Intermembrane inclusions induced by anoxia in heart and skeletal muscle mitochondria.

Heart and skeletal muscle from rats of different ages were incubated in vitro in an oxygen-free medium supplied with substrates in order to investigate the effect of anoxia on muscle fine structure, particulary on the mitochondria. In skeletal muscle fibers anoxia has been found to induce changes similar to those previously described in ischemic muscles in vivo namely giant mitochondria, apparently derived by mitochondrial fusion, and intermembrane inclusions with a paracrystalline structure. The plate-like inclusions are mostly located in the intracristal spaces and are closely associated to cristal membranes even in markedly swollen mitochondria. Identical inclusions have been observed in cardiac muscle cells following anoxic injury, whereas they are never found in non-muscle cells such as endothelia, fibroblasts and nerve fibers. Cardiac and skeletal muscle fibers from newborn rats maintained in an oxygen-free medium show mitochondrial swelling but no intermembrane inclusions. The different response of mitochondria from developing vs adult striated muscle to anoxia may be due to changes during postnatal development in the quality or quantity of the protein component(s) involved in paracrystal formation.     

L-lactate oxidation by skeletal muscle mitochondria

1. Mitochondria isolated from rat skeletal muscle possess lactate dehydrogenase which is involved in direct oxidation of L-lactate in the presence of external NAD. 2. L-lactate oxidation can be stimulated in a reversible manner by ADP. 3. Mitochondrial lactate oxidation is sensitive to oxamate-inhibitor of LDH, alpha-cyano-3-hydroxy-cinnamate-pyruvate translocase inhibitor and respiratory chain inhibitors (rotenone, antimycin A, KCN). 4. In the same conditions the mitochondria did not oxidize pyruvate in the absence of malate, whereas, oxidize pyruvate plus external NADH in an uncoupling manner.     

High‐fat diet affects skeletal muscle mitochondria comparable to pressure overload‐induced heart failure

In heart failure, high‐fat diet (HFD) may exert beneficial effects on cardiac mitochondria and contractility. Skeletal muscle mitochondrial dysfunction in heart failure is associated with myopathy. However, it is not clear if HFD affects skeletal muscle mitochondria in heart failure as well. To induce heart failure, we used pressure overload (PO) in rats fed normal chow or HFD. Interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) from gastrocnemius were isolated and functionally characterized. With PO heart failure, maximal respiratory capacity was impaired in IFM but increased in SSM of gastrocnemius. Unexpectedly, HFD affected mitochondria comparably to PO. In combination, PO and HFD showed additive effects on mitochondrial subpopulations which were reflected by isolated complex activities. While PO impaired diastolic as well as systolic cardiac function and increased glucose tolerance, HFD did not affect cardiac function but decreased glucose tolerance. We conclude that HFD and PO heart failure have comparable effects leading to more severe impairment of IFM. Glucose tolerance seems not causally related to skeletal muscle mitochondrial dysfunction. The additive effects of HFD and PO may suggest accelerated skeletal muscle mitochondrial dysfunction when heart failure is accompanied with a diet containing high fat.     

脊椎动物胚胎骨骼肌的生成

肌肉发生的起始和生肌节的形成是胚胎肌肉发育中的两个关键事件。研究表明,肌肉发生的起始有赖于体节周围组织所产生的分泌因子的影响。这些组织包括体轴结构,侧板中胚层及体节正上方的外胚层,代表性的分子有Ｗｎｔｓ家族的一些成员以及Ｓｈｈ和ＢＭＰ－４;而生肌节的形成则首先依赖与分节化相关的基因,如ｄｅｌｔａ,ｈｅｒ１等的正常功能,分节之后也同样需在周围环境的作用之下形成生肌节。两栖类非洲爪蟾的肌肉发生有其特殊性。本文对这一领域中最近的研究进展作一综合介绍。     

Water movement from intracristal spaces in isolated liver mitochondria

When analyzing mitochondria isolated in a sucrose medium that had been embedded for thin sectioning according to one low denaturation embedding technique, large intracristal spaces were present in close to 90% of the mitochondria. The two crista membranes were closely apposed in only 40% of all cristae. When the mitochondria were transferred to an incubation medium, the percentage of mitochondria with intracristal spaces was reduced to 40%. About 90% of all cristae were lacking any space separating the two crista membranes. The presence of inorganic phosphate in the medium was required for the closing of the intracristal spaces. The percentage of cristae lacking an intracristal space remained the same after addition of substrate for respiration (state 4) and of ADP (state 3). Inhibition or uncoupling of respiration led to an increase in the percentage of intracristal spaces, showing that oxidative phosphorylation is required to maintain the crista membranes closely apposed. The appearance and disappearance of the intracristal spaces was an indication of water movements across the crista membranes. The mean volume of the mitochondria increased 33% when they were transferred from the sucrose medium to the incubation medium, showing that the removal of water from the cristae was not caused by a passive osmotic effect. Addition of substrate made the volume decrease by 28%. After further addition of ADP, the volume decreased another 23%. No change in volume was associated with inhibition or uncoupling of respiration. The observations revealed that water can move into or out of the cristae independently of water movement out from the entire mitochondrion. Therefore, the water moving out from or into the cristae is translocated across the cristae membrane. The observations are interpreted to reveal the presence of a mechanism that actively prevents water from accumulating in the crista membrane. This mechanism allows for a low water activity to be maintained within the membrane. The variations in the frequency of intracristal spaces occurred without any simultaneous changes in the width of the space appearing between the two surface membranes after isolation of the mitochondria. The observations, therefore, do not agree with the concept that there is an outer compartment that communicates freely with intracristal spaces.     

