茶鹼衍生物KMUP-3的強心作用與緩解心肌梗塞後心臟重塑型之研究

The Inotropic Effect and Attenuation of Ventricular Remodeling after Myocardial Infarction of Theophylline-based KMUP-3

劉中平(Chung-Pin Liu)

指導教授 ： 賴文德 ； 葉竹來

高雄醫學大學/醫學院/醫學研究所/博士(2011年)

https://doi.org/10.6832/KMU.2011.00024

摘要

本研究為探討本實驗室新開發的藥物KMUP-3 (7-[2-[4-(4-Nitrobenzene)piperazinyl]-ethyl]-1,3-dimethyl-xanthine)在心臟方面的強心作用與心肌保護機轉。 KMUP-3具有磷酸二酯酶（phosphodiesterase, PDE）抑制效果，可以有效提升細胞內cAMP及cGMP，並且可以活化鉀離子通道（K+ channel）與eNOS，在人體臍帶靜脈內皮細胞（human umbilical vein endothelial cell，HUVEC）、主動脈平滑肌細胞（aortic smooth muscle cell），及氣管平滑肌細胞（tracheal smooth muscle cell）都有良好的藥效。 從文獻回顧中，我們瞭解cAMP及cGMP對心肌收縮力調節的重要性，而鉀離子通道與eNOS對心肌保護也是不可或缺的。 我們利用離體心房組織及活體大鼠實驗，檢測KMUP-3對心臟輸出指標與相關蛋白質表現的影響。 結果顯示：KMUP-3透過提升細胞內cAMP以達到提升離體心房與活體大鼠的心室收縮力的效果；在離體右心房組織，透過eNOS與副交感神經活化，KMUP-3可以降低心房收縮速率。 在離體心房組織，KMUP-3會顯著提升PKA、eNOS、RhoA及Rho kinase （ROCK）的表現。 使用ROCK抑制劑Y-27632會顯著抑制KMUP-3的強心效果，顯示ROCK有參與KMUP-3的強心作用。
本研究的第二部分測試KMUP-3在心肌梗塞的大鼠動物模式中的效果，並探討心肌梗塞後的心臟保護機轉。 Wistar大鼠隨機分為 3組：心肌梗塞組，心肌梗塞加KMUP-3組，假手術控制組（sham operation）。 心肌梗塞乃結紮左冠狀動脈前降支（left coronary artery anterior descending branch）來引發。 自手術恢復後，心肌梗塞加KMUP-3組接受KMUP-3（0.3 mg/kg/day）輸注 4週，而心肌梗塞組和假手術組只接受生理食鹽水。 結果顯示KMUP-3可以抑制心肌梗塞後的心臟肥厚與減少心肌梗塞的面積，改善心臟輸出量，同時心臟纖維化的面積也減少了。 從西方點墨法分析的結果，發現KMUP-3增強心肌中eNOS和TIMP-1的表現，並減少MMP-9的表現。 為進一步探討KMUP-3對心肌保護的機轉，我們在人類心臟纖維母細胞（human cardiac fibroblast）給予KMUP-3。 結果在給予TGF-β刺激的人類心臟纖維母細胞可以觀察到，KMUP-3對MMP-9抑制的效果可以被NOS抑制劑L-NAME逆轉，證實eNOS/NO路徑參與MMP-9的調控。 因此，我們在心肌組織和人類心臟纖維母細胞中都證明KMUP-3對MMP-9的抑制作用，而MMP-9的天然抑制物： TIMP-1的表現，也被KMUP-3提高了。顯示KMUP-3在心肌梗塞後減少心臟重塑型的效果，經由eNOS的提高而調整MMP-9與TIMP-1之間的平衡。

關鍵字

磷酸二酯酶心肌保護 ； 心肌梗塞

並列摘要

The aim of this study was to investigate the inotropic effect and cardioprotective mechanism of KMUP-3 (7-[2-[4-(4-Nitro-
benzene)-piperazinyl]-ethyl]-1,3-dimethyl-xanthine). KMUP-3 was a new drug synthesized in our laboratory and has been demonstrated to have phosphodiesterase inhibition, endothelial nitric oxide synthase (eNOS) enhancement, and KATP channel opening activities in human umbilical vein endothelial cells, aortic smooth muscle cells, and tracheal smooth muscle cells previously. It has been shown that cAMP and cGMP are involved in the cardiac inotropic effect, while KATP channel opening and eNOS activation have been shown to have cardioprotective effect. Here, we investigated the influence of KMUP-3 of cardiac output and protein expression in isolated atrium and Wistar rats in vivo. Our results demonstrated that Through cAMP enhancement, KMUP-3 increased contractility of isolated atrium and ventricule in Wistar rats. In isolated right atrium, KMUP-3 decreased the atrial contraction rate through activation of eNOS and parasympathetic nervous system. KMUP-3 increased the expression of PKA, eNOS, RhoA and Rho kinase (ROCK). The ROCK inhibitor, Y-27632 blocked the inotropic effect of KMUP-3, which suggested the involvement of RhoA/ROCK pathway in cardiac contractility.
In the second part of the study, we further investigated the cardioprotective effect of KMUP-3 in myocardial infarction (MI) rats. Wistar rats were randomized into three groups: MI, MI + KMUP-3 group, and sham group. MI was induced by ligation of the left anterior descending coronary artery. After recovery, MI + KMUP-3 group received KMUP-3 (0.3 mg/kg/day) infusion for 4 weeks, while MI and sham group received vehicle only. To further confirm the eNOS-dependent activity, KMUP-3 was applied in the culture of transforming growth factor-β (TGF-β)-stimulated human cardiac fibroblasts (HCFs).
KMUP-3 treatment attenuated cardiac hypertrophy with reduced infarction size after MI and improved cardiac output subsequently. The fibrotic area was reduced by KMUP-3 both in central, peri- and non-infarction area. KMUP-3 enhanced eNOS and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression, with reduction of matrix metalloproteinase-9 (MMP-9) expression in MI rats. In HCFs, the ability of KMUP-3 in reducing MMP-9 and enhancing TIMP-1 expression was blocked by pretreatment with eNOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME). Thus, KATP channel opener KMUP-3 preserved cardiac function after MI through eNOS enhancement. KMUP-3 restored the myocardial MMP-9/TIMP-1 balance and attenuated ventricular remodeling with an eNOS-dependent mechanism.

並列關鍵字

phosphodiesterase ； cardiac protection ； myocardial infarction

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