High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by mutations in the dystrophin gene. Loss of dystrophin prevents the formation of a critical connection between the muscle cell membrane and the extracellular matrix. Overexpression of sarcospan (SSPN) in the mouse model of DMD restores the membrane connection and reduces disease severity, making SSPN a promising therapeutic target for pharmacological upregulation. METHODS: Using a previously described cell-based promoter reporter assay of SSPN gene expression (hSSPN-EGFP), we conducted high-throughput screening on libraries of over 200,000 curated small molecules to identify SSPN modulators. The hits were validated in both hSSPN-EGFP and hSSPN-luciferase reporter cells. Hit selection was conducted on dystrophin-deficient mouse and human myotubes with assessments of (1) SSPN gene expression using quantitative PCR and (2) SSPN protein expression using immunoblotting and an ELISA. A membrane stability assay using osmotic shock was used to validate the functional effects of treatment followed by cell surface biotinylation to label cell surface proteins. Dystrophin-deficient mdx mice were treated with compound, and muscle was subjected to quantitative PCR to assess SSPN gene expression. RESULTS: We identified and validated lead compounds that increased SSPN gene and protein expression in dystrophin-deficient mouse and human muscle cells. The lead compound OT-9 increased cell membrane localization of compensatory laminin-binding adhesion complexes and improved membrane stability in DMD myotubes. We demonstrated that the membrane stabilizing benefit is dependent on SSPN. Intramuscular injection of OT-9 in the mouse model of DMD increased SSPN gene expression. CONCLUSIONS: This study identifies a pharmacological approach to treat DMD and sets the path for the development of SSPN-based therapies.

Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion. METHODS: Standard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting. RESULTS: We investigated the gene expression profiles of SSPN and its associated proteins during myoblast differentiation into myotubes, revealing an increase in expression after 3 days of differentiation. We created C2C12 muscle cells expressing an EGFP reporter for SSPN promoter activity and observed a comparable increase in reporter levels during differentiation. Assay conditions for high-throughput screening were optimized for a 384-well microplate format and a high-content imager for the visualization of reporter levels. We conducted a screen of 3200 compounds and identified seven hits, which include an overrepresentation of L-type calcium channel antagonists, suggesting that SSPN gene activity is sensitive to calcium. Further validation of a select hit revealed that the calcium channel inhibitor felodipine increased SSPN transcript and protein levels in both wild-type and dystrophin-deficient myotubes, without increasing differentiation. CONCLUSIONS: We developed a stable muscle cell line containing the promoter region of the human SSPN protein fused to a fluorescent reporter. Using the reporter cells, we created and validated a scalable, cell-based assay that is able to identify compounds that increase SSPN promoter reporter, transcript, and protein levels in wild-type and dystrophin-deficient muscle cells.

Bradycardia in a Man With Hypertension.

A man without cardiac symptoms was found to have a slow irregular pulse, and an electrocardiogram revealed sinus bradycardia with escape-capture bigeminy. He was taking verapamil, clonidine, and hydralazine for hypertension. The verapamil was discontinued; he returned to normal sinus rhythm and was discharged on the second hospital day.

Stabilization of the cardiac sarcolemma by sarcospan rescues DMD-associated cardiomyopathy.

In the current preclinical study, we demonstrate the therapeutic potential of sarcospan (SSPN) overexpression to alleviate cardiomyopathy associated with Duchenne muscular dystrophy (DMD) utilizing dystrophin-deficient mdx mice with utrophin haploinsufficiency that more accurately represent the severe disease course of human DMD. SSPN interacts with dystrophin, the DMD disease gene product, and its autosomal paralog utrophin, which is upregulated in DMD as a partial compensatory mechanism. SSPN transgenic mice have enhanced abundance of fully glycosylated α-dystroglycan, which may further protect dystrophin-deficient cardiac membranes. Baseline echocardiography reveals SSPN improves systolic function and hypertrophic indices in mdx and mdx:utr-heterozygous mice. Assessment of SSPN transgenic mdx mice by hemodynamic pressure-volume methods highlights enhanced systolic performance compared to mdx controls. SSPN restores cardiac sarcolemma stability, the primary defect in DMD disease, reduces fibrotic response and improves contractile function. We demonstrate that SSPN ameliorates more advanced cardiac disease in the context of diminished sarcolemma expression of utrophin and ß1D integrin that mitigate disease severity and partially restores responsiveness to ß-adrenergic stimulation. Overall, our current and previous findings suggest SSPN overexpression in DMD mouse models positively impacts skeletal, pulmonary and cardiac performance by addressing the stability of proteins at the sarcolemma that protect the heart from injury, supporting SSPN and membrane stabilization as a therapeutic target for DMD.

