Upregulation of PPARβ/δ Is Associated with Structural and Functional Changes in the Type I Diabetes Rat Diaphragm

Diabetes mellitus is associated with alterations in peripheral striated muscles and cardiomyopathy. We examined diaphragmatic function and fiber composition and identified the role of peroxisome proliferator-activated receptors (PPAR alpha and beta/delta) as a factor involved in diaphragm muscle plasticity in response to type I diabetes.Streptozotocin-treated rats were studied after 8 weeks and compared with their controls. Diaphragmatic strips were stimulated in vitro and mechanical and energetic variables were measured, cross bridge kinetics assessed, and the effects of fatigue and hypoxia evaluated. Morphometry, myosin heavy chain isoforms, PPAR alpha and beta/delta gene and protein expression were also assessed. Diabetes induced a decrease in maximum velocity of shortening (-14%, P<0.05) associated with a decrease in myosin ATPase activity (-49%, P<0.05), and an increase in force (+20%, P<0.05) associated with an increase in the number of cross bridges (+14%, P<0.05). These modifications were in agreement with a shift towards slow myosin heavy chain fibers and were associated with an upregulation of PPARbeta/delta (+314% increase in gene and +190% increase in protein expression, P<0.05). In addition, greater resistances to fatigue and hypoxia were observed in diabetic rats.Type I diabetes induced complex mechanical and energetic changes in the rat diaphragm and was associated with an up-regulation of PPARbeta/delta that could improve resistance to fatigue and hypoxia and favour the shift towards slow myosin heavy chain isoforms

EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription

Caveolae are small invaginated pits that function as dynamic mechanosensors to buffer tension variations at the plasma membrane. Here we show that under mechanical stress, the EHD2 ATPase is rapidly released from caveolae, SUMOylated, and translocated to the nucleus, where it regulates the transcription of several genes including those coding for caveolae constituents. We also found that EHD2 is required to maintain the caveolae reservoir at the plasma membrane during the variations of membrane tension induced by mechanical stress. Metal-replica electron microscopy of breast cancer cells lacking EHD2 revealed a complete absence of caveolae and a lack of gene regulation under mechanical stress. Expressing EHD2 was sufficient to restore both functions in these cells. Our findings therefore define EHD2 as a central player in mechanotransduction connecting the disassembly of the caveolae reservoir with the regulation of gene transcription under mechanical stress

Chapter 15 Determination of PPAR Expression by Western Blot

International audiencePeroxisome proliferator-activated receptors (PPARs) are key nuclear receptors which mediate the regulation of the transcription of many genes, especially those involved in lipid catabolism and inflammation. In mammals, three types of PPARs named PPARa, PPARb (also called PPARd), and PPARg have been identified. Up and down-regulations of one or more of these receptors are encountered during development , physiological adaptations, and in several diseases. Therefore, the ability to accurately measure PPAR expression in cells and tissues is an important aspect of PPAR research. This can be done by Western blot with specific antibodies. In this chapter, we describe the use of our techniques (Cardiovasc Res 84: 83-90, 2009; PLoS One 5: e11494, 2010) to measure PPARs expression, describe the adequate way to extract proteins, the best antibodies currently available and discuss potential misleading results in the absence of appropriate controls

Les pinces optiques en biologie et en médecine

Les lasers trouvent de continuelles applications en biologie, tant dans le domaine de la recherche que dans celui du diagnostic et de la thérapeutique. L’utilisation de la force optique produite par les lasers a permis de créer une pince optique capable d’attraper une cellule ou un fragment d’organite cellulaire, de les fixer et de les transporter dans un autre site cellulaire. Plus récemment, l’utilisation des pinces optiques a permis de mesurer des déplacements nanométriques et des forces de l’ordre de quelques picoNewtons exercées par un moteur moléculaire unique, comme la myosine ou la kinésine. La pince optique est un outil extraordinaire permettant de « nano-manipuler » un objet sans le toucher, c’est-à-dire sans qu’il y ait de contact matériel entre l’outil et l’objet.Optical trapping techniques provide unique means to manipulate biological particles such as virus, living cells and subcellular organelles. Another area of interest is the measurement of mechanical (elastic) properties of cell membranes, long strands of single DNA molecule, and filamentous proteins. One of the most attractive applications is the study of single motor molecules. With optical tweezers traps, one can measure the forces generated by single motor molecules such as kinesin and myosin, in the piconewton range and, for the first time, resolve their detailed stepping motion

Altered cross-bridge properties in skeletal muscle dystrophies

International audience† Co-first authors. Force and motion generated by skeletal muscle ultimately depends on the cyclical interaction of actin with myosin. This mechanical process is regulated by intracellular Ca 2+ through the thin filament-associated regulatory proteins i.e.; troponins and tropomyosin. Muscular dystrophies are a group of heterogeneous genetic affections characterized by progressive degeneration and weakness of the skeletal muscle as a consequence of loss of muscle tissue which directly reduces the number of potential myosin cross-bridges involved in force production. Mutations in genes responsible for skeletal muscle dystrophies (MDs) have been shown to modify the function of contractile proteins and cross-bridge interactions. Altered gene expression or RNA splicing or post-translational modifications of contractile proteins such as those related to oxidative stress, may affect cross-bridge function by modifying key proteins of the excitation-contraction coupling. Micro-architectural change in myofilament is another mechanism of altered cross-bridge performance. In this review, we provide an overview about changes in cross-bridge performance in skeletal MDs and discuss their ultimate impacts on striated muscle function

PCM1 labelling reveals myonuclear and nuclear dynamics in skeletal muscle across species

International audienceMyonuclei transcriptionally regulate muscle fibers during homeostasis and adaptation to exercise. Their cellular location and quantity are important when characterising phenotypes of myopathies, the effect of treatments and to understand the roles of satellite cells in muscle adaptation and muscle ‘memory’. Difficulties arise in identifying myonuclei due to their proximity to the sarcolemma and closely residing interstitial cell neighbours. We aimed to determine to what extent PCM1 is a specific marker of myonuclei in-vitro and in-vivo. Single isolated myofibers and cross-sections sections from mice and humans were studied from several models including Wild-type and Lamin A/C mutant mice after functional overload, and damage and recovery in humans following forced eccentric contractions. Fibers were immuno-labelled for PCM1, Pax7 and DNA. C2C12 myoblasts were also studied to investigate changes in PCM1 localisation during myogenesis. PCM1 labelled the nuclear envelope of myonuclei in mature myofibers and in newly formed myotubes, but also labelled centrosomes in proliferating myogenic precursors which may or may not fuse to join the myofiber syncytium. It also labelled non-myogenic nuclei near the sarcolemma especially in regenerating areas of the Lmna+/ΔK32 mouse and damaged human muscle. While PCM1 is not completely specific to myonuclei, the impact that PCM1+ macrophages and interstitial cells have on myonuclei counts would be small in healthy muscle. PCM1 may prove useful as a marker of satellite cell dynamics due to the distinct change in localisation during differentiation, revealing satellite cells in their quiescent (PCM1-), proliferating (PCM1+ centrosome), and pre-fusion states (PCM1+ nuclear envelope)

A potential link between PPAR signaling and the pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC) Time for Primary review: 50 Days Cardiovascular Research Advance

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Effects of Acute Respiratory and Metabolic Acidosis on Diaphragm Muscle Obtained from Rats

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