Force generation examined by laser temperature-jumps in shortening and lengthening mammalian (rabbit psoas) muscle fibres

We examined the tension change induced by a rapid temperature jump (T-jump) in shortening and lengthening active muscle fibres. Experiments were done on segments of permeabilized single fibres (length (L0) ∼2 mm, sarcomere length 2.5 μm) from rabbit psoas muscle; [MgATP] was 4.6 mm, pH 7.1, ionic strength 200 mm and temperature ∼9°C. A fibre was maximally Ca2+-activated in the isometric state and a ∼3°C, rapid (< 0.2 ms), laser T-jump applied when the tension was approximately steady in the isometric state, or during ramp shortening or ramp lengthening at a limited range of velocities (0–0.2 L0 s−1). The tension increased to 2- to 3 × P0 (isometric force) during ramp lengthening at velocities > 0.05 L0 s−1, whereas the tension decreased to about < 0.5 × P0 during shortening at 0.1–0.2 L0 s−1; the unloaded shortening velocity was ∼1 L0 s−1 and the curvature of the force–shortening velocity relation was high (a/P0 ratio from Hill's equation of ∼0.05). In isometric state, a T-jump induced a tension rise of 15–20% to a new steady state; by curve fitting, the tension rise could be resolved into a fast (phase 2b, 40–50 s−1) and a slow (phase 3, 5–10 s−1) exponential component (as previously reported). During steady lengthening, a T-jump induced a small instantaneous drop in tension, followed by recovery, so that the final tension recorded with and without a T-jump was not significantly different; thus, a T-jump did not lead to a net increase of tension. During steady shortening, the T-jump induced a pronounced tension rise and both its amplitude and the rate (from a single exponential fit) increased with shortening velocity; at 0.1–0.2 L0 s−1, the extent of fibre shortening during the T-jump tension rise was estimated to be ∼1.2% L0 and it was shorter at lower velocities. At a given shortening velocity and over the temperature range of 8–30°C, the rate of T-jump tension rise increased with warming (Q10 ≈ 2.7), similar to phase 2b (endothermic force generation) in isometric muscle. Results are discussed in relation to the previous findings in isometric muscle fibres which showed that a T-jump promotes an early step in the crossbridge–ATPase cycle that generates force. In general, the finding that the T-jump effect on active muscle tension is pronounced during shortening, but is depressed/inhibited during lengthening, is consistent with the expectations from the Fenn effect that energy liberation (and acto-myosin ATPase rate) in muscle are increased during shortening and depressed/inhibited during lengthening

Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.The High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics is funded by Wellcome Trust grant reference 090532/Z/09/Z and MRC Hub grant G0900747 91070. This study was supported by Wellcome Trust Strategic Award 082030 (CCG), Wellcome Trust Studentship 094446/Z/10/Z (KRB), the Oxford NIHR Biomedical Research Centre, and the MRC Human Immunology Unit (RJC). AJR and GL were supported by Wellcome Trust grant 090532/Z/ 09/Z, CCG and AE by a Major initiative Award from the Clive and Vera Ramaciotti Foundation, and AE by an NHMRC Career Development Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

An analysis of the temperature dependence of force, during steady shortening at different velocities, in (mammalian) fast muscle fibres

We examined, over a wide range of temperatures (10–35°C), the isometric tension and tension during ramp shortening at different velocities (0.2–4 L0/s) in tetanized intact fibre bundles from a rat fast (flexor hallucis brevis) muscle; fibre length (L0) was 2.2 mm and sarcomere length ~2.5 μm. During a ramp shortening, the tension change showed an initial inflection of small amplitude (P1), followed by a larger exponential decline towards an approximate steady level; the tension continued to decline slowly afterwards and the approximate steady tension at a given velocity was estimated as the tension (P2) at the point of intersection between two linear slopes, as previously described (Roots et al. 2007). At a given temperature, the tension P2 declined to a lower level and at a faster rate (from an exponential curve fit) as the shortening velocity was increased; the temperature sensitivity of the rate of tension decline during ramp shortening at different velocities was low (Q10 0.9–1.5). The isometric tension and the P2 tension at a given shortening velocity increased with warming so that the relation between tension and (reciprocal) temperature was sigmoidal in both. In isometric muscle, the temperature T0.5 for half-maximal tension was ~10°C, activation enthalpy change (∆H) was ~100 kJ mol−1 and entropy change (∆S) ~350 J mol−1 K−1. In shortening, these were increased with increase of velocity so that at a shortening velocity (~4 L0/s) producing maximal power at 35°C, T0.5 was ~28°C, ∆H was ~200 kJ mol−1 and ∆S ~ 700 J mol−1 K−1; the same trends were seen in the tension data from isotonic release experiments on intact muscle and in ramp shortening experiments on maximally Ca-activated skinned fibres. In general, our findings show that the sigmoidal relation between force and temperature can be extended from isometric to shortening muscle; the implications of the findings are discussed in relation to the crossbridge cycle. The data indicate that the endothermic, entropy driven process that underlies crossbridge force generation in isometric muscle (Zhao and Kawai 1994; Davis, 1998) is even more pronounced in shortening muscle, i.e. when doing external work

