Skinned

Skinned

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In comparison, torrent salamanders (Rhyacotriton spp.) differ in having costal grooves, smooth skin, large eyes, white speckling on the sides of the body and the males have squared cloacal lobes. Torrent salamanders are also much smaller (adult 1.8 to 2.2 inches snout to vent length) than rough-skinned newts.

The larvae are pond-type with large gills and a high tail fin. Rough-skinned newts are our only salamander larvae that have eyes on the margins of the head and a snout that narrows in front of the eyes. A faint horizontal stripe is present from the snout through the eye, and one or two distinct rows of white spots occur on the sides. A salmon-colored area is present on the underside of the chest. Metamorphosis takes place when larvae are approximately 1.2 to 2.75 inches in total length.

The rough-skinned newt is one of only three Washington amphibians (includes tiger salamander and long-toed salamander) that lays single eggs, but it is the only one that hides its eggs within vegetation. The ova are small (0.7 inches diameter). The ovum (egg) is tan above and cream below with a thin layer of jelly surrounding the capsule. The jelly retains its shape out of water.

In Washington, rough-skinned newts occur primarily west of the Cascade Crest in the Pacific Coast, Puget Trough and West Cascades Ecoregions. They also occur east of the Cascade Crest in some areas of the East Cascades Ecoregion.

This map from the Washington Herp Atlas illustrates the distribution of rough-skinned newt in Washington based on records in the WDFW database as of 2016. If you see this species in areas that are not indicated on the map or have more recent observations (less than 10 years), please share your observation using the WDFW wildlife reporting form.

Maria Garcia and Maria Hinojosa are both Mexican American, both mestiza, and both relatively light-skinned. But Maria Hinojosa strongly identifies as a woman of color, whereas Maria Garcia has stopped doing so. So in this episode, we're asking: How did they arrive at such different places? To find out, listen to our latest installment in this series about what it means to be Latino.

Myofilaments and their associated proteins, which together constitute the sarcomeres, provide the molecular-level basis for contractile function in all muscle types. In intact muscle, sarcomere-level contraction is strongly coupled to other cellular subsystems, in particular the sarcolemmal membrane. Skinned muscle preparations (where the sarcolemma has been removed or permeabilized) are an experimental system designed to probe contractile mechanisms independently of the sarcolemma. Over the last few decades, experiments performed using permeabilized preparations have been invaluable for clarifying the understanding of contractile mechanisms in both skeletal and cardiac muscle. Today, the technique is increasingly harnessed for preclinical and/or pharmacological studies that seek to understand how interventions will impact intact muscle contraction. In this context, intrinsic functional and structural differences between skinned and intact muscle pose a major interpretational challenge. This review first surveys measurements that highlight these differences in terms of the sarcomere structure, passive and active tension generation, and calcium dependence. We then highlight the main practical challenges and caveats faced by experimentalists seeking to emulate the physiological conditions of intact muscle. Gaining an awareness of these complexities is essential for putting experiments in due perspective.

To identify the ionic and molecular mechanisms that regulate the sarcomere, it is necessary to control the chemical environment it is exposed to. The biochemistry of the sarcomere proteins can be studied using in vitro biochemistry assays. However, these fail to account for the regular structure of the sarcomere, which is important for both biochemistry and function. Alternatively, the sarcomeres can be accessed by skinning the muscle, i.e., removing the sarcolemma membrane (or making it permeable to compounds and ions), while preserving sarcomere functionality (Curtin et al., 2015). Exposing the sarcomeres to tailored ionic conditions provides a means to observe and control molecular behavior in a setting that more closely resembles native structures. After skinning, the sarcomere system is effectively isolated from the other cellular subsystems (except in some skeletal muscle experiments that remove the sarcolemma while preserving intracellular organelles and structures; Donaldson, 1985; Fill and Best, 1988; Posterino et al., 2000). This facilitates the study of contraction and its regulation separately from the sarcolemma. The central assumption of skinned muscle experiments is that the response of the sarcomeres to changes in the natural cytosol can be reproduced artificially and controllably through analogous changes in the bathing solution.

The principal readouts of skinned-muscle experiments are contraction kinetics, adenosine triphosphatase (ATPase) activity, and generated force. Their value therefore rests on the premise that the structural integrity of the sarcomeres is preserved. Under this condition, skinned muscle may be viewed as an intermediary experimental system, straddling intact muscle and in vitro molecular experiments.

