The Relationship Between Cardiac Hypertrophy And Gene Expression Levels Of Ion Channels In Ventricular Fibroblast

Cardiac hypertrophy is an adaptive process against increased cardiac work loads. To adapt to mechanical overload, the heart produces myocardial hypertrophy, fibroblast proliferation and differentiation, and changes of expression and activities of a variety of ion channels and ion transporters. The early stage of cardiac hypertrophy, which maintains the normal systolic function of the heart, is compensatory. Because the heart changes in morphology during hypertrophy, the process is called remodelling, and therefore, the changes at the cellular and ion channel level are often called cellular and ion channel remodelling. In the long-term persistence of the overload, further cardiac remodelling can eventually lead to heart failure. In both stages of compensatory cardiac hypertrophy and heart failure, remodelling processes can increase the risk of arrhythmias and sudden death.Cardiac fibroblasts, which play a pivotal role in regulating normal myocardial function and in the adverse myocardial remodeling that occurs with hypertension, cardiac hypertrophy, myocardial infarction and heart failure, are the most widespread cell type in the heart. Cardiac fibroblasts can express contractile proteins and become migratory, proliferative and secretory when they go through differentiation to a myofibroblast phenotype, which are responsive to proinflammatory cytokines (e.g. TNFα, IL-1, IL-6, TGF-β), vasoactive peptides (e.g. AngII, ET-1, natriuretic peptides) and hormones (e.g. noradrenaline). And the levels of those cell cytokines and hormones are increased in the remodeling heart. Therefore, fibroblasts provide a potential therapeutic target for cardiac hypertrophy.Potassium channels are the most widely distributed type of ion channel. They are found in most cell types and control a wide variety of cell functions. K⁺ channels are targets for the actions of transmitters, hormones, or drugs that modulate cardiac functions. Profound physiological consequences occur when changes in densities and/or properties of these K⁺ channels occur during the normal development or as a result of damage or disease. Studies suggest that the change of K⁺ channel activity related to cardiac hypertrophy is the major factor for the electrophysiological remodeling and arrhythmia. And K⁺ channel remodeling is often related with calcium-dependent molecular pathway. Most past studies on the relationship between ion channel and cardiac hypertrophy are focused on the myocardial cells. However, little is known about the roles of ion channel in cardic fibroblast playing in the development of cardiac hypertrophy. In this experiment, we use the Real-Time PCR method to study gene expression levels of K⁺ channel, Ca²⁺ channel, Na⁺ channel, Cl- channel and Na⁺-K⁺-ATPase in ventricular fibroblast from normal adult rats and rats with cardiac hypertrophy. The comparion of the expression of ion channels between the normal and the hypertrophic heart will shed lights on which of these ion channels are possibly involved in the development of cardiac hypertrophy.Part 1 Estabolisment of rat cardiac hypertrophy model and evaluation of cardiac function and histologyObject: To establish rat cardiac hypertrophy models induced with isoproterenol and abdominal aorta coarctation.Method: For cardiac hypertrophy model induced by Isoproterenol (Iso). Iso 5mg/(kg.d) was injected in to the back subcutaneous for consecutive seven days. For cardiac hypertrophy model induced by abdominal aorta coarctation (AAC), following precdures were performed: Normal adult male rats were anesthetized by intraperitoneal injection of 1.2% sodium pentobarbital and laid in a left lateral position. An incision was made in ventral to expose abdominal aorta, which was ligated together with a self-made 8^# cured injection needle by a 5^# suture. The needle was then slowly withdrewn, and the organ were repositioned and the abdominal wall was sutured. Cardiac hypertrophy was established after 4 weeks. Results: Hemodynamic parameters: Iso group: compared with the normal group, LVSP was significantly decreased (P<0.01), LVEDP was significantly increasd (P<0.01), and±dp/dt_max was significantly decreased (P<0.05); AAC group: compared with the normal group, LVSP was significantly increasd (P<0.05), LVEDP was significantly increasd (P<0.01), and±dp/dt_max was significantly decreased (P<0.05). Heart weght index (HWI): compared with the control group, HWI was significantly increasd in model groups (P<0.01). Pathological examination: HE staining: light microscopy showed that in the control group, myocardial fibers arranged in neat rows, the myocardial structure was clearly evident and no