Early hemodynamic and biochemical changes in overloaded swine ventricle.

The present study was undertaken to investigate, in an animal model, the relationship between sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) activity, phospholamban phosphorylation, acylphosphatase activity, and hemodynamic changes that occur in the early phase of pressure overload. In 54 study-group pigs, weighing 40±5 kg each, an aortic stenosis was created with a band of umbilical tape tied around the aorta; 18 sham-operated pigs formed our control group. Eight animals (6 study and 2 control) were randomly assigned to each experimental time (0.5, 3, 6, 12, 24, 48, 72, 96, and 168 hr). All indices of left ventricular function declined significantly, with a peak at 6 hr and a return to baseline at 168 hr. At each observational time, SERCA2a activity, Ca2+ uptake, and acylphosphatase activity rose significantly, with a maximum increase at 6 hr. These changes indicated a higher expression of these proteins; conversely, phospholamban did not show significant changes in its concentration or in its phosphorylation status. Nuclear proto-oncogene c-fos expression rose at 6 hr. A strong inverse correlation was found when Ca2+-ATPase activity, Ca2+-ATPase expression, Ca2+ uptake, and acylphosphatase were compared with indices of systolic function. In our model of induced pressure overload, an initial phase of depressed myocardial contractility was accompanied by an increased sarcoplasmic reticulum function and by higher Ca2+-ATPase and Ca2+ uptake activities mediated by acylphosphatase. This new finding of Ca2+ homeostasis might indicate a compensatory mechanism for mechanical stress. Further studies are needed to confirm our findings.

Cellular retrograde cardiomyoplasty and relaxin therapy for postischemic myocardial repair in a rat model.

We sought to determine whether skeletal myoblasts, wild-type or engineered to express relaxin, might improve myocardial viability and performance in a rat model of chronic myocardial infarction. Our purpose was to investigate a potential new therapy for heart failure. From October 2005 through September 2009, we surgically induced acute myocardial infarction in 80 male Wistar rats. Thirty days after surgery, the rats underwent reoperation for the retrograde coronary venous infusion of skeletal myoblasts, relaxin, or both. The animals were randomly assigned to 4 experimental groups: R1 (the control group, which underwent saline-solution infusion), R2 (systemic relaxin therapy), R3 (myoblast infusion), and R4 (myoblast infusion and systemic relaxin therapy). Echocardiography, positron emission tomography, and cellular and histologic analysis were performed at 4 established time points. Mortality rates were similar among the groups. Postinfarction echocardiographic evaluation revealed similar left ventricular dysfunction. Viable myocardium, evaluated with positron emission tomography, was analogous. After therapy, the echocardiographic values of cardiac function improved significantly (P<0.05) in all groups except R1. Myocardial viability volume increased significantly in groups R3 and R4 (P<0.05) but was unchanged in groups R2 and R1. In group R4, the echocardiographic and positron emission tomographic results improved significantly (P<0.001). Histologic analysis showed that myoblasts settled in regions of ischemic scarring, especially when combined with relaxin. The retrograde venous route is safe, effective, and clinically feasible for cell delivery. Myoblasts and relaxin are better than either alone in terms of myocardial viability and performance improvement.

Skeletal myoblasts overexpressing relaxin improve differentiation and communication of primary murine cardiomyocyte cell cultures.

The possibility that resident myocardial progenitor cells may be re-activated by transplantation of exogenous stem cells into the post-infarcted heart has been suggested as a possible mechanism to explain the heart's functional improvement after stem cell therapy. Here we studied whether differentiation of mouse neonatal immature cardiomyocytes in vitro was influenced by mouse skeletal myoblasts C2C12, wild type or engineered to secrete the cardiotropic hormone relaxin. The cultured cardiomyocytes formed spontaneously beating clusters and temporally exhibited cardiac immunophenotypical (cKit, atrial natriuretic peptide, troponin T, connexin-43, HCN4) and electrical features (inward voltage-dependent Na(+), T- and L-type Ca(2+) currents, outward and inward K(+) currents, I(f) pacemaker current). These clusters were functionally connected through nanotubular structures and undifferentiated cardiac cells in the form of flattened stripes, bridging the clusters through connexin-43-containing gap junctions. These findings suggested the existence of long distance cell-to-cell communications among the cardiomyocyte aggregates involved in the intercellular transfer of Ca(2+) signals and organelles, likely required for coordination of myocardial differentiation. Co-presence of the myoblasts greatly increased cardiomyocyte differentiation and the amount of intercellular connections. In fact, these cells formed a structural support guiding elongation of nanotubules and stripe-like cells. The secretion of relaxin by the engineered myoblasts accelerated and enhanced the cardiomyogenic potential of the co-culture. These findings underscore the possibility that grafted myoblasts and cardiotropic factors, such as relaxin, may influence regeneration of resident immature cardiac cells, thus adding a tile to the mosaic of mechanisms involved in the functional benefits of cell transplantation for cardiac repair.

Sphingosine 1-phosphate induces Ca2+ transients and cytoskeletal rearrangement in C2C12 myoblastic cells.

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+ concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349-357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+ channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+ channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.

Concomitant effect of topical ubiquinone Q10 and vitamin E to prevent keratocyte apoptosis after excimer laser photoablation in rabbits.

PURPOSE: To investigate in vivo whether ubiquinone Q10 together with vitamin E protects rabbit corneas from keratocyte apoptosis after excimer laser irradiation. METHODS: Photorefractive keratectomy (PRK) was performed in both eyes of three New Zealand white rabbits. During 3 days before surgery, each right eye received four-times-daily instillation of an eye-drop solution containing ubiquinone Q10 0.20% and vitamin E 0.04%; each left eye was treated with a solution that did not contain ubiquinone or vitamin E. The central cornea was analyzed after surgery using the in situ end labelling (ISEL) technique of nicked DNA to detect DNA fragmentation. To determine the number of ISEL positive nuclei, an average of 70 random microscopic fields (five for each de-epithelialized tissue section) of 138,000 mu2 were examined in the right and left cornea samples at 250X by two different observers. RESULTS: Light microscopic examination of the sections from corneas treated before PRK showed that cells committed to apoptosis by PRK were about 50% compared to those of untreated controls. CONCLUSION: Treatment of rabbit eyes before PRK with ubiquinone Q10 lowered the number of apoptotic events.