Characterization of the hydroxycarboxylic acid receptor 2 in cats.

The hydroxycarboxylic acid receptor 2 (HCA2) belongs to a family of nutrient-sensing receptors that bind ß-hydroxybutyrate, an alternative fuel source produced during a negative energy balance. The HCA2 receptor has not been identified or characterized in cats. Therefore, the following were the objectives of this study: (1) identify the feline HCA2 receptor protein sequence and compare against known human and rodent sequences, (2) determine tissue distribution and relative expression in lean, healthy cats, and (3) demonstrate in vitro functionality in feline adipose tissue. Tissues (n = 6) and primary adipocytes (n = 4) were collected from lean, healthy, female cats. The published genomic sequence for cats was used to design primers for polymerase chain reaction isolation of HCA2. Relative tissue distribution was evaluated using reverse transcriptase-polymerase chain reaction with RNA isolated from 9 different tissues (spleen, pancreas, lymph node, jejunum, kidney, liver, heart, and subcutaneous and abdominal adipose tissue). Receptor function was evaluated in primary feline adipocyte culture, and changes were compared with basal lipolysis. The in silico predicted feline HCA2 protein sequence exhibited 83.1% and 86.5% amino acid similarity to human and mouse sequences, respectively. The feline HCA2 receptor is predominantly expressed in adipose tissue and spleen. Exposure of feline adipocytes to niacin, a pharmacologic ligand of HCA2, inhibited lipolysis to a similar degree as insulin, a potent lipolytic inhibitor. In conclusion, the feline HCA2 receptor is similar to human and murine receptors in sequence, distribution, and functionality. By gaining a better understanding of the HCA2 receptor in cats, we will be able to better manage feline patients.

Future of GPR109A agonists in the treatment of dyslipidaemia.

Abnormal blood lipids are the major modifiable risk factor underlying the development of cardiovascular disease. Niacin has a profound ability to reduce low-density lipoprotein-C, very low-density lipoprotein-C and triglycerides and is the most effective pharmacological agent to increase high-density lipoprotein-C. Recently, the receptor for niacin, GPR109A, was discovered. GPR109A in the adipocyte mediates a niacin-induced inhibition of lipolysis, which could play a crucial part in its role as a lipid-modifying drug. GPR109A in epidermal Langerhans cells mediates flushing, an unwanted side effect of niacin therapy. For the past decade, the functions of GPR109A have been studied and full or partial agonists have been developed in an attempt to achieve the beneficial effects of niacin while avoiding the unwanted flushing side effect. This review presents what is known to date about GPR109A biology and function and the future of GPR109A as a pharmacological target.

Changes in mitotic rate and GFAP expression in the primary olfactory axis of streptozotocin-induced diabetic rats.

Many diabetic individuals develop anosmia but the mechanism(s) causing the dysfunction in the olfactory system is (are) unknown. Glial fibrillary acidic protein expression is reduced in diabetic retinopathy and is also reduced, with unknown consequences, in other brain regions of diabetic rats. We used immunohistochemistry and immunoblotting from untreated control and streptozotocin-induced type 1 (insulin dependent) diabetic rats to investigate main olfactory epithelial mitotic rate and glial fibrillary acidic protein expression in the lamina propria of the sensory epithelium and in the olfactory bulb. Numbers of bromodeoxyuridine-positive cells were significantly lower in the diabetic sensory epithelium compared to non-diabetic controls. Immunohistochemical observations suggested a qualitative difference in glial fibrillary acidic protein expression in both regions examined especially in the olfactory bulb external plexiform layer and the lamina propria. Immunoblot analysis confirmed that the diabetic olfactory bulb and lamina propria expressed less glial fibrillary acidic protein compared to the non-diabetic control group. The lower expression levels in the olfactory bulb external plexiform layer suggested by immunohistochemistry do not reflect a change in the number of astrocytes since the numbers of S100B(+) cells were not different between the two groups.

