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Background: To clarify the mechanisms underlying physical activity (PA)-related cardioprotection, we examined the association of PA with plasma bioactive lipids (BALs) and cardiovascular disease (CVD) events. We additionally performed genome-wide associations. Methods: PA-bioactive lipid associations were examined in VITAL (VITamin D and OmegA-3 TriaL)-clinical translational science center (REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01169259; N=1032) and validated in JUPITER (Justification for the Use of statins in Prevention: an Intervention Trial Evaluating Rosuvastatin)-NC (REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT00239681; N=589), using linear models adjusted for age, sex, race, low-density lipoprotein-cholesterol, total-C, and smoking. Significant BALs were carried over to examine associations with incident CVD in 2 nested CVD case-control studies: VITAL-CVD (741 case-control pairs) and JUPITER-CVD (415 case-control pairs; validation). Results: We detected 145 PA-bioactive lipid validated associations (false discovery rate Conclusions: We identified a PA-related bioactive lipidome profile out of which 12 BALs also had opposite associations with incident CVD events.

Introduction: To better understand the cardioprotective mechanisms of regular physical activity (PA), we examined its association with plasma bioactive lipids (BAL) and prospective cardiovascular disease (CVD) events and evaluated the extent to which BMI might mediate this association. Hypothesis: 1) Higher PA associates with a bioactive lipid profile related to lower CVD risk; 2) BMI mediates the PA-BAL and BAL-CVD associations. Methods: Using linear models (adjusted for age, sex, race, smoking, LDL, and total cholesterol), self-reported PA was examined with BAL assayed by an untargeted metabolomics platform in VITAL (N=1032) with validation in JUPITER (N=589). After performing mediation analysis through BMI (same adjustments), we evaluated both BMI-mediated and non-mediated PA-BAL associations with incident CVD using similarly adjusted conditional logistic regression in two nested CVD case-control studies: VITAL-CVD (770 pairs, followed by mediation analysis) and JUPITER-CVD (415 case-control pairs - validation) Results: We detected 237 significant PA-BAL associations in VITAL with replication in JUPITER (FDRvice-versa . Conclusion: We identified a signature of 22 PA-related BAL, which were also associated with incident CVD in two independent case-control studies. Although BMI-mediated associations were predominant, non-mediated effects were also detected, implying that more than one pathway may play a role in the PA-CVD relationship.

Background Epidemiological and animal studies have associated systemic inflammation with blood pressure (BP). However, the mechanistic factors linking inflammation and BP remain unknown. Fatty acid–derived eicosanoids serve as mediators of inflammation and have been suggested to regulate renal vascular tone, peripheral resistance, renin‐angiotensin system, and endothelial function. We hypothesize that specific proinflammatory and anti‐inflammatory eicosanoids are linked with BP. Methods and Results We studied a population sample of 8099 FINRISK 2002 participants randomly drawn from the Finnish population register (53% women; mean age, 48±13 years) and, for external validation, a sample of 2859 FHS (Framingham Heart Study) Offspring study participants (55% women; mean age, 66±9 years). Using nontargeted liquid chromatography–mass spectrometry, we profiled 545 distinct high‐quality eicosanoids and related oxylipin mediators in plasma. Adjusting for conventional hypertension risk factors, we observed 187 (34%) metabolites that were significantly associated with systolic BP ( P Conclusions Plasma eicosanoids demonstrate strong associations with BP in the general population. As eicosanoid compounds affect numerous physiological processes that are central to BP regulation, they may offer new insights about the pathogenesis of hypertension, as well as serve as potential targets for therapeutic intervention.

