Protective effects of green tea against hypoxia-induced cytoskeletal alterations in cultured cardiomyocytes.

Introduction. Cardiac ischemia-reperfusion injury results in oxidative stress and poor physiological recovery. Episodes of hypoxia-reoxygenation (H/R) cause some subtle functional and structural alterations in sarcolemma, mithocondria, sarcoplasmic reticulum, nucleus, as well as cytoskeleton (1). In this report, by using cultured rat cardiomyocytes and laser confocal microscopy we have verified the possibility to counteract cytoskeleton alterations induced by H/R with the supplementation of an antioxidant agent, a green tea extract (GTE), and compared its effects to those of -tocopherol (-TC). Moreover the effects of GTE on cell viability and cytosolic antioxidant activity have been evaluated. H/R induced myocardial damage occurs as histological alterations such as degeneration and disorganization of cytoskeleton and nuclear structural integrity. GTE supplementation shows protective effects on cardiomyocyte cytoarchitecture and viability, due to GTE ability to increase cytosolic antioxidant activity. Materials and Methods. Primary heart cell cultures were obtained by isolation of cardiomyocytes from the ventricles of 2–4 day old Wistar rats. In some dishes, 50 μg/mL GTE or 20 μM -TC were added to the culture medium 24 hours before the induction of hypoxia. Cell viability and cellular damage elicited by H/R treatment was evaluated by measuring MTT reduction. In both normoxic condition and H/R, cytosolic antioxidant activity was determined in cardiomyocytes using the method based on the ability of the antioxidant molecules in the sample to reduce the radical cation of ABTS, and measured as quenching of the absorbance at 740 nm. The effects of hypoxia on the microtubule network (MT) and on the nuclear morphology were investigated by fluorescence and immunofluorescence methods. MT was stained and integrity, in the absence or presence of GTE or -TC, was observed by confocal microscopy. Results. The ability of cardiomyocytes to reduce MTT was drastically reduced after H/R (67.32 ± 4.52%, p<0.05). GTE supplementation completely maintained cell viability at values similar to that of normoxic cells. -TC supplementation showed a significant increase in cell survival following H/R, but the values were still lower than in normoxic cells. GTE and -TC significantly increased TEAA in control cells, but to a different extent, GTE being more effective than -TC. H/R caused a significant decrease in TEAA levels with respect to normoxic cardiomyocytes. GTE was able to completely restore the TEAA values at levels significantly different from H/R cardiomyocytes and comparable to normoxic cells. Alpha-TC supplementation was only able to partially restore TEAA levels after H/R, but the values were significantly lower than in normoxic cells. Furthermore, as shown by confocal microscopy, GTE supplementation protected the cells from the damages to nuclear morphology and cytoskeleton caused by oxygen readmission. In particular, in H/R cardiomyocytes GTE fully restored the organization of actin fibres and MT observed in normoxic conditions. On the other hand -TC showed only a partial protection of the cytoskeleton architecture.