Silencing of drpr leads to muscle and brain degeneration in adult Drosophila.

Mutations in the gene encoding the single transmembrane receptor multiple epidermal growth factor-like domain 10 (MEGF10) cause an autosomal recessive congenital muscle disease in humans. Although mammalian MEGF10 is expressed in the central nervous system as well as in skeletal muscle, patients carrying mutations in MEGF10 do not show symptoms of central nervous system dysfunction. drpr is the sole Drosophila homolog of the human genes MEGF10, MEGF11, and MEGF12 (JEDI, PEAR). The functional domains of MEGF10 and drpr bear striking similarities, and residues affected by MEGF10 mutations in humans are conserved in drpr. Our analysis of drpr mutant flies revealed muscle degeneration with fiber size variability and vacuolization, as well as reduced motor performance, features that have been observed in human MEGF10 myopathy. Vacuolization was also seen in the brain. Tissue-specific RNAi experiments demonstrated that drpr deficiency in muscle, but not in the brain, leads to locomotor defects. The histological and behavioral abnormalities seen in the affected flies set the stage for further studies examining the signaling pathway modulated by MEGF10/Drpr in muscle, as well as assessing the effects of genetic and/or pharmacological manipulations on the observed muscle defects. In addition, the absence of functional redundancy for Drpr in Drosophila may help elucidate whether paralogs of MEGF10 in humans (eg, MEGF11) contribute to maintaining wild-type function in the human brain.

Cardiotoxicity of the cancer therapeutic agent imatinib mesylate.

Imatinib mesylate (Gleevec) is a small-molecule inhibitor of the fusion protein Bcr-Abl, the causal agent in chronic myelogenous leukemia. Here we report ten individuals who developed severe congestive heart failure while on imatinib and we show that imatinib-treated mice develop left ventricular contractile dysfunction. Transmission electron micrographs from humans and mice treated with imatinib show mitochondrial abnormalities and accumulation of membrane whorls in both vacuoles and the sarco- (endo-) plasmic reticulum, findings suggestive of a toxic myopathy. With imatinib treatment, cardiomyocytes in culture show activation of the endoplasmic reticulum (ER) stress response, collapse of the mitochondrial membrane potential, release of cytochrome c into the cytosol, reduction in cellular ATP content and cell death. Retroviral gene transfer of an imatinib-resistant mutant of c-Abl, alleviation of ER stress or inhibition of Jun amino-terminal kinases, which are activated as a consequence of ER stress, largely rescues cardiomyocytes from imatinib-induced death. Thus, cardiotoxicity is an unanticipated side effect of inhibition of c-Abl by imatinib.

The beta-catenin/T-cell factor/lymphocyte enhancer factor signaling pathway is required for normal and stress-induced cardiac hypertrophy.

In cells capable of entering the cell cycle, including cancer cells, beta-catenin has been termed a master switch, driving proliferation over differentiation. However, its role as a transcriptional activator in terminally differentiated cells is relatively unknown. Herein we utilize conditional, cardiac-specific deletion of the beta-catenin gene and cardiac-specific expression of a dominant inhibitory mutant of Lef-1 (Lef-1Delta20), one of the members of the T-cell factor/lymphocyte enhancer factor (Tcf/Lef) family of transcription factors that functions as a coactivator with beta-catenin, to demonstrate that beta-catenin/Tcf/Lef-dependent gene expression regulates both physiologic and pathological growth (hypertrophy) of the heart. Indeed, the profound nature of the growth impairment of the heart in the Lef-1Delta20 mouse, which leads to very early development of heart failure and premature death, suggests beta-catenin/Tcf/Lef targets are dominant regulators of cardiomyocyte growth. Thus, our studies, employing complementary models in vivo, implicate beta-catenin/Tcf/Lef signaling as an essential growth-regulatory pathway in terminally differentiated cells.

The impact of Megf10/Drpr gain-of-function on muscle development in Drosophila.

