The Mitochondrial Ca(2+) Uniporter: Structure, Function, and Pharmacology.

Mitochondrial Ca(2+) uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca(2+) uptake and our current understanding of mitochondrial Ca(2+) homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca(2+) uniporter complex

Methods for Assessing Mitochondrial Function in Diabetes

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

Towards a personalised approach in exercise-based cardiovascular rehabilitation: How can translational research help? A ‘call to action’ from the Section on Secondary Prevention and Cardiac Rehabilitation of the European Association of Preventive Cardiology

The benefit of regular physical activity and exercise training for the prevention of cardiovascular and metabolic diseases is undisputed. Many molecular mechanisms mediating exercise effects have been deciphered. Personalised exercise prescription can help patients in achieving their individual greatest benefit from an exercise-based cardiovascular rehabilitation programme. Yet, we still struggle to provide truly personalised exercise prescriptions to our patients. In this position paper, we address novel basic and translational research concepts that can help us understand the principles underlying the inter-individual differences in the response to exercise, and identify early on who would most likely benefit from which exercise intervention. This includes hereditary, non-hereditary and sex-specific concepts. Recent insights have helped us to take on a more holistic view, integrating exercise-mediated molecular mechanisms with those influenced by metabolism and immunity. Unfortunately, while the outline is recognisable, many details are still lacking to turn the understanding of a concept into a roadmap ready to be used in clinical routine. This position paper therefore also investigates perspectives on how the advent of ‘big data’ and the use of animal models could help unravel inter-individual responses to exercise parameters and thus influence hypothesis-building for translational research in exercise-based cardiovascular rehabilitation

Physical and Functional Interaction of NCX1 and EAAC1 Transporters Leading to Glutamate-Enhanced ATP Production in Brain Mitochondria

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production

Mitochondrial function after global cardiac ischemia and reperfusion: Influences of organelle isolation protocols

Dog hearts were made globally ischemic for 1 hr at normothermia, at 28°C, or at normothermia after perfusion with a hyperkalemic cardioplegia solution. After 1 hr of reperfusion mitochondria were isolated from each heart using three protocols involving: processing (homogenization and centrifugation) exclusively in KCl, Tris-EDTA plus albumin (KEA) solution; homogenizing in KEA but washing mitochondria in EDTA-depleted media (KA); or processing exclusively in EDTA-free medium.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41745/1/395_2005_Article_BF01907770.pd

Initial Polymerization Sites Indicated During Mitochondrial Oxidation of Diaminobenzidine after Toluene Treatment

Numerous studies have dealt with the cytochemical localization of cytochrome oxidase via cytochrome c. More recent studies have dealt with indicating initial foci of this reaction by altering incubation pH (1) or postosmication procedure (2,3). The following study is an attempt to locate such foci by altering membrane permeability. It is thought that such alterations within the limits of maintaining morphological integrity of the membranes will ease the entry of exogenous substrates resulting in a much quicker oxidation and subsequently a more precise definition of the oxidative reaction.The diaminobenzidine (DAB) method of Seligman et al. (4) was used. Minced pieces of rat liver were incubated for 1 hr following toluene treatment (5,6). Experimental variations consisted of incubating fixed or unfixed tissues treated with toluene and unfixed tissues treated with toluene and subsequently fixed.</jats:p

A Na+-Ca2+ exchange process in isolated sarcolemmal membranes of mesenteric arteries from WKY and SHR rats

The existence of a Na+-Ca2+ exchange process in cell membrane vesicles isolated from mesenteric arteries of Wistar-Kyoto normotensive (WKY) and spontaneously hypertensive (SHR) rats was investigated. Membranes from cleaned mesenteric arteries were isolated by sucrose density gradient centrifugation, which yielded three distinct membrane fractions. The lighter membrane fraction of both WKY and SHR rats was enriched in 5'-nucleotidase activity, a marker for cell membrane, by about 10-fold, based on the activity in the homogenate, and was higher in membranes of SHR compared with WKY rats. Ouabain-sensitive Na+-K+-ATPase activity, another marker for cell membrane, was also concentrated in the lighter membrane fraction and was lower in the membranes of SHR compared with WKY rats. Higher activities of 5'-nucleotidase and Na+-K+-ATPase of both WKY and SHR rats was taken as evidence that the lighter membrane fraction was enriched in plasma membrane. Electron microscopic examination indicated that the membranes were in vesicular form. When the vesicles were loaded with Na+, a time-dependent uptake of Ca2+ was observed if the assay was carried out in high potassium to create a Na+ concentration gradient across the membrane of the vesicles. Very little Ca2+ uptake was observed when the vesicles were loaded with K+ or when the uptake of Ca2+ was carried out under conditions in which the Na+ gradient across the vesicle membranes was reduced. Ca2+ uptake in Na+-loaded vesicles of SHR rats was only slightly increased compared with WKY rats. The data indicate that a Na+-Ca2+ exchange process exists in the cell membrane of rat mesenteric arteries.</jats:p

An Attempt at the Cytochemical Localization of Citrate Synthase in Rat Heart Muscle

Most of the Krebs cycle enzymes are believed to exist in ‘soluble’ form in the mitochondrial matrix except succinate dehydrogenase which is a part of the inner membrane (1). Citrate synthase, one of the Krebs cycle enzymes, is present in rat liver, kidney and heart mitochondria proportional to the amount of the inner membrane area rather than the matrix volume (2). Recent studies on model systems support the idea that the ‘soluble’ Krebs cycle enzymes are probably localized on the inner surface (matrix side) of the inner membrane (3-5). Localization of these enzymes in mitochondria by cytochemical or immunocytochemical methods would be crucial to determine the validity of the above hypothesis. The present study describes cytochemical localization of citrate synthase in rat heart muscle.</jats:p