巨细胞病毒感染致室性心律失常与射频消融的研究
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摘要
第一部分巨细胞病毒感染诱导小鼠心肌炎室性心律失常及其机理的研究
背景鼠巨细胞病毒(MCMV)感染诱导小鼠心律失常的研究尚属空白。
目的探讨MCMV感染诱导BALB/c小鼠心肌炎室性心律失常及其可能机理。
方法60只雄性4周龄BALB/c小鼠随机分成二组：实验组(A组，36只，MCMV腹腔注射感染小鼠)和对照组(B组，24只，3T3细胞裂解液腹腔注射小鼠)。于病毒接种后3、7、14、21、28、35、42、49、56、63和70天，眼内眦静脉取血提取血清测定血清抗心肌β_1-受体抗体，记录心电图，用断颈法分批处死小鼠，摘取心脏，提取少量心肌组织检测MCMV基因表达，并进行病理学和炎性因子免疫组化检查。分离BALB/c小鼠心室肌细胞，在电流钳和电压钳模式下，观察抗β1-受体抗体对心肌细胞动作电位及I_(Ca-L)电流的影响；运用激光共聚焦技术，观察抗β1-受体抗体对心肌细胞胞浆游离Ca~(2+)浓度([Ca~(2+)]_I)的影响。
结果A组36只小鼠70天心肌炎累积发病25只(25/36，69．4％)；累积死亡4只(4/36，11.1％)；B组无1只死亡。显微病理显示，A组接种病毒后7-14天，心肌病理改变最明显，表现为心肌细胞肿胀变性，单个心肌细胞核固缩、小灶性坏死，心肌组织局灶性或弥散性炎性细胞浸润；即使病毒感染后70天，心肌间质仍有散在炎性细胞浸润。病理学半定量分析发现，A组心肌组织在病毒感染后7-14天炎性细胞浸润和心肌小灶性坏死达高峰；心肌病理积分均在2分以下，属轻度病毒性心肌炎。免疫组化结果显示，感染病毒后3天，心肌组织炎性因子IL-1β和TNF-α蛋白表达呈阳性；而感染病毒后7天，心肌IL-1β和TNF-α蛋白呈强阳性表达。运用PCR技术，A组接种病毒后第3天和第7天心肌组织可检测到MCMV特异基因片段表达；但在病毒接种后第14天至第70天，心肌组织却不能检测到MCMV特异基因片段的表达；B组心肌始终不能检测到MCMV表达。B组小鼠前7周血清抗β1受体抗体滴度为0，而在第8-10周滴度轻微升高；A组小鼠前5周血清抗β1抗体滴度为0，但从第6周开始升高直至第10周；接种病毒后6-10周，二组血清抗β1受体抗体滴度比较，具有极显著性差异，P＜0.001。A组小鼠心电图异常累计发生率达50％；感染病毒后3天即可记录到心律失常；前5周心律失常主要是以窦性心律失常、房性心律失常和传导阻滞为主；而从第6周至第10周，虽然心肌组织不能检测到MCMV基因片段，但仍有心律失常发生，主要表现为室性心律失常和房性心律失常，此时血清抗β1受体抗体滴度明显升高。分离小鼠心室肌细胞，在电流钳模式下可见，1：100抗β1-受体抗体可明显延长动作电位平台期，预先给予美托洛尔阻断β1-受体，1：100抗β1-受体抗体基本没有延长动作电位平台期的作用；在电压钳模式下，1：100抗β1受体抗体可显著增加I_(Ca-L)电流，预先加入美托洛尔，则I_(Ca-L)基本不变。运用激光共聚焦技术可见，1：100抗β1受体抗体可显著增加心肌细胞胞内钙荧光强度，预先加入美托洛尔，抗β1-受体抗体仅能轻微增加胞内钙荧光强度。
结论MCMV感染可引起小鼠急慢性心肌炎，导致各种类型心律失常；感染早期的心肌病理改变及心律失常发生可能与MCMV感染直接损伤心肌有关，而慢性期的心肌病理改变和心律失常发生，很可能与MCMV诱导产生的自身抗体-抗β1受体抗体的作用相关；抗β1受体抗体可能通过增加I_(Ca-L)电流，增加心肌胞内钙浓度，诱发早期后除极，导致室性心律失常的发生。
第二部分巨细胞病毒感染与特发性右室心律失常及其右室壁异常相关性的研究
背景特发性右室心律失常(IRVA)的病因不明。
目的探讨巨细胞病毒(CMV)感染与IRVA发生及其MRI右室影像异常的相关性。
方法进行病例对照研究。分为四组，即IRVA组(45例)、特发性左室心动过速(ILVT)组(50例)、其他心脏病平行对照组(Heart-Disease-Control，50例)和健康对照组(Healthy-Control，50例)，每组性别年龄匹配。接受常规检查(心电图、X线胸片、心脏超声等)后，进行病毒血清学、心脏磁共振(MRI)、血清抗心肌自身抗体和心肌损伤标志物检查，随访6-12个月。
结果IRVA组中，单纯右室频发早搏12例、右室频发早搏伴反复性阵发性右室心动过速33例；根据心电图判断，心律失常起源于右室流出道37例、右室游离壁或心尖8例。ILVT组中，起源于左室间隔45例、左室心尖3例、左室游离壁2例。MRI结果显示，IRVA组所有患者左室均未发现异常，其中31例(31/45，68.9％)存在右室壁局灶性和散在性病变；ILVT组中2例(2/50，4％)左室间隔部存在微小室壁瘤，3例(3/50，6％)右室游离壁近间隔处变薄；Heart-Disease-Control组中5例(5/50，10％)右室壁存在局灶性病变；Healthy-Control组中6例(6/50，12％)右室存在上述局灶性病变；IRVA组MRI右室异常阳性率显著高于其它三组，差异具有极显著性(P＜0.01)。四组血清柯萨奇B组病毒(CVB)IgM抗体阳性率均低，组间比较无显著性差异。但IRVA组血清巨细胞病毒(CMV)IgM抗体阳性率(73.3％)显著高于其他三组(P＜0.01)；即使随访6-12个月，IRVA组CMV IgM阳性率仍然持续增高(66.7％)。四组血清CK、CK-MB、cTNI的测值结果均在正常范围，组间比较无显著性差异(P＞0.05)。相关性分析发现，CVB感染与IRVA发生及其MRI右室壁异常的关联强度较低(P＞0.05)，而CMV感染与IRVA发生及其MRI右室壁异常的关联强度较高(P＜0.001)。比较四组血清抗心肌自身抗体可见，四组的抗ANT抗体、抗M_2抗体及抗MHC抗体阳性率均低，组间差异无显著性(P＞0.05)；但抗β1受体抗体阳性率在四组间却存在显著性差异，表现为IRVA组的抗β1受体抗体阳性率显著高于其他三组(P＜0.01)，即使随访6-12个月，IRVA组的抗β1受体抗体阳性率依然增高。
