| Ryanodine receptor type 2 (RyR-2) mediates Ca2+ release from sarcoplasmic reticulum (SR) and plays a key role in the pathogenesis of contractile dysfunction that is associated with altered intracellular Ca2+ handling. However, the role of RyR-2 in the development of cardiac hypertrophy remains unclear. Here, mice with reduction of RyR-2gene (RyR-2+/-) and their littermate wild type (WT) mice were analyzed. At baseline, there was no difference in cardiomyocyte size, heart morphology and cardiac contractile function between WT and RyR-2+/- mice, although Ca2+ release from SR was impaired in isolated RyR-2+/- cardiomyocytes. When these animals were subjected to the pressure overload stimulation, comparing to WT ones, RyR-2+/- mice exhibited attenuated cardiac hypertrophy, myocardial fibrosis and contractile functions associated with increased apoptosis and decreased autophagy of cardiomyocyte, but unrelated to the impaired cardiac angiogenesis. Pressure overload-induced increases in activation of calcineurin, extracellular signal-regulated protein kinases (ERKs) and protein kinase B/Akt in WT heart was disappeared in RyR-2+/- mice, but that of Ca2+/calmodulin-dependent protein kinase II (CaMKII) was similar between RyR-2+/- and WT mice, suggesting that RyR-2 is a regulator for calcineurin, ERK and Akt but not for CaMKII during pressure overload. Although the detailed mechanism is unclear, our data indicate that RyR-2 contributes to the development of cardiac hypertrophy and adaptation of cardiac function during pressure overload through regulation of Ca2+ handing, activation of calcineurin, ERKs and Akt and cardiomyocyte survival. PART 1:Reduction of RyR-2 gene results in altered Ca2+ handling in adult cardiac myocytes and attenuated cardiac hypertrophy and contractility in response to pressure overloadObjective:To examined the impact of heterozygous RyR-2 gene disruption on Ca2+ handling and contractility in isolated adult cardiac myocytes and to evaluate cardiac morphology and contractile function of WT and RyR-2+/- mice after TAC by echocardiography and catheterization.Method:Adult cardiomyocytes were isolated from mice by Langendorff perfusion method. The intracellular Ca2+ transients were electrically ctivatedat0.25Hz,stabilized within 2 min, and then 10 mmol/L caffeine was rapidly applied to the cardiomyocytes just afte the pacing was turned off. The cells were excited by UV light of 485 nm, and the emission of 530 nm was collected. TAC was performed on WT and RyR-2+/- mice for 2 days and 3 weeks respectively. The right carotid blood pressure was measure and the thickness of left ventricular wall and heart function were measured by echocardiography. H-E and V-G staining analysis were used to reveal the myofiber size and the extent of fibrosis.Results:1)The amplitude of Ca2+ tntracellular Ca2+ concentration during systole, and percentile fractional shortening were significantly reduced in myocytes isolated from RyR-2+/- mice compared to those isolated from WT mice.2) The half time of relaxation from caffeine-induced contracture was comparable between WT and RyR-2+/- myocytes.3)There was no significant difference in dimension and contractility between WT and RyR-2+/(?) mice.4) 3 weeks after TAC, thickness of interventricular septum in diastole (IVSTd) and LV ejection fraction (LVEF) in RyR-2+/- mice were significantly reduced compared to those of WT mice and hemodynamic studies revealed increased LVEDP、decreased dP/dtmax in RyR-2+/- mice. 5) Morphological studies showed heart weight/body weight ratio of RyR-2+/- mice 3 weeks after TAC was significantly smaller than that of WT mice.6) Histological analysis revealed that myofiber size and the extent of fibrosis were significantly reduced in RyR-2+/- mice compared to WT mice.The size of isolated cardiomocytes was comparable between WT and RyR-2+/- cells at baseline but was significantly reduced in RyR-2+/- cells compared to WT cells after TAC.7) AngⅡ(10 nmol/L) could not induce increases in cytosolic free Ca2+ levels at both the systolic and diastolic phases in cultured cardiomyocytes of neonatal rats.Conclusion:1) The heterozygous deletion of RyR-2 gene resulted in altered Ca2+ handling in isolated cardiac myocytes.2) These observations suggest that, although there is no apparent cardiac phenotype at baseline, heterozygous deletion of RyR-2 gene resulted in attenuated cardiac hypertrophy and impaired contractility in response to pressure overload.PART 2:Reduction of RyR-2 gene results in the alteration of hemodynamic load-responsive gene expression programObjective:To examine induction of hypertrophic responses of the heart to hemodynamic overload in the heart at 2 days and 3 weeks after TAC.Method:Mice were divided in to WT and RyR-2+/- groups. TAC and sham operation was performed respectively. The