Although, for many years, skinned muscle experiments have served as an invaluable method for investigating fundamental physiology, they are increasingly inspiring more ambitious practical applications. At a practical level, live human cells are inevitably a highly scarce resource, with facilities for collecting, storing, and measuring samples often being displaced both geographically and temporally. These issues are more realistically resolved with skinned cells, which can be preserved frozen for several months (Mosqueira et al., 2019). The development of new sarcomere drugs, including omecamtiv mecarbil and mavacamten, demonstrate that the sarcomere is a viable drug target (Tsukamoto, 2019). Similarly, Ca2+-sensitizing drugs (which act by increasing either the sensitivity to [Ca2+] or the magnitude of the generated force) such as levosimendan (Edes et al., 1995), pimobendan (Fitton and Brogden, 1994; Scheld et al., 1989), sulmazole (Solaro and Rüegg, 1982), isomazole (Lues et al., 1988), and EMD-57033 (Gross et al., 1993; Lee and Allen, 1997) have all been assessed using measurements on skinned fibers. Identifying further novel sarcomere modulator compounds requires large high-throughput screening, which is unrealistic using intact muscle.

In both of these evolving applications, the relevance and value of skinned-muscle experiments hinges on their ability to reliably emulate the intact system (Land et al., 2017; Margara et al., 2021; Mijailovich et al., 2021). Skinned-muscle experiments conducted over the past decades confirm the fidelity, in many respects, of these preparations as valid experimental models. However, they also highlight caveats and significant interpretational challenges. Gaining an awareness of these issues is becoming all the more essential to avoid misinterpretations that may have practical consequences. This review therefore aims to highlight these challenges, to help users of skinned-based measurements put them in an appropriate perspective.

The present review is structured as follows. We first compare measurements of the principal physiological properties of skinned and intact muscle, highlighting similarities and discrepancies. We focus primarily on chemical skinning, and in particular Triton X-100 (the predominantly used chemical agent). We then describe practical challenges involved in conducting experiments, insofar as they impact on measurement outcomes. We conclude with a summary of recommendations and main caveats.

Skinned muscle experiments aim to reveal and controllably reproduce features of the physiological function of sarcomeres. However, notable discrepancies arise between skinned- and intact-muscle measurements of basic muscle properties that govern overall muscle function. To establish these differences rigorously at the single-cell level encounters significant methodological challenges. Although it might seem obvious that this would require doing measurements systematically on both preparation types in tandem, many early experiments were done predominantly on skinned rather than on intact cells (King et al., 2011). This stems largely from the specific challenges of noninjurious cell attachment and performing small-force measurement on intact single cells (Brady, 1991). More recently, technical developments (e.g., involving the use of flexible carbon fibers to hold the cells at opposite ends; Iribe et al., 2007; Le Guennec et al., 1990; Yasuda et al., 2001) have made these measurements more practicable. Despite these advances, however, only a fraction of studies in the literature have systematically made direct comparisons between skinned and intact systems taken from the same species under optimally similar conditions (see the selection listed in Table 1). Although, as discussed below, this optimization encounters many hurdles, these comparisons are essential for a reliable interpretation of the measurements.

The expansion of the myofilament spacing in skinned preparations can be reversed by increasing the osmotic pressure of the solution using dextran (Cazorla et al., 2001; Konhilas et al., 2002). However, this compressive effect does not by itself return the myofilaments fully to their intact physiological state (Konhilas et al., 2002). Recent x-ray diffraction experiments have identified an alteration of the detailed molecular structure of the thick filaments below physiological temperatures (Caremani et al., 2019, 2021). Although this effect is overlooked in many experiments, it may significantly affect cross-bridge kinetics.

Skinning may also impact sarcomere morphology on larger scales. While measuring the effect of skinning on the sarcomere length in rat heart trabeculae using laser diffraction, Kentish et al. (1986) observed an increase in the diffraction intensity and a decrease in the dispersion of the first-order diffraction. Although this effect might result from the loss of intracellular scatterers (mitochondria, cytosolic proteins, etc.) upon skinning, the authors hypothesize that the skinning process might effectively enhance the homogenization of the sarcomere environment of the skinned tissue, relative to the intact one, where individual cells may display spontaneous and uncoordinated contractions. Nonetheless, the relative homogeneity of the skinned tissue degrades rapidly after successive contractions, possibly due to a loss of integrity of the cellular structure and content, in both cardiac (Kentish et al., 1986) and skeletal muscle (Fabiato and Fabiato, 1978b). This reflects a degree of irreproducibility inherent to skinned systems. 781b155fdc