增龄引起犬心房L型电压依赖型钙通道离子重构的分子机制

Aging-related ionic remodeling of L-type voltage dependent calcium channel in left atria of canine

目的探讨增龄导致犬心房L型电压依赖型钙通道离子重构的分子机制。方法采用全细胞膜片钳技术记录犬左心房肌细胞L型电压依赖型钙通道动作电位时限（APD。）、动作电位平台期电压和L型钙离子电流（ICn-L）特性。应用实时定量逆转录聚合酶链反应（RT-PCR）法测定犬左心耳L型电压依赖型钙通道仅1亚单位（CaV1.2）、钙离子释放通道兰尼碱受体（RYR2）、肌浆网钙调控-Ca2＋ATP酶基因（SERCA2）、钙激活蛋白酶-I（Calpain—I）、磷酸受钠蛋白（PLN1）等的mRNA表达，用Westernblot检测蛋白表达。结果老年犬与成年犬比较，心房肌细胞L型电压依赖型钙通道APD90较长[（340．5±10．1）ms比（320．0±7．9）ms，P〈0．05]；动作电位平台期电压较低[（-9．5-4-1．7）mV比（-6．4±1．1）mV，P〈0．05]；ICa-L电流密度较低[（-14．04±0．82）pA／pF比（-8．11±0．54）pA／pF，P〈0．05]。老年犬与成年犬比较，CaV1.2基因表达明显下调（0．90±0．35比2．38±0．40，P〈0．05），RYR2基因表达明显上调（4．39±4．68比1．49±1．69，P〈0．05），两组犬SERCA2、Calpain—I、PLN】基因表达差异无统计学意义；Cavll2蛋白表达明显下调（0．13±0．10比0．29±0．12，P〈0．05），RYR2蛋白明显上调（0．18±0．21比0．08±0．36，P〈0．05），两组犬SERCA2、Calpain-I、PLN，蛋白表达无明显改变。结论增龄导致犬心房肌细胞钙通道CaV1.2和RYR2基因和蛋白表达的改变是L型电压依赖型钙通道离子重构的分子机制，可能是老年相关性心房颤动的潜在机制之一。

Objective To investigate aging-related ionic remodeling of L-type voltage dependent calcium channel （LVDCC） in left atria of canine. Methods Seven adult （2. 0 - 2.5 years） and 10 aged （ 〉 8 years） dogs were used. The current of LVDCC was recorded by patch clamp technique in the whole cell mode. The action potential duration （APD90）, amplitude of action potential plateau （APA）, ICa-L peak current density of LVDCC were recorded. The mRNA and protein expressions of txlc subunit （ CaV1.2 ）, sarcoplasmic reticulum Ca2＋ -ATPase （SECRA2） , Calpain-I, ryanodine receptor （RYR2 ） were detected by quantiative RT-PCR and Western blot, respectively. Results /Co-L peak current density [ （ - 8.11 ± 0. 54） pA/pF vs. （ - 14. 04 ± 0. 82） pA/pF, P 〈 0. 05 ] was significantly reduced and action potential duration to 90% repolarization （ APD90 ） significantly prolonged [ （ 340. 5 ± 10. 1 ） ms vs. （ 320. 0 ± 7.9 ） ms, P 〈 0.05 ] in aged group than in adult group. The mRNA gene expression level of Carl.2 was significantly lower （0. 90 ±0. 35 vs. 2. 38 ± 0. 40, P 〈 0. 05 ） while mRNA expression of RYR2 was significantly higher （4.39 ±4. 68 vs. 1.49 ±1.69 ,P 〈 0. 05 ） in the aged dogs than in the adult dogs. mRNA expression of SECRA2 and Calpain-I was similar between the two groups. Similarly, the protein expression level of Caw.2 was significantly lower （0. 13 ± 0. 10 vs. 0. 29 ± 0. 12, P 〈 0. 05 ） while the protein expression level of RYR2 was significantly higher （0. 18 ±0. 21 vs. 0. 08 ±0. 36, P 〈0. 05） in the aged dogs than in the adult dogs. Again, protein expression of SECRA2, PLNI and Calpain-I was similar between the two groups. Conclusion These data suggest that aging could induce mRNA and protein expression changes of CaV1.2 and RYR2 of LVDCC which might serve as the molecular basis of ICa-L remodeling in aged dogs and might be linked to the increased likelihood of developing atrial fibrillation （AF） in aged dogs.