1. Cardiac myocyte hypertrophy and impaired cytokinesis in nonmuscle myosin II-B-ablated and -deficient mice Nonmuscle myosin IIs are cytoskeletal proteins that play a role in cytokinesis, cell motility and cell morphology. Most vertebrate cells express nonmuscle myosin heavy chain II-A (NMHC II-A) and NMHC II-B, both of which are involved in cytokinesis. However cardiac myocytes only express NMHC II-B. We report evidence for a defect in cytokinesis in cardiac myocytes from mice in which NMHC II-B was ablated using homologous recombination. Cardiac myocytes. were hypertrophied as early as embryonic day (E) 12.5 (7.4 vs 4.9 m in diameter, B-/B- vs B+/B+, B+/B- controls). E12 mice showed increased binucleation (23 vs 1.2 % in situ and 55 vs 3.2 % in culture for B-/B- mice vs controls). In tissue sections of B-/B- hearts, binucleated myocytes were twice as large as the mononucleated cells (13 vs 6.8 micron), suggesting a failure in cytokinesis but not karyokinesis. This binucleation and cellular enlargement are reminiscent of those seen in Dictyostelium after ablation of NMHC II-B. Although there was no difference in the thickness of the ventricular wall between B-/B- and control hearts, the number of myocytes/unit area was lower in the former. Apoptosis (TUNEL method) was not different in the two groups. Of note was a lack of binucleation among the non-cardiac cells in B-/B- hearts, which contain NMHC II-A. In adult cardiac myocytes, NMHC II-B is localized in the Z-line and intercalated discs. We also generated two strains of hypomorphic mice which develop myocyte hypertrophy by either 11 months (88% reduction in NMHC II-B) or 1 months after birth (95% reduction). Unlike B-/B- mice, these mice show no increased binucleation. Myocyte hypertrophy in these hypomorphic mice most likely reflects defects in the integrity of the Z-line and intercalated discs and is manifested by increased myocyte disarray in mice with a 95% decrease in NMHC II-B. Thus two causes of hypertrophy are seen, depending on the occurrence of either deficiency or absence of NMHC II-B. B-/B- mice develop a novel form of myocyte hypertrophy due to a defect in cytokinesis. 2. Nitric oxide mediates myocyte apoptosis in experimental acute Chagasic myocarditis Apoptosis has been implicated in the pathogenesis of acute chagasic myocarditis. We have previously showed that in myocarditis, the extent of apoptosis in intracellular T. cruzi correlated with the intensity for inducible nitric oxide synthase (iNOS). iNOS is greatly increased in areas of inflammation in acute Chagasic myocarditis, suggesting that it plays a role (by generating NO) in inducing apoptosis in T. cruzi and host cells. To evaluate the role of NO in the pathogenesis and consequences of myocyte apoptosis in acute chagasic myocarditis, we infected inducible NO synthase knock-out (KO) and C57BL/6x 129sv (WT) mice with the Tulahuen strain of T. cruzi. Serial transthoracic were performed to measure left ventricular (LV) end-diastolic diameter (EDD), relative wall thickness (RWT) and fractional shortening (FS). At day 19 post-infection, compared with the KO infected mice, the WT infected mice showed larger LV EDD (3.5+/-0.3 vs 2.4+/-0.2 mm, p=0.01), lower RWT (0.4+/-0.1 vs 0.8+/-0.1, p=0.01), and lower SF (43+/-5 vs 56+/-4%, NS). The differences in LV EDD, RWT and SF between infected animals and their corresponding control mice were significantly greater in the WT than in the KO mice: EDD (+65+/-7 vs +7+/-5%, p less than 0.001), RWT (-42+/-5 vs +29+/-9, p less than 0.001) and SF (-30+/-5 vs -7+/-4%, p=0.01). At necropsy, myocardial inflammation and necrosis were more severe in WT than in KO mice. In addition, higher numbers of apoptotic LV myocytes by TUNEL assay were seen in WT than in KO mice (1.12+/-0.16 vs 0.61+/-0.09 per HPF 200X, p less than 0.01). In conclusion, apoptosis is a major form of myocyte death in acute chagasic myocarditis. It contributes to the adverse cardiac functional consequences of the disease and is mediated, in part, by NO. 3. Remodeling of cardiac collagen and extracellular matrix in Keshan disease Keshan disease is a cardiomyopathy related to deficiency of selenium in China and occurs in acute, subacute and chronic forms. The latter resembles dilated cardiomyopathy clinically. The present study explores the mechanisms of remodeling of the cardiac extracellular matrix in Keshan disease. Immunohistochemical staining techniques were performed on paraffin sections of formalin-fixed hearts from patients with subacute (n=5) and chronic (n=7) Keshan disease. Dual fluorescent labeling techniques were used to demonstrate the immunoreactivity (IR) for type I, III and IV collagen, matrix metalloproteinases (MMPs), including MMP-1, MMP-2, MMP-9, and membrane-type-1 MMP (MT-1-MMP), tissue inhibitors of MMPs (TIMP-1 and TIMP-2) and angiotensin-converting enzyme (ACE), followed by nuclear counterstaining with DAPI and examination by confocal microscopy. In subacute Keshan disease, the cardiac lesions were focal and characterized by myocyte loss and developing fibrosis. IR for collagen types I and IV and MMP-1, MMP-2, MMP-9, MT1-MMP and ACE was stronger than in normal control heart. The IR for TIMP-2 was increased in fibrotic areas. Staining for type III collagen was decreased, and the proportion of type I collagen was increased in the lesions. Type IV collagen was increased in some areas and partially lysed in others. In chronic Keshan disease, cardiac fibrosis was very severe and myocyte damage was less evident. The IR for type IV collagen was normal, but that for type I and type III collagen was still stronger than normal. However, the ratio of type I to type III collagen was similar to that in normal hearts. Staining for ACE, MMP-1, MMP-2, MMP-9 and MT-1-MMP was decreased, but still stronger than normal. In conclusion, remodeling of cardiac connective tissue in Keshan disease proceeds through a stage of MMP activation (induced by excessive amounts of ROS) and lysis of collagens, and a subsequent stage of fibrosis, normalization of MMP activity and increased activity of TIMPs.