Recessive mutations in multiple epidermal growth factor-like domains 10 (MEGF10) underlie a rare congenital muscle disease known as MEGF10 myopathy. MEGF10 and its Drosophila homolog Draper (Drpr) are transmembrane receptors expressed in muscle and glia. Drpr deficiency is known to result in muscle abnormalities in flies. In the current study, flies that ubiquitously overexpress Drpr, or mouse Megf10, display developmental arrest. The phenotype is reproduced with overexpression in muscle, but not in other tissues, and with overexpression during intermediate stages of myogenesis, but not in myoblasts. We find that tubular muscle subtypes are particularly sensitive to Megf10/Drpr overexpression. Complementary genetic analyses show that Megf10/Drpr and Notch may interact to regulate myogenesis. Our findings provide a basis for investigating MEGF10 in muscle development using Drosophila.

Evidence for premature aging in a Drosophila model of Werner syndrome.

Werner syndrome (WS) is an autosomal recessive progeroid disease characterized by patients' early onset of aging, increased risk of cancer and other age-related pathologies. WS is caused by mutations in WRN, a RecQ helicase that has essential roles responding to DNA damage and preventing genomic instability. While human WRN has both an exonuclease and helicase domain, Drosophila WRNexo has high genetic and functional homology to only the exonuclease domain of WRN. Like WRN-deficient human cells, Drosophila WRNexo null mutants (WRNexoΔ) are sensitive to replication stress, demonstrating mechanistic similarities between these two models. Compared to age-matched wild-type controls, WRNexoΔ flies exhibit increased physiological signs of aging, such as shorter lifespans, higher tumor incidence, muscle degeneration, reduced climbing ability, altered behavior, and reduced locomotor activity. Interestingly, these effects are more pronounced in females suggesting sex-specific differences in the role of WRNexo in aging. This and future mechanistic studies will contribute to our knowledge in linking faulty DNA repair mechanisms with the process of aging.

Fatty infiltration of right ventricle (adipositas cordis): an unrecognized cause of early graft failure after cardiac transplantation.

Fatty infiltration of the right ventricle is usually an incidental finding at post-mortem, but may have clinical significance at times of physiologic stress. We report a case of fatal right ventricular dysfunction immediately after cardiac transplantation secondary to massive fatty infiltration of the donor right ventricle. Ante-mortem diagnosis of fatty infiltration may be difficult to determine despite non-invasive cardiac evaluation. If the diagnosis of fatty infiltration is suspected at time of donor harvest, the relative risks and benefits of proceeding with transplantation should be carefully assessed.

High prevalence of herpes simplex virus DNA in temporal arteritis biopsy specimens.

Giant cell arteritis (GCA) affecting the cranial arteries is a disease of unknown cause that causes blindness, stroke, and other morbidity. Its sudden onset and segmental distribution are suggestive of diseases that involve viral reactivation, and cranial arteries are known to be innervated by ganglia that harbor herpes simplex virus (HSV). We used a high-sensitivity polymerase chain reaction assay to test for HSV DNA in specimens from 39 consecutive temporal artery biopsies performed for suspected GCA. HSV DNA was detected in 21 (88%) of 24 histologically positive and 8 (53%) of 15 histologically negative specimens (P = .027; Fisher exact test). Analysis of 10 renal artery samples from age-matched control subjects using the same assay showed no detectable HSV DNA. We conclude that detectable HSV DNA is correlated with histologically confirmed GCA in this patient population.

Blood cyst of the mitral valve apparatus in a woman with a history of orthotopic liver transplantation.

Blood cysts within the heart are rare findings in adults. We describe a case of a woman with a history of orthotopic liver transplantation who presented for an echocardiogram to evaluate a potential source of cerebrovascular embolism. A cystic mass attached to the submitral valve apparatus was identified by transthoracic and confirmed by transesophageal echocardiography. Surgical exploration and pathologic examination confirmed the mass to be a blood cyst attached to the chordal apparatus of the posterior papillary muscle. This case report highlights the use of echocardiography in diagnosing intracardiac blood cysts.

Transfer of fetal cells with multilineage potential to maternal tissue.