strategy of northern blotting was used to test the fetal-type cardiac genes such as ANP, BNP and a-SKA and also the expression of genes encoding Ca2+ handling proteins such as SERCA2, LCC, NCX.Results:1) TAC-mediated induction of ANP and BNP genes was downregulated whereas induction of a-SKA gene was not altered at day 2 and enhanced 3 weeks after TAC in the heart of RyR-2+/- mice.2) In WT hearts,3 weeks of pressure overload induced downregulation of RyR-2 and SERCA2 genes and upregulation of LCC and NCX genes.3) In RyR-2+/- hearts, the expression levels of SERCA2 and LCC genes were not altered whereas that of NCX gene was downregulated.4) Association of FKBP with RyR-2 was further decreased and phosphorylation of RyR-2 by PKA was further increased in the WT heart compared to WT hearts after TAC.Conclusion:1) Pressure overload-responsive gene expression program is impaired or altered by heterozygous deletion of RyR-2 gene.2) Hypertrophic responses of the gene program regulating the expression of Ca2+ handling proteins are also altered by heterozygous deletion of RyR-2 gene.PART 3:Reduction of RyR-2 gene results in the attenuated activation of hypertrophic signaling pathways in response to pressure overloadObjective:To examine whether the load-induced activation of hypertrophic signaling pathways are impaired in RyR-2+/- hearts.Method:Since pressure overload-induced cardiac hypertrophy is attenuated in RyR-2+/- mice and CaMKII and calcineurin are critical regulators of Ca2+ -mediated cardiac hypertrophy, histochemical staining. The expression of CaMKⅡ、calcineurin、ERK and AKT in myocardium were detected by Western blotting.Results:1) Although AngⅡrapidly (at 1 min) increased activities of CaMKII in a dose-dependent manner, it could not induce an increase in calcineurin activation.2) Pressure overload activated CaMKII and calcineurin in WT hearts.3) Activation of calcineurin in response to pressure overload was impaired in RyR-2+/- hearts whereas the activation of CaMKII was comparable between WT and RyR-2+/- hearts. 4) TAC for 3 weeks induced robust activation of ERK1/2 and Akt in WT hearts but not in RyR-2+/- hearts.Conclusion:These observations indicate that heterozygous deletion of RyR-2 gene led to the attenuated activation of hypertrophic signaling pathways in response to pressure overload, which may account for the reduced growth of the heart in RyR-2+/ mice after TAC.PART 4:Reduction of RyR-2 gene results in increased apoptosis and decreased autophagy but unchanged angiogenesis in pressure overloaded heartObjective:To find out the possible additional mechanisms that contribute to impaired cardiac function in RyR-2+/- mice independently of the alteration in Ca2+ homeostasisMethod:Human failing hearts were obtained from 3 end-stage heart failure patients with dilated cardiomyopathy (DCM). Two donor hearts that could not be transplanted for technical reasons were used for controls. The expression of RyR-2 at the mRNA level was evaluated using RT-PCR. Mice were divided in to RyR-2+/+ and RyR-2+ groups. TAC and sham operation was performed respectively.The density of capillaries in the myocardium was examined by CD31 immunostaining, and the number of CD31-positive microvessels per 100 cardiomyocytes was calculated. Apoptotic death of cardiomyocytes was detected in situ by TUNEL kit in paraffin-embedded heart tissue sections. Autophagy of cardiomyocytes was evaluated by LC3b immunostaining on paraffin sections. Immunostaining was performed using rabbit anti-LC3b antibody after minimal antigen retrieval.Results:1) Comparing with controls, the DCM hearts showed a decrease of RyR-2 gene expression, and the decrease seemed to be more significant in patients with worse contractile functions.2) At baseline condition, the number of apoptotic cardiomyocytes in the heart as evidenced by TUNEL positivity was comparable between WT and RyR-2+/- mice, however, after chronic pressure overload, cardiomyocyte apoptosis in RyR-2+/- heart was significantly increased compared to that in WT hearts.3) The extent of autophagy in the myocardium after pressure overload as evidenced by LC3b positive dots or increased expression of beclinl was attenuated in RyR-2+/-hearts compared to that inWT hearts.4) The number of capillaries and the expression of vascular endothelial growth factor (VEGF) were not altered between WT and RyR-2+/- hearts after chronic pressure overload.Conclusion:1) The heterozygous deletion of RyR-2 gene led to increased myocyte apoptosis after chronic pressure overload.2) The heterozygous deletion of RyR-2 gene led to decreased autophagy in the heart after chronic pressure overload.3) The myocardial ischemia does not contribute to contractile dysfunction of RyR-2+/- hearts after pressure overload. |
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