Exposure to rosiglitazone, a PPAR-gamma agonist, in late gestation reduces the abundance of factors regulating cardiac metabolism and cardiomyocyte size in the sheep fetus

It is unknown whether cardiomyocyte hypertrophy and the transition to fatty acid oxidation as the main source of energy after birth is dependent on the maturation of the cardiomyocytes' metabolic system, or on the limitation of substrate availability before birth. This study aimed to investigate whether intrafetal administration of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, during late gestation can stimulate the expression of factors regulating cardiac growth and metabolism in preparation for birth, and the consequences of cardiac contractility in the fetal sheep at ∼140 days gestation. The mRNA expression and protein abundance of key factors regulating growth and metabolism were quantified using quantitative RT-PCR and Western blot analysis, respectively. Cardiac contractility was determined by measuring the Ca(2+) sensitivity and maximum Ca(2+)-activated force of skinned cardiomyocyte bundles. Rosiglitazone-treated fetuses had a lower cardiac abundance of insulin-signaling molecules, including insulin receptor-β, insulin receptor substrate-1 (IRS-1), phospho-IRS-1 (Tyr-895), phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, PI3K catalytic subunit p110α, phospho-3-phosphoinositide-dependent protein kinase 1 (Ser-241), protein kinase B (Akt-1), phospho-Akt (Ser-273), PKCζ, phospho-PKCζ(Thr-410), Akt substrate 160 kDa (AS160), phospho-AS160 (Thr-642), and glucose transporter type-4. Additionally, cardiac abundance of regulators of fatty acid β-oxidation, including adiponectin receptor 1, AMPKα, phospho-AMPKα (Thr-172), phospho-acetyl CoA carboxylase (Ser-79), carnitine palmitoyltransferase-1, and PGC-1α was lower in the rosiglitazone-treated group. Rosiglitazone administration also resulted in a decrease in cardiomyocyte size. Rosiglitazone administration in the late-gestation sheep fetus resulted in a decreased abundance of factors regulating cardiac glucose uptake, fatty acid β-oxidation, and cardiomyocyte size. These findings suggest that activation of PPAR-γ using rosiglitazone does not promote the maturation of cardiomyocytes; rather, it may decrease cardiac metabolism and compromise cardiac health later in life.Shervi Lie, Melisa Hui, I. Caroline McMillen, Beverly S. Muhlhausler, Giuseppe S. Posterino, Stacey L. Dunn, Kimberley C. Wang, Kimberley J. Botting, Janna L. Morriso

Effects of oxidation and cytosolic redox conditions on excitation–contraction coupling in rat skeletal muscle

In this study the effects of oxidation and reduction on various steps in the excitation-contraction (E–C) coupling sequence was examined in mammalian skeletal muscle. In mechanically skinned fast-twitch fibres, electric field stimulation was used to generate action potentials in the sealed transverse-tubular (T-) system, thereby eliciting twitch responses, which are a sensitive measure of Ca2+ release. Treatment of fibres with the oxidant H2O2 (200 μm and 10 mm) for 2–5 min markedly potentiated caffeine-induced Ca2+ release and the force response to partial depolarisation of the T-system (by solution substitution). Importantly, such H2O2 treatment had no effect at all on any aspect of the twitch response (peak amplitude, rate of rise, decay rate constant and half-width), except in cases where it interfered with the T-system potential or voltage-sensor activation, resulting in a reduction or abolition of the twitch response. Exposure to strong thiol reductants, dithiothreitol (DTT, 10 mm) and reduced glutathione (GSH, 5 mm), did not affect the twitch response over 5 min, nor did varying the glutathione ratio (reduced to oxidised glutathione) from the level present endogenously in the cytosol of a rested fibre (30:1) to the comparatively oxidised level of 3:1. In fibres that had been oxidised by H2O2 (10 mm) (or by 2,2′-dithiodipyridine, 100 μm), exposure to GSH (5 mm) caused potentiation of twitch force (by ∼20 % for H2O2); this effect was due to the increase in the Ca2+ sensitivity of the contractile apparatus that occurs under such circumstances and was fully reversed by subsequent exposure to 10 mm DTT. We conclude that: (a) the redox potential across the sarcomplamsic reticulum has no noticeable direct effect on normal E–C coupling in mammalian skeletal muscle, (b) oxidising the Ca2+-release channels and greatly increasing their sensitivity to Ca2+-induced Ca2+ release does not alter the amount of Ca2+ released by an action potential and (c) oxidation potentiates twitches by a GSH-mediated increase in the Ca2+ sensitivity of the contractile apparatus