Limits of detection of regional differences in vasodilated flow in viable myocardium by first-pass magnetic resonance perfusion imaging.

BACKGROUND: Perfusion imaging techniques intended to identify regional limitations in coronary flow reserve in viable myocardium need to identify 2-fold differences in regional flow during coronary vasodilation consistently. This study evaluated the suitability of current first-pass magnetic resonance approaches for evaluating such differences, which are 1 to 2 orders of magnitude less than in myocardial infarction. METHODS AND RESULTS: Graded regional differences in vasodilated flow were produced in chronically instrumented dogs with either left circumflex (LCx) infusion of adenosine or partial LCx occlusion during global coronary vasodilation. First-pass myocardial signal intensity-time curves were obtained after right atrial injection of gadoteridol (0.025 mmol/kg) with an MRI inversion recovery true-FISP sequence. The area under the initial portion of the LCx curve was compared with that of a curve from a remote area of the ventricle. Relative LCx and remote flows were assessed simultaneously with microspheres. The ratio of LCx and remote MRI curve areas and the ratio of LCx and remote microsphere concentrations were highly correlated and linearly related over a 5-fold range of flow differences (y=0.96 x+/-0.07, P<0.0001, r(2)=0.87). The 95% confidence limits for individual MRI measurements were +/-35%. Regional differences of >/=2-fold were consistently apparent in unprocessed MR images. CONCLUSIONS: Clinically relevant regional reductions in vasodilated flow in viable myocardium can be detected with 95% confidence over the range of 1 to 5 times resting flow. This suggests that MRI can identify and quantify limitations in perfusion reserve that are expected to be produced by stenoses of >/=70%.

Transmural extent of acute myocardial infarction predicts long-term improvement in contractile function.

BACKGROUND: Previous animal studies have demonstrated that the transmural extent of acute myocardial infarction defined by contrast-enhanced MRI (ceMRI) relates to early restoration of flow and future improvements in contractile function. We tested the hypothesis that ceMRI would have similar predictive value in humans. METHODS AND RESULTS: Twenty-four patients who presented with their first myocardial infarction and were successfully revascularized underwent cine and ceMRI of their heart within 7 days (scan 1) of the peak MB band of creatine kinase. Cine MRI was repeated 8 to 12 weeks later (scan 2). The transmural extent of infarction on scan 1 and wall thickening on both scans were determined using a 72-segment model. A total of 524 of 1571 segments (33%) were dysfunctional on scan 1. Improvement in segmental contractile function on scan 2 was inversely related to the transmural extent of infarction on scan 1 (P=0.001). Improvement in global contractile function, as assessed by ejection fraction and mean wall thickening score, was not predicted by peak creatine kinase-MB (P=0.66) or by total infarct size, as defined by MRI (P=0.70). The best predictor of global improvement was the extent of dysfunctional myocardium that was not infarcted or had infarction comprising <25% of left ventricular wall thickness (P<0.005 for ejection fraction, P<0.001 for mean wall thickening score). CONCLUSION: In patients with acute myocardial infarction, the transmural extent of infarction defined by ceMRI predicts improvement in contractile function.

Theory of high-speed MR imaging of the human heart with the selective line acquisition mode.

Selective line acquisition mode (SLAM) reduces magnetic resonance imaging time by a factor n relative to conventional techniques. Seventeen patients with cardiac disease and three volunteers were examined with SLAM and two-frame interpolation (2FI). SLAM images were sharper than 2FI images and showed well-defined endocardial borders. SLAM is best suited for fast imaging of moving objects, such as the heart, confined to 1/n of the field of view.

