the brain. These results demonstrate the role of neuronal Htt-PQ in dysfunction in models of HD, suggest that brain-periphery crosstalk could be important to the pathogenesis of HD, and show that rapamycin reduces mutant huntingtin aggregation in the brain.

Time-restricted feeding regulates lipid metabolism under metabolic challenges.

Dysregulation of lipid metabolism is a commonly observed feature associated with metabolic syndrome and leads to the development of negative health outcomes such as obesity, diabetes mellitus, non-alcoholic fatty liver disease, or atherosclerosis. Time-restricted feeding/eating (TRF/TRE), an emerging dietary intervention, has been shown to promote pleiotropic health benefits including the alteration of diurnal expression of genes associated with lipid metabolism, as well as levels of lipid species. Although TRF likely induces a response in multiple organs leading to the modulation of lipid metabolism, a majority of the studies related to TRF effects on lipids have focused only on individual tissues, and furthermore there is a lack of insight into potential underlying mechanisms. In this review, we summarize the current insights regarding TRF effects on lipid metabolism and the potential mechanisms in adipose tissue, liver, skeletal muscle, and heart, and conclude by outlining possible avenues for future exploration.

Automated evaluation of cardiac contractile dynamics and aging prediction using machine learning in a Drosophila model.

The Drosophila model has proven tremendously powerful for understanding pathophysiological bases of several human disorders including aging and cardiovascular disease. Relevant high-speed imaging and high-throughput lab assays generate large volumes of high-resolution videos, necessitating next-generation methods for rapid analysis. We present a platform for deep learning-assisted segmentation applied to optical microscopy of Drosophila hearts and the first to quantify cardiac physiological parameters during aging. An experimental test dataset is used to validate a Drosophila aging model. We then use two novel methods to predict fly aging: deep-learning video classification and machine-learning classification via cardiac parameters. Both models suggest excellent performance, with an accuracy of 83.3% (AUC 0.90) and 77.1% (AUC 0.85), respectively. Furthermore, we report beat-level dynamics for predicting the prevalence of cardiac arrhythmia. The presented approaches can expedite future cardiac assays for modeling human diseases in Drosophila and can be extended to numerous animal/human cardiac assays under multiple conditions.

Time-restricted feeding promotes muscle function through purine cycle and AMPK signaling in Drosophila obesity models.

Obesity caused by genetic and environmental factors can lead to compromised skeletal muscle function. Time-restricted feeding (TRF) has been shown to prevent muscle function decline from obesogenic challenges; however, its mechanism remains unclear. Here we demonstrate that TRF upregulates genes involved in glycine production (Sardh and CG5955) and utilization (Gnmt), while Dgat2, involved in triglyceride synthesis is downregulated in Drosophila models of diet- and genetic-induced obesity. Muscle-specific knockdown of Gnmt, Sardh, and CG5955 lead to muscle dysfunction, ectopic lipid accumulation, and loss of TRF-mediated benefits, while knockdown of Dgat2 retains muscle function during aging and reduces ectopic lipid accumulation. Further analyses demonstrate that TRF upregulates the purine cycle in a diet-induced obesity model and AMPK signaling-associated pathways in a genetic-induced obesity model. Overall, our data suggest that TRF improves muscle function through modulations of common and distinct pathways under different obesogenic challenges and provides potential targets for obesity treatments.

Mitochondrial epigenetic modifications and nuclear-mitochondrial communication: A new dimension towards understanding and attenuating the pathogenesis in women with PCOS.

Mitochondrial DNA (mtDNA) epigenetic modifications have recently gained attention in a plethora of complex diseases, including polycystic ovary syndrome (PCOS), a common cause of infertility in women of reproductive age. Herein we discussed mtDNA epigenetic modifications and their impact on nuclear-mitochondrial interactions in general and the latest advances indicating the role of mtDNA methylation in the pathophysiology of PCOS. We highlighted epigenetic changes in nuclear-related mitochondrial genes, including nuclear transcription factors that regulate mitochondrial function and may be involved in the development of PCOS or its related traits. Additionally, therapies targeting mitochondrial epigenetics, including time-restricted eating (TRE), which has been shown to have beneficial effects by improving mitochondrial function and may be mediated by epigenetic modifications, have also been discussed. As PCOS has become a major metabolic disorder and a risk factor for obesity, cardiometabolic disorders, and diabetes, lifestyle/behavior intervention using TRE that reinforces feeding-fasting rhythms without reducing caloric intake may be a promising therapeutic strategy for attenuating the pathogenesis. Furthermore, future perspectives in the area of mitochondrial epigenetics are described.