The cardiac extracellular matrix is a complex network of proteins and plays a key role in cardiac remodeling after myocardial infarction. One of the extracellular matrix proteins, asporin, is a member of the SLRP family and inhibits TGFβ signaling. However, very little is known about the function and regulation of asporin in cardiac remodeling. Using bioinformatics approach, microarray data analysis from publicly available datasets identified asporin as a top candidate showing an increase in left ventricle tissue with ischemic cardiomyopathy as compared to control donor hearts. To confirm, we detected an increase in asporin expression in paired heart biopsies taken before and after cardiopulmonary bypass surgery by western blot analysis. Our group showed that glucagon like receptor 1 agonist (GLP-1Ra) is beneficial for cardiac remodeling and prevents fibrosis. In a permanent coronary artery ligation model, we found increased extracellular asporin expression after 30 days in border zone and scar area with GLP-1Ra treatment compared to vehicle-treatment. To further elucidate the mechanism of asporin secretion, we used H9c2 cells as well as left ventricle tissue from mice and found that asporin was predominantly present in the heavy membrane enriched fraction (8,000xg pellet from postnuclear supernatant). Sequence analysis of asporin protein using SecretomeP 2.0 server predicted asporin might use the non-conventional form of secretion (secretory autophagy). Differentiation of H9c2 cells is accompanied by autophagy and mitochondrial biogenesis, and we found that the abundance of asporin in total lysate and heavy membrane fraction decreased suggesting it could be released via secretory autophagy. Using recombinant human asporin, we demonstrated that asporin stimulates an increase in LC3 and p62 in parallel to a decrease in COX IV and SMAD2 phosphorylation, indicating mitophagy induction and a decrease in TGFβ signaling. Based on our preliminary data and sequence comparison to a previously reported human peptide, we developed a rodent asporin peptide which is predicted to induce mitophagy while decreasing TGFβ signaling to prevent fibrosis. Studies are ongoing to establish the clinical relevance of asporin in animal models of ischemic injury.

Pioglitazone (PIO) and GLP-1R receptor agonist (GLP1Ra) have been shown to be cardioprotective against ischemic cardiac injury. Much less is known about the effects of these agents on adverse post-MI LV remodeling. Based on our earlier findings that PIO and a GLP1Ra stimulate mitochondrial turnover, we evaluated the effects of these agents on post-MI remodeling. Lean mice underwent permanent coronary artery ligation (PCAL) to ensure that remodeling would be independent of changes in infarct size. Vehicle, PIO and GLP1Ra (Sigma) were administered i.p. 2h after the infarct and then every other day for a total of 6 doses. Echo and histology (Masson’s trichrome) were used to assess LV remodeling 30 days post-MI (late phase); immune cell infiltration was assessed 7 days after PCAL (early phase) via hematoxylin and eosin (H&E) staining. Both PIO and GLP1Ra were associated with significant improvements in ejection fraction and fractional shortening (A); however, at the dose used, neither drug restored function to that of the sham-operated mice (EF 60% and FS 30%, data not shown). GLP1Ra-treated mice exhibited a marked reduction in immune cell infiltration at 7d after PCAL (B), and fibrosis at 30d (C) compared to vehicle or PIO. While PIO had no effect on immune-cell infiltration and fibrosis, it was effective in attenuating post-MI remodeling. While sharing a common endpoint of attenuating adverse remodeling, the finding that PIO stimulates mitochondrial biogenesis and GLP1Ra induces mitophagy may help explain their disparate findings with respect to early phase remodeling.

MicroRNAs (miRs) regulate post-transcriptional gene expression and protein translation. There is now increasing evidence that miRs regulate mitochondrial turnover. miR-33 regulates cholesterol metabolism and mitochondrial function in cardiac fibroblasts and macrophages and has been implicated in promoting fibrosis. Excessive fibrosis contributes to the development of adverse LV remodeling. Given our recent findings that a glucagon-like peptide-1 receptor agonist (GLP1Ra) [Sigma] induces mitophagy and mitigates post-MI LV remodeling, the purpose of this study was to determine its effects on miR-33 expression after MI. We analyzed miR-33 expression in the viable regions of hearts in lean mice after permanent coronary arterial ligation (PCAL) and subsequent treatment with GLP1Ra administered 2h and 48h after the infarction (total of 2 doses). PCAL was associated with a marked increase in miR-33 that peaked at day 3 (data not shown). In the presence of GLP1Ra, miR-33 expression was suppressed 3 days post-PCAL ( A ). Polysome profiling revealed that miR-33 was decreased in the translating fractions of GLP1Ra-treated mice and increased in the non-translating fraction (80S free ribosomes) compared to vehicle ( B and C ). Decreased association of miR-33 with polysomes in GLP1Ra-treated mice would allow for increased target translation. Given that suppression of miR-33 is implicated in promoting fibrosis, a suppression of this miR by acute GLP1Ra treatment could explain the beneficial effects of this drug in limiting adverse cardiac remodeling.

