Knockdown of the E3 ubiquitin ligase UBR5 and its role in skeletal muscle anabolism.

UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5's role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3ß/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (-9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3ß activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (-4.6%) and larger reductions in fiber CSA (-18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.

Surface electrical stimulation for facial paralysis is not harmful.

INTRODUCTION: Does electrical stimulation (ES) of denervated muscles delay or prevent reinnervation, or increase synkinesis? In this retrospective study we evaluate the outcome, with and without ES, of patients with acutely denervated facial muscles. METHODS: The effect of ES was analyzed in two experiments. In the first experiment, 39 patients (6 with home-based ES, median 17.5 months) underwent facial nerve reconstruction surgery. Time to recovery of volitional movements was analyzed. The second experiment involved 13 patients (7 with ES, median 19 months) during spontaneous reinnervation. Sunnybrook and eFACE scores provided functional outcome measures. RESULTS: No difference in time of reinnervation after facial nerve reconstruction surgery was seen between the patients with and without ES (median [interquartile range]: 4.5 [3.0-5.25] vs 5.7 [3.5-9.5] months; P = .2). After spontaneous reinnervation, less synkinesis was noted (Sunnybrook synkinesis score: 3.0 [2.0-3.0] vs 5.5 [4.75-7.0]; P = .02) with ES. DISCUSSION: We find no evidence that ES prevents or delays reinnervation or increases synkinesis in facial paralysis.

Dietary restriction of tyrosine and phenylalanine lowers tyrosinemia associated with nitisinone therapy of alkaptonuria.

Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone-treated AKU mice, and in an observational study of dietary intervention in AKU patients. Nitisinone-treated AKU mice were fed tyrosine/phenylalanine-free and phenylalanine-free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre-nitisinone, post-nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700 µmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. Elevated tyrosine (813 µmol/L) was significantly reduced in nitisinone-treated AKU mice fed a tyrosine/phenylalanine-free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3, 274.8, and 144.3 µmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3, 530.2, and 656.2 µmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (P = .002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine <700 µmol/L. Tyrosine/phenylalanine dietary restriction significantly reduced nitisinone-induced tyrosinemia in mice, with phenylalanine restriction alone proving ineffective. Similarly, protein restriction significantly reduced circulating tyrosine in AKU patients.

Morphological Substrates for Atrial Arrhythmogenesis in a Heart With Atrioventricular Septal Defect.

Due to advances in corrective surgery, congenital heart disease has an ever growing patient population. Atrial arrhythmias are frequently observed pre- and post-surgical correction. Pharmaceutical antiarrhythmic therapy is not always effective, therefore many symptomatic patients undergo catheter ablation therapy. In patients with atrioventricular septal defects (AVSD), ablation therapy itself has mixed success; arrhythmogenic recurrences are common, and because of the anatomical displacement of the atrioventricular node, 3-degree heart block post-ablation is a real concern. In order to develop optimal and safe ablation strategies, the field of congenital cardiac electrophysiology must combine knowledge from clinical electrophysiology with a thorough understanding of the anatomical substrates for arrhythmias. Using image-based analysis and multi-cellular mathematical modeling of electrical activation, we show how the anatomical alterations characteristic of an AVSD serve as arrhythmogenic substrates. Using ex-vivo contrast enhanced micro-computed tomography we imaged post-mortem the heart of a 5 month old male with AVSD at an isometric spatial resolution of 38 µm. Morphological analysis revealed the 3D disposition of the cardiac conduction system for the first time in an intact heart with this human congenital malformation. We observed displacement of the compact atrioventricular node inferiorly to the ostium of the coronary sinus. Myocyte orientation analysis revealed that the normal arrangement of the major atrial muscle bundles was preserved but was modified in the septal region. Models of electrical activation suggest the disposition of the myocytes within the atrial muscle bundles associated with the "fast pathway," together with the displaced atrioventricular node, serve as potential substrates for re-entry and possibly atrial fibrillation. This study used archived human hearts, showing them to be a valuable resource for the mathematical modeling community, and opening new possibilities for the investigations of arrhythmogenesis and ablation strategies in the congenitally malformed heart.