Circadian-mediated regulation of cardiometabolic disorders and aging with time-restricted feeding.

Circadian rhythms are present throughout biology, from the molecular level to complex behaviors such as eating and sleeping. They are driven by molecular clocks within cells, and different tissues can have unique rhythms. Circadian disruption can trigger obesity and other common metabolic disorders such as aging, diabetes, and cardiovascular disease, and circadian genes control metabolism. At an organismal level, feeding and fasting rhythms are key drivers of circadian rhythms. This underscores the bidirectional relationship between metabolism and circadian rhythms, and many metabolic disorders have circadian disruption or misalignment. Therefore, studying circadian rhythms may offer new avenues for understanding the etiology and management of obesity. This review describes how circadian rhythm dysregulation is linked with cardiometabolic disorders and how the lifestyle intervention of time-restricted feeding (TRF) regulates them. TRF reinforces feeding-fasting rhythms without reducing caloric intake and ameliorates metabolic disorders such as obesity and associated cardiac dysfunction, along with reducing inflammation. TRF optimizes the expression of genes and pathways related to normal metabolic function, linking metabolism with TRF's benefits and demonstrating the molecular link between metabolic disorders and circadian rhythms. Thus, TRF has tremendous therapeutic potential that could be easily adopted to reduce obesity-linked dysfunction and cardiometabolic disorders.

A Skeletal Muscle-Centric View on Time-Restricted Feeding and Obesity under Various Metabolic Challenges in Humans and Animals.

Nearly 50% of adults will suffer from obesity in the U.S. by 2030. High obesity rates can lead to high economic and healthcare burdens in addition to elevated mortality rates and reduced health span in patients. Emerging data demonstrate that obesity is a multifactorial complex disease with various etiologies including aging, a lifestyle of chronic high-fat diets (HFD), genetic predispositions, and circadian disruption. Time-restricted feeding/eating (TRF; TRE in humans) is an intervention demonstrated by studies to show promise as an effective alternative therapy for ameliorating the effects of obesity and metabolic disease. New studies have recently suggested that TRF/TRE modulates the skeletal muscle which plays a crucial role in metabolism historically observed to be impaired under obesity. Here we discuss recent findings regarding potential mechanisms underlying TRF's modulation of skeletal muscle function, metabolism, and structure which may shed light on future research related to TRF as a solution to obesity.

Author Correction: Time-restricted feeding restores muscle function in Drosophila models of obesity and circadian-rhythm disruption.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Time-Restricted Eating to Prevent and Manage Chronic Metabolic Diseases.

Molecular clocks are present in almost every cell to anticipate daily recurring and predictable changes, such as rhythmic nutrient availability, and to adapt cellular functions accordingly. At the same time, nutrient-sensing pathways can respond to acute nutrient imbalance and modulate and orient metabolism so cells can adapt optimally to a declining or increasing availability of nutrients. Organismal circadian rhythms are coordinated by behavioral rhythms such as activity-rest and feeding-fasting cycles to temporally orchestrate a sequence of physiological processes to optimize metabolism. Basic research in circadian rhythms has largely focused on the functioning of the self-sustaining molecular circadian oscillator, while research in nutrition science has yielded insights into physiological responses to caloric deprivation or to specific macronutrients. Integration of these two fields into actionable new concepts in the timing of food intake has led to the emerging practice of time-restricted eating. In this paradigm, daily caloric intake is restricted to a consistent window of 8-12 h. This paradigm has pervasive benefits on multiple organ systems.