CONTEXT: During pregnancy, fetal CD34+ cells enter the maternal circulation, persist for decades, and create a state of physiologic microchimerism. Many studies have confirmed the residual presence of fetal cells in maternal blood and tissues following pregnancy. Fetal cells may respond to maternal injury by developing multilineage capacity in maternal organs. OBJECTIVE: To verify that fetal microchimeric cells express markers of epithelial, leukocyte, and hepatocyte differentiation within maternal organs. DESIGN, SETTING, AND PATIENTS: Archived paraffin-embedded tissue section specimens from 10 women who had male offspring and were previously found to have high numbers of microchimeric cells, and 11 control women who had no prior male pregnancies. Male cells were identified by fluorescence in situ hybridization, using X and Y chromosome-specific probes, followed by histologic and immunochemical studies using anticytokeratin (AE1/AE3) as a marker of epithelial cells, anti-CD45 as a leukocyte marker, and heppar-1 as a hepatocyte marker. MAIN OUTCOME MEASURE: Percentage of microchimeric cells expressing nonhematopoietic markers. RESULTS: A total of 701 male (XY+) microchimeric cells were identified (mean [SD], 227 [128] XY+ cells per million maternal cells). In maternal epithelial tissues (thyroid, cervix, intestine, and gallbladder), 14% to 60% of XY+ cells expressed cytokeratin. Conversely, in hematopoietic tissues, such as lymph nodes and spleen, 90% of XY+ cells expressed CD45. In 1 liver sample, 4% of XY+ cells expressed heppar-1. Histologic and immunochemical evidence of differentiation, as assessed by independent observers, was highly concordant (kappa = 0.72). CONCLUSION: The detection of microchimeric male cells, bearing epithelial, leukocyte, or hepatocyte markers, in a variety of maternal tissue specimens suggests the presence of fetal cells that may have multilineage capacity.

Loss of the bloom syndrome helicase increases DNA ligase 4-independent genome rearrangements and tumorigenesis in aging Drosophila.

BACKGROUND: The BLM DNA helicase plays a vital role in maintaining genome stability. Mutations in BLM cause Bloom syndrome, a rare disorder associated with cancer predisposition and premature aging. Humans and mice with blm mutations have increased frequencies of spontaneous mutagenesis, but the molecular basis of this increase is not well understood. In addition, the effect of aging on spontaneous mutagenesis in blm mutants has not been characterized. To address this, we used a lacZ reporter system in wild-type and several mutant strains of Drosophila melanogaster to analyze mechanisms of mutagenesis throughout their lifespan. RESULTS: Our data show that Drosophila lacking BLM have an elevated frequency of spontaneous genome rearrangements that increases with age. Although in normal flies most genome rearrangements occur through DNA ligase 4-dependent classical end joining, most rearrangements that accumulate during aging in blm mutants do not require DNA ligase 4, suggesting the influence of an alternative end-joining mechanism. Adult blm mutants also display reduced lifespan and ligase 4-independent enhanced tumorigenesis in mitotically active tissues. CONCLUSIONS: These results suggest that Drosophila BLM suppresses error-prone alternative end-joining repair of DNA double-strand breaks that can result in genome instability and tumor formation during aging. In addition, since loss of BLM significantly affects lifespan and tumorigenesis, the data provide a link between error-prone end joining, genome rearrangements, and tumor formation in a model metazoan.

The evolutionarily conserved RNA binding protein SMOOTH is essential for maintaining normal muscle function.

The Drosophila smooth gene encodes an RNA binding protein that has been well conserved through evolution. To investigate the pleiotropic functions mediated by the smooth gene, we have selected and characterized two sm mutants, which are viable as adults yet display robust phenotypes (including a significant decrease in lifespan). Utilizing these mutants, we have made the novel observation that disruption of the smooth/CG9218 locus leads to age-dependent muscle degeneration, and motor dysfunction. Histological characterization of adult sm mutants revealed marked abnormalities in the major thoracic tubular muscle: the tergal depressor of the trochanter (TDT). Corresponding defects include extensive loss/disruption of striations and nuclei. These pathological changes are recapitulated in flies that express a smooth RNA interference construct (sm RNAi) in the mesoderm. In contrast, targeting sm RNAi constructs to motor neurons does not alter muscle morphology. In addition to examining the TDT phenotype, we explored whether other muscular abnormalities were evident. Utilizing physiological assays developed in the laboratory, we have found that the thoracic muscle defect is preceded by dysmotility of the gastrointestinal tract. SMOOTH thus joins a growing list of hnRNPs that have previously been linked to muscle physiology/pathophysiology. Our findings in Drosophila set the stage for investigating the role of the corresponding mammalian homolog, hnRNP L, in muscle function.

Tumors of testis and midgut in aging flies.