Visualization of discrete microinfarction after percutaneous coronary intervention associated with mild creatine kinase-MB elevation.

BACKGROUND: Mild elevations in creatine kinase-MB (CK-MB) are common after successful percutaneous coronary interventions and are associated with future adverse cardiac events. The mechanism for CK-MB release remains unclear. A new contrast-enhanced MRI technique allows direct visualization of myonecrosis. METHODS AND RESULTS: Fourteen patients without prior infarction underwent cine and contrast-enhanced MRI after successful coronary stenting; 9 patients had procedure-related CK-MB elevation, and 5 did not (negative controls). The mean age of all patients was 61 years, 36% had diabetes, 43% had multivessel coronary artery disease, and all had a normal ejection fraction. Twelve patients (86%) received an intravenous glycoprotein IIb/IIIa inhibitor; none underwent atherectomy, and all had final TIMI 3 flow. Of the 9 patients with CK-MB elevation, 5 had a minor side branch occlusion during stenting, 2 had transient ECG changes, and none developed Q-waves. The median CK-MB was 21 ng/mL (range, 12 to 93 ng/mL), which is 2.3x the upper limit of normal. Contrast-enhanced MRI demonstrated discrete regions of hyperenhancement within the target vessel perfusion territory in all 9 patients. Only one developed a new wall motion abnormality. The median estimated mass of myonecrosis was 2.0 g (range, 0.7 to 12.2 g), or 1.5% of left ventricular mass (range, 0.4% to 6.0%). Hyperenhancement persisted in 5 of the 6 who underwent a repeat MRI at 3 to 12 months. No control patient had hyperenhancement. CONCLUSIONS: Contrast-enhanced MRI provides an anatomical correlate to biochemical evidence of procedure-related myocardial injury, despite the lack of ECG changes or wall motion abnormalities. Mild elevation of CK-MB after percutaneous coronary intervention is the result of discrete microinfarction.

Posttransplantation lymphoproliferative disorder initially seen as iris mass and uveitis.

Primary ocular posttransplantation lymphoproliferative disorder is rare. Epstein-Barr virus is implicated as the cause as a result of systemic immunosuppression after transplant surgery. We studied a patient who developed ocular posttransplantation lymphoproliferative disorder after orthotopic liver transplantation. Slitlamp and light microscopic photographs confirmed the diagnosis.

Visualisation of presence, location, and transmural extent of healed Q-wave and non-Q-wave myocardial infarction.

BACKGROUND: A technical advance in contrast-enhanced magnetic resonance imaging (MRI) has significantly improved image quality. We investigated whether healed myocardial infarction can be visualised as hyperenhanced regions with this new technique, and whether assessment of the transmural extent of infarction yields new physiological data. METHODS: 82 MRI examinations were carried out in three groups: patients with healed myocardial infarction; patients with non-ischaemic cardiomyopathy; and healthy volunteers. Patients with healed myocardial infarction were prospectively enrolled after enyzmatically proven necrosis and imaged 3 months (SD 1) or 14 months (7) later. The MRI procedure used a segmented inversion-recovery gradient-echo sequence after gadolinium administration. Findings were compared with those of coronary angiography, electrocardiography, cine MRI, and creatine kinase measurements. FINDINGS: 29 (91%) of 32 patients with infarcts imaged at 3 months (13 non-Q-wave) and all of 19 imaged at 14 months (eight non-Q-wave) showed hyperenhancement. In patients in whom the infarct-related-artery was identified by angiography, 24 of 25 imaged at 3 months and all of 14 imaged at 14 months had hyperenhancement in the appropriate territory. None of the 20 patients with non-ischaemic cardiomyopathy or the 11 healthy volunteers showed hyperenhancement. Irrespective of the presence or absence of Q waves, the majority of patients with hyperenhancement had only non-transmural involvement. Normal left-ventricular contraction was shown in seven patients examined at 3 months and three examined at 14 months, but in these cases hyperenhancement was limited to the subendocardium. INTERPRETATION: The presence, location, and transmural extent of healed Q-wave and non-Q-wave myocardial infarction can be accurately determined by contrast-enhanced MRI.

