Zhi-ping Zong

Amiodarone minimizes experimental autoimmune myocarditis in rats

Amiodarone, a promising drug for the treatment of tachyarrythmias, was recently found to have immunomodulatory effects in vitro. We hypothesized that amiodarone would affect the immune system in vivo and examined the effect of amiodarone on myocarditis in rats. We induced experimental autoimmune myocarditis in rats by cardiac myosin immunization and treated the animals with an intraperitoneal injection of amiodarone at 25 mg/kg/every other day, 10 times after the induction of experimental autoimmune myocarditis. In the treated group, both microscopic and macroscopic examinations showed reduced heart weights, a mild and localized infiltration of inflammatory cells and fibrosis in the myocardium, and a mild congestion in the liver and lungs as compared with the control group. The phenotypic distribution of lymphocytes in peripheral blood showed a significant decrease in the CD4/CD8a ratio in the treated group, but not in the control group. The proportion of mast cells involved in inflammatory cell infiltration was lower in the treated group than the control group. In vitro, amiodarone inhibited the proliferation of mast cells by arresting them in the G2 phase of the cell cycle. These results indicated that amiodarone minimized the progression of experimental autoimmune myocarditis, suggesting a potential therapeutic role for amiodarone treatment in patients suffering from myocarditis, especially myocarditis complicated by cardiac arrhythmias. One possible mechanism by which amiodarone minimizes the progression of experimental autoimmune myocarditis may be to affect the immune system via the immunomodulatory effects on T cell and mast cell functions.

Involvement of protein kinase C in taxol-induced polyploidization in a cultured sarcoma cell line

Taxol was found to inhibit the proliferation and to induce the polyploidization of cultured methylcholanthrene-induced sarcoma cells (Meth-A cells). To investigate whether protein kinase C is involved in taxol-induced polyploidization, phorbol 12-myristate 13-acetate (PMA), which regulates the activity of protein kinase C, was used along with taxol to treat the cells. We found that PMA did not interfere with the proliferation and did not induce polyploidization by itself. However, at low concentration, taxol, which by itself did not induce polyploidization, clearly induced polyploidization in the presence of PMA. To explore the mechanism by which PMA potentiates polyploidization, the levels of the G1 checkpoint-related proteins cyclin E and cdk2, and those of the G2 checkpoint-related proteins cyclin B and cdc2 were determined by flow cytometry. We found that both G1 and G2 checkpoint-related proteins increased during the induction of polyploidization. To verify the relationship between protein kinase C and tubulin polymerization, flow cytometry was used to determine the total content of tubulin protein, and morphological observation was used to examine spindle organization. PMA did not affect the taxol-induced increase in tubulin protein, but markedly potentiated taxol-induced spindle disorganization. These findings suggest that protein kinase C plays an important role in regulating the induction of polyploidization in Meth-A cells.

Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells polyploidized by demecolcine

Abstract. The nuclear morphology of polyploidized cells was examined in V79 Chinese hamster cells polyploidized by demecolcine or K‐252a, inhibitors of spindle fibre formation and protein kinases, respectively. A variety of nuclear morphologies, including multinuclei, were observed in V79 cells polyploidized by demecolcine but not by K‐252a, which produced mononuclear cells. A lack of synchrony in the nuclear cycle was observed among nuclei in multinuclear polyploidized cells. Partial DNA fragmentation, defined as DNA fragmentation of a nucleus in a multinuclear cell, was detected using the TUNEL method in V79 cells polyploidized by demecolcine but not by K‐252a. Apoptosis occurred earlier in cell populations treated with demecolcine than in these treated with K‐252a once the drugs were removed from the medium, suggesting that polyploidized cells with separate nuclei tend to apoptose earlier than those with mononuclei.

Amelioration of myocarditis by statin through inhibiting cross-talk between antigen presenting cells and lymphocytes in rats

Statins, inhibitors of 3-hydroxy-3-methylglutary-coenzyme A (HMG-CoA) reductase, have been recognized as a new type of immunomodulator and reported to have anti-inflammatory effect. To investigate the effect of simvastatin, a lipophilic statin, on myocarditis, we explored whether simvastatin is able to inhibit experimental autoimmune myocarditis (EAM) and adoptive transfer of EAM in rats. We found that administration of simvastatin not only interfered with the development of EAM, but also inhibited the transfer. Antigen presenting cells (APCs) were proved to be important for the development of EAM. The ability of myocarditic splenocytes to transfer myocarditis was enhanced after co-culture with APCs. During co-culture of the myocarditic splenocytes and the APCs, simvastatin not only decreased percentages of CD28 expression in CD4-positive myocarditic splenocytes, and CD80 and CD86 expressions in APCs, but also inhibited the production of tumor necrosis factor (TNF)-partial differential in the CD4-positive myocarditic splenocytes and the APCs. These results indicate that simvastatin was able to ameliorate EAM through the inhibition of cross-talk between lymphocytes and APCs, suggesting beneficial role of simvastatin in the treatment of autoimmune myocarditis.