In order to better understand the pathology of aging in the fly we used standard techniques of surgical pathology to conduct a histologic screen of approximately 1400 adult male flies ranging in age from one to five weeks. We found that flies developed tumors of the testis and gut and that the incidence of these tumors increased with age. Aging is the greatest single risk factor for the development of tumors in the general human population. Here, we show for the first time that aging is also a risk factor for tumor development in flies. These findings in one of the world's best-studied and genetically tractable model organisms open up opportunities for deeper experimental exploration of the relationship between aging and neoplasia.

Low power ultrasound delivered through a PTCA-like guidewire: preclinical feasibility and safety of a novel technology for intracoronary thrombolysis.

BACKGROUND: Low power ultrasound delivered through an angioplasty-like guidewire may be effective for intracoronary thrombolysis. We evaluated the preclinical feasibility and safety of such wire. METHODS AND RESULTS: In 15 anesthetized Yucatan minipigs, the ultrasonic wire was advanced percutaneously into all three coronaries. Each coronary was randomized to long activation (6 minutes), short activation (3 minutes), or control (3 minutes indwelling, no activation). The energy delivered was 0.14 +/- 0.01 W/cm of active length (20 kHz). No changes in heart rate, rhythm, or arterial pressure occurred during wire positioning or activation. Mean lumen diameter (MLD) by quantitative angiography was not significantly different pre- and postintervention (2.36 +/- 0.12 mm vs 2.36 +/- 0.11 mm for long activation, P = 0.96; 2.33 +/- 0.15 mm vs 2.34 +/- 0.14 mm for short activation, P = 0.54; 2.30 +/- 0.12 mm vs 2.33 +/- 0.12 mm for control, P = 0.21). There were no angiographic stenoses at 60 or 90 days follow-up. Compared with baseline, MLD at follow-up increased in all the three groups (2.40 +/- 0.13 mm vs 2.53 +/- 0.11 mm, P = 0.004 for long activation; 2.37 +/- 0.17 mm vs 2.52 +/- 0.14 mm, P = 0.023 for short activation; 2.20 +/- 0.12 mm vs 2.33 +/- 0.11 mm, P = 0.001 for the control group). By histology, there were no clinically significant pathologic changes in coronary morphology. CONCLUSION: Use of a transverse cavitation therapeutic wire is feasible and well tolerated acutely in the normal porcine coronary. At 60 and 90 days, no angiographically apparent damage, no clinically significant pathologic changes, and no adverse events were seen. This technology may be safely used during percutaneous coronary intervention. Further studies are justified to evaluate its efficacy for intracoronary thrombus ablation.

Vibrio cholerae infection of Drosophila melanogaster mimics the human disease cholera.

Cholera, the pandemic diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, continues to be a major public health challenge in the developing world. Cholera toxin, which is responsible for the voluminous stools of cholera, causes constitutive activation of adenylyl cyclase, resulting in the export of ions into the intestinal lumen. Environmental studies have demonstrated a close association between V. cholerae and many species of arthropods including insects. Here we report the susceptibility of the fruit fly, Drosophila melanogaster, to oral V. cholerae infection through a process that exhibits many of the hallmarks of human disease: (i) death of the fly is dependent on the presence of cholera toxin and is preceded by rapid weight loss; (ii) flies harboring mutant alleles of either adenylyl cyclase, Gsalpha, or the Gardos K channel homolog SK are resistant to V. cholerae infection; and (iii) ingestion of a K channel blocker along with V. cholerae protects wild-type flies against death. In mammals, ingestion of as little as 25 mug of cholera toxin results in massive diarrhea. In contrast, we found that ingestion of cholera toxin was not lethal to the fly. However, when cholera toxin was co-administered with a pathogenic strain of V. cholerae carrying a chromosomal deletion of the genes encoding cholera toxin, death of the fly ensued. These findings suggest that additional virulence factors are required for intoxication of the fly that may not be essential for intoxication of mammals. Furthermore, we demonstrate for the first time the mechanism of action of cholera toxin in a whole organism and the utility of D. melanogaster as an accurate, inexpensive model for elucidation of host susceptibility to cholera.

Primary angioplasty of an anomalous left main coronary artery: diagnostic and technical considerations.

In this case of primary angioplasty of an anomalous left main coronary artery, a method of identifying the course of these vessels is reviewed. Unique electrocardiographic findings relative to left main occlusion, aiding in suspecting this anomaly before angiographic study, are presented and the technical approach to revascularization is discussed.