Carbonic anhydrase in guinea pig skeletal muscle mitochondria

The presence of carbonic anhydrase activity was demonstrated in guinea pig skeletal muscle mitochondria purified by Percoll gradient centrifugation such that contamination by sarcoplasmic reticulum vesicles was less than 5%. Assay of purified heavy sarcoplasmic reticulum vesicles for carbonic anhydrase activity showed these to have somewhat less activity than the mitochondria, so that any contribution by sarcoplasmic reticulum vesicles to mitochondrial activity would be negligible. In agreement with this observation, rabbit skeletal muscle mitochondria prepared by the Percoll method had no detectable activity. Assay of the guinea pig muscle mitochondrial enzyme activity in the presence of Triton X-100 showed a sixfold greater activity than in its absence, indicating a matrix location for the carbonic anhydrase. The enzyme is highly sensitive to the sulfonamide inhibitor ethoxzolamide, with Ki = 8.7 nM. The activation energy obtained from the rate constant for CO2 hydration, kenz with units (mg/ml)-1 s-1, over the range 4 to 37 degrees C was 12.8 kcal/mol. These properties are those expected for a carbonic anhydrase of the CA II class of isozymes, rather than for CA I, CA III, and the liver mitochondrial enzyme CA V.     

A novel potassium channel in skeletal muscle mitochondria

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.     

The effect of oncotic pressure on heart muscle mitochondria

The role of oncotic pressure (i.e. pressure created by non-penetrants of high molecular weight) in structural responses of mitochondria has been studied.

It has been found that treatment of beef of rabbit heart mitochondria by a synthetic non-penetrant of high molecular weight, polyvinyl pyrrolidone, induces a decrease in the intermembrane (intracristal) space and an increase in the matrix space of mitochondria. As a result, the appearance of the in vitro mitochondria proves to be similar to that of the in situ ones. If a Waring blender is used to homogenize the tissue, only a portion of the mitochondria respond to polyvinyl pyrrolidone. If a glass-Teflon homogenizer is used instead all the mitochondria prove responsive. The addition of 0.5 mM polyvinyl pyrrolidone is found to be sufficient for the effect to be observable.

In the presence of polyvinyl pyrrolidone, energy-dependent changes in mitochondrial structure can be demonstrated. The increase in matrix space by polyvinyl pyrrolidone treatment enlarges even more when an energy source, a penetrating weak acid and a penetrating cation are added. The size of the matrix increases in the following order: (1) de-energized mitochondria without polyvinyl pyrrolidone, (2) de-energized + polyvinyl pyrrolidone, (3) energized + polyvinyl pyrrolidone, (4) as (3) + phosphate (“twisted” configuration of cristae), (5) as (3) + phosphate + Ca²⁺. Structural changes resembling those indicated in points (2)–(5) are shown for mitochondria in the tissue, when pieces of rat diaphragm muscle treated with an uncoupler, phosphate, and Ca²⁺ were studied in conditions excluding anaerobiosis.

The effect of polyvinyl pyrrolidone is suggested to be due to it balancing the oncotic pressure created by high molecular weight compounds dissolved in the intermembrane water, which are incapable of penetrating the outer mitochondrial membrane. A concept is discussed considering mitochondrial structure changes as a function of the osmotic gradient across the inner membrane and the oncotic gradient across the outer membrane of mitochondria.     

Intramedullary pressure and matrix strain induced by oscillatory skeletal muscle stimulation and its potential in adaptation

Intramedullary pressure (ImP) and low-level bone strain induced by oscillatory muscle stimulation (MS) has the potential to mitigate bone loss induced by disuse osteopenia, i.e., hindlimb suspension (HLS). To test this hypothesis, we evaluated (a) MS-induced ImP and bone strain as function of stimulation frequency and (b) the adaptive responses to functional disuse, and disuse plus 1 and 20 Hz stimulation in vivo. Femoral ImP and bone strain generated by MS were measured in the frequencies of 1–100 Hz in four rats. Forty retired breeder rats were used for the in vivo HLS study. The quadriceps muscle was stimulated at frequencies of 1 and 20 Hz, 10 min/d for four weeks. The metaphyseal trabecular bone quantity and microstructure at the distal femur were evaluated using μCT, while bone formation indices were analyzed using histomorphometric technique. Oscillatory MS generated a maximum ImP of 45±9 mmHg at 20 Hz and produced a maximum matrix strain of 128±19 με at 10 Hz. Our analyses from the in vivo study showed that MS at 20 Hz was able to attenuate trabecular bone loss and partially maintain the microstructure induced by HLS. Conversely, there was no evidence of an adaptive effect of stimulation at 1 Hz on disused skeleton. The results suggested that oscillatory MS regulates fluid dynamics and mechanical strain in bone, which serves as a critical mediator of adaptation. These results clearly demonstrated the ability of MS in attenuating bone loss from the disuse osteopenia, which may hold potential in mitigating skeletal degradation imposed by conditions of disuse, and may serve as a biomechanical intervention in clinic application.