Twitch and tetanic force responses and longitudinal propagation of action potentials in skinned skeletal muscle fibres of the rat

Transverse electrical field stimulation (50 V cm−1, 2 ms duration) of mechanically skinned skeletal muscle fibres of the rat elicited twitch and tetanic force responses (36 ± 4 and 83 ± 4 % of maximum Ca2+-activated force, respectively; n = 23) closely resembling those in intact fibres. The responses were steeply dependent on the field strength and were eliminated by inclusion of 10 μm tetrodotoxin (TTX) in the (sealed) transverse tubular (T-) system of the skinned fibres and by chronic depolarisation of the T-system.Spontaneous twitch-like activity occurred sporadically in many fibres, producing near maximal force in some instances (mean time to peak: 190 ± 40 ms; n = 4). Such responses propagated as a wave of contraction longitudinally along the fibre at a velocity of 13 ± 3 mm s−1 (n = 7). These spontaneous contractions were also inhibited by inclusion of TTX in the T-system and by chronic depolarisation.We examined whether the T-tubular network was interconnected longitudinally using fibre segments that were skinned for only ∼2/3 of their length, leaving the remainder of each segment intact with its T-system open to the bathing solution. After such fibres were exposed to TTX (60 μm), the adjacent skinned region (with its T-system not open to the solution) became unresponsive to subsequent electrical stimulation in ∼50 % of cases (7/15), indicating that TTX was able to diffuse longitudinally inside the fibre via the tubular network over hundreds of sarcomeres.These experiments show that excitation–contraction coupling in mammalian muscle fibres involves action potential propagation both transversally and longitudinally within the tubular system. Longitudinal propagation of action potentials inside skeletal muscle fibres is likely to be an important safety mechanism for reducing conduction failure during fatigue and explains why, in developing skeletal muscle, the T-system first develops as an internal longitudinal network

Examination of the subsarcolemmal tubular system of mammalian skeletal muscle fibers

A subsarcolemmal tubular system network (SSTN) has been detected in skeletal muscle fibers by confocal imaging after the removal of the sarcolemma. Here we confirm the existence and resolve the fine architecture and the localization of the SSTN at an unprecedented level of detail by examining extracellularly applied tubular system markers in skeletal muscle fiber preparations with a combination of three imaging modalities: confocal fluorescence microscopy, direct stochastic optical reconstruction microscopy, and tomographic electron microscopy. Three-dimensional reconstructions showed that the SSTN was a dense two-dimensional network within the subsarcolemmal space around the fiber, running ∼500-600 nm underneath and parallel to the sarcolemma. The SSTN is composed of tubules ∼95 nm in width with ∼60% of the tubules directed transversely and >30% directed longitudinally. The deeper regular transverse tubules located at each A-I boundary of the sarcomeres branched from the SSTN, indicating individual transverse tubules that form triads are continuous with, but do not directly contact the sarcolemma. This suggests that the SSTN plays an important role in affecting the exchange of deeper tubule lumina with the extracellular space

Depletion "skraps" and dynamic buffering inside the cellular calcium store

Ca2+ signals, produced by Ca2+ release from cellular stores, switch metabolic responses inside cells. In muscle, Call sparks locally exhibit the rapid start and termination of the cell-wide signal. By imaging Call inside the store using shifted excitation and emission ratioing of fluorescence, a surprising observation was made: Depletion during sparks or voltage-induced cell-wide release occurs too late, continuing to progress even after the Ca2+ release channels have closed. This finding indicates that Call is released from a '' proximate '' compartment functionally in between store lumen and cytosol. The presence of a proximate compartment also explains a paradoxical surge in intrastore Ca2+, which was recorded upon stimulation of prolonged, cell-wide Call release. An intrastore surge upon induction of Ca2+ release was first reported in subcellular store fractions, where its source was traced to the store buffer, calsequestrin. The present results update the evolving concept, largely due to N. Ikemoto and C. Kang, of calsequestrin as a dynamic store. Given the strategic location and reduction of dimensionality of Ca2+-adsorbing linear polymers of calsequestrin, they could deliver Ca2+ to the open release channels more efficiently than the luminal store solution, thus constituting the proximate compartment. When store depletion becomes widespread, the polymers would collapse to increase store [Ca2+] and sustain the concentration gradient that drives release flux