Randomized clinical trials have demonstrated that pioglitazone (PIO), a thiazolidinedione, and liraglutide and semaglutide, glucagon-like peptide-1 receptor agonists (GLP1Ra), are effective in treating Type 2 diabetes mellitus. Of considerable interest is the additional observation that they may also be effective in reducing the incidence of major adverse cardiovascular events (MACE) independent of glycemic control. The mechanism underlying this protection is unknown. To test the hypothesis that enhanced mitochondrial turnover (mitophagy and mitochondrial biogenesis) might play a role, obese mice with insulin resistance were subjected to permanent coronary artery ligation (PCAL). Two hours later they were administered an i.p. bolus of vehicle (50μL DMSO), PIO (10mg/kg), or a GLP1Ra (10nmoles/25g) (Sigma). This was repeated every other day for 2 weeks. Hearts were probed by western blot for markers of mitochondrial biogenesis and mitophagy. As shown, PIO was a strong inducer of mitochondrial biogenesis evidenced by increased mitochondrial markers in the whole lysate (mitochondrial biogenesis) whereas GLP1Ra stimulated mitophagy, indicated by Parkin, p62 and optineurin translocation to the mitochondrial fraction. These findings suggest that enhanced mitochondrial biogenesis and mitophagy may be important therapeutic targets for the prevention of adverse cardiovascular outcomes. If confirmed, enhanced mitochondrial turnover may be one of the mechanisms underlying the reduced incidence of MACE that was observed in the PROactive (PIO) and the LEADER and SUSTAIN-6 (GLP1Ra) clinical trials.

Background: Wound drains that are left in place for a prolonged period of time have a higher rate of bacterial contamination. Following spinal surgery, a drain is often left in place for a longer period of time if it maintains a high output. Given the major consequences of an infection following spinal surgery and the lack of data with regard to the use of antibiotics and drains, we performed a study of patients with a drain following spinal surgery to compare infection rates between those who were treated with antibiotics for twenty-four hours and those who received antibiotics for the duration for which the drain was in place. Methods: We performed a prospective randomized trial of 314 patients who underwent multilevel thoracolumbar spinal surgery followed by use of a postoperative drain. The patients were randomized into two groups, one of which received perioperative antibiotics for twenty-four hours (twenty-four-hour group) and the other of which received antibiotics for the duration that the drain was in place (drain-duration group). Data collected included demographic characteristics, medical comorbidities, type of spinal surgery, and surgical site infection. Results: Twenty-one (12.4%) of the 170 patients in the twenty-four-hour group and nineteen (13.2%) of the 144 in the drain-duration group developed a surgical site infection (p = 0.48). There were no significant differences between the twenty-four-hour and drain-duration groups with respect to demographic characteristics (except for the American Society of Anesthesiologists [ASA] classification), operative time, type of surgery, drain output, or length of hospital stay. Conclusions: Continuing perioperative administration of antibiotics for the entire duration that a drain is in place after spinal surgery did not decrease the rate of surgical site infections. Level of Evidence: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Introduction: Premenopausal women, as well as females in animal studies, have a reduced risk of cardiovascular diseases and a reduced myocardial susceptibility to ischemia/reperfusion (I/R) injury. However, with constantly increasing prevalence of obesity, the impact of diet-induced obesity on females remains unclear. Mechanisms of autophagy and mitophagy, differentially regulated between males and females, have been shown to be cardioprotective and impacted under nutritional overload. Hypothesis: Autophagy and mitophagy pathways can be involved in both female cardio protection and deleterious effect of obesity. Methods: Male and female C57Bl/6J mice (n =20 per group) of 8 weeks old were fed a low-fat (LFD, 10% fat) or high-fat (HFD, 60% fat) diet for 12 weeks. At 12 weeks, hearts from mice were studied under basal conditions and after global no-flow ischemia and reperfusion on a Langendorff perfusion system. Results: In both males and females, HFD significantly increases body weight (31.4g vs 48.3g and 22.1g vs 39.9g respectively), fat mass (+200% in males and +300% in females) and blood glucose (+79mg/dl and + 54mg/dl, respectively) (all p Conclusion: All together, these results suggest an impairment of autophagy and mitophagy pathways under HFD. A more drastic change occurs in female mice and may imply a more important response of female mice to HFD, although further studies are needed. However, this result suggests that cardiovascular disease may be more deleterious in women despite their lower cardiovascular risk; additional studies examining sex differences in the response to metabolic syndrome and ischemic heart disease are warranted.

