Disrupted Membrane Structure and Intracellular Ca2+ Signaling in Adult Skeletal Muscle with Acute Knockdown of Bin1

Efficient intracellular Ca2+ ([Ca2+]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca2+ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short –hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca2+ release. Voltage-induced Ca2+ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca2+ current and SR Ca2+ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca2+ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca2+ sparks' amplitude that was attributed to decreased total Ca2+ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca2+]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy

Enhancement of Cardiac Store Operated Calcium Entry (SOCE) within Novel Intercalated Disk Microdomains in Arrhythmic Disease

Store-operated Ca2+ entry (SOCE), a major Ca2+ signaling mechanism in non-myocyte cells, has recently emerged as a component of Ca2+ signaling in cardiac myocytes. Though it has been reported to play a role in cardiac arrhythmias and to be upregulated in cardiac disease, little is known about the fundamental properties of cardiac SOCE, its structural underpinnings or effector targets. An even greater question is how SOCE interacts with canonical excitation-contraction coupling (ECC). We undertook a multiscale structural and functional investigation of SOCE in cardiac myocytes from healthy mice (wild type; WT) and from a genetic murine model of arrhythmic disease (catecholaminergic ventricular tachycardia; CPVT). Here we provide the first demonstration of local, transient Ca2+ entry (LoCE) events, which comprise cardiac SOCE. Although infrequent in WT myocytes, LoCEs occurred with greater frequency and amplitude in CPVT myocytes. CPVT myocytes also evidenced characteristic arrhythmogenic spontaneous Ca2+ waves under cholinergic stress, which were effectively prevented by SOCE inhibition. In a surprising finding, we report that both LoCEs and their underlying protein machinery are concentrated at the intercalated disk (ID). Therefore, localization of cardiac SOCE in the ID compartment has important implications for SOCE-mediated signaling, arrhythmogenesis and intercellular mechanical and electrical coupling in health and disease

The German Music@Home: Validation of a questionnaire measuring at home musical exposure and interaction of young children.

The present study introduces the German version of the original version of the Music@Home questionnaire developed in the UK, which systematically evaluates musical engagement in the home environment of young children. Two versions are available, an Infant version for children aged three to 23 months and a Preschool version for children aged two to five and a half years. For the present study, the original Music@Home questionnaire was translated from English into German and 656 caregivers completed the questionnaire online. A confirmatory factor analysis showed moderate to high fit indices for both versions, confirming the factor structure of the original questionnaire. Also, the reliability coefficients for the subscales (Parental beliefs, Child engagement with music, Parent initiation of singing, Parent initiation of music-making for the Infant version and Parental beliefs, Child engagement with music, Parent initiation of music behavior and Breadth of musical exposure for the Preschool version) ranged from moderate to high fits. Furthermore, the test-retest analysis (N = 392) revealed high correlations for the general factor and all subscales confirming their internal reliability. Additionally, we included language questionnaires for children of two and three years of age. Results showed that higher scores on the Music@Home questionnaire were moderately associated with better language skills in two-year-olds (N = 118). In sum, the study presents the validated German Music@Home questionnaire, which shows good psychometric properties. The two versions of the questionnaire are available for use in order to assess home musical engagement of young children, which could be of interest in many areas of developmental research

The Apoptosome: Emerging Insights and New Potential Targets for Drug Design

Apoptosis plays a crucial role in tissue homeostasis, development and many diseases. The relevance of Apaf1, the molecular core of apoptosome, has been underlined in mitochondria-dependent apoptosis, which according to a growing body of evidence, is involved in various pathologies where the equilibrium of life-and-death is dysregulated, such as heart attack, stroke, liver failure, cancer and autoimmune diseases. Consequently, great interest has emerged in devising therapeutic strategies for regulating the key molecules involved in the life-and-death decision. Here we review recent progress in apoptosis-based pharmacological therapies and, in particular, we point out a possible role of the apoptosome as an emerging and promising pharmacological target

L-Type Ca2+ Channel Function Is Linked to Dystrophin Expression in Mammalian Muscle

