Agonist specific L-type Ca(2+)-current stimulation in ventricular myocytes by a novel steroid-like compound

In cardiac muscle the amplitude of Ca(2+) transients can be increased by enhancing Ca(2+) influx. Among the processes leading to increased Ca(2+) influx, agonists of the L-type Ca(2+)-channel can play an important role. Known pharmacological Ca(2+)-channel agonists act on different binding sites on the channel protein, which may lead not only to enhanced peak currents, but also to distinct changes in other biophysical characteristics of the current. In this study, membrane currents were recorded with the patch-clamp technique in the whole-cell configuration in guinea pig isolated ventricular myocytes in combination with confocal fluorescence Ca(2+) imaging techniques and a variety of pharmacological tools. Testing a new positive inotropic steroid-like compound, we found that it increased the L-type Ca(2+)-current by 2.5-fold by shifting the voltage-dependence of activation by 20.2 mV towards negative potentials. The dose-response relationship revealed two vastly different affinities (EC(50(high-affinity))=4.5+/-1.7 nM, EC(50(low-affinity))=8.0+/-1.1 microM) exhibiting differential pharmacological interactions with three classes of Ca(2+)-current antagonists, suggesting more than one binding site on the channel protein. Therefore, we identified and characterized a novel positive inotropic compound (F90927) as a member of a new class of Ca(2+)-channel agonists exhibiting unique features, which set it apart from other presently known L-type Ca(2+)-channel agonists.

The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

Treatment of osteoporosis in a mouse model of Duchenne muscular dystrophy using black bear parathyroid hormone

Duchenne muscular dystrophy (DMD) is a progressive disease affecting skeletal and cardiac muscle, as well as bone. Long term disuse and glucocorticoid treatments cause progressive osteoporosis in DMD patients, leading to an increase in fracture incidence. Treatments for osteoporosis in these patients have not been widely explored. Parathyroid hormone (PTH), an anabolic treatment for post-menopausal osteoporosis, could benefit DMD patients by improving skeletal properties and reducing fracture risk. Other PTH analogues are not currently FDA approved to treat osteoporosis, but may have improved osteogenic effects compared to the human analogue. Black bear PTH is especially promising as an osteoporosis treatment for the DMD population. Black bears are unique models of bone maintenance during disuse, since during six months of inactivity (hibernation), they maintain skeletal properties, unlike other hibernators. Additionally, black bear PTH has been correlated to bone formation markers during hibernation, indicating it may be, at least in part, the mechanism by which bears maintain bone during disuse. Employing black bear PTH as a treatment for osteoporosis in DMD patients could greatly improve quality of life for these individuals, and reduce the pain and expense associated with frequent fractures.

Novel cell-free strategy for therapeutic angiogenesis: in vitro generated conditioned medium can replace progenitor cell transplantation

BACKGROUND: Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy. METHODOLOGY/PRINCIPAL FINDINGS: EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2+/-2.9% and 83.7+/-3.0% vs. 53.5+/-2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62+/-0.03 and 1.68+/-0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6+/-0.3 and 8.1+/-0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7+/-44.1 vs. 340.0+/-29.1 CD34(+)/CD45(-) cells/1x10(5) mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9+/-0.7 vs. 2.6+/-0.4 CD34(+) cells/HPF, P<0.001) 3 days after the last injection. CONCLUSIONS/SIGNIFICANCE: Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.

Relationships between structural and functional measures of nutritional status in a normally nourished population

Both anthropometric and functional measurements have been used in nutritional assessment and monitoring. Hand dynamometry is a predictor of surgical outcome and peak expiratory flow rate has been used as an index of respiratory muscle function. This study aims to measure in normal subjects the relationship between anthropometric measurements, voluntary muscle strength by hand grip dynamometry and respiratory muscle function by peak expiratory flow rate.

A defective SERCA1 protein is responsible for congenital pseudomyotonia in Chianina cattle

Recently, a muscular disorder defined as "congenital pseudomyotonia" was described in Chianina cattle, one of the most important Italian cattle breeds for quality meat and leather. The clinical phenotype of this disease is characterized by an exercise-induced muscle contracture that prevents animals from performing muscular activities. On the basis of clinical symptoms, Chianina pseudomyotonia appeared related to human Brody's disease, a rare inherited disorder of skeletal muscle function that results from a sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) deficiency caused by a defect in the ATP2A1 gene that encodes SERCA1. SERCA1 is involved in transporting calcium from the cytosol to the lumen of the sarcoplasmic reticulum. Recently, we identified the genetic defect underlying Chianina cattle pseudomyotonia. A missense mutation in exon 6 of the ATP2A1 gene, leading to an R164H substitution in the SERCA1 protein, was found. In this study, we provide biochemical evidence for a selective deficiency in SERCA1 protein levels in sarcoplasmic reticulum membranes from affected muscles, although mRNA levels are unaffected. The reduction of SERCA1 levels accounts for the reduced Ca(2+)-ATPase activity without any significant change in Ca(2+)-dependency. The loss of SERCA1 is not compensated for by the expression of the SERCA2 isoform. We believe that Chianina cattle pseudomyotonia might, therefore, be the true counterpart of human Brody's disease, and that bovine species might be used as a suitable animal model.

