The Relationship Between Cardiac Hypertrophy And TRP Channel Expression Levels

Cardiac hypertrophy is the common pathology of a variety of cardiovascular diseases. The pathological changes include myocardial hypertrophy,?myocardial interstitial cell proliferation and extracellular matrix modification, namely myocardial remodeling. Characters of cardiac hypertrophy include increase myocardial protein synthesis, increased size of cardiac myocytes with larger dimeter or increased length, accompanied by an increase in fibrous tissue hyperplasia. Cardiac hypertrophy is caused by a variety of risk factors for cardiovascular disease. Although hypertrophy is initially a compensatory mechanism that helps sustain cardiac output, prolonged hypertrophy will inevitably give rise to heart failure and even sudden death. The increase in intracellular Ca2+ ([Ca2+]i) plays an important role in the development of hypertrophy. Hypertrophic stimulation by pressure overload and neurohormonal factors(including AngⅡ, ET-1 and PE et al) lead to the SOC (store-operated calcium channels), ROC (receptor-operated calcium channels) or VDCC (voltage-dependent calcium channels) opening. These Ca2+-entry channels induce a prolonged, low-amplitude rise in Ca2+. This sustained Ca2+ entry dominantly activates the calcineurin/NFAT pathway. The calcineurin/ NFAT pathway produces long-term hypertrophic changes in cardiac myocytes and subsequent cardiac hypertrophy.TRP channels are plasma membrane cation channels. They are expressed in almost every tissue, including the heart and vasculature. In the heart, evidences are accumulating that TRPC channels, and possibly TRPM and TRPV, are present and functional, Most TRP channels are Ca2+ permeable, Moreover, some of the Ca2+ channels such as ROC and SOC may have molecular basis related to some TRPC channel subtypes. ROC and SOC are believed to play important role for Ca2+ influx during cardiac hypertrophy, As thus, TRPC channels may also be important for occurrence of cardiac hypertrophy. Indeed some TRPC channels have been indicated in cardiac hypertrophy. However, little is known about other role of TRP channels subtypes play in cardiac hypertrophy.In this experiment, we use the Real Time PCR method to study expression profiles of the subtypes of TRPC, TRPV snd TRPM channels, as well as STIM1 and Orai1 in myocardial tissue from normal adult rats, neonatal rats and rats with cardiac hypertrophy. Such a comparion between the normal and hypertrophic heart will give us indications which of these TRP channels are possibly involved in cardiac hypertrophy development. And moreover, comparison between neonatal rats and normal adult rat and hypertrophyic rats would also shed light on relationship between cardiac development and hypertrophy.Part 1 Establishment of rat cardiac hypertrophy model and evaluation of cardiac function and histologyObject: To establish rat cardiac hypertrophy models by means of isoproterenol injection and abdominal aorta coarctation. Method: Isoproterenol (Iso)-induced cardiac hypertrophy model: Iso 5mg/(kg·d), back subcutaneous injection for consecutive seven days, was used to induce cardiac hypertrophy; Abdominal aorta coarctation (AAC)-induced cardiac hypertrophy model: intraperitoneal injection of 1% sodium pentobarbital was used to anesthetize rats. The anetsthetized rats were laid in a left-lateral position. Abdominal was cut open to expose abdominal aorta. A 4# suture was used to tight the abdominal aorta together with a self-made 8# cured injection needle, causing abdominal aorta coarctation. After this operation, abdominal fascia and skin were sutured. Intraperitoneal injection of benzylpenicillin potassium (16U, a week) was applied to prevent infection. After 12 weeks rat cardiac hypertrophy were established.Results: Hemodynamic parameters: compare with the control group, LVEDP was significantly higher (P<0.01), whereas LVSP and±dp/dtmax were significantly decreased in model groups (P<0.01). However, LVSP, LVEDP and±dp/dtmax were not significantly different within two model groups (P>0.05). Heart weght index (HWI): compare with control