HSP90 and eNOS partially co-localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes.

BACKGROUND INFORMATION: Cultivation techniques promoting three-dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co-cultures to evaluate, on both two- and three-dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat-shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). RESULTS: Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co-culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localization coefficients, which indicated the highest correlation with respect to the other substrates. CONCLUSIONS: Our three-dimensional adult cardiomyocyte cultures are suitable for both analysing cell-ECM interactions at electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocyte phenotype.

Human recombinant vasostatin-1 may interfere with cell-extracellular matrix interactions.

Vasostatin-1 (VS-1), the N-terminal fragment derived from the cleavage of chromogranin A (CgA), has been shown to exert several biological activities on several tissues and organs. Recently, it has been reported that human recombinant VS-1 (STA-CGA1-78) may alter myocardial contractility in eel, frog, and rat hearts. In this article we have explored if STA-CGA1-78 can induce intracellular cascades interacting both with adhesion molecules and/or extracellular matrix (ECM), components, that is, involvement of the heat shock protein 90 (HSP90) and the endothelial NOS (eNOS), known to be implicated in signal transduction mechanisms affecting myocardial contractility. We used 3D cultured adult rat cardiomyocytes cultivated over fibronectin or fibroblasts or embedded in matrigel or collagen type I. Aurion-conjugated VS-1 (Au-STA-CGA1-78) has been used to identify possible sites of interaction of this molecule with the cell membrane. We found that in our 3D culture, cell-ECM interactions played a crucial role in the cellular localization of HSP90 as well as in the expression of eNOS. VS-1 appeared to modulate cell-ECM interactions, thereby remarkably leading to a different cellular localization of HSP90. Moreover, Au-STA-CGA1-78 was never detected inside the cell nor overlapping the plasma membrane, but nearby the outer side of the cardiomyocyte plasmalemma, at a particular distance, typical of integrins. On the whole, these data suggest that VS-1 does not have a classic receptor on the membrane but that integrins may represent a nonconventional VS-1 receptor modulating eNOS signaling pathway.

Cardiac stem cell research: an elephant in the room?

Heart disease is the leading cause of death in the industrialized world, and stem cell therapy seems to be a promising treatment for injured cardiac tissue. To reach this goal, the scientific community needs to find a good source of stem cells that can be used to obtain new myocardium in a very period range of time. Since there are many ethical and technical problems with using embryonic stem cells as a source of cells with cardiogenic potential, many laboratories have attempted to isolate potential cardiac stem cells from several tissues. The best candidates seem to be cardiac "progenitor" and/or "stem" cells, which can be isolated from subendocardial biopsies from the same patient or from embryonic and/or fetal myocardium. Regardless of the technique used to isolate and characterize these cells, it appears that the different cells isolated from adult myocardium to date are all phenotypic variations of a unique cell type that expresses several markers, such as c-Kit, CD34, MDR-1, Sca-1, CD45, nestin, or Isl-1, in various combinations.

The synovial joints of the human foot.

The human foot is considered an organ with an assortment of tissues with different morphological characteristics and well defined limits, but effectively has a simple functionality when static that becomes extremely complex when in movement. Its complex structure, comprised of an elastic and resistant skin covering a bone framework, joints, muscles, tendons, veins and nerves, can be compared to an efficient mechanical assembly. After a long and extraordinary evolutive journey, the human foot has undergone numerous changes to perfect its function; it has lost most of its grabbing function whilst gaining new characteristics that have ultimately allowed the modern man to stand upright. The complex articular structure of the human foot consists of thirty four synovial joints, of which eighteen have curved surfaces and sixteen plane surfaces. Following the criteria set by the systematic, radiological and clinical anatomy, the Authors contribute further to the current knowledge on the ankle, tarsal (anatomic subtalar, transverse tarsal, cuneonavicular, intercuneiform and cuneocuboid), tarsometatarsal, intermetatarsal, metatarsophalangeal and interphalangeal joints and dorsal, plantar and interosseous ligaments of the human foot. The articular lines of the transverse tarsal (Chopart) and tarsometatarsal (Lisfranc) joints are particularly interesting and not only from a surgical point of view; through a straightforward identification of few reference points, it is possible to find the medial and lateral extremities of the Chopart's and Lisfranc's lines, the former pinpoints the boundary between the hindfoot and midfoot and the latter between the midfoot and forefoot.

Effects of conjugated linoleic acid and endurance training on peripheral blood and bone marrow of trained mice.

Fat supplements, especially conjugated linoleic acid (CLA), are increasingly popular ergogenic aids among endurance athletes. To evaluate the importance of fat supplementation in the practice of endurance sports, we investigated the effects of CLA supplementation on body weight, muscle hypertrophy, peripheral blood composition, and bone marrow composition in healthy, young, endurance-trained mice. Young, healthy mice were subdivided into control, trained, and treated groups, according to their running attitudes. Training was performed over a period of 6 weeks on a treadmill, at a gradually increasing duration and speed. CLA-treated groups were gavaged with 0.425 mg x d(-1) CLA supplement for the entire training period. The exercise protocol induced a significant decrease in body weight (p < 0.003) and a consistent muscle hypertrophy (p < 0.003). A morphological evaluation of bone marrow from trained mice revealed an accelerated turnover of the erythroid lineage, i.e., a relative increase in proerythroblasts. Conjugated linoleic acid supplementation did not induce a further decrease in total body weights in either untrained or trained mice (p = 0.747), but induced a further increase in muscle hypertrophy in trained mice (p = 0.009). Furthermore, CLA feeding induced an important lymphopenia in peripheral blood of CLA-fed trained mice (p < or = 0.05). These findings suggest that CLA may improve the performance of endurance athletes by increasing muscle hypertrophy, and, at the same time, that it may cause oxidative stress damage, leading to a peripheral blood lymphopenia and a consequent neutrophilia as a defensive response. Despite the positive increase in muscle hypertrophy claimed by the pharmaceutics companies, we suggest that endurance athletes and those looking to improve their own skeletal muscle mass refrain from CLA supplementation, because it seems to intensify the oxidative stress caused by exhaustive exercise.