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Rationale Dysfunctional Parkin-mediated mitophagic culling of senescent or damaged mitochondria is a major pathological process underlying Parkinson disease and a potential genetic mechanism of cardiomyopathy. Despite epidemiological associations between Parkinson disease and heart failure, the role of Parkin and mitophagic quality control in maintaining normal cardiac homeostasis is poorly understood. Objective We used germline mutants and cardiac-specific RNA interference to interrogate Parkin regulation of cardiomyocyte mitochondria and examine functional crosstalk between mitophagy and mitochondrial dynamics in Drosophila heart tubes. Methods and Results Transcriptional profiling of Parkin knockout mouse hearts revealed compensatory upregulation of multiple related E3 ubiquitin ligases. Because Drosophila lack most of these redundant genes, we examined heart tubes of parkin knockout flies and observed accumulation of enlarged hollow donut mitochondria with dilated cardiomyopathy, which could be rescued by cardiomyocyte-specific Parkin expression. Identical abnormalities were induced by cardiomyocyte-specific Parkin suppression using 2 different inhibitory RNAs. Parkin-deficient cardiomyocyte mitochondria exhibited dysmorphology, depolarization, and reactive oxygen species generation without calcium cycling abnormalities, pointing to a primary mitochondrial defect. Suppressing cardiomyocyte mitochondrial fusion in Parkin-deficient fly heart tubes completely prevented the cardiomyopathy and corrected mitochondrial dysfunction without normalizing mitochondrial dysmorphology, demonstrating a central role for mitochondrial fusion in the cardiomyopathy provoked by impaired mitophagy. Conclusions Parkin deficiency and resulting mitophagic disruption produces cardiomyopathy in part by contamination of the cardiomyocyte mitochondrial pool through fusion between improperly retained dysfunctional/senescent and normal mitochondria. Limiting mitochondrial contagion by inhibiting organelle fusion shows promise for minimizing organ dysfunction produced by defective mitophagic signaling.

Age-related locomotor impairment in humans is important clinically because it is associated with several co-morbidities and increased risk of death. One of the hallmarks of age-related locomotor impairment in humans is a decrease in walking speed with age. Genetically tractable model organisms such as Drosophila are essential for delineating mechanisms underlying age-related locomotor impairment and age-related decreases in locomotor speed. Negative geotaxis, the ability of flies to move vertically when startled, is a common measure of locomotor behavior that declines with age in Drosophila. Toward further developing Drosophila as a model for age-related locomotor impairment, we investigated whether negative geotaxis reflects climbing or a combination of climbing and other behaviors such as flying and jumping. Additionally, we investigated whether locomotor speed in negative geotaxis assays declines with age in flies as found for walking speed in humans. We find that the vast majority of flies climb during negative geotaxis assays and that removal of hind legs, but not wings, impairs the behavior. We also find that climbing speed decreases with age in four wild type genetic backgrounds, in flies housed at different temperatures, and in control and long-lived flies harboring a mutation in OR83b. The decreases in climbing speed correlate with the age-related impairments in the distance climbed. These studies establish negative geotaxis in Drosophila as a climbing behavior that declines with age due to a decrease in climbing speed. Agerelated decreases in locomotor speed are common attributes of locomotor senescence in flies and humans.

Salt-induced overexpression of genes cloned downstream of the phage T7 10 promoter was demonstrated in an Escherichia coli strain (GJ1158) which carries a single chromosomally integrated copy of the gene for phage T7 RNA polymerase under transcriptional control of the cis-regulatory elements of the osmoresponsive proU operon. Plasmids that have been constructed to obtain overproduction of individual target gene products in strain BL21(DE3) (by addition of isopropyl-␤-D-thiogalactopyranoside as an inducer) can directly be transformed into GJ1158. The NaCl induction regimen was also shown to be associated with a decreased propensity for sequestration of overexpressed target proteins within insoluble inclusion bodies.Escherichia coli is widely used in basic research studies as a host strain for the overproduction of proteins from cloned genes, and several proteins of pharmaceutical value are also produced on a commercial scale in E. coli. In the two most commonly used strategies, a target gene is overexpressed (directly or indirectly) by temperature induction of a phage promoter or induction with isopropyl-␤-D-thio-galactopyranoside (IPTG) of the lac promoter and its variants (reviewed in reference 13).Some problems associated with temperature-induced overexpression of recombinant gene products include (i) difficulty in achieving rapid temperature upshift, particularly when handling larger culture volumes, (ii) increased likelihood of formation of insoluble inclusion bodies at higher incubation temperatures (20), and (iii) induction of several proteases in E. coli upon heat shock (4). With the lac promoter-based systems as well, the problem of inclusion body formation may be significant, in addition to the high cost and toxicity of IPTG (13).In this study, we have developed a simple, efficient, and generally applicable method for the overproduction of recombinant proteins in E. coli that makes use of NaCl as an inducer. For this purpose, we have constructed a host strain in which synthesis of the RNA polymerase (RNAP) of bacteriophage T7 has been placed under control of the osmotically inducible proU promoter of E. coli (6). Any target gene which is cloned downstream of a phage T7 promoter (e.g., 10) and introduced into this strain will be overexpressed, through a two-tier amplification strategy, upon salt addition. We also demonstrate that the propensity for inclusion body formation may be significantly reduced with the NaCl induction protocol.Methods. Most of the genetic and recombinant DNA procedures used have been described earlier (5, 19). Induction of target genes by 1 mM IPTG in derivatives of strain BL21(DE3) was done as previously described (21,22). NaCl induction experiments were done as follows. Ten-milliliter cultures were grown at 37°C to an A 600 of around 0.8 in low-osmolarity LBON medium (Luria-Bertani medium [19] with NaCl omitted, containing [per liter] 10 g of tryptone and 5 g of yeast extract [pH adjusted to 7.0 with NaOH]) supplemented with appropriate antibiotics. Each culture was then divided...