Effects Of Angâ…¡ And NE On Ca²⁺ Signal In Gαq Over Expressing Neonate Rat Vetricule Myocytes

Cardiac hypertrophy occurs as an adaptive response to different cardiac diseases such as hypertension, valvular heart disease, and myocardial ischemia. Structural and electrophysiological remodeling induced by sustained stimulus will increase the risk of arrhythmia and heart failure. To understand the mechanism of hypertrophy development and to prevent the process of hypertrophy happening are important for reducing the risk of heart failure. Accumulative studies have suggested that the activation of Gq pathway might be a critical factor in the development of myocardial hypertrophy. The agonists of Gq protein-coupled receptors, including Angâ…¡, NE, and ET-1 can induce the hypertrophy of neonate rat vetricule myocytes (NRVMs).Overexpression of the wild-type Gαq in NRVMs causes myocardial hypertrophy, while overexpression of constitutively activated Gαq mutant produces hypertrophy initially, and eventually results in cardiomyocytes death. Cardiac specific Gαq transgenic mice leads to heart overweight, cardiomyocyte enlargement, as well as increasement of ANP,α-action andβ-MHC expression.So far, the mechanism of Gαq-stimulated myocardial hypertrophy is unclear. For the past few years, accumulative evidence suggested that Ca²⁺ play a critical role in the development of myocardial hypertrophy. It is suggested that increased [Ca²⁺] i will activate CaN, and initiate a series of events, which activate the NFAT and increase the transcription of hypertrophy related genes. This pathway might be essential for the development of hypertrophy. However, the link between Ca²⁺ signal and hypertrophy induced by the GPCRs (including Angâ…¡, NE) and overexpression of Gαq is still missing. In this study, we investigated the effects of overexpression of Gαq on NE and Ang II induced Ca²⁺ signal in rat cardiomyocytes. A recombinant adenovirus carrying mice wild-type Gαq was first constructed and was then used to infect the NRNMs. The intracellular Ca²⁺ signal induced by Angâ…¡and NE in NRVMs with and wihout overexpressed Gαq was compared in order to understand the role of Ca²⁺ signal in hypertrophy induced by the GPCRs.Part1 Construction of the recombinant adenovirus carrying mice wild-type Gαq gene.Objective: To construct recombinant adenovirus adenovirus carrying mice wild-type Gαq (pAd-Gαq-GFP), and to amplify the pAd-Gαq-GFP in the HEK293A cells.Methods: (1) Gαq cDNA was amplified by PCR from vector pGEMHE- Gαq and the restriction end nuclease site of Pvuâ… and Speâ… were added to the ends of Gαq cDNA. The PCR products were analyzed by agarose gel electrophoresis, and purified by DNA gel extraction Kit. (2) The PCR fragments were cutted with Speâ… and Pvuâ… , and were ligated into pshuttle-IRES-hrGFP-1, which was also cutted by the same enzymes. The recombinant plasmid was analyzed by agarose gel electrophoresis and confirmed by sequencing. (3) pshuttle-Gαq was linerized by Pme I, followed by purification and dephosphorylation. Subsequently, linerized pshuttle-Gαq was electrotransformated into BJ-5183 electroporated competent cells carrying adenovirus backbone plasmid pAdEasy-1 to generate recombinant pAd-Gαq-GFP. (4) Positive recombinant adenovirus plasmids identified by restriction endonuclease digestion were used to transform DH5αcompetent cells so that the recombinant adenovirus plasmid DNA could be amplified. Then the newly constructed pAd-Gαq-GFP was linearized by Pacâ… , and transfected into HEK293A cells through Lipofectamine TM 2000. (5) The viruses were amplified and packaged in HEK293A cells, then the viruses particles were liberated by three freeze/thaw cycles. Results:(1) PCR products of Gαq cDNA were analyzed through agarose gel electrophoresis. The size of fragments was as large as expected (1089bp).(2) pShuttle- Gαq-GFP was generated by subcloning Gαq PCR products into pShuttle vector. The correctness of the insert was confirmed by restriction digestion analysis of the plasmid DNA using Pvuâ… and Spe. Two bands with expected sizes were seen on agarose gel electrophoresis (0.8%) and the pShuttle- Gαq-GFP was also verified by DNA sequencing.(3) pshuttle-Gαq was linerized by Pme I. After purifization and dephosphorylation, linerized pshuttle-Gαq DNAs were electrotransformated into BJ-5183-pAdeasy to generate recombinant pAd-Gαq-GFP. Positively constructed vector was identified by restriction digestion analysis with Pacâ… . Fragments with sizes of 30 Kb and 3Kb or 4.5 Kb were identified, which suggested a successful recombinent.(4) HEK293A cells were infected with linearized pAd-Gαq-GFP and showed a typical cytopathic effect (CPE) including roundind up and detachment of the cells from the plate. These results indicated successfully package of virus.(5) Western-blot results showed the expression of Gαq protein in infected HEK293A cells was significantly increased compared with control HEK293A cells.Conclusion: The recombinant adenovirus carrying mice wild-type Gαq gene was established successfully. This would be the fundament for the further study.Part2 Effect of overexpression of Gαq on the intracellular Ca²⁺ signal of NRVMs induced by Ang II and NEObjective: To overexpress Gαq in NRVMs through adenovirus infection and to observe effect of overexpression of Gαq on the intracellular Ca²⁺ signal of NRVMs induced by Ang II and NE.Methods: (1) Cell dissociation and culture: cardiac muscle of 2 or 3 days neonate rattus was digested repeatedly 6-7 times by 0.04% trypsin and 0.06% collagenaseâ…¡. The cell suspension were collected in DMEM medium, which contains 10% FBS. Myocardial cells were purified by the technique of differential anchoring velocity within a time of 80 min and Brdu, and were then placed into CO2 incubator. (2) NRVMs were infected by adenovirus carrying mice wild-type Gαq. For this, the medium incubating NRVMs was replaced with adenoviruses which had been diluted by the medium, and then the cardiomyocytes were placed into CO2 incubator for 48h and the overexpression of Gαq was confirmed by western-blot analysis. (3) The change of intracellular Ca²⁺ was studied through confocal microscope. NRVMs, infected with pAd-Gαq-GFP or control GFP, were loaded with Rhod2-AM and treated by Ang II or NE.Results:(1) NRVMs progressivly stretch out with parapodium and congregate together and form the cell clumps 48h after dissociation. Cell clumps rhythmically beat in a synchronized and spontaneous fashion, indication of healthy NRVMs.(2) NRVMs were infected by pAd-Gαq-GFP with high efficiency, and the expression of Gαq protein in NRVMs infected with pAd-Gαq-GFP was about 3.5 times as that of the NRVMs infected with control virus pAd-GFP.(3) Overexpression of Gαq in NRVMs markedly increased the frequency of calcium oscillation induced by 5μM Ang II or NE, which was not observed in NRVMs without Gαq overexpression. Meanwhile, the diastolic Ca²⁺ increasement caused by 5μM Ang II was also more profound in NRVMs overexpressing Gαq than in control NRVMs.Conclusion: Overexpression of Gαq in myocardial cells markedly affect the Ca²⁺ signal caused by stimulating factor of hypertrophy, and the enhanced Ca²⁺ signal may be the critical factor for hypertrophy induced by Gq pathway.