Girish C. Melkani

Time-restricted feeding attenuates age-related cardiac decline in Drosophila

Midnight snacks are bad for the heart Circadian clocks help animals coordinate their active and rest periods with the daily cycles of light and darkness. As anyone who has suffered jet lag or worked night shifts knows, losing this coordination can have deleterious effects. Gill et al. compared fruit flies that were allowed to eat at any time with flies that were only allowed to eat during the day (when they are active). The flies with restricted feeding times slept better and had a slower decline in heart function as they aged. They also showed less weight gain, even though both groups of flies consumed about the same amount. Science, this issue p. 1265 Restricting feeding to daytime has health benefits for fruitflies. Circadian clocks orchestrate periods of rest or activity and feeding or fasting over the course of a 24-hour day and maintain homeostasis. To assess whether a consolidated 24-hour cycle of feeding and fasting can sustain health, we explored the effect of time-restricted feeding (TRF; food access limited to daytime 12 hours every day) on neural, peripheral, and cardiovascular physiology in Drosophila melanogaster. We detected improved sleep, prevention of body weight gain, and deceleration of cardiac aging under TRF, even when caloric intake and activity were unchanged. We used temporal gene expression profiling and validation through classical genetics to identify the TCP-1 ring complex (TRiC) chaperonin, the mitochondrial electron transport chain complexes, and the circadian clock as pathways mediating the benefits of TRF.

Huntington's Disease Induced Cardiac Amyloidosis Is Reversed by Modulating Protein Folding and Oxidative Stress Pathways in the Drosophila Heart

Amyloid-like inclusions have been associated with Huntingtons disease (HD), which is caused by expanded polyglutamine repeats in the Huntingtin protein. HD patients exhibit a high incidence of cardiovascular events, presumably as a result of accumulation of toxic amyloid-like inclusions. We have generated a Drosophila model of cardiac amyloidosis that exhibits accumulation of PolyQ aggregates and oxidative stress in myocardial cells, upon heart-specific expression of Huntingtin protein fragments (Htt-PolyQ) with disease-causing poly-glutamine repeats (PolyQ-46, PolyQ-72, and PolyQ-102). Cardiac expression of GFP-tagged Htt-PolyQs resulted in PolyQ length-dependent functional defects that included increased incidence of arrhythmias and extreme cardiac dilation, accompanied by a significant decrease in contractility. Structural and ultrastructural analysis of the myocardial cells revealed reduced myofibrillar content, myofibrillar disorganization, mitochondrial defects and the presence of PolyQ-GFP positive aggregates. Cardiac-specific expression of disease causing Poly-Q also shortens lifespan of flies dramatically. To further confirm the involvement of oxidative stress or protein unfolding and to understand the mechanism of PolyQ induced cardiomyopathy, we co-expressed expanded PolyQ-72 with the antioxidant superoxide dismutase (SOD) or the myosin chaperone UNC-45. Co-expression of SOD suppressed PolyQ-72 induced mitochondrial defects and partially suppressed aggregation as well as myofibrillar disorganization. However, co-expression of UNC-45 dramatically suppressed PolyQ-72 induced aggregation and partially suppressed myofibrillar disorganization. Moreover, co-expression of both UNC-45 and SOD more efficiently suppressed GFP-positive aggregates, myofibrillar disorganization and physiological cardiac defects induced by PolyQ-72 than did either treatment alone. Our results demonstrate that mutant-PolyQ induces aggregates, disrupts the sarcomeric organization of contractile proteins, leads to mitochondrial dysfunction and increases oxidative stress in cardiomyocytes leading to abnormal cardiac function. We conclude that modulation of both protein unfolding and oxidative stress pathways in the Drosophila heart model can ameliorate the detrimental PolyQ effects, thus providing unique insights into the genetic mechanisms underlying amyloid-induced cardiac failure in HD patients.

The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

UNC-45 is a UCS (UNC-45/CRO1/She4P) class chaperone necessary for myosin folding and/or accumulation, but its requirement for maintaining cardiac contractility has not been explored. Given the prevalence of myosin mutations in eliciting cardiomyopathy, chaperones like UNC-45 are likely to be equally critical in provoking or modulating myosin-associated cardiomyopathy. Here, we used the Drosophila heart model to examine its role in cardiac physiology, in conjunction with RNAi-mediated gene silencing specifically in the heart in vivo. Analysis of cardiac physiology was carried out using high-speed video recording in conjunction with movement analysis algorithms. unc-45 knockdown resulted in severely compromised cardiac function in adults as evidenced by prolonged diastolic and systolic intervals, and increased incidence of arrhythmias and extreme dilation; the latter was accompanied by a significant reduction in muscle contractility. Structural analysis showed reduced myofibrils, myofibrillar disarray, and greatly decreased cardiac myosin accumulation. Cardiac unc-45 silencing also dramatically reduced life-span. In contrast, third instar larval and young pupal hearts showed mild cardiac abnormalities, as severe cardiac defects only developed during metamorphosis. Furthermore, cardiac unc-45 silencing in the adult heart (after metamorphosis) led to less severe phenotypes. This suggests that UNC-45 is mostly required for myosin accumulation/folding during remodeling of the forming adult heart. The cardiac defects, myosin deficit and decreased life-span in flies upon heart-specific unc-45 knockdown were significantly rescued by UNC-45 over-expression. Our results are the first to demonstrate a cardiac-specific requirement of a chaperone in Drosophila, suggestive of a critical role of UNC-45 in cardiomyopathies, including those associated with unfolded proteins in the failing human heart. The dilated cardiomyopathy phenotype associated with UNC-45 deficiency is mimicked by myosin knockdown suggesting that UNC-45 plays a crucial role in stabilizing myosin and possibly preventing human cardiomyopathies associated with functional deficiencies of myosin.

Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase.

UNC-45 belongs to the UCS (UNC-45, CRO1, She4p) domain protein family, whose members interact with various classes of myosin. Here we provide structural and biochemical evidence that Escherichia coli-expressed Drosophila UNC-45 (DUNC-45) maintains the integrity of several substrates during heat-induced stress in vitro. DUNC-45 displays chaperone function in suppressing aggregation of the muscle myosin heavy meromyosin fragment, the myosin S-1 motor domain, alpha-lactalbumin and citrate synthase. Biochemical evidence is supported by electron microscopy, which reveals the first structural evidence that DUNC-45 prevents inter- or intra-molecular aggregates of skeletal muscle heavy meromyosin caused by elevated temperatures. We also demonstrate for the first time that UNC-45 is able to refold a denatured substrate, urea-unfolded citrate synthase. Overall, this in vitro study provides insight into the fate of muscle myosin under stress conditions and suggests that UNC-45 protects and maintains the contractile machinery during in vivo stress.

Drosophila UNC-45 accumulates in embryonic blastoderm and in muscles, and is essential for muscle myosin stability

UNC-45 is a chaperone that facilitates folding of myosin motor domains. We have used Drosophila melanogaster to investigate the role of UNC-45 in muscle development and function. Drosophila UNC-45 (dUNC-45) is expressed at all developmental stages. It colocalizes with non-muscle myosin in embryonic blastoderm of 2-hour-old embryos. At 14 hours, it accumulates most strongly in embryonic striated muscles, similarly to muscle myosin. dUNC-45 localizes to the Z-discs of sarcomeres in third instar larval body-wall muscles. We produced a dunc-45 mutant in which zygotic expression is disrupted. This results in nearly undetectable dUNC-45 levels in maturing embryos as well as late embryonic lethality. Muscle myosin accumulation is robust in dunc-45 mutant embryos at 14 hours. However, myosin is dramatically decreased in the body-wall muscles of 22-hour-old mutant embryos. Furthermore, electron microscopy showed only a few thick filaments and irregular thick–thin filament lattice spacing. The lethality, defective protein accumulation, and ultrastructural abnormalities are rescued with a wild-type dunc-45 transgene, indicating that the mutant phenotypes arise from the dUNC-45 deficiency. Overall, our data indicate that dUNC-45 is important for myosin accumulation and muscle function. Furthermore, our results suggest that dUNC-45 acts post-translationally for proper myosin folding and maturation.

The UNC-45 myosin chaperone: from worms to flies to vertebrates.

UNC-45 (uncoordinated mutant number 45) is a UCS (UNC-45, CRO1, She4p) domain protein that is critical for myosin stability and function. It likely aides in folding myosin during cellular differentiation and maintenance, and protects myosin from denaturation during stress. Invertebrates have a single unc-45 gene that is expressed in both muscle and nonmuscle tissues. Vertebrates possess one gene expressed in striated muscle (unc-45b) and another that is more generally expressed (unc-45a). Structurally, UNC-45 is composed of a series of α-helices connected by loops. It has an N-terminal tetratricopeptide repeat domain that binds to Hsp90 and a central domain composed of armadillo repeats. Its C-terminal UCS domain, which is also comprised of helical armadillo repeats, interacts with myosin. In this chapter, we present biochemical, structural, and genetic analyses of UNC-45 in Caenorhabditis elegans, Drosophila melanogaster, and various vertebrates. Further, we provide insights into UNC-45 functions, its potential mechanism of action, and its roles in human disease.

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.

Mapping interactions between myosin relay and converter domains that power muscle function.

Background: Dissecting the molecular mechanism of muscle myosin function in vivo has proved difficult. Results: Transgenic fruit flies expressing mutant myosin and potential suppressor mutations suggest interacting amino acids. Conclusion: Communication between a relay domain residue and one in the converter is likely critical to myosin motor function. Significance: The transgenic system allows mapping of putative protein interactions essential for muscle contraction. Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile508, Asn509, and Asp511) in communicating with converter domain residue Arg759. We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.

Two Drosophila Myosin Transducer Mutants with Distinct Cardiomyopathies Have Divergent ADP and Actin Affinities

Two Drosophila myosin II point mutations (D45 and Mhc5) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc5) could stabilize (D45) or destabilize (Mhc5) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1A261T and S1G200D) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1A261T is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin·S1), and reduced ATP-induced dissociation of actin·S1), whereas S1G200D shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin·S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1A261T (dilated) and S1G200D (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1A261T weakens the affinity of S1·ADP for actin, whereas S1G200D increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up2 mutation in Drosophila.