Background Heart failure （HF） is a major cause of morbidity and mortality worldwide, but current treatment modalities cannot reverse the underlying pathological state of the heart. Gene-based therapies are emerging ...Background Heart failure （HF） is a major cause of morbidity and mortality worldwide, but current treatment modalities cannot reverse the underlying pathological state of the heart. Gene-based therapies are emerging as promising therapeutic modalities in HF patients. Our previous studies have shown that recombinant adeno-associated viral （rAAV） gene transfer of Sarco-endoplasmic reticulum calcium ATPase （SERCA2a） can be effective in treating rats with chronic heart failure （CHF）. The aim of this study was to examine the effects of SERCA2a gene transfer in a large HF animal model. Methods HF was induced in beagles by rapid right ventricular pacing （230 beats/min） for 30 days. A reduced rate ventricular pacing （180 beats/min） was continued for another 30 days. The beagles were assigned to four groups： （a） control group （n=4）; （b） HF group （n=4）; （c） enhanced green fluorescent protein group （n=4）; and （d） SERCA2a group （n=4）. rAAV1-EGFP （1×10^12 μg） and rAAV1-SERCA2a （1×10^12 μg） were delivered intramyocardially. SERCA2a expression was assessed by Western blotting and immunohistochemistry. Results Following 30 days of SERCA2a gene transfer in HF beagles its protein expression was significantly higher than in the HF group than in the control group （P 〈0.05）. Heart function improved along with the increase in SERCA2a expression. Left ventricular systolic function significantly improved, including the ejection fraction, left ventricular systolic pressure, maximal rate of rise of left ventricular pressure （＋dp/dtmax）, and the maximal rate of decline of left ventricular pressure （-dp/dtmax） （P 〈0.05）. Left ventricular end-diastole pressure significantly decreased （P 〈0.05）. The expression of SERCA2a in the myocardial tissue was higher in the SERCA2a group than in the HF group （P〈0.05）. Conclusions Intramyocardial injection of rAAV1-SERCA2a can improve the cardiac function in beagles induced with HE We expect further studies on SERCA2a＇s long-term safety, efficacy, dosage and the optimization before using it in humans with HF.展开更多
Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum...Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum" hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2 b and Ry R1 are preferentially targeted to the sarcoplasmic reticulum(SR) proximal to the plasma membrane(PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2 a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. Ry R3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of Ry R2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear Ry R3 s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via Ry R2 in order to elicit contraction. Data also suggest that unique SERCA and Ry R are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.展开更多
AIM:To investigate serotonergic Ca 2+ signaling and the expression of 5-hydroxytryptamine(5-HT) receptors,as well as Ca 2+ transporting proteins,in hepatic stellate cells(HSCs) . METHODS:The intracellular Ca 2+ concen...AIM:To investigate serotonergic Ca 2+ signaling and the expression of 5-hydroxytryptamine(5-HT) receptors,as well as Ca 2+ transporting proteins,in hepatic stellate cells(HSCs) . METHODS:The intracellular Ca 2+ concentration([Ca 2+ ]i) of isolated rat HSCs was measured with a fluorescence microscopic imaging system.Quantitative PCR was per-formed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum(ER) proteins involved in Ca 2+ storage and release in cultured rat HSCs. RESULTS:Distinct from quiescent cells,activated HSCs exhibited[Ca 2+ ]i transients following treatment with 5-HT,which was abolished by U-73122,a phospholipase C inhibitor.Upregulation of 5-HT2A and 5-HT2B receptors,but not 5-HT3,was prominent during trans-differentiation of HSCs.Pretreatment with ritanserin,a 5-HT2 antagonist,inhibited[Ca 2+ ]i changes upon application of 5-HT.Expression of type 1 inositol-5'-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca 2+ storage and release in activated HSCs.Ca 2+ binding chaperone proteins of the ER,including calreticulin,calnexin and calsequestrin,were up-regulated following activation of HSCs. CONCLUSION:The appearance of 5-HT-induced[Ca 2+ ]i response accompanied by upregulation of metabotropic 5-HT2 receptors and Ca 2+ transporting/chaperone ER proteins may participate in the activating process of HSCs.展开更多
基金This work was supported by grants from the China ＂973＂ plan （No. 2006CB503806）, and the National Science Foundation of China （No. 30600236）.