An improved MR imaging technique for the visualization of myocardial infarction.

PURPOSE: To design a segmented inversion-recovery turbo fast low-angle shot (turboFLASH) magnetic resonance (MR) imaging pulse sequence for the visualization of myocardial infarction, compare this technique with other MR imaging approaches in a canine model of ischemic injury, and evaluate its utility in patients with coronary artery disease. MATERIALS AND METHODS: Six dogs and 18 patients were examined. In dogs, infarction was produced and images were acquired by using 10 different pulse sequences. In patients, the segmented turboFLASH technique was used to acquire contrast material-enhanced images 19 days +/- 7 (SD) after myocardial infarction. RESULTS: Myocardial regions of increased signal intensity were observed in all animals and patients at imaging. With the postcontrast segmented turboFLASH sequence, the signal intensity of the infarcted myocardium was 1,080% +/- 214 higher than that of the normal myocardium in dogs-nearly twice that of the next best sequence tested and approximately 10-fold greater than that in previous reports. All 18 patients with myocardial infarction demonstrated high signal intensity at imaging. On average, the signal intensity of the high-signal-intensity regions in patients was 485% +/- 43 higher than that of the normal myocardium. CONCLUSION: The segmented inversion-recovery turboFLASH sequence produced the greatest differences in regional myocardial signal intensity in animals. Application of this technique in patients with infarction substantially improved differentiation between injured and normal regions.

Role of glucose and insulin in thiazolidinedione-induced alterations in hepatic gluconeogenesis.

Previous studies from our laboratory as well as from others have suggested that the thiazolidinediones have the capacity to act as insulinomimetic agents, especially in the liver. In order to further characterize this insulinomimetic action, we evaluated the effect of troglitazone, a representative thiazolidinedione, on lactate- and glucagon-stimulated gluconeogenesis, in the presence or absence of insulin (10 nM) in isolated rat hepatocytes. The antigluconeogenic effect of troglitazone under basal (lactate-stimulated) conditions was found to be due to an elevation in the fructose 2,6-bisphosphate content, which was, however, not mediated by an activation of 6-phosphofructo 2-kinase. Troglitazone (125 and 250 microM) in the absence of insulin, produced a dose-dependent reduction in glucagon-stimulated gluconeogenesis, thereby suggesting an insulinomimetic effect. In addition, troglitazone (125 and 250 microM), in combination with insulin, produced an additive inhibition of gluconeogenesis during glucagon-stimulated conditions. However, unlike insulin, the metabolic mechanism responsible for these effects (in the presence or absence of insulin) does not involve fructose 2,6-bisphosphate.

Contrast-enhanced magnetic resonance imaging of myocardium at risk: distinction between reversible and irreversible injury throughout infarct healing.

OBJECTIVES: We sought to determine the relationship of delayed hyperenhancement by contrast magnetic resonance imaging (MRI) to viable and nonviable myocardium within the region at risk throughout infarct healing. BACKGROUND: The relationship of delayed MRI contrast enhancement patterns to injured but viable myocardium within the ischemic bed at risk has not been established. METHODS: We compared in vivo and ex vivo MRI contrast enhancement to histopathologic tissue sections encompassing the entire left ventricle in dogs (n = 24) subjected to infarction with (n = 12) and without (n = 12) reperfusion at 4 h, 1 day, 3 days, 10 days, 4 weeks and 8 weeks. In vivo MR imaging was performed 30 min after contrast injection. RESULTS: The sizes and shapes of in vivo myocardial regions of elevated image intensity (828+/-132% of remote) were the same as those observed ex vivo (241 slices, r = 0.99, bias = 0.05+/-1.6% of left ventricle [LV]). Comparison of ex vivo MRI to triphenyltetrazolim chloride-stained sections demonstrated that the spatial extent of hyperenhancement was the same as the spatial extent ofinfarction at every stage of healing (510 slices, lowest r = 0.95, largest bias = 1.7+/-2.9% of LV). Conversely, hyperenhanced regions were smaller than the ischemic bed at risk defined by fluorescent microparticles at every stage of healing (239 slices, 35+/-24% of risk region, p<0.001). Image intensities of viable myocardium within the risk region were the same as those of remote, normal myocardium (102+/-9% of remote, p = NS). CONCLUSIONS: Delayed contrast enhancement by MRI distinguishes between viable and nonviable regions within the myocardium at risk throughout infarct healing.

