The Mechanism Of Calcium Handling In Hypertrophic Cardiomyocytes Induced By NPY

BackgroundLeft ventricular hypertrophy(LVH) is the most complication in patients with hypertension. Cardiomyocyte hypertrophy is the main feature of a series of cardiac structure remodeling. Cardiomyocyte hypertrophy is not only the risk factor of heart failure but also a marker for determining the prognosis of cardiovascular diseases. Mechanism of cardiomyocyte hypertrophy has been one of the most popular research topicses in cardiovascular disease field .Intracellular calcium is one of the most extensive and important messengers in vivo. It plays a central role in network of cell signal to regulate information of activities of life, including systolic function, rhythm changes, cell growth and death.Calcium signal codes and transmits external stimulation information through the changes of calcium concentration, spatial distribution and phase. Calcium signal involved in the occurrence and development of cardiomyocyte hypertrophy.Neuropeptide Y(NPY) is the most abundant peptide in the mammalian heart and brain. NPY is released from sympathetic neurons with norepinephrine. NPY exerts an acute action as a neurotransmitter on cardiac function that occurs over a period of seconds or minutes. On the other hand, NPY plays long-term effect (defined as several hours or days) as a trophic factor responsible for cardiac hypertrophy and angiogenesis. Both of them are closely related to calcium signal. However, the relationship and mutual influence of them are poorly understood.The most of researchs about NPY's effect on calcium signal were acute test.It was suggested that the effects of NPY on calcium signal and cardiac contractility were temporary,as the enhance of calcium transcient was disappeared 4~5min after NPY stimulation. However, in our previous study, NPY had sustained effect on calcium signals and correlated with cardiomyocyte hypertrophy. Therefore, it is necessary to explore the alterations of calcium activity and homeostasis indued by sustained NPY stimulation and the relationships with cardiac hypertrophy.Sarcoplasmic reticulum (SR) Ca2+ handling is central in intracellular Ca2+ homeostasis. The Ca2+ handling protein RyR2 and SERCA2a,control the release and uptake of calcium respectively. The coupling of auxiliary proteins PLN and CASQ2 seem to mediate the functions of calcium handling. However, the alteration of SR Ca2+ handling in hypertrophic myocytes induced by NPY remains unclear. CaMKⅡplays a important role in cardiovascular system, which regulats SR calcium handling protein activity through phosphorylation and influences on SR calcium release and uptake. It is unclear that the role of CaMKⅡplays in the alteration of SR Ca2+ handling in hypertrophic myocytes induced by NPY.This study was the first investigation about above questions. We explored Ca2+ mobilizations, distribution, SR calcium handling and CaMKⅡin hypertrophic cardiomyocytes induced by 24 hr stimulation of NPY. All of them were to explore the relating mechanism for cardiac hypertrophy and find effective drug prevention.Part one The alteration of calcium signals in hypertrophic cardiomyocytes induced by NPY1 ObjectivesTo investigate the changes of distribution of intracellular calcium and calcium transient in hypertrophic cardiomyocytes induced by NPY.2 Methods2.1 Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY 100 nmol/L for 24 h. The cardiomyocytes were randomly divided into two groups(n=6) :ⅰcontrol group :Without any treatment ;ⅱNPY group:treated with 100 nmol/L NPY.2.2 24 hours after incubation with NPY, the protein synthesis rate was determinded by 3H-Leu incorporation, the ANF andβ-MHC mRNA expression were determinded by FQ-PCR..2.3 Fluorescent indicator Fluo-4 AM was used to detect [Ca2+] in plasma and calcium transient. Fluo-5N AM was used to detect [Ca2+] in sarcoplasmic reticulum .Calcium images were recorded by laser scanning confocal microscope. The SR Ca2+ load was estimated by Caffeine-induced Ca2+ transient(CCT).3 Results3.1 Compared with control group, 3H-Leu incorporation was significantly elevated in NPY group (P<0.05).3.2 Compared with control group, the ANF andβ-MHC mRNA expression were significantly elevated in NPY group (P<0.05).3.3 Compared with control group, the concentration of free Ca2+ in plasma([Ca2+]i) was significantly elevated in NPY group (P<0.05); and the concentration of free Ca2+ in SR([Ca2+]SR) was significantly decreased in NPY group (P<0.05).3.4 Under the field stimulation ,the evoked Ca2+ transient was of higher amplitude and of faster decay in the presence of NPY(P< 0.01).The time-to-peak-Ca2+ was no significant change.The peak of CCT was slightly attenuated by NPY(P).4 Brief summary4.1 NPY(100 nmol/L) can induce hypertrophy of cardiomyocytes, which appeared upregulation of ANF andβ-MHC mRNA expression and the increasing of myocardial protein synthesis rate.4.2 NPY stimulation