Time-restricted feeding restores muscle function in Drosophila models of obesity and circadian-rhythm disruption.

Pathological obesity can result from genetic predisposition, obesogenic diet, and circadian rhythm disruption. Obesity compromises function of muscle, which accounts for a majority of body mass. Behavioral intervention that can counteract obesity arising from genetic, diet or circadian disruption and can improve muscle function holds untapped potential to combat the obesity epidemic. Here we show that Drosophila melanogaster (fruit fly) subject to obesogenic challenges exhibits metabolic disease phenotypes in skeletal muscle; sarcomere disorganization, mitochondrial deformation, upregulation of Phospho-AKT level, aberrant intramuscular lipid infiltration, and insulin resistance. Imposing time-restricted feeding (TRF) paradigm in which flies were fed for 12 h during the day counteracts obesity-induced dysmetabolism and improves muscle performance by suppressing intramuscular fat deposits, Phospho-AKT level, mitochondrial aberrations, and markers of insulin resistance. Importantly, TRF was effective even in an irregular lighting schedule mimicking shiftwork. Hence, TRF is an effective dietary intervention for combating metabolic dysfunction arising from multiple causes.

Suppression of myopathic lamin mutations by muscle-specific activation of AMPK and modulation of downstream signaling.

Laminopathies are diseases caused by dominant mutations in the human LMNA gene encoding A-type lamins. Lamins are intermediate filaments that line the inner nuclear membrane, provide structural support for the nucleus and regulate gene expression. Drosophila melanogaster models of skeletal muscle laminopathies were developed to investigate the pathological defects caused by mutant lamins and identify potential therapeutic targets. Human disease-causing LMNA mutations were modeled in Drosophila Lamin C (LamC) and expressed in indirect flight muscle (IFM). IFM-specific expression of mutant, but not wild-type LamC, caused held-up wings indicative of myofibrillar defects. Analyses of the muscles revealed cytoplasmic aggregates of nuclear envelope (NE) proteins, nuclear and mitochondrial dysmorphology, myofibrillar disorganization and up-regulation of the autophagy cargo receptor p62. We hypothesized that the cytoplasmic aggregates of NE proteins trigger signaling pathways that alter cellular homeostasis, causing muscle dysfunction. In support of this hypothesis, transcriptomics data from human muscle biopsy tissue revealed misregulation of the AMP-activated protein kinase (AMPK)/4E-binding protein 1 (4E-BP1)/autophagy/proteostatic pathways. Ribosomal protein S6K (S6K) messenger RNA (mRNA) levels were increased and AMPKα and mRNAs encoding downstream targets were decreased in muscles expressing mutant LMNA relative controls. The Drosophila laminopathy models were used to determine if altering the levels of these factors modulated muscle pathology. Muscle-specific over-expression of AMPKα and down-stream targets 4E-BP, Forkhead box transcription factors O (Foxo) and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), as well as inhibition of S6K, suppressed the held-up wing phenotype, myofibrillar defects and LamC aggregation. These findings provide novel insights on mutant LMNA-based disease mechanisms and identify potential targets for drug therapy.

Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy.

K146N is a dominant mutation in human ß-cardiac myosin heavy chain, which causes hypertrophic cardiomyopathy. We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical, physiological and mechanical foundations of the disease. ATPase assays, actin motility, and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment, yielding prolonged binding. This increases isometric force generation, but also resistive force and work absorption during cyclical contractions, resulting in decreased work, power output, flight ability and degeneration of flight muscle sarcomere morphology. Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes, 146N/+ hearts are hypercontractile with increased tension generation periods, decreased diastolic/systolic diameters and myofibrillar disarray. This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments.

Increasing autophagy and blocking Nrf2 suppress laminopathy-induced age-dependent cardiac dysfunction and shortened lifespan.

Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age-dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A-type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age-dependent heart defects are not well understood. To address this issue, we modeled human disease-causing mutations in the Drosophila melanogaster Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1 redox sensing pathway was activated, mitochondria exhibited abnormal morphology, and the autophagy cargo receptor Ref2(P)/p62 was upregulated. Genetic analyses demonstrated that simultaneous over-expression of the autophagy kinase Atg1 gene and an RNAi against CncC eliminated the cytoplasmic protein aggregates, restored cardiac function, and lengthened lifespan. These data suggest that simultaneously increasing rates of autophagy and blocking the Nrf2/Keap1 pathway are a potential therapeutic strategy for cardiac laminopathies.

TRiC/CCT chaperonins are essential for maintaining myofibril organization, cardiac physiological rhythm, and lifespan.

We recently reported that CCT chaperonin subunits are upregulated in a cardiac-specific manner under time-restricted feeding (TRF) [Gill S et al. (2015) Science 347, 1265-1269], suggesting that TRiC/CCT has a heart-specific function. To understand the CCT chaperonin function in cardiomyocytes, we performed its cardiac-specific knock-down in the Drosophila melanogaster model. This resulted in disorganization of cardiac actin- and myosin-containing myofibrils and severe physiological dysfunction, including restricted heart diameters, elevated cardiac dysrhythmia and compromised cardiac performance. We also noted that cardiac-specific knock-down of CCT chaperonin significantly shortens lifespans. Additionally, disruption of circadian rhythm yields further deterioration of cardiac function of hypomorphic CCT mutants. Our analysis reveals that both the orchestration of protein folding and circadian rhythms mediated by CCT chaperonin are critical for maintaining heart contractility.

A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects.

Individuals with inclusion body myopathy type 3 (IBM3) display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K) in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in decreased in vitro motility, reduced muscle power output and focal myofibrillar disorganization similar to that seen in individuals with IBM3.

Time-restricted feeding for prevention and treatment of cardiometabolic disorders.

The soaring prevalence of obesity and diabetes is associated with an increase in comorbidities, including elevated risk for cardiovascular diseases (CVDs). CVDs continue to be among the leading causes of death and disability in the United States. While increased nutritional intake from an energy-dense diet is known to disrupt metabolic homeostasis and contributes to the disease risk, circadian rhythm disruption is emerging as a new risk factor for CVD. Circadian rhythms coordinate cardiovascular health via temporal control of organismal metabolism and physiology. Thus, interventions that improve circadian rhythms are prospective entry points to mitigate cardiometabolic disease risk. Although light is a strong modulator of the neural circadian clock, time of food intake is emerging as a dominant agent that affects circadian clocks in metabolic organs. We discovered that imposing a time-restricted feeding (TRF) regimen in which all caloric intakes occur consistently within ≤ 12 h every day exerts many cardiometabolic benefits. TRF prevents excessive body weight gain, improves sleep, and attenuates age- and diet-induced deterioration in cardiac performance. Using an integrative approach that combines Drosophila melanogaster (fruit fly) genetics with transcriptome analyses it was found that the beneficial effects of TRF are mediated by circadian clock, ATP-dependent TCP/TRiC/CCT chaperonin and mitochondrial electron transport chain components. Parallel studies in rodents have shown TRF reduces metabolic disease risks by maintaining metabolic homeostasis. As modern humans continue to live under extended periods of wakefulness and ingestion events, daily eating pattern offers a new potential target for lifestyle intervention to reduce CVD risk.

Using Drosophila as an integrated model to study mild repetitive traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of morbidity and mortality worldwide. In addition, there has been a growing appreciation that even repetitive, milder forms of TBI (mTBI) can have long-term deleterious consequences to neural tissues. Hampering our understanding of genetic and environmental factors that influence the cellular and molecular responses to injury has been the limited availability of effective genetic model systems that could be used to identify the key genes and pathways that modulate both the acute and long-term responses to TBI. Here we report the development of a severe and mild-repetitive TBI model using Drosophila. Using this system, key features that are typically found in mammalian TBI models were also identified in flies, including the activation of inflammatory and autophagy responses, increased Tau phosphorylation and neuronal defects that impair sleep-related behaviors. This novel injury paradigm demonstrates the utility of Drosophila as an effective tool to validate genetic and environmental factors that influence the whole animal response to trauma and to identify prospective therapies needed for the treatment of TBI.