The use of contrast-enhanced magnetic resonance imaging to identify reversible myocardial dysfunction.

BACKGROUND: Recent studies indicate that magnetic resonance imaging (MRI) after the administration of contrast material can be used to distinguish between reversible and irreversible myocardial ischemic injury regardless of the extent of wall motion or the age of the infarct. We hypothesized that the results of contrast-enhanced MRI can be used to predict whether regions of abnormal ventricular contraction will improve after revascularization in patients with coronary artery disease. METHODS: Gadolinium-enhanced MRI was performed in 50 patients with ventricular dysfunction before they underwent surgical or percutaneous revascularization. The transmural extent of hyperenhanced regions was postulated to represent the transmural extent of nonviable myocardium. The extent of regional contractility at the same locations was determined by cine MRI before and after revascularization in 41 patients. RESULTS: Contrast-enhanced MRI showed hyperenhancement of myocardial tissue in 40 of 50 patients before revascularization. In all patients with hyperenhancement the difference in image intensity between hyperenhanced regions and regions without hyperenhancement was more than 6 SD. Before revascularization, 804 of the 2093 myocardial segments analyzed (38 percent) had abnormal contractility, and 694 segments (33 percent) had some areas of hyperenhancement. In an analysis of all 804 dysfunctional segments, the likelihood of improvement in regional contractility after revascularization decreased progressively as the transmural extent of hyperenhancement before revascularization increased (P<0.001). For instance, contractility increased in 256 of 329 segments (78 percent) with no hyperenhancement before revascularization, but in only 1 of 58 segments with hyperenhancement of more than 75 percent of tissue. The percentage of the left ventricle that was both dysfunctional and not hyperenhanced before revascularization was strongly related to the degree of improvement in the global mean wall-motion score (P<0.001) and the ejection fraction (P<0.001) after revascularization. CONCLUSIONS: Reversible myocardial dysfunction can be identified by contrast-enhanced MRI before coronary revascularization.

Early assessment of myocardial salvage by contrast-enhanced magnetic resonance imaging.

BACKGROUND: Myocardial salvage after acute myocardial infarction is defined clinically by early restoration of flow and long-term improvement in contractile function. We hypothesized that contrast-enhanced magnetic resonance imaging (MRI), performed early after myocardial infarction, indexes myocardial salvage. We studied the relationship between the transmural extent of hyperenhancement by contrast-enhanced MRI, restoration of flow, and recovery of function. METHODS AND RESULTS: The left anterior descending coronary artery was occluded in dogs (n=15) for either 45 minutes, 90 minutes, or permanently. Cine and contrast-enhanced MRI were performed 3 days after the procedure; cine MRI was also done 10 and 28 days after the procedure. The transmural extent of hyperenhancement and wall thickening were determined using a 60-segment model. The mean transmural extent of hyperenhancement for the 45-minute occlusion group was 22% of the 90-minute group and 18% of the permanent occlusion group (P:<0.05 for both). The transmural extent of hyperenhancement on day 3 was related to future improvement in both wall thickening score and absolute wall thickening at 10 and 28 days (P:<0.0001 for each). For example, of the 415 segments on day 3 that were dysfunctional and had <25% transmural hyperenhancement, 362 (87%) improved by day 28. Conversely, no segments (0 of 9) with 100% hyperenhancement improved. The transmural extent of hyperenhancement on day 3 was a better predictor of improvement in contractile function than occlusion time (P:<0.0001). CONCLUSIONS: A reduction in the transmural extent of hyperenhancement by contrast-enhanced MRI early after myocardial infarction is associated with an early restoration of flow and future improvement in contractile function.