OBJECTIVES The aim of the study was to evaluate indications, results, and clinical and neurological evolution in children who have undergone liver transplantation for classical maple syrup urine disease (MSUD). METHODS Descriptive study of liver transplantation for MSUD between 1991 and 2012. Eight patients were transplanted. RESULTS Indications for transplant were poor metabolic control expressed as significant psychomotor disabilities (4 had psychomotor delays, 5 had spasticity, and 5 had epilepsy) and poor quality of life (mean number of acute metabolic decompensations and mean number of total hospitalizations before transplantation 5 and 12, respectively). Four required nasogastric tube, with a maximum 4 g/day protein-restricted diet in all of them. Seven sustained significant alterations in brain magnetic resonance imaging. Mean leucine and alloisoleucine levels were 608 (standard deviation [SD] 516) and 218 μmol/L (SD 216), respectively. All of the patients received transplants with deceased-donor livers, with ages between 1.5 and 2.5 years (mean 1.78 years). Mean posttransplantation follow-up period was 12.2 years (range 5-21 years). Final patient and graft survival was 87.5% and 75%, respectively. Following transplantation, none required hospitalization in the last 3 years nor did any have new acute metabolic decompensations following a normal diet. Five followed normal schooling, 2 had motor disabilities, and 2 had convulsive crises. Brain magnetic resonance imaging was taken in 4 patients, showing neuroimage improvement in 3 of them. Mean leucine levels were

Surgical decompression of calcific tendonitis of the shoulder can be difficult because identification of the lesion is prob- lematic, as the lesion is usually within the supraspinatus and cannot be directly visualized. We demonstrate a technique to identify and mark the calcific lesion using portable 2-D ultrasound to guide the placement of a breast-biopsy localization wire into the calcific lesion, followed by arthroscopic decompression of the calcific lesion. After regional an- esthesia, portable ultrasound is used to identify the calcific lesion. The introducer needle of the breast-lesion localization wire is then ad- vanced into the calcific lesion. The localization wire, with 2 barbs at its tip, is advanced through the introducer needle into the lesion. The barbs secure the wire into the lesion. After diagnostic arthroscopy, the arthroscope is placed into the subacromial space. The localization wire is identified and followed to the calcific lesion. The lesion is debrided with a motorized shaver, removing as much calcific material as pos- sible. The supraspinatus tendon is then examined. If a significant defect is present, it is repaired. Early clinical results show good subjective and objective improvement.

Duchenne muscular dystrophy, a crippling genetic disease leading to premature death, affects the heart as well as skeletal muscle. Indeed, cardiomyopathy is the leading cause of death in Duchenne patients. There are no approved treatments for the cardiomyopathy, and novel Duchenne-specific experimental approaches such as exon skipping do not benefit the heart. Here we demonstrate that cardiosphere-derived cells (CDCs), which are in advanced clinical testing for therapeutic regeneration after myocardial infarction, reverse the key pathophysiological hallmarks of Duchenne cardiomyopathy (oxidative stress, inflammation, fibrosis and mitochondrial dysfunction) in mdx mice. Exosomes secreted by human CDCs reproduce the benefits of CDCs in mdx mice, and reverse mitochondrial dysfunction in human Duchenne cardiomyocytes. Both CDCs and their exosomes improve heart function in mdx mice (P