can significantly impact excitation-contraction coupling process of dynamic activity of calcium, namely the enhancement of amplitude and the decrease of decay time in Ca2+ transient, which can contributed to promote cardiomyocytes contractility and was related with enhancement pumping function in cardiomyocyte hypertrophy.4.3 NPY stimulation caused redistribution of free calcium in cardiomyocytes, namely the elevation in [Ca2+]i and decline in [Ca2+]SR. The elevation in [Ca2+]i may activate intracellular signaling pathways responsible for cardiac hypertrophy. Reduce SR calcium content can occur during the development of cardiac failure.Part two Effects of sarcoplasmic reticulum Ca2+ handling in hypertrophic cardiomyocytes induced by NPY1 ObjectivesTo investigate the effects of NPY on sarcoplasmic reticulum Ca2+ handling in hypertrophic cardiomyocytes..2 Methods2.1 Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY 100 nmol/L for 24 h. The Cardiomyocytes were randomly divided into two groups(n=6) :ⅰcontrol group :Without any treatment ;ⅱNPY group:treated with 100 nmol/L NPY.2.2 24 hours after incubation with NPY, SERCA2a, RyR2, PLB and CASQ2 mRNA expression were determinded by FQ-PCR..2.3 The changes of the expression of SERCA2a and RyR2 were detected with Western blot and double immunofluorescence method.3 Results3.1 Compared with control group, SERCA2a, RyR2, PLB and CASQ2 mRNA expression were significantly elevated in NPY group (P<0.05).3.2 According to the analyze of Western blot, SERCA2a and RyR2 protein expression in NPY group were significantly elevated compared to control group (P<0.05).3.3 By the detection of double immunofluorescence, SERCA2a and RyR2 protein expressions in NPY group were elevated sychronously compared to control group (P<0.05). 4 Brief summaryBy sustained stimulation of NPY, the levels of Serca2, RyR2 in myocytes were elevated synchronically, accompanied with enhances of CASQ2 and PLN mRNA. These alterations were consistent with the changes of calcium transcient. However, the promotions of Ca2+ release and Ca2+ uptake in SR by NPY could not be imbalanced.Part three CaMKⅡparticipates in alteration of Ca2+ handling induced by NPY1 ObjectivesTo investigate the activation of CaMKⅡin hypertrophic cardiomyocytes induced by NPY and its roles in sarcoplasmic reticulum Ca2+ handling.2 Methods2.1 Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY 100 nmol/L for 24 h. The Cardiomyocytes were randomly divided into two groups(n=6) :ⅰcontrol group :Without any treatment ;ⅱNPY group:treated with 100 nmol/L NPY;ⅲKN- 93group:Co-treated with NPY+KN-93(10μmol/L)。2.2 The activity of CaMKⅡwas determined.2.3 Fluorescent indicator Fluo-4 AM was used to detect [Ca2+] in plasma and calcium transient. Fluo-5N AM was used to detect [Ca2+] in sarcoplasmic reticulum .Calcium images were recorded by laser scanning confocal microscope.2.4 SERCA2a, RyR2, PLB and CASQ2 mRNA expression were determinded by FQ-PCR..2.5 The changes of the expression of SERCA2a and RyR2 were detected with Western blot and immunofluorescence method.3 Results3.1 Compared with control group, the activity of CaMKⅡwas elevated in NPY group (P<0.05) and the effect was blunted by KN-93,a inhibitor of CaMKⅡ.3.2 Compared with control group, the concentration of free Ca2+ in plasma([Ca2+]i) was significantly elevated in npy group(P<0.05), the concentration of free Ca2+ in SR([Ca2+]SR) was significantly decreased in npy group (P<0.05) and the effects were attenuated by KN-93.3.3 Compared with control group, SERCA2a, RyR2, PLB and CASQ2 mRNA expression were significantly elevated in NPY group (P<0.05) and the effects were blunted by KN-93.3.4 SERCA2a and RyR2 protein expression were the same result of mRNA expression.4 Brief summary Conclusions4.1 NPY activated CaMKⅡin cardiomyocytes.4.2 The activated CaMKⅡcan influence calcium mobilizations in SR by regulating calcium handling proteins in SR, including SERCA2a,RyR2 , PLB and CASQ2.4.3 CaMKⅡis responsible critically for Ca2+ redistribution induced by NPY, which can activate intracellular signaling pathways of cardiac hypertrophy.ConclusionsCalcium signals were altered remarkably in hypertrophic cardiomyocytes induced by 24 hr stimulation,including:1.NPY stimulation can significantly impact excitation-contraction coupling process of dynamic activity of calcium, which can contributed to the enhancing pump function in cardiomyocyte hypertrophy.2.NPY stimulation caused redistribution of free calcium in cardiomyocytes. The elevation in [Ca2+]i may activate intracellular signaling pathways responsible for cardiac hypertrophy. Reduce SR calcium content can occur during the development of cardiac failure.3.By sustained stimulation of NPY, the levels of Serca2, RyR2 in myocytes were elevated synchronically, accompanied with enhances of CASQ2 and PLN mRNA. 4. NPY activated CaMKⅡin cardiomyocytes, which takes critical roles in alterations of Ca2+ signal induced by NPY.