The end of the unique myocardial band: Part I. Anatomical considerations.

The concept of the 'unique myocardial band', which proposes that the ventricular myocardial cone is arranged like skeletal muscle, provides an attractive framework for understanding haemodynamics. The original idea was developed by Francisco Torrent-Guasp. Using boiled hearts and blunt dissection, Torrent-Guasp created a single band of ventricular myocardium extending from the pulmonary trunk to the aortic root, with the band thus constructed encircling both ventricular cavities. Cooked hearts can, however, be dissected in many ways. In this review, we show that the band does not exist as an anatomical entity with defined borders. On the contrary, the ventricular cardiomyocytes are aggregated end to end and by their branching produce an intricate meshwork. Across the thickness of the left ventricular wall, the chains of cardiomyocytes exhibit a gradually changing helical angle, with a circumferential zone formed in the middle. There is no abrupt change in helical angle, as could be expected if the wall was constructed of opposing limbs of a single wrapped band, nor does the long axis of the cardiomyocytes consistently match with the long axis of the unique myocardial band. There are, furthermore, no connective tissue structures that could be considered to demarcate its purported boundaries. The unique myocardial band should be consistent with evolution, and although the ventricular wall of fish and reptiles has one or several distinct layers, a single band is not found. In 1965, Lev and Simpkins cautioned that the ventricular muscle mass of a cooked heart can be dissected almost at the whim of the anatomist. We suggest that the unique myocardial band should have ended there.

The end of the unique myocardial band: Part II. Clinical and functional considerations.

Two of the leading concepts of mural ventricular architecture are the unique myocardial band and the myocardial mesh model. We have described, in an accompanying article published in this journal, how the anatomical, histological and high-resolution computed tomographic studies strongly favour the latter concept. We now extend the argument to describe the linkage between mural architecture and ventricular function in both health and disease. We show that clinical imaging by echocardiography and magnetic resonance imaging, and electrophysiological studies, all support the myocardial mesh model. We also provide evidence that the unique myocardial band model is not compatible with much of scientific research.

High resolution 3-Dimensional imaging of the human cardiac conduction system from microanatomy to mathematical modeling.

Cardiac arrhythmias and conduction disturbances are accompanied by structural remodelling of the specialised cardiomyocytes known collectively as the cardiac conduction system. Here, using contrast enhanced micro-computed tomography, we present, in attitudinally appropriate fashion, the first 3-dimensional representations of the cardiac conduction system within the intact human heart. We show that cardiomyocyte orientation can be extracted from these datasets at spatial resolutions approaching the single cell. These data show that commonly accepted anatomical representations are oversimplified. We have incorporated the high-resolution anatomical data into mathematical simulations of cardiac electrical depolarisation. The data presented should have multidisciplinary impact. Since the rate of depolarisation is dictated by cardiac microstructure, and the precise orientation of the cardiomyocytes, our data should improve the fidelity of mathematical models. By showing the precise 3-dimensional relationships between the cardiac conduction system and surrounding structures, we provide new insights relevant to valvar replacement surgery and ablation therapies. We also offer a practical method for investigation of remodelling in disease, and thus, virtual pathology and archiving. Such data presented as 3D images or 3D printed models, will inform discussions between medical teams and their patients, and aid the education of medical and surgical trainees.

Transcriptomic and epigenetic regulation of disuse atrophy and the return to activity in skeletal muscle.