cell swelling was seen. In Iso group and AAC group, myocardial fibers were disordered and the cardiomyocytes were significant hypertrophy. Masson staining: light microscopy showed that in the control group, myocardial fibers arranged in neat rows, and no cell swelling and proliferation of collagen fibers were seen. In Iso group and AAC group, myocardial fibers were disordered, cardiomyocytes were significant hypertrophy, and collagen fibers were significant proliferated and associated with fibrosis of vascular wall.Conclusion: The cardiac hypertrophy was successfully estabolished with Iso and AAC treatment. The Iso method is relatively simple and can be estabolished in a short time and has high successful rate. AAC method mimicks the overloading process of the pathophysiology of clinical cardiac hypertrophy but takes longer time. Both models were successfully estabolished and can be used for further study.Part 2 Study of the relationship between cardiac hypertrophy and ion channel mRNA expression levels in ventricular fibroblast.Object: To study mRNA expression levels of the K⁺ channels, Ca²⁺ channels, Na⁺ channels, Cl^- channels and Na⁺-K⁺-ATPase in ventricular fibroblast of normal rats and cardiac hypertrophy model rats.Method: The rat ventricular fibroblasts were isolated with the methed of enzymatic digestion and culturing for three days. The total RNA from ventricular fibroblasts was prepared by the Trizol methed. The single-strand cDNA from total RNA was synthesized by using Promega Reverse Transcription System. cDNA was synthesized using Takara SYBR Premix Ex Taq^TM and performance of Real-Time PCR. 2^-ΔCt methed was used in the data analysis. Ct value is the number of cycles when genes reach the exponential growth period. AndΔCt is the difference between target gene Ct values of the treatment group and the control group.Results:1 The mRNA expression levels ofβ-actin in all groups were not significantly different (P>0.05). Compared with normal rats, the mRNA expression levels of BNP andα-SMA in the Iso-induced and AAC-induced cardiac hypertrophy models were significantly increased (P<0.01).2 Compared with normal rats, the mRNA expression levels of Kv2.1, Kv4.1 and Kv4.2 channel in the cardiac hypertrophy models were significantly increased. Compared with normal rats, the mRNA expression levels of Kv1.2 channel in the AAC-induced but not in the Iso-induced cardiac hypertrophy models were significantly reduced (P<0.05). Compared with normal rats, the mRNA expression levels of Kv1.6 channel in the Iso-induced but not in the AAC-induced cardiac hypertrophy models were significantly increased (P<0.05). The mRNA expression levels of Kv1.1, Kv1.3, Kv1.5, Kv3.1 and Kv4.3 channel in all groups were not significantly changed (P>0.05).3 Compared with normal rats, the mRNA expression levels of Kir2.2 channel in the cardiac hypertrophy models were significantly reduced, and the mRNA expression levels of Kir6.1 channel were significantly increased (P<0.01). Compared with normal rats, the mRNA expression levels of Kir2.1 channel in the AAC-induced cardiac hypertrophy models were significantly increased (P<0.05), but there was no significantly change in the Iso-induced cardiac hypertrophy models (P>0.05). The mRNA expression levels of Kir3.4 and Kir6.2 channel in all groups were not significantly changed (P>0.05).4 Compared with normal rats, the mRNA expression levels of Nav2.1 channel in the cardiac hypertrophy models were significantly reduced (P<0.01), and the mRNA expression levels of Cav3.1 channel and Na⁺-K⁺-ATPaseβ1 channel were significantly increased (P<0.01). Compared with normal rats, the mRNA expression levels of Nav1.1 channel in the AAC-induced but not in the Iso-induced cardiac hypertrophy models were significantly reduced (P<0.05). Compared with normal rats, the mRNA expression levels of Cav1.2, CLC-3 channel and Na⁺-K⁺-ATPaseα1 in the Iso-induced but not in the AAC-induced cardiac hypertrophy models were significantly increased. The mRNA expression levels of Na⁺-K⁺-ATPaseα2 in all groups were not significantly changed (P>0.05).5 The channels (α-SMA, Kv1.6, Kv4.1, Kir6.1 and Nav2.1) which have altered expression levels of mRNA in cardiac hypertrophy models normally have higher mRNA expression levels compared with other channel and transporter genes tested in normal rats.Conclusion: mRNA expression levels of some of K⁺ channels, Ca⁺ channels, Na⁺ channels, Cl- channels and Na⁺-K⁺-ATPase were altered in cardiac hypertrophy of rats. These data suggest that these channels may play an important role in the occurrence and development of cardiac hypertrophy. The results of this study indicate that ion channels may represent an important therapeutic target for the treatment of cardiac hypertrophy.