Reduced life span with heart and muscle dysfunction in Drosophila sarcoglycan mutants.

In humans, genetically diverse forms of muscular dystrophy are associated with a disrupted sarcoglycan complex. The sarcoglycan complex resides at the muscle plasma membrane where it associates with dystrophin. There are six known sarcoglycan proteins in mammals whereas there are only three in Drosophila melanogaster. Using imprecise P element excision, we generated three different alleles at the Drosophila delta-sarcoglycan locus. Each of these deletions encompassed progressively larger regions of the delta-sarcoglycan gene. Line 840 contained a large deletion of the delta-sarcoglycan gene, and this line displayed progressive impairment in locomotive ability, reduced heart tube function and a shortened life span. In line 840, deletion of the Drosophila delta-sarcoglycan gene produced disrupted flight muscles with shortened sarcomeres and disorganized M lines. Unlike mammalian muscle where degeneration is coupled with ongoing regeneration, no evidence for regeneration was seen in this Drosophila sarcoglycan mutant. In contrast, line 28 was characterized with a much smaller deletion that affected only a portion of the cytoplasmic region of the delta-sarcoglycan protein and left intact the transmembrane and extracellular domains. Line 28 had a very mild phenotype with near normal life span, intact cardiac function and normal locomotive activity. Together, these data demonstrate the essential nature of the transmembrane and extracellular domains of Drosophila delta-sarcoglycan for normal muscle structure and function.

Processing and assembly of the dystrophin glycoprotein complex.

The assembly, processing and translocation of proteins occur constantly in all cells, and these processes also take place during the genesis, maintenance and repair of skeletal muscle. Skeletal muscle fibers are composed of myofibrils and are surrounded by a muscle plasma membrane, the sarcolemma. The sarcolemma serves as a docking location for many proteins. These proteins are important for establishing the physical connection between the extracellular matrix and the cytoskeleton and play a role in transmitting force related to muscle contraction. This physical connection is maintained through a myriad of proteins including the dystrophin glycoprotein complex (DGC). Normal sarcolemmal function requires proper DGC synthesis and positioning, and perturbation of the DGC leads to muscle membrane instability and disease.

Doxycycline-regulated over-expression of hsp22 has negative effects on stress resistance and life span in adult Drosophila melanogaster.

Drosophila hsp22 is a member of the small heat shock proteins family (shsps). The hsp22 is expressed in a tissue-general pattern in response to heat stress and during normal aging, and localizes to the mitochondrial matrix, however, its exact function and targets are unknown. Hsp22 was found to be rapidly induced in response to oxidative stress, indicating that hsp22 is also an oxidative stress response gene. To assay for effects of hsp22, a ubiquitous pattern of hsp22 gene expression was generated in young flies using the "tet-on" doxycycline-regulated promoter system. The hsp22 over-expression made flies more sensitive to heat and oxidative stress, while resistance to coumarin poisoning was not affected. Life span was also reduced, particularly at higher culture temperatures. Members of other hsp families have been shown to feedback-inhibit their own expression by interacting with the heat shock transcription factor (HSF) and preventing binding to the HSEs. Induction of hsp22:lacZ and hsp70:lacZ reporter transgenes in response to acute stress was normal in the presence of hsp22 protein over-expression and in old flies, indicating that the negative effects of hsp22 are downstream of the HSF/HSE pathway and the transcriptional heat shock response. The data demonstrate a specific over-expression phenotype for hsp22 and suggest that hsp22 interacts with heat and oxidative stress resistance pathways.

Genetic compensation for sarcoglycan loss by integrin alpha7beta1 in muscle.