group, HWI was significantly higher in model groups (P<0.01). Histopathological examination: the myocardial structure in the normal rat group was clearly evident; myofibrils are the main components of cytoplasm, and they appeared orderly, with bright and dark areas clearly evident; In Iso group and the AAC group, myocardial cells appeared hypertrophy, with an increase in cell size and deposition of fibrous tissue.Conclusion: Isoproterenel injection and abdominal aorta coarctation successfully induced rat cardiac hypertrophy. The Isoproteronel method is relatively simple and had high successful rate. AAC method mimics better the pathophysiology of clinical cardiac hypertrophy with overload. Both models can be used for further study.Part 2 Study of the relationship between cardiac hypertrophy and TRP channel mRNA expression levels.Object: To Study the mRNA expression levels of the TRP channels, STIM1 and Orai1 in rat myocardium of normal adult rats, neonatal rat and cardiac hypertrophy model rats.Method: Using Promega total RNA Isolation Syetem method to isolate total RNA from myocardial tissues of normal adult rats, neonatal rats and model rats. Using Promega Reverse Transcription System to synthesize single-strandea cDNA from total RNA. Using Takara SYBR Premix Ex TaqTM to perform Real-Time PCR from synthesized cDNA. The difference in amount of original cDNA from different experimental rat groups were calculated using expression of 2-ΔCt; -ΔCt is the difference of PCR circle number in threshold between different groups.Results:1. Compared with normal adult rats, the BNP mRNA expression levels in the Iso-induced cardiac hypertrophy and AAC-induced cardiac hypertrophy models were increased by 7.80±0.33-fold and 8.11±0.37-fold, respectively (P<0.01), and the neonatal rat mRNA expression level was increased by 8.08±0.39-fold (P<0.01).2. Compared with normal adult rats, TRPC1, TRPC3 and TRPC6 channel mRNA expression levels in the cardiac hypertrophy model rats and neonatal rats were higher (P<0.01, For detailed data, see the Part 2 Table3 and Table4). However, there was no significant difference in mRNA expression levels (TRPC1, TRPC3 and TRPC6) between model rats and neonatal rats. The mRNA expression levels of TRPC2, TRPC4 and TRPC5 in all groups were not significantly different (P>0.05). TRPC7 mRNA expression level in neonatal rats was higher than other groups (P<0.01), and the normal adult rats groups and model groups were not significantly different (P>0.05).3. Among all TRPM family members, only TRPM4 and TRPM7 expression were detected. The mRNA expression level of TRPM4 in neonatal rats group and model rats group was higher than normal rats group (P<0.01, for detailed data, see Part 2 Table5 and Table6). The TRPM7 channel mRNA expression level was not significant different in all groups.4. Among all TRPV family members, only TRPV2 and TRPV6 expression were detected. The mRNA expression levels of TRPV2 and TRPV6 in cardiac hypertrophy model rats group were higher than normal rats group and neonatal rats groups (P<0.01, for detailed data, see Part 2 Table5 and Table6). There was no significant difference between hypertrophy model groups.5. Compared with normal adult rats, mRNA expression levels of STIM1 and Orai1 in neonatal rats group and cardiac hypertrophy model rats group were significantly higher (P<0.01, for detailed data, see part 2 Table1 and Table2).Conclusion: Expression levels of some TRP channels subtypes were selectively increased in cardiac hypertrophy of rats. These data suggest that these TRP channels may play an important role in the occurrence and development of cardiac hypertrophy. Furthermore, general expression profile of TRP channels in myocardial tissue from neonatal rats is similar to that from cardiac hypertrophy models rats. Thus, compared with normal rats groups, the expression levels of BNP, TRPC1, TRPC3, TRPC6, TRPM4, STIM1 and Orai1 in neonatal rats and cardiac hypertrophy model rats were increased. The results of this study indicate that TRP channels may represent an important therapeutic target for the treatment of cardiac hypertrophy.