Mysterious Ca2+ -independent muscular contraction: Déjà vu

Cardiac Surgery Research Laboratory, Department of Heart Surgery, Innsbruck Medical University, Innsbruck, Austria.
Biochemical Journal (Impact Factor: 4.4). 05/2012; 445(3):333-6. DOI: 10.1042/BJ20120439
Source: PubMed


The permeabilized cells and muscle fibres technique allows one to study the functional properties of mitochondria without their isolation, thus preserving all of the contacts with cellular structures, mostly the cytoskeleton, to study the whole mitochondrial population in the cell in their natural surroundings and it is increasingly being used in both experimental and clinical studies. The functional parameters (affinity for ADP in regulation of respiration) of mitochondria in permeabilized myocytes or myocardial fibres are very different from those in isolated mitochondria in vitro. In the present study, we have analysed the data showing the dependence of this parameter upon the muscle contractile state. Most remarkable is the effect of recently described Ca(2+)-independent contraction of permeabilized muscle fibres induced by elevated temperatures (30-37°C). We show that very similar strong spontaneous Ca(2+)-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibres that result in a disorganization of mitochondrial arrangement, leading to a significant increase in affinity for ADP. These data show that Ca(2+)-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore the use of their inhibitors is strongly advised at the permeabilization step with careful washing of fibres or cells afterwards. A possible physiologically relevant relationship between Ca(2+)-regulated ATP-dependent contraction and mitochondrial functional parameters is also discussed.

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    • "Using electron microscopy it was shown that 100 μg/mL of saponin used to permeabilize cells for 30 min did not alter MOM connections with cytoskeleton [45]. However, taking into account that by removing cholesterol from lysosomal membranes, saponin hypothetically could increase cytoskeleton proteolysis, all experiments were lead in the presence of protease inhibitors [29] [33]. Low MtCK expression in glycolytic muscles is another factor capable of influencing Cr-stimulated respiration. "
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    ABSTRACT: The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers, focusing also on the role of cytoskeletal protein tubulin βII isotype in mitochondrial metabolism and organization. By analyzing accessibility of mitochondrial ADP, using respirometry and pyruvate kinase - phosphoenolpyruvate trapping system for ADP, we show that the apparent affinity of respiration for ADP can be directly linked to the permeability of the mitochondrial outer membrane (MOM). Previous studies have shown that MOM permeability in cardiomyocytes can be regulated by VDAC interaction with cytoskeletal protein, βII tubulin. We found that in oxidative soleus skeletal muscle the high apparent Km for ADP is associated with low MOM permeability and high expression of non-polymerized βII tubulin. Very low expression of non-polymerized form of βII tubulin in glycolytic muscles is associated with high MOM permeability for adenine nucleotides (low apparent Km for ADP).
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    • "Neither inhibition of potential cycling of residual endogenous ADP/ATP between creatine kinase and myosin-ATPase, nor limiting exposure to calcium (i.e., inclusion of high concentrations of the Ca2+-chelator EGTA up to 5 mmol/L) prevents the spontaneous contraction (54), suggesting that the effect is independent of endogenous adenine nucleotide cycling or calcium. Treatment with trypsin induces a similar rapid shortening of permeabilized cardiomyocytes due to degradation of the cytoskeleton (68), leading Kuznetsov et al. (129) to raise the intriguing possibility that the presence of endogenous proteases may account for the apparent calcium-independent contraction in permeabilized muscle fibers. We recently tested this possibility in both rodent (Fig. 1) and human (115) permeabilized skeletal muscle fiber bundles treated with protease inhibitors in three different respiration buffers, including Mitomed as recommended by Kuznetsov et al. (129). "
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    ABSTRACT: A growing body of research is investigating the potential contribution of mitochondrial function to the etiology of type 2 diabetes. Numerous in vitro, in situ, and in vivo methodologies are available to examine various aspects of mitochondrial function, each requiring an understanding of their principles, advantages, and limitations. This review provides investigators with a critical overview of the strengths, limitations and critical experimental parameters to consider when selecting and conducting studies on mitochondrial function. In vitro (isolated mitochondria) and in situ (permeabilized cells/tissue) approaches provide direct access to the mitochondria, allowing for study of mitochondrial bioenergetics and redox function under defined substrate conditions. Several experimental parameters must be tightly controlled, including assay media, temperature, oxygen concentration, and in the case of permeabilized skeletal muscle, the contractile state of the fibers. Recently developed technology now offers the opportunity to measure oxygen consumption in intact cultured cells. Magnetic resonance spectroscopy provides the most direct way of assessing mitochondrial function in vivo with interpretations based on specific modeling approaches. The continuing rapid evolution of these technologies offers new and exciting opportunities for deciphering the potential role of mitochondrial function in the etiology and treatment of diabetes.
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    • "As demonstrated in Supplementary videos 2–3, the addition of protease inhibitors to permeabilization medium (Supplementary video 2) or both permeabilization and assay media (Supplementary video 3) does not prevent ADP-triggered PmFB contraction at 37 • C. Furthermore, inclusion of protease inhibitors during permeabilization, wash and assay medium did not prevent contraction-related reductions in apparent K m for ADP in human skeletal muscle PmFBs when assessed with MiR05, Buffer Z (Perry et al. 2011) or Mitomed (Kuznetsov et al. 2012) (data not shown). These data provide evidence that ADP triggers contraction per se in skeletal muscle PmFBs independent of the proteolysis reported for cardiomyocytes (Kuznetsov et al. 2012). This contraction is especially apparent given that the effect is prevented by myosin inhibition (present study and Perry et al. 2011). "
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    ABSTRACT: Energy transfer between mitochondrial and cytosolic compartments is predominantly achieved by creatine-dependent phosphate shuttling (PCr/Cr) involving miCK. However ADP/ATP diffusion through adenine nucleotide translocase (ANT) and voltage-dependent anion carriers (VDAC) are also involved in this process. To determine if exercise alters the regulation of this system, ADP-stimulated mitochondrial respiratory kinetics were assessed in permeabilized muscle fibre bundles (PmFB) taken from biopsies before and after 2hr of cycling exercise (60% VO(2)peak) in 9 lean males. Concentrations of creatine (Cr) and phosphocreatine (PCr) as well as the contractile state of PmFB were manipulated in situ. In the absence of contractile signals (relaxed PmFB) and miCK activity (no Cr), post-exercise respiratory sensitivity to ADP was reduced in situ (up to 126% higher apparent K(m) to ADP) suggesting inhibition of ADP/ATP diffusion between matrix and cytosolic compartments (possibly ANT and VDAC). However this effect was masked in the presence of saturating Cr (no effect of exercise on ADP sensitivity). Given the role of ANT is thought to be independent of Cr, these findings suggest ADP/ATP, but not PCr/Cr, cycling through the outer mitochondrial membrane (VDAC) may be attenuated in resting muscle after exercise. In contrast, in contracted PmFB, post-exercise respiratory sensitivity to ADP increased with miCK activation (saturating Cr; 33% lower apparent (Km) to ADP), suggesting prior exercise increases miCK sensitivity in situ. These observations demonstrate that exercise increases miCK-dependent respiratory sensitivity to ADP, promoting mitochondrial-cytosolic energy exchange via PCr/Cr cycling, possibly through VDAC. This effect may mask an underlying inhibition of Cr-independent ADP/ATP diffusion. This enhanced regulation of miCK-dependent phosphate shuttling may improve energy homeostasis through more efficient coupling of oxidative phosphorylation to perturbations in cellular energy charge during subsequent bouts of contraction.
    Full-text · Article · Aug 2012 · The Journal of Physiology
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