Background: Previously we showed that the homeostatic intracellular repair response (HIR2) is activated in the hearts of patients undergoing cardiac surgery. Autophagy is a principal component of this beneficial response that clears fragile mitochondria and protein aggregates. Moreover, we have previously shown that mitochondrial elimination through autophagy (mitophagy) is a key element in ischemic preconditioning. Thus, an important mechanism of cardioprotection appears to involve the upregulation of autophagy which facilitates the clearance of vulnerable mitochondria to limit I/R injury. We hypothesized that this protective action leads to turnover of the existing mitochondrial population in the heart during the resolution of I/R injury. The purpose of this study was to examine if the mechanism of HIR2 extends to remodeling the existing mitochondrial population of the heart. Study Design: Autophagy and mitochondrial turnover were assessed in 10 patients undergoing coronary artery bypass or valve surgery requiring cardiopulmonary bypass. Biopsies of the right atrial appendage obtained before initiation and after weaning from cardiopulmonary bypass were processed to yield whole tissue lysates and mitochondria-enriched heavy membrane fractions. Samples were analyzed for autophagy by immunoblotting for LC3, Beclin-1, ATG5-12, and p62. Mitochondrial turnover was assessed by monitoring Tom70, Cox4, Drp1, p62 and Parkin in tissue lysates and heavy membrane fractions. Results: Heart surgery was associated with a robust increase in autophagy indicated by depletion of LC3, Beclin-1, ATG5-12 and p62, as well as the mitophagy and fission regulator Drp1. Parkin increased in the mitochondrial fraction after bypass. Surprisingly, post-bypass tissue lysates showed a marked increase in mitochondrial markers Tom70 and Cox4, suggesting mitochondrial biogenesis. Conclusions: These findings provide evidence for the first time in humans that coordinated mitophagy and biogenesis are part of the homeostatic response to I/R, pointing to the importance of studying this aspect of HIR2. Strategies designed to amplify HIR2 during cardiac stress may represent an entirely new approach to myocardial protection in patients undergoing heart surgery.

Myogenesis is a crucial process governing muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNML1/DRP1-mediated fragmentation and subsequent removal of mitochondria via p62/SQSTM-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α-mediated biogenesis. Mitochondrial fusion protein OPA1 is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that blocking autophagy with various inhibitors during differentiation results in a blockade in myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

The Internet, with established and newly appearing websites, is becoming a more integral part of orthopaedic education every day. We review some of the well-known resources on the Internet (Orthogate, OrthoNet, American Academy of Orthopaedic Surgeons site, Orthopedic Hyperguide, WorldOrtho, Wheeless's Textbook of Orthopaedics, Orthoteers, AO North America site, University of Iowa Virtual Hospital texts, and South Australian Orthopaedic Registrars' Notebook) and reports the results of a survey of 35 orthopaedic surgery residents and 24 attending orthopaedic surgeons on the use of orthopaedic knowledge resources on the World Wide Web. The top three most commonly used online sites by the residents were Wheeless's Textbook of Orthopaedics, the American Academy of Orthopaedics Surgeons website, and Orthopedics Hyperguide. According to the survey, all 35 residents used online resources for learning, giving preference to online textbooks when looking for clinical information and online practice examinations when preparing for the in-training (OITE) or board examinations. Among the attending physicians, 19 (79%) used the Internet. Their most commonly used online sites were the American Academy of Orthopaedics Surgeons website, AO North America site, and Wheeless's Textbook of Orthopaedics. The attending physicians also preferred online textbooks to other online resources. Also provided in this article is a discussion of our idea for a future comprehensive, accurate, and constantly updated orthopaedic online resource that uses already available technology.

To make recommendations for the selection of digital imaging equipment and its use in the typical orthopaedic surgery practice, we investigated four digital cameras with maximal resolution ranging from 1.3-3.34 megapixels. We took images of a plain radiograph, a magnetic resonance image, a hand model, and a minifragment plate with all four digital cameras and a 35-mm film camera. Several variables were evaluated to determine their effect on image quality, including adjusting maximal camera resolution, using a flash, using a camera stand, and using a macromode. Images were graded on a 3-point scale on the computer desktop and as 5 x 7 inch prints by two blinded observers. A maximal camera resolution of 2.1 megapixels was required to make photograph-quality 5 x 7 inch prints of all subjects imaged. No difference in print quality was seen between images taken in the highest quality JPEG format and the uncompressed TIFF format. A macromode with closest focal length less than 5 inches was needed for imaging small subjects. The 1.3-megapixel camera was comparable with the higher resolution cameras for imaging radiographic studies and when viewing color images on the computer desktop.

Proteus syndrome is a polymorphic hamartomatous disorder associated with a broad spectrum of rare congenital malformations. Many neoplasms have been linked to this condition, including lipomas, lymphangiomas, and hemangiomas. The authors describe a case of a congenital muscular lesion in a child with Proteus syndrome. This mass was painless but was growing out of proportion to her leg musculature. A magnetic resonance imaging scan and incisional biopsy showed findings consistent with focal myositis. The clinical, radiographic, and histologic characteristics of this previously undescribed lesion are discussed, with a brief review of the literature.