BACKGROUND: In dystrophic mdx skeletal muscle, aberrant Ca2+ homeostasis and fibre degeneration are found. The absence of dystrophin in models of Duchenne muscular dystrophy (DMD) has been connected to altered ion channel properties e.g. impaired L-type Ca2+ currents. In regenerating mdx muscle, 'revertant' fibres restore dystrophin expression. Their functionality involving DHPR-Ca2+-channels is elusive. METHODS AND RESULTS: We developed a novel 'in-situ' confocal immuno-fluorescence and imaging technique that allows, for the first time, quantitative subcellular dystrophin-DHPR colocalization in individual, non-fixed, muscle fibres. Tubular DHPR signals alternated with second harmonic generation signals originating from myosin. Dystrophin-DHPR colocalization was substantial in wt fibres, but diminished in most mdx fibres. Mini-dystrophin (MinD) expressing fibres successfully restored colocalization. Interestingly, in some aged mdx fibres, colocalization was similar to wt fibres. Most mdx fibres showed very weak membrane dystrophin staining and were classified 'mdx-like'. Some mdx fibres, however, had strong 'wt-like' dystrophin signals and were identified as 'revertants'. Split mdx fibres were mostly 'mdx-like' and are not generally 'revertants'. Correlations between membrane dystrophin and DHPR colocalization suggest a restored putative link in 'revertants'. Using the two-micro-electrode-voltage clamp technique, Ca2+-current amplitudes (i(max)) showed very similar behaviours: reduced amplitudes in most aged mdx fibres (as seen exclusively in young mdx mice) and a few mdx fibres, most likely 'revertants', with amplitudes similar to wt or MinD fibres. Ca2+ current activation curves were similar in 'wt-like' and 'mdx-like' aged mdx fibres and are not the cause for the differences in current amplitudes. i(max) amplitudes were fully restored in MinD fibres. CONCLUSIONS: We present evidence for a direct/indirect DHPR-dystrophin interaction present in wt, MinD and 'revertant' mdx fibres but absent in remaining mdx fibres. Our imaging technique reliably detects single isolated 'revertant' fibres that could be used for subsequent physiological experiments to study mechanisms and therapy concepts in DMD

Poloxamer 188 does not Alter Performance Improvements Associated with Equivalent Exercise Regimens in Mice

1Krajek, A., 2Weisleder, N., 1Orange, M. 1Gettysburg College, Gettysburg PA, 2The Ohio State University, Columbus, Ohio Purpose: Poloxamers are synthetic amphipathic co-block polymers that may insert themselves into the plasma membrane and have been shown to protect multiple cell types from various injuries. Our goal is to investigate the possible utility of poloxamer 188 (P188) to improve performance outcomes associated with regular exercise. The aim of this study was to determine the effect of P188 treatment among groups of equivalent exercise loads. Methods: Thirty-one adult C57Bl6/J mice were randomly distributed amongst four groups: P188 treated and exercised (P188-E), saline injected and exercised (Sal-E), P188 treated and no exercise (P188-N), saline injected and no exercise (Sal-N). Mice received intraperitoneal injection of P188 solution (10 µL/g of 100 mg P188/mL in 0.9% saline; 1000 mg P188/kg dose) or vehicle (0.9% saline solution) three times weekly. At 30 minutes post-injection mice were run on a treadmill for 45 minutes at speeds increasing to a maximum of 16 m/min. After eight weeks of this training and treatment program, average treadmill exhaustion time was determined for each group. Results: Exercise groups displayed significantly higher exhaustion times relative to non-trained control groups: P188-E 25.24 min, P188-N 21.43min, Sal-E 26.60 min, Sal-N 22.38 min (main effect, p = 0.002, 2X2 analysis of variance). The difference between P188 treated and vehicle injected groups was not statistically significant (main effect, p = 0.333). Conclusion: These observations suggest that the cellular mechanism of P188 membrane protection has no impact on performance improvement when treated and non-treated groups are exposed to equivalent exercise regimens. Future experimentation will investigate the impact of P188 on recovery from acute bouts of intense exercise. Research funded in part by a grant to Gettysburg College from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Progra