| Heart diseases are the leading causes of morbidity and mortality throughout the world. Accumulating costs from heart damage especially heart failure are no more than the quality of human life. Heart failure, as the terminal phase of cardiovascular diseases has complicated etiology and long-lasting course of disease. Myocardial remodeling is a major process linked closely to the heart failure, which mainly consists of cardiac hypertrophy, cardiac apoptosis and cardiac fibrosis. The first two pathological processes dominantly determine the development of heart failure. As a characteristic stage of myocardial remodeling, compensatory cardiac hypertrophy that maintains cardiac output by regulating volume-overloading and stress-overloading is characterized by pathological alterations in cardiomyocytes such as the extension of cell sizes, the increase of cellular protein synthesis and embryonic gene re-expression. On the other hand, many observations point to the cardiomyocytes death of apoptosis as an essential factor in the transition from compensatory cardiac hypertrophy to heart failure.At early phase of heart dysfunction, B-type natriuretic peptide, as the peptide secreted by ventricle to plasma of patient, was elevated in response to many pathological stimuli such as mechanical stretch and ischema. B-type natriuretic peptide is involved not only in regulating blood pressure and body fluid homeostasis, but also infacilitating diuresis and natriuresis. Currently, B-type natriuretic peptide has been proved as a drug for treatment of acute heart failure in clinic. Several previous studies report that B-type natriuretic peptide inhibits the proliferation and / or hypertrophy of rat vascular smooth muscle cells and endothelial cells as its potential therapeutic benefit. Nevertheless, the prognosis of long-tem treatment of B-type natriuretic peptide within heart is still not well-established. Commonly, the growth-promoting agents tend to promote survival; B-type natriuretic peptide was therefore hypothesized to exhibit the positive effect on cardiomyocyte apoptosis. On the other hand, low oxygen is widely involved in triggering and regulating myocardial remodeling in many ischemic heart episodes. Since natriuretic peptide receptors are expressed on cardiomyocytes, the effect of B-type natriuretic peptide on mild low oxygen-induced cardiomyocytes hypertrophy and apoptosis, as well as the intracellular mechanism are our great interest to explore.Present research consists of the following two sections:Firstly, in order to comfirm the model of mild hypoxia (10% O2)-induced cardiac hypertrophy in vitro, we measured the intracellular protein content, [3H]-leucine incorporation of protein synthesis and statistically analyzed the cell surface size. Based on above, the mechanism of BNP on hypertrophic cardiomyocytes was further investigated. Using the microplate reader and the laser scanning confocal microscopy, we found that BNP inhibited the hypertrophy of cardiomyocytes and increased the intracellular cGMP level and nitrite accumulation concentration-dependently. To our best knowledge, three kinds of nitric oxide synthase are expressed in cardiomyocytes. Among them, endothelial nitric oxide synthase (eNOS) in the present study wasn't involved in the late stage of hypoxia since eNOS specific inhibitor; L-NNA didn't influence nitric oxide accumulation. Moreover, present research also found inhibition of induced nitric oxide synthase (iNOS) specific inhibitor aminoguanidine (AG) on the effect of BNP. Using real-time RT PCR technology, we further demonstrated the BNP also upregulated the iNOS mRNA expression in the presence of mild hypoxia. 8-bromo-cGMP which increased cGMP independent of BNP mimicked the effects of BNP. Whereas Rp-8-br-cGMP, cGMP dependent protein specific kinase inhibitor inhibited that.Secondly, apoptosis was induced by mild hypoxia (3% O2) in cardiomyocytes and the chromosome condensation of apoptotic cardiomyocytes stained by specific dye (Hoechst 33342 / PI) was utilized to confirm the pahological cell model. We next measured the mitochondrial transmembrane potential and PS evagination by flow cytometry and laser scanning confocal microscope. Microplate reader was utilized to determine the caspase-3 enzyme activity in cardiomyocytes. Collectively, in the present study, we found that mild hypoxia triggered the dissipation of mitochondrial transmembrane potential, the depletion of ATP, PS transition to the outside of membrane and the increase of caspase-3 activity. These manifestations were further enhanced by BNP in a concentration-dependent manner. Furthermore, the results of real-time RT PCR revealed the down-regulation of Bcl-2 mRNA expression by BNP in the presence of mild hypoxia. 8-bromo-cGMP mimicked the effect of BNP on the cardiomyocytes apoptosis.Generally, it is reasonable to draw conclusion as following: (1) BNP, the reliable prognostic of heart pathology inhibited cardiomyocytes hypertrohpy, which was induced by mild low oxygen (10%O2, 12h). This growth-suppression was via up-regulation of iNOS-derived NO after cGMP generation. NO is known forinhibition of hypertrophy. (2) In the presence of mild low oxygen (3% O2, 12h), BNP could aggravate the cardiomyocytes apoptosis by activation of GC followed by elevation of cGMP. These actions were via the mitochondrial-dependent cell death of apoptosis pathway.Mild hypoxia could induce cardiac hypertrophy and apoptosis which are the major features of myocardial remodeling. In the present study, we gave the novel evidence that BNP inhibited cardiac hypertrophy and enhanced cardiomyocytes apoptosis, which suggests that BNP as a promising drug of long-term heart disease therapy is left to further discussion. |
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