Introduction: Macroautophagy (autophagy) is highly responsive to nutrient/energy status and cellular stress. It plays an adaptive role in limiting ischemia/reperfusion (I/R) injury in the heart. Nutrient depletion is a potent inducer of autophagy and is mediated by reciprocal regulation of AMPK and mTOR pathways which in turn regulate autophagy initiation via ULK1/Atg1. Autophagy is reduced in the setting of obesity, dyslipidemia, and insulin resistance associated with metabolic syndrome (MetS). The purpose of this study was determine whether impaired autophagy in MetS is due to altered upstream signaling or a disruption in the autophagic pathway. Methods: Mice were ad-lib fed a chow (Lean) or a high fat diet (HFD) (D12492, 60% fat) for 18 weeks. In both groups, half of the mice were freely fed with the other half fasted for 24 hours prior to cardiac harvest. Protein levels were determined with Western blotting and gene expression with quantitative PCR. Results: Consistent with previous data, HFD animals exhibited obesity and insulin resistance as reflected by increased body weight, and elevated serum insulin/HOMA compared to Lean animals. Fasting resulted in a marked reduction in serum insulin in both Lean (p=0.013) and HFD (p =0.002) animals. This was accompanied by marked increases in cardiac AMPK activation as reflected by increased phosphorylation in both Lean (p =0.002) and HFD (p=0.002) groups with parallel reciprocal suppression of mTOR activity as reflected by decreased pS6K and pS6. As expected, the Lean group exhibited a fasting-induced increase in LC3-II (p=0.020) indicating an increase in autophagy. This did not occur in the HFD group despite a similar induction in LC3B and p62 gene expression. This lack of autophagy stimulation with fasting in HFD animals was not explained by differences in autophagy initiation as the phosphorylation status of ULK1 decreased similarly with fasting in both groups. Conclusions: These data indicate that the impairment of cardiac autophagy in the presence of MetS is not attributable to abnormal upstream nutrient sensing pathways. The findings suggest a MetS-associated defect in the autophagy/lysosomal fusion pathway. This could explain the increased vulnerability of the heart to I/R injury in the setting of MetS.

Injury to articular cartilage is increasingly diagnosed as a cause of knee pain. New surgical treatment options are now available to treat this clinical entity. Diagnosis is made easier by improved magnetic resonance imaging protocols that better visualize articular cartilage. On physical examination, patients at any age can have joint surface damage and primarily experience joint line tenderness and pain with activity. Treatment options include debridement of nonviable cartilage with abrasion or microfracture of the subchondral bone, transplantation of autologous osteochondral plugs, autologous chondrocyte transplantation, transplantation of cadaveric allogenic osteochondral grafts, and, for irreparable damage, total knee arthroplasty. These newer treatment options show promising early and intermediate results.

Introduction: Over the last decade, stem/progenitor cell therapy has emerged as an innovative approach to promote cardiac repair and regeneration. However, the therapeutic prospects of are currently limited by inadequate means to regulate cell proliferation, homing, engraftment, and differentiation. Autophagy, a lysosome-mediated degradation pathway for recycling organelles and protein aggregates, is recognized as important for facilitating cell differentiation. Studies have shown that induced pluripotent stem cells (iPCs), which exhibit a predominantly glycolytic metabolism, shift towards oxidative mitochondrial metabolism as a prerequisite for the formation of sarcomeres and differentiation into cardiomyocytes. C2C12 myoblasts are a mouse-derived myogenic progenitor cell line which can be induced to differentiate into myotubes. We hypothesize that autophagy is essential in coordinating transcription factor activity and metabolic reprogramming of mitochondria to support myocyte differentiation. Methods: C2C12 myoblasts were cultured in DMEM (10% FBS) and induced to differentiate into myotubes with DMEM (2% horse serum) for 6d. To disrupt autophagy, cells were: 1) transfected with 50nM Atg5 siRNA for 8h twice over a 48h period, or 2) treated with 10nM bafilomycin A1 or vehicle control for a 3h/day for the first 3d of differentiation. GFP-LC3 adenovirus was employed to visualize autophagy. Western blot and real-time qPCR were used to examine proteins and transcripts of interest. Results: We observed increased LC3-II levels and GFP-LC3 puncta during the differentiation of C2C12 cells, suggesting the involvement of autophagy in this process. Transient inhibition of autophagy during the early stages of differentiation with either Atg5 siRNA or bafilomycin A1 interfered with myotube formation and attenuated the upregulation of myogenic transcription factors MyoD and myogenin. Differentiation was accompanied by an increase in PGC1α mRNA, mitochondrial mass, and oxygen consumption, all of which were blocked by disruption of autophagy. Conclusions: Autophagy coordinates transcription factor expression and mitochondrial turnover essential for cell differentiation.

