Effects of aging on the cardiac remodeling induced by chronic high-altitude hypoxia in rat.

Effects of chronic high-altitude hypoxia on the remodeling of right ventricle were examined in three age groups of rats: 2, 6, and 18 mo. The extent of right ventricular (RV) hypertrophy (RVH) showed an age-associated diminution. RV cell size and pericellular fibrosis showed a significant increase in the 2- and 6-mo-old exposed rats but not in the 18-mo-old exposed rats compared with control. A hyperplasic response was underscored in the three exposed age groups but appeared less pronounced in the 18-mo-old rats. A significant decrease in the transient outward potassium current (Ito) density was observed in RV cell only in the 2-mo-old exposed group compared with the control group. In the control group, there was a clear tendency for Ito density to decrease as a function of age. The sustained outward current density was modified neither by the hypoxia condition nor by the age. Neither the cytochrome c oxidase activity nor the heat shock protein 72 content in the RV was altered after hypoxic exposure regardless of age. The norepinephrine content in the RV was significantly decreased in each age group exposed to hypoxia when compared with their age-matched control group. Our findings indicate that the remodeling (at morphological and electrophysiological levels) induced by chronic hypoxia in the RV can be decreased by the natural aging process.

Reversibility of electrophysiological changes induced by chronic high-altitude hypoxia in adult rat heart.

Recent studies indicate that regression of left ventricular hypertrophy normalizes membrane ionic current abnormalities. This work was designed to determine whether regression of right ventricular hypertrophy induced by permanent high-altitude exposure (4,500 m, 20 days) in adult rats also normalizes changes of ventricular myocyte electrophysiology. According to the current data, prolonged action potential, decreased transient outward current density, and increased inward sodium/calcium exchange current density normalized 20 days after the end of altitude exposure, whereas right ventricular hypertrophy evidenced by both the right ventricular weight-to-heart weight ratio and the right ventricular free wall thickness measurement normalized 40 days after the end of altitude exposure. This morphological normalization occurred at both the level of muscular tissue, as shown by the decrease toward control values of some myocyte parameters (perimeter, capacitance, and width), and the level of the interstitial collagenous connective tissue. In the chronic high-altitude hypoxia model, the regression of right ventricular hypertrophy would not be a prerequisite for normalization of ventricular electrophysiological abnormalities.

Voltage-gated sodium channel (SkM1) content in dystrophin-deficient muscle.

The membrane cytoskeleton is increasingly considered as both an anchor and a functional modulator for ion channels. The cytoskeletal disruptions that occur in the absence of dystrophin led us to investigate the voltage-gated sodium channel (SkM1) content in the extensor digitorum longus (EDL) muscle of the dystrophin-deficient mdx mouse. Levels of SkM1 mRNA were determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). A C-terminal portion of the mouse-specific SkM1 alpha-subunit cDNA (mScn4a) was identified first. SkM1 mRNA levels were as abundant in mdx as in normal muscle, thus suggesting that the transcriptional rate of SkM1 remains unchanged in mdx muscle. However, SkMI density in the extrajunctional sarcolemma was shown to be significantly reduced in mdx muscle, using confocal immunofluorescence image analysis. This decrease was found to be associated with a reduction in the number of SkM1-rich fast-twitch IIb fibres in mdx muscle. In addition, lowered SkM1 sarcolemmal labelling was found in all mdx fibres regardless of their metabolic type. These results suggest the existence of a perturbation of SkM1 anchorage to the plasma membrane. Such an alteration is likely to be related to the 50% decrease in mdx muscle of the dystrophin-associated syntrophins, which are presumed to be involved in SkM1 anchorage. However, the moderate reduction in SkM1 density (-12.7%) observed in mdx muscle argues in favour of a non-exclusive role of syntrophins in SkM1 anchorage and suggests that other membrane-associated proteins are probably also involved.

Supramolecular organization of the subsarcolemmal cytoskeleton of adult skeletal muscle fibers. A review.

This review is focused on the composition and organization of the junctional subsarcolemmal cytoskeleton of adult muscle fibers. The cytoskeleton of muscle fibers is organized in functionally distinct compartments and the subsarcolemmal cytoskeleton itself can be broadly divided into junctional (myotendinous junction, neuromuscular junction and costameres) and non-junctional domains. In junctional zones three different multimolecular cytoskeletal complexes coexist: the focal adhesion-type, the spectrin-based and the dystrophin vs utrophin-based membrane skeleton systems. These complexes extend over several levels, from intracytoplasmic to subsarcolemmal and transmembranous; their common feature is the anchorage of actin filaments emanating from the intracytoplasmic level. The different cytoskeletal proteins, their putative roles and their interactions with various signaling pathways are presented here in detail. The subsarcolemmal cytoskeleton complexes are thought to play distinct physiological roles (membrane stabilization, force transmission to extracellular matrix, ionic channel anchorage, etc) but their colocalization on the three sarcolemmal junctional domains strongly suggests interrelated or common functions.

Visualization of the subsarcolemmal cytoskeleton network of mouse skeletal muscle cells by en face views and application to immunoelectron localization of dystrophin.

The ultrastructural organization of the highly interconnected filamentous network underneath the sarcolemma as well as the role played by the muscle protein dystrophin within this cytoskeleton remain yet unclear. More accurate information has been obtained by using a method which provides three-dimensional en face views of large membrane areas applied to mouse cultured myotubes and isolated adult skeletal muscle fibres. Two levels have been distinguished in the cytoskeleton underlying the sarcolemma: the submembranous level, partly integrated into the membrane, and the cortical level, invading the proximal cytoplasmic space. Few differences have been found between the membrane cytoskeletons of myotubes issued from 14-day-old cultures and those of adult fibres. The comparison was done with cells where dystrophin is missing (mdx mouse muscle): surprisingly, the lack of dystrophin does not induce obvious or dramatic ultrastructural disorganization, either in the cortical cytoskeletal network or in the submembranous one. Immunogold labelling of either the central-rod or the C-terminal domain of dystrophin is not located among the cortical network. This study provides additional data on the spatial ordering of subsarcolemmal cytoskeletal elements: dystrophin does not appear as a filamentous structure entirely located among subsarcolemmal cytoskeleton but seems partly embedded in membranous material.

Inward barium current and excitation-contraction coupling in frog twitch muscle fibres.

The role of barium ions in excitation-contraction coupling was studied in single isolated frog semitendinosus fibres. Simultaneous recordings of membrane currents and contraction under voltage-clamp conditions in a sucrose-vaseline gap device show that barium ions have a reversible inhibiting effect on contraction. This inhibiting action was correlated to the entry of barium ions via the DHP-sensitive tubular calcium channel. Cytological observations and X-ray microanalysis performed on the fibres used in the electrophysiological experiments indicate that barium ions do not accumulate in the junctional sarcoplasmic reticulum; they can freely diffuse in the intermyofibrillar space and they accumulate in mitochondria. Calcium release experiments performed on isolated sarcoplasmic reticulum vesicles show that barium ions are not able to induce calcium release from calcium-loaded vesicles, they behave as calcium release inhibitors. These results are discussed in relation with the possible role of the slow Ca current in excitation-contraction coupling.

Degradation of metal-labeled collagen implants: ultrastructural and X-ray microanalysis.

Three different metal salts, silver nitrate, uranyl acetate and lead citrate, are mixed with a collagen gel to produce 3 metal/collagen sponges. These sponges were implanted subcutaneously in the rat and samples harvested after 5 days of implantation. TEM observation shows that sponges are degraded and digested by macrophages, polymorphonuclear cells (PMN) and fibroblasts. We have observed that the location of the precipitates differs according to the metal added to the collagen. Lead precipitates stay longer on the collagen mesh while silver precipitates, after 5 days, are soon digested and are found in phagosomes of macrophages. Uranium precipitates are digested with the collagen and uranium/collagen associated pictures are seen in phagolysosomes. Metal precipitates accumulated in phagolysosomes of macrophagic cells are recognized by X-ray microanalysis. The degradation process of implanted collagen is discussed.

X-ray microanalysis of calcium containing organelles in resin embedded tissue.

The localization of calcium in cell organelles at the electron microscope level is often achieved through cytochemical techniques, and verified by X-ray microanalysis. Various methods have been used to cytochemically detect calcium or calcium-binding sites: calcium loading, calcium substitution by strontium, barium, or even lead, and calcium precipitation by oxalate, phosphate, fluoride, or pyroantimonate. Their results may have heuristic value, particularly in preliminary studies of poorly known cell types. A complementary and more physiological approach is offered by quantitative measurement of the total calcium content of organelles after cryofixation. Resin embedding is less demanding than cryomicrotomy and gives better images: it can be used after cryosubstitution in the presence of oxalic acid. This technique was tested, and applied to several cell types.

Increase in the calcium content of cardiac tissue after postfixation with osmium tetroxide.

The concentration of osmium has been measured by destructive chemical analysis in glutaraldehyde fixed heart tissue postfixed with osmium tetroxide and embedded in epoxy resin. After such treatment, the mean atomic number of the specimen (Z) is close to 10, which permits a quantitative analysis of calcium (Ca) by the continuum method, using Z2/A as a correcting factor (A: atomic weight). Wavelength-dispersive X-ray microanalysis has been used to determine the Ca concentration of frog cardiac tissue fixed in glutaraldehyde and embedded in resin. These measurements have been repeated on tissue postfixed in osmium tetroxide; contrary to expectations, the apparent Ca concentration is much higher in osmium treated than in nontreated tissue. However, this result is observed with OsO4 solutions prepared in glass, not with solutions prepared in plastic. It is shown by energy dispersive X-ray analysis of droplets that OsO4 solutions prepared in glass contain large amounts of calcium, potassium and silicon. Care must be taken in preparing OsO4 fixatives when the fixed tissues are to be subjected to X-ray microanalysis of such elements as Ca or Si.

Quantitative X-ray microanalysis of calcium with the Camebax-TEM system in frozen, freeze-substituted and resin-embedded tissue sections. Application to molluscan glio-interstitial granules.

The relevance of the continuum method for a quantitative X-ray microanalysis of epon embedded tissue sections in the particular conditions offered by the Camebax-TEM system was tested and an improved model of specimen holder is proposed. The absolute calcium concentration [Ca] of membrane-bound intracellular glio-interstitial granules was determined by X-ray microanalysis in transmission electron microscopy of Mytilus retractor muscle. The Ca peak and background values were measured by the wavelength-dispersive spectrometer of the Camebax; the mass thickness of the section was recorded simultaneously with an added energy-dispersive detector. The tissue was frozen at approximately equal to 77 K in a mixture of liquid propane and butane, freeze-substituted in the presence of oxalic acid and embedded in epoxy resin. The calcium concentration of glio-interstitial granules can be as high as 180 mmol.kg-1 of epoxy-embedded tissue, with an average of 40 mmol.kg-1. The sampling of the data through repeated experiments is discussed and it is proposed that the cell would be the main level of variation. The Ca content of glio-interstitial granules is significantly lower in the tissues of animals submitted to high-potassium artificial sea-water for 10 min. This finding was predicted by the hypothesis that glio-interstitial tissue is a regulator of calcium concentration in extracellular spaces.