Structure and superorganization of acetylcholine receptor-rapsyn complexes

The scaffolding protein at the neuromuscular junction, rapsyn, enables clustering of nicotinic acetylcholine receptors in high concentration and is critical for muscle function. Patients with insufficient receptor clustering suffer from muscle weakness. However, the detailed organization of the receptor-rapsyn network is poorly understood: it is unclear whether rapsyn first forms a wide meshwork to which receptors can subsequently dock or whether it only forms short bridges linking receptors together to make a large cluster. Furthermore, the number of rapsyn-binding sites per receptor (a heteropentamer) has been controversial. Here, we show by cryoelectron tomography and subtomogram averaging of Torpedo postsynaptic membrane that receptors are connected by up to three rapsyn bridges, the minimum number required to form a 2D network. Half of the receptors belong to rapsyn-connected groups comprising between two and fourteen receptors. Our results provide a structural basis for explaining the stability and low diffusion of receptors within clusters.

Contrast-enhanced ultrasound for myocardial perfusion imaging.

Ultrasound contrast agents are gas-filled microbubbles that enhance visualization of cardiac structures, function and blood flow during contrast-enhanced ultrasound (CEUS). An interesting cardiovascular application of CEUS is myocardial contrast echocardiography, which allows real-time myocardial perfusion imaging. The intraoperative use of this technically challenging imaging method is limited at present, although several studies have examined its clinical utility during cardiac surgery in the past. In the present review we provide general information on the basic principles of CEUS and discuss the methodology and technical aspects of myocardial perfusion imaging.

In vivo electroporation-mediated gene delivery to the beating heart

Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. Here, we describe two protocols involving in vivo electroporation for gene transfer to the beating heart.

A novel animal model for external anal sphincter insufficiency

Abstract PURPOSE: Reliable animal models are essential to evaluate future therapeutic options like cell-based therapies for external anal sphincter insufficiency. The goal of our study was to describe the most reliable model for external sphincter muscle insufficiency by comparing three different methods to create sphincter muscle damage. METHODS: In an experimental animal study, female Lewis rats (200-250 g) were randomly assigned to three treatment groups (n = 5, each group). The external sphincter muscle was weakened in the left dorsal quadrant by microsurgical excision, cryosurgery, or electrocoagulation by diathermy. Functional evaluation included in vivo measurements of resting pressure, spontaneous muscle contraction, and contraction in response to electrical stimulation of the afferent nerve at baseline and at 2, 4, and 6 weeks after sphincter injury. Masson's trichrome staining and immunofluorescence for skeletal muscle markers was performed for morphological analysis. RESULTS: Peak contraction after electrical stimulation was significantly decreased after sphincter injury in all groups. Contraction forces recovered partially after cryosurgery and electrocoagulation but not after microsurgical excision. Morphological analysis revealed an incomplete destruction of the external sphincter muscle in the cryosurgery and electrocoagulation groups compared to the microsurgery group. CONCLUSIONS: For the first time, three different models of external sphincter muscle insufficiency were directly compared. The animal model using microsurgical sphincter destruction offers the highest level of consistency regarding tissue damage and sphincter insufficiency, and therefore represents the most reliable model to evaluate future therapeutic options. In addition, this study represents a novel model to specifically test the external sphincter muscle function.

