Sanford I. Bernstein

Molecular Genetic Analysis of Muscle Development, Structure, and Function in Drosophila

In this chapter we discuss the ultrastructural and physiological properties of the diverse muscle types of Drosophila melanogaster and how genetic studies permit an understanding of muscle cell determination, myofibril assembly, and protein isoform function.

Myosin heavy chain isoforms regulate muscle function but not myofibril assembly.

Myosin heavy chain (MHC) is the motor protein of muscle thick filaments. Most organisms produce many muscle MHC isoforms with temporally and spatially regulated expression patterns. This suggests that isoforms of MHC have different characteristics necessary for defining specific muscle properties. The single Drosophila muscle Mhc gene yields various isoforms as a result of alternative RNA splicing. To determine whether this multiplicity of MHC isoforms is critical to myofibril assembly and function, we introduced a gene encoding only an embryonic MHC into Drosophila melanogaster. The embryonic transgene acts in a dominant antimorphic manner to disrupt flight muscle function. The transgene was genetically crossed into an MHC null background. Unexpectedly, transformed flies expressing only the embryonic isoform are viable. Adult muscles containing embryonic MHC assemble normally, indicating that the isoform of MHC does not determine the dramatic ultrastructural variation among different muscle types. However, transformed flies are flightless and show reduced jumping and mating ability. Their indirect flight muscle myofibrils progressively deteriorate. Our data show that the proper MHC isoform is critical for specialized muscle function and myofibril stability.

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head.

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light‐chain binding site, the α‐helical rod and the C‐terminal tailpiece. Both isoforms of headless myosin co‐assemble with endogenous full‐length myosin in wild‐type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full‐length myosin heavy chain is produced. These filaments show the characteristic hollow cross‐section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.

The myosin converter domain modulates muscle performance

Myosin is the molecular motor that powers muscle contraction as a result of conformational changes during its mechanochemical cycle. We demonstrate that the converter, a compact structural domain that differs in sequence between Drosophila melanogaster myosin isoforms, dramatically influences the kinetic properties of myosin and muscle fibres. Transgenic replacement of the converter in the fast indirect flight muscle with the converter from an embryonic muscle slowed muscle kinetics, forcing a compensatory reduction in wing beat frequency to sustain flight. Conversely, replacing the embryonic converter with the flight muscle converter sped up muscle kinetics and increased maximum power twofold, compared to flight muscles expressing the embryonic myosin isoform. The substitutions also dramatically influenced in vitro actin sliding velocity, suggesting that the converter modulates a rate-limiting step preceding cross-bridge detachment. Our integrative analysis demonstrates that isoform-specific differences in the myosin converter allow different muscle types to meet their specific locomotion demands.

Determining structure/function relationships for sarcomeric myosin heavy chain by genetic and transgenic manipulation of Drosophila

Drosophila melanogaster is an excellent system for examining the structure/function relationships of myosin. It yields insights into the roles of myosin in assembly and stability of myofibrils, in defining the mechanical properties of muscle fibers, and in dictating locomotory abilities. Drosophila has a single gene encoding muscle myosin heavy chain (MHC), with alternative RNA splicing resulting in stage‐ and tissue‐specific isoform production. Localization of the alternative domains of Drosophila MHC on a three‐dimensional molecular model suggests how they may determine functional differences between isoforms. We are testing these predictions directly by using biophysical and biochemical techniques to characterize myosin isolated from transgenic organisms. Null and missense mutations help define specific amino acid residues important in actin binding and ATP hydrolysis and the function of MHC in thick filament and myofibril assembly. Insights into the interaction of thick and thin filaments result from studying mutations in MHC that suppress ultrastructural defects induced by a troponin I mutation. Analysis of transgenic organisms expressing engineered versions of MHC shows that the native isoform of myosin is not critical for myofibril assembly but is essential for muscle function and maintenance of muscle integrity. We show that the C‐terminus of MHC plays a pivotal role in the maintenance of muscle integrity. Transgenic studies using headless myosin reveal that the head is important for some, but not all, aspects of myofibril assembly. The integrative approach described here provides a multi‐level understanding of the function of the myosin molecular motor. Microsc. Res. Tech. 50:430–442, 2000.

Spatially and temporally regulated expression of myosin heavy chain alternative exons during Drosophila embryogenesis

We used alternative exon-specific probes to determine the accumulation of transcripts encoding myosin heavy chain (MHC) isoforms in Drosophila melanogaster embryos. Six isoforms accumulate in body wall muscles. Transverse (external) muscles express a different major form than intermediate and internal muscles, suggesting different physiological properties. Cardioblasts express one of the somatic muscle transcripts; visceral muscles express at least two transcript types. The pharyngeal muscle accumulates a unique Mhc transcript, suggesting unique contractile abilities. Mhc transcription begins in stage 12 in visceral and somatic muscles, but as late as stage 15 in cardioblasts. This is the first study of myosin isoform localization during insect embryogenesis, and forms the basis for transgenic and biochemical experiments designed to determine how MHC domains regulate muscle physiology.

A mighty small heart: the cardiac proteome of adult Drosophila melanogaster.

Drosophila melanogaster is emerging as a powerful model system for the study of cardiac disease. Establishing peptide and protein maps of the Drosophila heart is central to implementation of protein network studies that will allow us to assess the hallmarks of Drosophila heart pathogenesis and gauge the degree of conservation with human disease mechanisms on a systems level. Using a gel-LC-MS/MS approach, we identified 1228 protein clusters from 145 dissected adult fly hearts. Contractile, cytostructural and mitochondrial proteins were most abundant consistent with electron micrographs of the Drosophila cardiac tube. Functional/Ontological enrichment analysis further showed that proteins involved in glycolysis, Ca2+-binding, redox, and G-protein signaling, among other processes, are also over-represented. Comparison with a mouse heart proteome revealed conservation at the level of molecular function, biological processes and cellular components. The subsisting peptidome encompassed 5169 distinct heart-associated peptides, of which 1293 (25%) had not been identified in a recent Drosophila peptide compendium. PeptideClassifier analysis was further used to map peptides to specific gene-models. 1872 peptides provide valuable information about protein isoform groups whereas a further 3112 uniquely identify specific protein isoforms and may be used as a heart-associated peptide resource for quantitative proteomic approaches based on multiple-reaction monitoring. In summary, identification of excitation-contraction protein landmarks, orthologues of proteins associated with cardiovascular defects, and conservation of protein ontologies, provides testimony to the heart-like character of the Drosophila cardiac tube and to the utility of proteomics as a complement to the power of genetics in this growing model of human heart disease.

Genetic and Biochemical Analysis of Alternative RNA Splicing

Publisher Summary This chapter discusses the genetic and biochemical analysis of alternative RNA splicing. The chapter focuses on the biochemical studies and in vitro mutagenesis experiments for the understanding of alternative splicing. These analyses provide insight by identifying important cis-acting sequences and a few trans-acting factors that permit selective use of splice junctions. Such analyses shows that RNA secondary structure, steric constraints, splice junction affinity, and splice site competition play important regulatory roles. The chapter also discusses genetic approaches to understand the alternative RNA splicing along with the discussion that covers the studies of several other genes in Drosophila and yeast that illustrate the success of applying genetics to alternative RNA splicing. The observation includes that several human diseases result from exon skipping or the activation of cryptic splice junctions. The genetic approach may prove a useful future direction for eventually understanding the basis of alternative RNA splicing in a wider variety of pre-mRNAs.

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.

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.