The cardioprotective effects of statins are well known yet the mechanism is unclear. Previously we showed that autophagy is required for cardioprotection from ischemia/reperfusion injury. More recently, we reported that ischemic preconditioning involves Parkin-mediated mitophagy. We hypothesized that the molecular basis of statin-mediated cardioprotection may involve mitochondrial quality control through mitophagy. HL-1 cardiomyocytes treated with simvastatin for 24hr exhibited diminished Akt/mTOR signaling, increased activation of ULK1, and upregulation of autophagy (n=3, p Figure: Statin-induced cardioprotection against I/R injury: solid bars/diamonds = wild-type; open bars/diamonds = Parkin knockout mice.

Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, dsRed, developed by Terskikh and colleagues. Its fluorescence shifts over time from green to red as the protein matures. This molecular clock gives temporal and spatial information on protein turnover. To visualize mitochondrial turnover, we targeted Timer to the mitochondrial matrix with a mitochondrial targeting sequence (coined “MitoTimer”) and cloned it into a tetracycline-inducible promoter construct to regulate its expression. Here we report characterization of this novel fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were transfected with pTRE-tight-MitoTimer and induced production with doxycycline. Mitochondrial distribution was demonstrated by fluorescence microscopy and verified by subcellular fractionation and western blot analysis. Doxycycline addition for as little as 1hr was sufficient to label mitochondria. MitoTimer was detected as early as 4hr following doxycycline addition, and persisted in mitochondria for at least 72hr. The color-specific conformation of MitoTimer was stable after fixation with 4% paraformaldehyde. MitoTimer matured to red fluorescence within 48hr, at which time a second pulse of doxycycline induced expression of green (immature) MitoTimer which was selectively incorporated into a subset of mitochondria actively engaged in protein import. The extent of new protein incorporation during a second pulse was increased under conditions of mito-biogenesis and reduced if mitochondrial membrane potential was dissipated. We conclude that MitoTimer can be used to monitor mitophagy and biogenesis.

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

In a pilot study, the polymerase chain reaction was found to be more sensitive than standard viral culture methods for the detection of cytomegalovirus, particularly from blood and tissues. We therefore applied this technique to 71 serially collected liver biopsies from 16 orthotopic liver transplant patients. All patients were CMV-seropositive (n = 15) or seroconverted (n = 1). Seven patients (9 biopsies) had histologically proved CMV hepatitis, and all these biopsies were CMV PCR-positive. Six of these 7 patients had a prior liver biopsy that was CMV PCR-positive, but culture and histology-negative, an average of 13.2 +/- 6.9 days before the histologically positive biopsy. The 7th patient was not biopsied prior to the diagnostic biopsy. Three patients had 7 liver biopsies that were CMV PCR-positive, but histologically negative for CMV hepatitis. Two of these three had CMV infection confirmed by viral culture of blood or liver biopsy. The remaining 6 patients had a total of 26 liver biopsies that were negative for CMV by PCR, culture, and histology. Among liver transplant patients, CMV PCR performed on liver biopsy specimens correctly identified all histologically proven cases of CMV hepatitis. CMV PCR positivity in liver tissue did not correlate with latent infection and preceded the development of CMV hepatitis or other meaningful CMV infection in 8 of 10 patients.