Nerve Transfers for Persistent Traumatic Peroneal Nerve Palsy: The Inselspital Bern Experience

BACKGROUND Patients in whom conventional peroneal nerve repair surgery failed to reconstitute useful foot lift need to be evaluated for their suitability to undergo a concomitant tendon transfer procedure or nerve transfers. OBJECTIVE To report our first clinical experience with nerve transfers for persistent traumatic peroneal nerve palsy. METHODS Between 2007 and 2013, 8 patients were operated on for foot drop after unsuccessful nerve surgery. Six patients without fatty degeneration of the anterior tibial muscle and proximal lesion of the peroneal nerve were oriented for tibial to peroneal nerve transfer. In the other 2 cases where the anterior and lateral compartments were destructed, the anterior tibial muscle function was reconstructed with a neurotized lateral gastrocnemius transfer. For each patient, we graded postoperative results using the Bureau of Meteorology Research Centre scheme and the Ninkovic assessment scale. RESULTS Of the 6 patients who underwent nerve transfer of the anterior tibial muscle, 2 patients had excellent results, 1 patient had good results, 1 patient had fair results, and 2 patients had poor results. Of the 2 patients that underwent neurotized lateral gastrocnemius transfer, 1 patient achieved excellent results after tenolysis, whereas 1 patient achieved poor results. After the nerve transfer, 5 patients did not wear an ankle-foot orthosis. Four patients did not limp. Four patients were able to walk barefoot, navigate stairs, and participate in activities. CONCLUSION Early clinical results after tibial to peroneal nerve transfer and neurotized lateral gastrocnemius transfer appear mixed. The results of nerve transfer seem, on the whole, less reliable than the literature reports on tendon transfer. ABBREVIATIONS EMG, electromyographyNAP, nerve action potential.

Identification of a missense mutation in the bovine ATP2A1 gene in congenital pseudomyotonia of Chianina cattle: an animal model of human Brody disease

Congenital pseudomyotonia in Chianina cattle is a muscle function disorder very similar to that of Brody disease in humans. Mutations in the human ATP2A1 gene, encoding SERCA1, cause Brody myopathy. The analysis of the collected Chianina pedigree data suggested monogenic autosomal recessive inheritance and revealed that all 17 affected individuals traced back to a single founder. A deficiency of SERCA1 function in skeletal muscle of pseudomyotonia affected Chianina cattle was observed as SERCA1 activity in affected animals was decreased by about 70%. Linkage analysis showed that the mutation was located in the ATP2A1 gene region on BTA25 and subsequent mutation analysis of the ATP2A1 exons revealed a perfectly associated missense mutation in exon 6 (c.491G>A) leading to a p.Arg164His substitution. Arg164 represents a functionally important and strongly conserved residue of SERCA1. This study provides a suitable large animal model for human Brody disease.

MODULATION OF HOST INTESTINAL MYOELECTRIC ACTIVITY BY LOCAL ANAPHYLAXIS: A COMPONENT OF PROTECTIVE IMMUNITY TO AN ENTERIC PARASITE (TRICHINELLA SPIRALIS, EIMERIA NIESCHULZI, BOWEL MOTILITY)

The hypothesis tested was that rapid rejection of Trichinella spiralis infective larvae from immunized rats following a challenge infection is associated with a local anaphylactic reaction, and this response should be reflected in altered small intestinal motility. The objective was to determine if altered gut smooth muscle function accompanies worm rejection based on the assumption that anaphylaxis in vivo could be detected by changes in intestinal smooth muscle contractile activity (ie. an equivalent of the Schultz-Dale reaction or in vitro anaphylaxis). The aims were to (1) characterize motility changes by monitoring intestinal myoelectric activity in conscious rats during the enteric phase of T. spiralis infection in immunized hosts, (2) detect the onset and magnitude of myoelectric changes caused by challenge infection in immunized rats, (3) determine the parasite stimulus causing changes, and (4) determine the specificity of host response to stimulation. Electrical slow wave frequency, spiking activity, normal interdigestive migrating myoelectric complexes and abnormal migrating action potential complexes were measured. Changes in myoelectric parameters induced by larvae inoculated into the duodenum of immune hosts differed from those associated with primary infection with respect to time of onset, magnitude and duration. Myoelectric changes elicited by live larvae could not be reproduced by inoculation of hosts with dead larvae, larval excretory-secretory products, or by challenge with a heterologous parasite, Eimeria nieschulzi. These results indicate that (1) local anaphylaxis is a component of the initial response to T. spiralis in immune hosts, since the rapid onset of altered smooth muscle function parallels in time the expression of rapid rejection of infective larvae, and (2) an active mucosal penetration attempt by the worm is necessary to elicit this host response. These findings provide evidence that worm rejection is a consequence of, or sequel to, an immediate hypersensitivity reaction elicited when parasites attempt to invade the gut mucosa of immunized hosts. ^

Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type1a receptor (AT1aR)-dependent mechanism. To understand how the level of AT1aRs is altered in this pathological state, we studied the expression of an injected AT1aR promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the α-skeletal actin promoter 1.7-fold whereas the α-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT1aR promoter by 1.6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT1aR promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT1aR promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.