Risposta cellulare allo stress e proteine dello stress nei tessuti muscolari striati. Ruolo di Grp94

The purpose of this thesis was to study the stress response of muscle cells using in vivo and in vitro experimental procedures. Animal models which replicate cytoprotective (mild exercise training) or pathologic (muscle disuse atrophy) conditions were used to analyze the extent of the stress response and the cellular distribution of stress proteins in muscle tissues. Here we show that mild exercise training (up to 30m/min treadmill run for 1h/day) significantly increased Hsp70 expression in cardiac and skeletal muscles. Studies on the cellular distribution of this protein showed that the percentage of Hsp70-positive myofibers increased about 3-fold in trained fast muscles of the posterior rat hindlimb, compared to the sedentary ones (P<0.001), and involved a larger subset of both type 2A and intermediate type 2A/2X-myofibers (P<0.001), and vascular smooth muscle cells. Therefore, chronic induction of Hsp70 expression in rat skeletal muscles is not obligatory related to the slow fiber phenotype, but reveals the occurrence of a stress-response (Tarricone et al. 2008). In addition, training significantly increased protein levels of HO-1 in the myocardium, which contributed to the reduced infarct size occurring after ischemia-reperfusion (Marini et al. 2007). Muscle disuse atrophy is characterized by increased ROS production. Experimental muscle disuse atrophy was achieved in the rat soleus muscles by means of hindlimb unloading by tail-suspension for 4 to 21 days. Our results showed significant and transient upregulation of heme oxygenase-1 at 7 days of unloading, whereas the relative amounts of the Glucose- regulated proteins Grp94 and Grp78 decreased significantly after 14 days of unloading to about 50% and 75%, respectively, of the protein levels of the non-suspended age- and sexmatched controls. In addition, protein oxidation was significantly increased in unloaded atrophic muscles. It is therefore possible that the reduced levels of the ER chaperones which bind calcium, such as Grp94 or Grp78, might adversley affect calcium homeostasis and potentially enhance oxidative stress of disused myofibers and favour progression of muscle atrophy. In vitro studies were then performed in order to analyze more precisely the contribution of Grp94 protein levels to cytoprotection of muscle cells againstapoptosis and oxidative damage, and mechanistichally to the control of calcium homeostasis. Grp94 overexpression was achieved by means of both stable and transient transfer of Grp94 cDNA into the myogenic cell line C2C12. The effects of pharmacological treatments, which selectively increased Grp94 cellular levels, were also explored. Results show that Grp94 overexpression obtained by gene transfer, protected myogenic C2C12 cells against apoptosis induced by exposure to 1µM staurosporin. Caspase-3 activation was reduced by 40-80% compared to control clones (p<0.05). Additional experiments showed that the percentage of apoptotic death, revealed by TUNEL assay, in C2C12 cells transiently transfected with grp94 cDNA was reduced by 50%, compared to empty vector-transfected cells. Furthermore, Grp94 overexpression reduced the degree of protein oxidation induced by exposure to hydrogen peroxide. A comparable effect was obtained when cells were transiently exposed, 24 hour in advance, to curcumin, an antioxidant which is also a SERCA inhibitor. Through its latter property, curcumin was effective in inducing a mild ER response, which apparently upregulated Grp94, in a selective manner, without changing protein levels of other ER chaperones, such as Grp78, calreticulin and HO-1. Subsequent exposure of curcumin- pretreated cells to hydrogen peroxide indeed showed reduced degree of protein oxidation. In order to identify the molecular mechanism through which Grp94 exerts antiapoptotic and anti-oxidant cytoprotection, we investigated changes in calcium homeostasis occurring in Grp94 overexpressing cells. By means of the fluorescent Ca2+ probe fura-2, Grp94- overexpressing clones showed a reduction ranging from 40 to 80% in ciclopiazonic acid-released Ca2+ compared to control clones. Transient co-transfection of C2C12 with both Grp94 and the cDNA coding for the recombinant luminescent Ca2+ sensor aequorin specifically targeted to the ER showed a slight (20%), but significant, decrease in [Ca2+]er (p<0.01), compared to control, void vector-transfected cells. However, when transient co-transfections were performed in HeLa or HEK-293 cells, no significant difference in [Ca2+]er was observed between Grp94 overexpressing and control cells, suggesting that Grp94 overexpression did not directly affect ER Ca2+ load. Experiments of immunoprecipitation from wild type C2C12 cells, processed with chemical cross-linking before lysis, showed that SERCA2 co-immunoprecipitated with Grp94. The Grp94-SERCA2 co-immunoprecipitation was clearly evident in C2C12 cells, whereas it was barely detectable in HeLa and HEK-293 cells, despite the higher relative amount of both Grp94 and SERCA2 in these two latter cell lines. The possibility that Grp94 overexpression affected [Ca2+]er by interaction with SERCA2 was further suggested by the analysis of the ER calcium refilling traces: the SERCA2 activity showed a significant decrease (20%) (p<0.01) in Grp94 overexpressing C2C12 cells compared to controls, while no differences were found in HeLa and HEK293. The possibility that Grp94 overexpression exerts a cytoprotective role by reducing [Ca2+]er load of myogenic cells through a specific inhibitory interaction with SERCA2 is supposed.