As orthopaedic surgeons, we commonly see patients with complaints of pain in and around tendons and/or tendon insertions. How often do we see runners with a painful Achilles, swimmers or throwers with a painful shoulder, or tennis players with a painful elbow? Despite the frequency of these conditions, our understanding of their etiology and optimal treatment remains limited. Several terms have been used to describe these conditions such as tendonitis, tendinosis, epicondylitis or bursitis. Many of these terms imply that the tendon pain is associated with an inflammatory process. Accordingly, traditional treatment options such as rest, ice, nonsteroidal antiinflammatory medications and corticosteroid injections have focused on reducing this inflammation. Recently the terms tendonitis, epicondylitis and bursitis are falling out of favor, as evidence of inflammation of the tendon and/or bursa is not typically present in these conditions. A more accurate term is tendinopathy. Tendinopathy is an all-encompassing term describing the pathologic, “degenerative” changes that occur within tendons in response to overuse and repetitive trauma. Histopathologic changes associated with tendinopathy include degeneration and disorganization of collagen fibers with increased cellularity, but minimal inflammation. Macroscopic changes include tendon thickening, loss of mechanical properties, and pain. Our lack of comprehension of the pathologic processes that result in tendinopathy has been a major impediment to progress in this field. A better understanding of the etiology of tendinopathy is essential in order to develop better treatment options for this condition. We are at an exciting time in this field as research in the last 5 years has improved our understanding of tendinopathy and has set the stage for further breakthroughs in the years to come. Advances in molecular biology, tissue culture, and the development of animal models of tendinopathy and tendon repair have facilitated the recent growth in this field. The goal of this symposium is to provide the orthopaedic community with a picture of our current understanding of the pathogenesis of tendinopathy and to provide a framework that may guide treatment strategies in the future. The cutting-edge work of many leaders in this field is presented here. Through the use of molecular biology, cell and tissue culture, and sophisticated animal models, the pathologic changes associated with tendinopathy are being elucidated. Work presented in this symposium and elsewhere suggests tendinopathy develops in response to tendon overuse. This overuse results in the induction of molecular events that lead to “degeneration” as well as protective, regenerative changes. As the repetitive trauma of overuse continues, pathologic changes such as the release of matrix metalloproteinases and tendon cell apoptosis begin to outweigh the regenerative ones. The net result is degradation and disorganization of the extracellular matrix and a loss of viable tenocytes. This produces a biomechanically inferior tendon that is painful and prone to tearing. Maintaining adequate tension on the tendon also seems to play an important role in tendon homeostasis as the loss of tension appears to magnify the changes seen in response to overuse. This may explain why the early degenerative changes seen in response to overuse can be reversed by cessation of activity, but chronic tendinopathy does not typically resolve quickly through activity modification alone. The ultimate goal of this work is to find ways of preventing tendinopathy and better treatment options once tendinopathy has developed. As Guest Editors, we are grateful to the staff of Clinical Orthopaedics and Related Research and to all of the authors and reviewers who have contributed to this symposium. We have been impressed by the quality of the manuscripts submitted and feel that it represents the highest level, cutting edge research in the field of tendinopathy.

Five adolescents, 13-18 years of age, underwent esophageal manometric studies because of chronic symptoms suggestive of esophageal dysfunction. Four of five patients had episodic nonexertional midchest pain; two patients experienced intermittent dysphagia. The manometric findings for these adolescents were consistent with a primary motility disorder known as diffuse esophageal spasm, a condition not previously reported in this age group. This represents approximately 1% of all pediatric patients undergoing esophageal manometry at our institution for the past 5 years. They have been followed for at least 2 years and three have experienced gradual resolution of their symptoms with normalization of manometric findings. Our report emphasizes two main points: (a) Diffuse esophageal spasm may cause chest pain and dysphagia in adolescents; and (b) the clinical history and esophageal manometric findings establish the diagnosis of diffuse esophageal spasm.

A 14-year-old boy experienced multiple hospitalizations because of symptoms due to Crohn's disease involving the stomach, duodenum, and ileum. He maintained that oral corticosteroids were not effective for control of his symptoms. However, i.v. corticosteroids always relieved his symptoms. To resolve the question of noncompliance versus altered corticosteroid absorption or metabolism, our patient underwent an oral prednisone absorption study. Prednisolone, the active metabolite of prednisone, was measured in his plasma using a high-pressure liquid chromatography technique. The results led to the discovery of an elaborate deception by the patient and his subsequent need for psychotherapy. This report documents the importance of measuring plasma prednisolone concentrations to diagnose noncompliance, especially in adolescents who are overly concerned about their body image.

A 7-year-old boy developed recurrent abdominal pain. He was eventually discovered to have an inverted appendix via colonoscopy. At surgery, a polyploid intracecal mass was palpated at the base of the partially invaginated appendix. Resection of the cecal mass (histologically, a juvenile polyp) and appendix was easily accomplished. The types of clinical presentation and treatment of children with appendiceal intussusception are discussed.