Physical inactivity and disuse are major contributors to age-related muscle loss. Denervation of skeletal muscle has been previously used as a model with which to investigate muscle atrophy following disuse. Although gene regulatory networks that control skeletal muscle atrophy after denervation have been established, the transcriptome in response to the recovery of muscle after disuse and the associated epigenetic mechanisms that may function to modulate gene expression during skeletal muscle atrophy or recovery have yet to be investigated. We report that silencing the tibialis anterior muscle in rats with tetrodotoxin (TTX)-administered to the common peroneal nerve-resulted in reductions in muscle mass of 7, 29, and 51% with corresponding reductions in muscle fiber cross-sectional area of 18, 42, and 69% after 3, 7, and 14 d of TTX, respectively. Of importance, 7 d of recovery, during which rodents resumed habitual physical activity, restored muscle mass from a reduction of 51% after 14 d TTX to a reduction of only 24% compared with sham control. Returning muscle mass to levels observed at 7 d TTX administration (29% reduction). Transcriptome-wide analysis demonstrated that 3714 genes were differentially expressed across all conditions at a significance of P ≤ 0.001 after disuse-induced atrophy. Of interest, after 7 d of recovery, the expression of genes that were most changed during TTX had returned to that of the sham control. The 20 most differentially expressed genes after microarray analysis were identified across all conditions and were cross-referenced with the most frequently occurring differentially expressed genes between conditions. This gene subset included myogenin (MyoG), Hdac4, Ampd3, Trim63 (MuRF1), and acetylcholine receptor subunit α1 (Chrna1). Transcript expression of these genes and Fboxo32 (MAFbx), because of its previously identified role in disuse atrophy together with Trim63 (MuRF1), were confirmed by real-time quantitative RT-PCR, and DNA methylation of their promoter regions was analyzed by PCR and pyrosequencing. MyoG, Trim63 (MuRF1), Fbxo32 (MAFbx), and Chrna1 demonstrated significantly decreased DNA methylation at key time points after disuse-induced atrophy that corresponded with significantly increased gene expression. Of importance, after TTX cessation and 7 d of recovery, there was a marked increase in the DNA methylation profiles of Trim63 (MuRF1) and Chrna1 back to control levels. This also corresponded with the return of gene expression in the recovery group back to baseline expression observed in sham-surgery controls. To our knowledge, this is the first study to demonstrate that skeletal muscle atrophy in response to disuse is accompanied by dynamic epigenetic modifications that are associated with alterations in gene expression, and that these epigenetic modifications and gene expression profiles are reversible after skeletal muscle returns to normal activity.-Fisher, A. G., Seaborne, R. A., Hughes, T. M., Gutteridge, A., Stewart, C., Coulson, J. M., Sharples, A. P., Jarvis, J. C. Transcriptomic and epigenetic regulation of disuse atrophy and the return to activity in skeletal muscle.

Recent advances in management of alkaptonuria (invited review; best practice article).

Alkaptonuria (AKU) is an autosomal recessive condition arising as a result of a genetic deficiency of the enzyme homogentisate 1,2 dioxygenase and characterised by accumulation of homogentisic acid (HGA). Oxidative conversion of HGA leads to production of a melanin-like polymer in a process termed ochronosis. The binding of ochronotic pigment to the connective tissues of the body leads to multisystem disorder dominated by premature severe spondylo-arthropathy. Other systemic features include stones (renal, prostatic, salivary, gall bladder), renal damage/failure, osteopenia/fractures, ruptures of tendons/muscle/ligaments, respiratory compromise, hearing loss and aortic valve disease. Detection of these features requires systematic investigation. Treatment in AKU patients is palliative and unsatisfactory. Ascorbic acid, low protein diet and physiotherapy have been tried but do not alter the underlying metabolic defect. Regular surveillance to detect and treat complications early is important. Palliative pain management is a crucial issue in AKU. Timely spinal surgery and arthroplasty are the major treatment approaches at present. A potential disease modifying drug, nitisinone, inhibits 4-hydroxy-phenyl-pyruvate-dioxygenase and decreases formation of HGA and could prevent or slow the progression of disease in AKU. If nitisinone therapy is able to complement the biochemical 'cure' with improved outcomes, it will completely alter the way we approach the management of this disease. Greater efforts to improve recognition and registration of the disease will be worthwhile. Improved laboratory diagnostics to monitor the tyrosine metabolic pathway that includes plasma metabolites including tyrosine to monitor efficacy, toxicity and safety postnitisinone will also be required.

