Mss51 deletion increases endurance and ameliorates histopathology in the mdx mouse model of Duchenne muscular dystrophy.

Mitochondrial derangement is an important contributor to the pathophysiology of muscular dystrophies and may be among the earliest cellular deficits. We have previously shown that disruption of Mss51, a mammalian skeletal muscle protein that localizes to the mitochondria, results in enhanced muscle oxygen consumption rate, increased endurance capacity, and improved limb muscle strength in mice with wildtype background. Here, we investigate whether Mss51 deletion in the mdx murine model of Duchenne muscular dystrophy (mdx-Mss51 KO) counteracts the muscle pathology and mitochondrial irregularities observed in mdx mice. We found that mdx-Mss51 KO mice had increased myofiber oxygen consumption rates and an amelioration of muscle histopathology compared to mdx counterparts. This corresponded with greater treadmill endurance and less percent fatigue in muscle physiology, but no improvement in forelimb grip strength or limb muscle force production. These findings suggest that although Mss51 deletion ameliorates the skeletal muscle mitochondrial respiration defects in mdx and improves fatigue resistance in vivo, the lack of improvement in force production suggests that this target alone may be insufficient for a therapeutic effect.

Chemical Contaminants from Plastics in the Animal Environment.

Accidental exposure of our mice to bisphenol A (BPA) from damaged polycarbonate cages 20 y ago provided some of the first evidence of the harmful effects of exposure to this common chemical. Recently we found that housing mice in damaged polysulfone cages resulted in similar harmful effects due to exposure to bisphenol S (BPS). This problem was unexpected for 2 reasons. First, polysulfone is a far more chemically resistant polymer than polycarbonate. Second, BPS is not a component in the manufacture of polysulfone. We report here our efforts to verify the source of the BPS and eliminate the exposure. Our analysis of new polysulfone caging materials confirmed that BPS is a breakdown product of damaged polysulfone plastic. Furthermore, we found that BPS can cross-contaminate new or undamaged cages in facilities that process damaged caging materials. Neither the use of disposable cages nor replacement of caging materials used solely for our colony was sufficient to eliminate exposure effects. Only the replacement of all cages and water bottles in the facility corrected the problem and allowed us to resume our studies. Taken together, our previous and current findings underscore the concern that chemicals from plastics are harmful environmental contaminants for both humans and animals. Furthermore, our results provide strong evidence that the presence of damaged plastic in a facility may be sufficient to affect research results and, by exten- sion, animal health.

Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower ß-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases ß-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of ß-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and ß-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that ß-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

Replacement Bisphenols Adversely Affect Mouse Gametogenesis with Consequences for Subsequent Generations.

20 years ago, accidental bisphenol A (BPA) exposure caused a sudden increase in chromosomally abnormal eggs from our control mice [1]. Subsequent rodent studies demonstrated developmental effects of exposure with repercussions on adult health and fertility (e.g., [2-9]; reviewed in [10-17]). Studies in monkeys, humans, fish, and worms suggest BPA effects extend across species (e.g., [18-30]; reviewed in [31-33]). Widespread use has resulted in ubiquitous environmental contamination and human BPA exposure. Consumer concern resulted in "BPA-free" products produced using structurally similar bisphenols that are now detectable environmental and human contaminants (e.g., [34-41]). We report here studies initiated by meiotic changes mirroring our previous BPA experience and implicating exposure to BPS (a common BPA replacement) from damaged polysulfone cages. Like with BPA [1, 2, 5], our data show that exposure to common replacement bisphenols induces germline effects in both sexes that may affect multiple generations. These findings add to growing evidence of the biological risks posed by this class of chemicals. Rapid production of structural variants of BPA and other EDCs circumvents efforts to eliminate dangerous chemicals, exacerbates the regulatory burden of safety assessment, and increases environmental contamination. Our experience suggests that these environmental contaminants pose a risk not only to reproductive health but also to the integrity of the research environment. EDCs, like endogenous hormones, can affect diverse processes. The sensitivity of the germline allows us to detect effects that, although not immediately apparent in other systems, may induce variability that undermines experimental reproducibility and impedes scientific advancement.

Increased Titin Compliance Reduced Length-Dependent Contraction and Slowed Cross-Bridge Kinetics in Skinned Myocardial Strips from Rbm (20ΔRRM) Mice.

Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20 (ΔRRM) ) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20 (ΔRRM) animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20 (ΔRRM) on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca(2+)-activated, skinned papillary muscle strips from Rbm20 (ΔRRM) and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca(2+)-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20 (ΔRRM) vs. WT mice. As sarcomere length increased from 1.9 to 2.2 µm, length-dependent activation of contraction was eliminated in the Rbm20 (ΔRRM) myocardium, even though myosin MgADP release rate decreased ~20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.

Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 µm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 µm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles.