Ultrastructural remodelling of slow skeletal muscle fibres in creatine kinase deficient mice: a quantitative study.

Creatine kinase content, isoform distribution, and participation in energy transfer are muscle type specific. We analysed ultrastructural changes in slow muscle fibres of soleus due to invalidation of creatine kinase (CK) to reveal a difference in the remodelling strategy in comparison with fast muscle fibres of gastrocnemius published previously. We have employed the stereological method of vertical sections and electron microscopy of soleus muscles of wild type (WT) and CK-/- mice. The mitochondrial volume density was 1.4× higher but that of sarcoplasmic reticulum (SR) was almost 5× lower in slow CK-/- muscles fibres than in WT fibres. The volume density of terminal cisterns and of t-tubules was also lower in CK-/- than in WT fibres. The analysis of organelle environment revealed increased neighbourhood of mitochondria and A-bands that resulted from the decreased volume density of SR, from relocation of mitochondria along myofibrils, and from intrusion of mitochondria to myofibrils. These processes direct ATP supply closer to the contractile machinery. The decreased interaction between mitochondria and SR suggests reduced dependence of calcium uptake on oxidative ATP production. In conclusion, the architecture of skeletal muscle cells is under control of a cellular program that optimizes energy utilization specifically for a given muscle type.

Role of the HBx oncoprotein in carbonic anhydrase 9 induction.

Carbonic anhydrase 9 (CA9), as one of the most hypoxia-responsive genes, has been associated almost exclusively with hypoxic tumors. Its principal role is in pH regulation which helps tumor cells overcome intracellular acidosis and survive extended periods of time with low oxygen. Hypoxia-inducible factor 1 (HIF-1) is the main transcriptional activator of CA9. Hepatitis B virus X protein (HBx) has been shown to increase the transcriptional activity of HIF-1. HBx is often expressed from the gene integrated in the hepatocytes infected persistently and contributes significantly to alterations in host gene expression that can lead to the development of hepatocellular carcinoma (HCC) associated with Hepatitis B virus (HBV). The aim of this study was to determine the effect of HBx on expression of CA9. Transient transfection of HBx led to an increase in the expression of CA9 as assessed by RT-PCR and Western blotting. HBx was able to increase CA9 promoter activity significantly in several cell lines. The effect was mediated via HIF-1 and a functional HRE element located -10/-3 bp upstream of the CA9 transcription initiation site. These data suggest that CA9 may be involved in the development of HCC by contributing to the survival of hepatocytes infected with HBV in liver tissue with fibrosis.

Architectural and functional remodeling of cardiac and skeletal muscle cells in mice lacking specific isoenzymes of creatine kinase.

Muscle is the major consumer of fuels and ATP in the body. Mitochondria and glycolytic complexes serve as the main energy production locations, while the highest energy demands are associated with the sarcoplasmic reticulum, myofibrillar compartments and plasma membrane. Creatine kinase (CK) is a dimeric protein, which is deeply involved in the production of high energy storage compounds. This enzyme reversibly phosphorylates creatine (Cr) to phosphocreatine (PCr), and it is also highly adapted to specialized muscle function. To date, four major isoenzymes of CK have been identified, two of which occur in the cytosol and two in mitochondria. Disruption of the phosphotransfer system induced by an absence of either the sarcomeric mitochondrial CK or cytosolic CK or both isoenzymes of CK (CK(-/-)) in muscle cells leads to morphological and functional adaptations towards preservation of muscle contractile abilities. Remodeling of the cell ultrastructure observed in CK(-/-) cardiomyocytes and glycolytic fibers was associated with direct transfer of energy from places of energy production to locations of energy utilization. This direct interaction among the organelles can maintain a high ATP/ADP ratio near the cellular ATPases when CK is not functionally active. This review summarizes the function and role of CK across different muscle cells in knockout mice.

Myocardial remodelling induced by repeated low doses of isoproterenol.

In the present work, the effect of isoproterenol on the electrical properties of the rat heart and on the cytoarchitecture of the surviving cardiomyocytes was studied. Myocardial remodelling was induced by the daily administration of 5 mg/kg isoproterenol (Iso) for 7 days. Administration resulted in a significant increase (52%) in the ratio of left ventricular weight to body weight. ECG voltage criteria confirmed the presence of left ventricular hypertrophy. QT interval prolongation by 23% and 58% was found in Iso rats and in the corresponding isolated hearts, respectively. Spontaneously beating Iso hearts had a higher incidence of dysrhythmias. The surviving cardiomyocytes showed an irregular shape with cytoplasmic processes rich in ribosomes and rough endoplasmic reticulum. In these regions, myofibril disorganization and mitochondrial fission were observed. A greatly increased incidence of caveolae was seen in the plasma membrane and in the mouth of t-tubules. The membranes of t-tubules showed vesiculation, especially near the dyads. Repeated administration of isoproterenol led to hypertrophy, characterized by the existence of myocytes with simultaneous signs of both mature and postnatally developing cardiomyocytes. Structural microheterogeneities at the level of individual cells may represent one of the factors leading to electrical imbalance in the myocardial tissue remodelled by isoproterenol.

Vincristine-induced overexpression of P-glycoprotein in L1210 cells is associated with remodeling of cell surface saccharides.

Multidrug resistance of murine leukemic cell line L1210/VCR (R), obtained by adaptation of parental L1210 cells (S) on vincristine, is associated with overexpression of P glycoprotein (P-gp, the ATP-dependent drug efflux pump). Previously, we found that cytochemical staining of negatively charged cell surface binding sites (probably sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of S cells. This is in contrast to R cells and L1210/VCR cells cultured in the presence of vincristine during the last cultivation prior to the experiment (V cells), where the RR layer was either reduced or absent. In the current paper, we observed differences in the interactions of S, R and V cells with Concanavalin A (ConA) and tomato lectin (lycopersicum esculentum agglutinin, LEA). ConA bound and induced cell damage more effectively in S cells than in R or V cells. Both of these effects could be prevented by methyl-manopyranose, but not by N-acetylglucosamine. In contrast, LEA lectin preferentially bound to R and V cells. While LEA agglutinated cells more effectively than ConA, it did not cause cell damage comparable to ConA. Binding of LEA to the cell surface could be prevented by chitooligosaccharides. Both LEA and ConA failed to identify P-gp in lectin blots. Thus, changes in ConA and LEA interactions are not caused by massive expression of P-gp in the plasma membrane and the consequent exposure of the inner saccharides to the external side of the plasma membrane.Taken together, the above facts suggest that S cells differ from R and V cells in the composition of cell surface glycosides not directly linked to P-gp.

Evaluation of changes in secretory granules of atrial myocytes: a morphometric approach.

OBJECTIVE: To evaluate the sensitivity and applicability of stereologic analysis for estimating changes in the secretory granule content of atrial myocytes. STUDY DESIGN: The content of secretory granules in the right atrial myocytes of Sprague-Dawley (SD) and Lewis (LW) rats was assessed using a stereologic analysis of electron microscopic images under control conditions and in response to forced wheel running. RESULTS: Volume density analysis revealed significant heterogeneity in the granule content of different strains of rats. The content of the dark secretory granules was significantly lower in control LW compared with control SD rats. The difference in pale granule content was opposite but less pronounced. Forced wheel running did not elicit statistically significant changes in the granule volume density. However, it changed significantly the number of dark granules in each rat strain, albeit in opposite directions, most likely due to changes in the number of small dark granules. No change was observed in the case of pale granules. This suggests higher sensitivity of dark granules to enhanced physical load. CONCLUSION: Both the volume and the number of secretory granules should be estimated in parallel to reveal responses of the atrial secretory system to different internal or external stimuli.