Rasgrp1 mutation increases naïve T-cell CD44 expression and drives mTOR-dependent accumulation of Helios+ T cells and autoantibodies

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1Anaef, with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1Anaef mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44hi Helios+ PD-1+ CD4+ T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1Anaef is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1Anaef naïve CD4+ T cells. CD44 expression, CD4+ T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1AnaefMtorchino double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1Anaef T cell dysregulation

Rasgrp1 mutation increases naïve T-cell CD44 expression and drives mTOR-dependent accumulation of Helios⁺ T cells and autoantibodies

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1ᴬⁿᵃᵉᶠ, with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1ᴬⁿᵃᵉᶠ mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44hi Helios⁺ PD-1⁺ CD4⁺ T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1ᴬⁿᵃᵉᶠ is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1ᴬⁿᵃᵉᶠ naïve CD4⁺ T cells. CD44 expression, CD4⁺ T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1ᴬⁿᵃᵉᶠMtorᶜʰⁱⁿᵒ double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1ᴬⁿᵃᵉᶠ T cell dysregulation

Composition and Function of Haemolymphatic Tissues in the European Common Shrew

BACKGROUND: Studies of wild animals responding to their native parasites are essential if we are to understand how the immune system functions in the natural environment. While immune defence may bring increased survival, this may come at a resource cost to other physiological traits, including reproduction. Here, we tested the hypothesis that wild common shrews (Sorex araneus), which produce large numbers of offspring during the one breeding season of their short life span, forgo investment in immunity and immune system maintenance, as increased longevity is unlikely to bring further opportunities for mating. In particular, we predicted that adult shrews, with shorter expected lifespans, would not respond as effectively as young animals to infection. METHODOLOGY/PRINCIPAL FINDINGS: We examined haemolymphatic tissues from wild-caught common shrews using light and transmission electron microscopy, applied in conjunction with immunohistology. We compared composition and function of these tissues in shrews of different ages, and the extent and type of inflammatory reactions observed in response to natural parasitic infections. All ages seemed able to mount systemic, specific immune responses, but adult shrews showed some signs of lymphatic tissue exhaustion: lymphatic follicles in adults (n = 21) were both smaller than those in sub-adults (n = 18; Wald = 11.1, p<0.05) and exhibited greater levels of depletion (Wald = 13.3, p<0.05). CONCLUSIONS/SIGNIFICANCE: Contrary to our expectations, shrews respond effectively to their natural parasites, and show little indication of immunosenescence as adults. The pancreas of Aselli, a unique lymphoid organ, may aid in providing efficient immune responses through the storage of large numbers of plasma cells. This may allow older animals to react effectively to previously encountered parasites, but infection by novel agents, and eventual depletion of plasma cell reserves, could both still be factors in the near-synchronous mortality of adult shrews observed shortly after breeding

Rasgrp1 mutation increases naïve T-cell CD44 expression and drives mTOR-dependent accumulation of Helios+ T cells and autoantibodies

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1(Anaef), with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1(Anaef) mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44(hi) Helios(+) PD-1(+) CD4(+) T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1(Anaef) is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1(Anaef) naïve CD4(+) T cells. CD44 expression, CD4(+) T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1(Anaef)Mtor(chino) double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1(Anaef) T cell dysregulation. DOI: http://dx.doi.org/10.7554/eLife.01020.00