Development of an in vitro model to investigate joint ochronosis in alkaptonuria.

OBJECTIVES: Alkaptonuria (AKU) is a genetic disorder caused by lack of the enzyme responsible for breaking down homogentisic acid (HGA), an intermediate in tyrosine metabolism. HGA is deposited as a polymer, termed ochronotic pigment, in collagenous tissues. Pigmentation is progressive over many years, leading to CTDs including severe arthropathies. To investigate the mechanism of pigmentation and to determine how it leads to arthropathy, we aimed to develop an in vitro model of ochronosis. METHODS: Osteosarcoma cell lines MG63, SaOS-2 and TE85 were cultured in medium containing HGA from 0.1 µM to 1 mM. Cultures were examined by light microscopy and transmission electron microscopy, and Schmorl's stain was used to detect pigment deposits in vitro, following the observation that this stain identifies ochronotic pigment in AKU tissues. The effects of HGA on cell growth and collagen synthesis were also determined. RESULTS: There was a dose-related deposition of pigment in cells and associated matrix from 33 µM to 0.33 mM HGA. Pigmentation in vitro was much more rapid than in vivo, indicating that protective mechanisms exist in tissues in situ. Pigment deposition was dependent on the presence of cells and was observed at HGA concentrations that were not toxic. There was an inhibition of cell growth and a stimulation of type I collagen synthesis up to 0.33 mM HGA, but severe cell toxicity at 1 mM HGA. CONCLUSION: We have developed an in vitro model of ochronosis that should contribute to understanding joint destruction in AKU and to the aetiology of OA.

Assisted Fontan procedure: animal and in vitro models and computational fluid dynamics study.

Fontan connection with intermittent compression by wrapped latissimus dorsi (LD) was tested in vivo, in vitro and by means of computational fluid dynamics (CFD). Experimental study: LD was conditioned in four pigs for three weeks before Fontan connection by valved conduit wrapped with LD. Mock circuit: Inflatable cuff wrapped around valved conduit provided intermittent external compression, with pressure and flow measured at driving pressure of 8 or 16 mmHg. CFD study: A circuit was tested for possible increase above basal flow (4 l/min) with intermittent external compression. Experimental study: Intermittent conduit compression by LD provided mean 7% decrease of baseline PA pressure, with simultaneous flow increase of 2%. Mock circuit: By raising the driving pressure from 8 to 16 mmHg, the flow increased with baseline PVR (56%) and with elevated PVR (80%). Total pulmonary flow was reduced during intermittent external compression with both baseline and elevated PVR. CFD study: Compression with 13.0 mmHg provided 4.9% increase of total pulmonary flow with substantial increase of the peak flow (92%). In vivo and in vitro, the increased flow produced by compressing a conduit was confounded by the inevitable intermittent flow restriction. Mathematical model using lower pressure for intermittent external compression showed potential for increase in pulmonary flow.

Real-time polymerase chain reaction to follow the response of muscle to training.

The adaptive response of muscle to changes in activity or loading can take many weeks. Changes in the levels of RNA within a muscle fiber can give an early indication of the nature of the response of that fiber to changes in activity or loading. We have designed a new primer set for quantitative polymerase chain reaction (PCR) that will allow us to follow these early transcriptional changes in rat muscle, and have shown that analysis can be performed by standard techniques on as little as 5 mg of muscle, an amount that can be obtained by needle biopsy.

Skeletal muscle ventricles with a single-limb conduit: the importance of hemodynamic design.