Disruption of the sarcoglycan complex leads to muscle membrane instability and muscular dystrophy in humans and mice. Through the dystrophin glycoprotein complex, sarcoglycan participates in connecting the internal cytoskeleton to the membrane and the extracellular matrix. Integrin alpha7beta1 is also a transmembrane protein of skeletal and cardiac muscle that similarly links the cytoskeleton to the extracellular matrix. Mice lacking integrin alpha7 develop mild muscle degeneration, while sarcoglycan mutant mice display overt muscle degeneration and muscular dystrophy. In sarcoglycan-deficient muscle, integrin alpha7 protein was upregulated at the plasma membrane. To ascertain whether integrin alpha7 upregulation compensates for the loss of the transmembrane sarcoglycan linkage in sarcoglycan-deficient muscle, we generated mice lacking both integrin alpha7 and gamma-sarcoglycan (gxi). These double-mutant gxi mice exhibit profound, rapid muscle degeneration leading to death before one month of age consistent with a weakened cellular attachment to the extracellular matrix. The regenerative capacity of gxi muscle was intact with increased embryonic myosin heavy chain expression, myofiber central nucleation and normal in vivo myoblast differentiation. Therefore, upregulation of integrin alpha7beta1 compensates as a transmembrane muscle cell attachment for sarcoglycan consistent with overlapping roles for sarcoglycan and integrins in mediating cytoskeletal-membrane-extracellular matrix interaction.

Smooth muscle cell-extrinsic vascular spasm arises from cardiomyocyte degeneration in sarcoglycan-deficient cardiomyopathy.

Vascular spasm is a poorly understood but critical biomedical process because it can acutely reduce blood supply and tissue oxygenation. Cardiomyopathy in mice lacking gamma-sarcoglycan or delta-sarcoglycan is characterized by focal damage. In the heart, sarcoglycan gene mutations produce regional defects in membrane permeability and focal degeneration, and it was hypothesized that vascular spasm was responsible for this focal necrosis. Supporting this notion, vascular spasm was noted in coronary arteries, and disruption of the sarcoglycan complex was observed in vascular smooth muscle providing a molecular mechanism for spasm. Using a transgene rescue strategy in the background of sarcoglycan-null mice, we replaced cardiomyocyte sarcoglycan expression. Cardiomyocyte-specific sarcoglycan expression was sufficient to correct cardiac focal degeneration. Intriguingly, successful restoration of the cardiomyocyte sarcoglycan complex also eliminated coronary artery vascular spasm, while restoration of smooth muscle sarcoglycan in the background of sarcoglycan-null alleles did not. This mechanism, whereby tissue damage leads to vascular spasm, can be partially corrected by NO synthase inhibitors. Therefore, we propose that cytokine release from damaged cardiomyocytes can feed back to produce vascular spasm. Moreover, vascular spasm feeds forward to produce additional cardiac damage.

Doxycycline-induced expression of sense and inverted-repeat constructs modulates phosphogluconate mutase (Pgm) gene expression in adult Drosophila melanogaster.

BACKGROUND: A tetracycline-regulated (conditional) system for RNA interference (RNAi) would have many practical applications. Such a strategy was developed using RNAi of the gene for phosphogluconate mutase (Pgm). Pgm is a candidate lifespan regulator: PgmS allele frequency is increased by selection for increased lifespan, whereas PgmM and PgmF allele frequencies are decreased. RESULTS: The Pgm alleles were cloned and sequenced and were found to differ by amino-acid substitutions consistent with the relative electrophoretic mobilities of the proteins. The 'tet-on' doxycycline-regulated promoter system was used to overexpress PgmS in a wild-type (PgmM) background. Enzyme activity increases of two- to five-fold were observed in five independent transgenic lines. Tet-on was also used to drive expression of an inverted-repeat fragment of Pgm coding region. The inverted-repeat transcript was expected to form a dsRNA hairpin, induce RNAi, and thereby reduce endogenous Pgm gene expression at the RNA level. Endogenous Pgm RNA levels in adult flies were found to be reduced or eliminated by doxycycline treatment in five independent inverted-repeat transgenic lines. Our results show that doxycycline-regulated expression of inverted-repeat constructs can cause a conditional reduction in specific gene expression. The effect of sense and inverted-repeat construct expression on lifespan was assayed in multiple transgenic lines. Under the conditions tested, altered Pgm gene expression had no detectable effect on adult Drosophila lifespan. CONCLUSIONS: A system for conditional RNAi in Drosophila adults shows promise for assay of gene functions during aging. Our results indicate that Pgm does not have a simple strong effect on longevity.