Methimazole Interferes with the Progression of Experimental Autoimmune Myocarditis in Rats

In order to ascertain whether methimazole, a drug commonly used for the treatment of hyperthyroidsim, interferes with the progression of autoimmune-mediated myocardial injury, we investigated the effect of methimazole on experimental autoimmune myocarditis (EAM) in rats. EAM was induced by immunization with porcine cardiac myosin. Methimazole administration markedly slowed the body weight growth in both normal and EAM rats, but did not induce morphologic change of cardiac tissue in normal rats. In EAM rats, macroscopic examination revealed discoloration of the cardiac surface, and histopathological examination by light microscopy showed extensive myocardial necrosis, infiltration by inflammatory cells and myocardial fibrosis. In the EAM rats treated with methimazole, the discolored areas on the cardiac surface were markedly diminished in size, and the myocardial necrosis, cellular infiltration and fibrosis were significantly less severe. To identify the mechanism responsible of this effect, we investigated the change of regulatory lymphocyte subsets in peripheral blood using an immunfluorescence technique with a flow cytometer. A decrease in the helper/suppressor T cell ratio as a result of the increased proportion of suppressor T cells and a decrease in the proportion of B cells were observed in normal rats after methimazole administration, and similar findings were made in the EAM rats treated with methimazole. These results indicate that methimazole interferes with the progression of EAM, and immunosuppression may, at least in part, be involved in the inhibitory effect of methimazole on EAM in rats.

Cell membrane changes of structure and function in protein kinase inhibitor-induced polyploid cells.

Exogenous cyclic AMP has been thought to be a chemical without marked pharmacological effect until now, as it is not capable of penetrating the cell membrane in most eucaryotic cells. The present study obtained results consistent with those of most previous studies, showing that exogenous cyclic AMP itself did not interfere with the cell cycle even at the high dose of 100 μM. However, it was found that K252a, a potent inhibitor of protein kinases including protein kinase C, induced DNA re‐replication, i.e. DNA synthesis at a elevated DNA ploidy in cells that had not undergone cytokinesis (leading to polyploidization), and that exogenous cyclic AMP markedly potentiated the K252a‐induced polyploidization at a very low dose similar to the effective dose of membrane‐permeable cyclic AMP analogue dibutyryl cyclic AMP. These findings suggested that the cell membrane changed during the formation of polyploid cells. This supposition was confirmed by scanning electron microscopy to observe structural changes and by determination of cellular attachment to investigate functional changes.

Growth hormone interferes with the progression of myocarditis in rats.

In this study, we investigated whether recombinant human growth hormone (rhGH) influences the progression of myocarditis. We induced experimental autoimmune myocarditis in F344 rats by subcutaneous injection of cardiac myosin, and divided the rats into three groups: (1) control group, saline injection; (2) pre-treated group, subcutaneous injection of rhGH (100 mIU/rat/day for 10 days) before induction of experimental autoimmune myocarditis; and (3) post-treated group, subcutaneous injection of rhGH (100 mIU/rat/day for 10 days) after induction of experimental autoimmune myocarditis. On the 35th day after induction of experimental autoimmune myocarditis, all rats were sacrificed and the hearts were examined. The increase in body weight was smaller in the control group than the pre-treated group and the rate of heart weight/body weight was larger in the control group than in the two treated groups. Histopathologically, rats in the control group showed multifocal infiltration by inflammatory cells, mainly neutrophils, lymphocytes and macrophages, extensive fibrosis, and a higher proportion of mast cells in the inflamed region. In contrast, rats in the two treated groups showed only minor changes. We found that rhGH did not influence the distribution of lymphocytes in peripheral blood in the three groups, and that rhGH induced G1 checkpoint dysfunction, thereby arresting the cell cycle in G1 and inhibiting the proliferation of mast cells in vitro. These findings suggest a possible role for mast cells in the progression of myocarditis and the rhGH may be a candidate for use as a new tool to treat myocarditis.