文摘Background Heart failure （HF） is a major cause of morbidity and mortality worldwide, but current treatment modalities cannot reverse the underlying pathological state of the heart. Gene-based therapies are emerging as promising therapeutic modalities in HF patients. Our previous studies have shown that recombinant adeno-associated viral （rAAV） gene transfer of Sarco-endoplasmic reticulum calcium ATPase （SERCA2a） can be effective in treating rats with chronic heart failure （CHF）. The aim of this study was to examine the effects of SERCA2a gene transfer in a large HF animal model. Methods HF was induced in beagles by rapid right ventricular pacing （230 beats/min） for 30 days. A reduced rate ventricular pacing （180 beats/min） was continued for another 30 days. The beagles were assigned to four groups： （a） control group （n=4）; （b） HF group （n=4）; （c） enhanced green fluorescent protein group （n=4）; and （d） SERCA2a group （n=4）. rAAV1-EGFP （1×10^12 μg） and rAAV1-SERCA2a （1×10^12 μg） were delivered intramyocardially. SERCA2a expression was assessed by Western blotting and immunohistochemistry. Results Following 30 days of SERCA2a gene transfer in HF beagles its protein expression was significantly higher than in the HF group than in the control group （P 〈0.05）. Heart function improved along with the increase in SERCA2a expression. Left ventricular systolic function significantly improved, including the ejection fraction, left ventricular systolic pressure, maximal rate of rise of left ventricular pressure （＋dp/dtmax）, and the maximal rate of decline of left ventricular pressure （-dp/dtmax） （P 〈0.05）. Left ventricular end-diastole pressure significantly decreased （P 〈0.05）. The expression of SERCA2a in the myocardial tissue was higher in the SERCA2a group than in the HF group （P〈0.05）. Conclusions Intramyocardial injection of rAAV1-SERCA2a can improve the cardiac function in beagles induced with HE We expect further studies on SERCA2a＇s long-term safety, efficacy, dosage and the optimization before using it in humans with HF.
基金supported by the British Heart Foundation(29885)
文摘Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum" hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2 b and Ry R1 are preferentially targeted to the sarcoplasmic reticulum(SR) proximal to the plasma membrane(PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2 a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. Ry R3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of Ry R2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear Ry R3 s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via Ry R2 in order to elicit contraction. Data also suggest that unique SERCA and Ry R are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.
基金Supported by Grants from the Korean National Research Foun-dation(2010-0014617)the Myung Sun Kim Memorial Founda-tion(2009)the Yonsei University Faculty Research Grant(2004)
文摘AIM:To investigate serotonergic Ca 2+ signaling and the expression of 5-hydroxytryptamine(5-HT) receptors,as well as Ca 2+ transporting proteins,in hepatic stellate cells(HSCs) . METHODS:The intracellular Ca 2+ concentration([Ca 2+ ]i) of isolated rat HSCs was measured with a fluorescence microscopic imaging system.Quantitative PCR was per-formed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum(ER) proteins involved in Ca 2+ storage and release in cultured rat HSCs. RESULTS:Distinct from quiescent cells,activated HSCs exhibited[Ca 2+ ]i transients following treatment with 5-HT,which was abolished by U-73122,a phospholipase C inhibitor.Upregulation of 5-HT2A and 5-HT2B receptors,but not 5-HT3,was prominent during trans-differentiation of HSCs.Pretreatment with ritanserin,a 5-HT2 antagonist,inhibited[Ca 2+ ]i changes upon application of 5-HT.Expression of type 1 inositol-5'-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca 2+ storage and release in activated HSCs.Ca 2+ binding chaperone proteins of the ER,including calreticulin,calnexin and calsequestrin,were up-regulated following activation of HSCs. CONCLUSION:The appearance of 5-HT-induced[Ca 2+ ]i response accompanied by upregulation of metabotropic 5-HT2 receptors and Ca 2+ transporting/chaperone ER proteins may participate in the activating process of HSCs.