BACKGROUND: Skeletal muscle ventricles (SMVs) connected to the descending thoracic aorta have the potential for providing long-term diastolic augmentation. A successful existing design employs a bifurcated conduit, but aortic constriction between the limbs of the conduit is required to ensure obligatory flow-through. Here we evaluate an alternative approach in which connection to the aorta is made by a single-limb conduit. METHODS: In two groups of dogs SMVs were constructed from the left latissimus dorsi muscle and connected to the circulation via a single-limb conduit of length 110-120 mm (Group 1, n = 5) or 70 mm (Group 2, n = 5). The animals were followed over 10 weeks. RESULTS: Although all animals showed significant augmentation of diastolic aortic pressure at the outset, substantial thrombus developed in the SMVs of both groups. The results were analyzed by reference to design criteria for a single-limb conduit SMV, developed from empirical, in-vitro flow studies and formulated mathematically. CONCLUSION: The SMVs constructed in this experiment appeared to meet the criteria for adequate mixing of blood within the ventricle. They did not, however, achieve adequate exchange of blood with the circulation. Thrombosis was therefore attributable to excessive residence time of blood in the SMV and conduit. Both the experimental study and the mathematical analysis point to the need for SMVs of this configuration to be constructed closer to the aorta. Preliminary results are reported for such an experiment in the pig, in which the SMV was thrombus-free when terminated electively after 1 week.

Avoiding ischemia in latissimus dorsi muscle grafts: electrical prestimulation versus vascular delay.

BACKGROUND: Surgical mobilization of the latissimus dorsi muscle produces regional ischemic damage that may compromise its function in clinical applications such as cardiomyoplasty. We compared the effectiveness of two procedures designed to maintain blood flow throughout the mobilized muscle. METHODS: Adult pigs were assigned to two experimental groups: an electrically prestimulated group (n = 10) and a vascular delay group (n = 10). In the prestimulated group the left latissimus dorsi muscle was activated in situ at 2 Hz for 24 h/d. In the vascular delay group, the intercostal perforating arteries to the left latissimus dorsi muscle were divided. Two weeks later, hyperemic blood flow was measured by means of fluorescent microspheres immediately before and after mobilizing the latissimus dorsi muscle and again after recovery for a further 2 days. RESULTS: In the prestimulated group, blood flow was not significantly depressed in any region of the muscle immediately after mobilization, and blood flow increased significantly in proximal (p = 0.01), middle (p = 0.02), and distal (p = 0.007) regions following recovery. In muscles subjected to vascular delay the proximal and middle regions showed no significant changes in blood flow after mobilization or recovery, but flow in the distal region was 50% lower after mobilization (p = 0.003), and it remained significantly depressed even after recovery (p = 0.008). CONCLUSIONS: Prestimulation was significantly more effective than vascular delay in preserving distal blood flow. Because it is also less invasive and initiates metabolic transformation before mobilization, this technique should allow cardiac assistance to be introduced at an earlier postoperative stage without compromising the viability of the grafted muscle.

The hemodynamic function of intrathoracic skeletal muscle ventricles after recovery from surgery in pigs.

The shortage of donor organs for heart transplantation highlights the need for new approaches to end-stage heart failure. A promising experimental technique is the use of pumping chambers formed from the latissimus dorsi muscle. We formed such skeletal muscle ventricles (SMVs) and connected them to the descending thoracic aorta in a single surgical procedure in pigs. Activation of conditioned SMVs from the end of systole for 80% of diastole increased mean aortic diastolic blood pressure by 11.2 +/- 1.6% in 1 animal and by 15.8 +/- 0.3% in another. The left-ventricular stroke work in the postassisted beat was decreased by 8.7 +/- 5.8% and 10.1 +/- 2.2% and the overall stroke work by 7.4 +/- 1.2% and 9.4 +/- 0.8%. The key to forming and connecting the SMV in a single procedure was the use of a composite homograft lining. In future clinical practice, this component could be replaced by a synthetic composite or by a tissue lining produced in vitro.