结论IRVA患者CMV感染阳性率高，多数右室壁存在局限性病变；CMV感染与IRVA的发生及其右室壁异常相关，可能是引起IRVA并导致右室病变的病因；CMV感染引起IRVA可能与自身抗体-抗β1受体抗体作用有关。
第三部分巨细胞病毒持续感染状态特发性右室心律失常的射频消融疗效观察
背景射频消融(RFCA)广泛地应用于治疗快速性心律失常。
目的探讨RFCA对于伴有巨细胞病毒(CMV)持续感染状态的特发性右室心律失常(IRVA)的治疗效果。
方法符合入选标准的IRVA患者45例，其中男17例、女28例，年龄21-53(37.5±17)岁；IRVA病史3-25(9.7±8)年；IRVA表现为单纯右室频发早搏12例、右室频发早搏伴反复性阵发性右室心动过速33例；根据心电图判断，心律失常起源于右室流出道37例、右室游离壁或心尖8例。术前接受常规检查(心电图、X线胸片、心脏超声等)，并进行CMV病毒血清学、心脏磁共振(MRI)、血清抗心肌自身抗体、血清心肌损伤标志物以及心内电生理等项目检查。所有患者射频消融术后随访6-12个月，并再次接受CMV病毒血清学及血清抗心肌自身抗体检测。
结果45例IRVA患者术前均检测了血清CMV IgM抗体，33例(33/45，73.3％)阳性，随访6-12个月，仍有30例CMV IgM抗体阳性(30/45，66.7％)，提示多数IRVA患者处于CMV病毒持续感染状态。术前MRI影像显示，31例(31/45，68.9％)右室壁存在局灶性和散在性病变，其中右室游离壁变薄27例(27/45，60％)、右室流出道壁变薄9例(9/45，20％)、局限性脂肪沉积25例(25/45，55.6％)、右室游离壁微小膨胀瘤1例(1/45，2.2％)；MRI右室病变部位与IRVA部位不一致。术前4种血清抗心肌自身抗体中，只有抗心肌β_1受体抗体阳性率较高(62.2％)，而其他3种抗心肌自身抗体的阳性率较低；随访以后，抗心肌β_1受体抗体阳性率仍然持续增高(64.4％)，提示多数IRVA患者血清持续存在抗心肌β_1受体抗体。在CMVIgM抗体阳性患者中，多数抗心肌β_1受体抗体为阳性(25/33，75.8％)。所有患者术前均检测了血清心肌损伤标志物，测值结果在正常范围。心内电生理检查显示，35例(35/45，77.7％)有自发IRVA，QRS波形态与平时发作时一致；9例(9/45，20％)静脉滴注异丙肾上腺素方可诱发出与平素发作ORS波形态一致的IRVA；1例(1/45，2.2％)不能诱发IRVA并放弃RFCA。在44例接受RFCA中，43例(43/44，97.7％)术后即刻成功，1例放电时出现剧烈胸痛终止手术；随访6-12个月，5例(5/44，11.6％)复发，再次消融成功；RFCA总成功率为95.6％(43/45)。33例CMV IgM抗体阳性病例中，31例(31/33，93.9％)消融成功；28例抗心肌β_1受体抗体阳性病例中，27例(27/28，96.4％)消融成功。
结论对于伴有CMV持续感染状态及血清抗β_1受体抗体阳性的IRVA患者，射频消融仍然是治疗IRVA的有效方法。
Part I
Evaluation of myocarditis and cardiac arrhythmias induced by murine cytomegalovirus and their mechanisms
Bacground Lack of study of murine cardiac arrhythmias induced by cytomegalovirus Objective To evaluate the myocarditis and the cardiac arrhythmias induced by murine cytomegalovirus and to investigate their mechanisms.
Methods sixty male four weeks Balb/c mice were random divided into two groups: one was experiment group (A group, abdominal injection of murine cytomegalovirus) and control group (B group, abdominal injection of 3T3 liquid). At 3,7,14,21,28,35,42,49,56,63 and 70 day after cytomegalovirus infection, blood taken from canthus vessels was used to detect serum antibodies against myocardial β_1 receptor. Meantime, electrocardiographies of mice were recorded. After killing mice, getting out their hearts, tested gene express of murine cytomegalovirus, took pathological examination and immunohistochemical examination. After separating ventricular muscle cells of mice, tested effects of above antibodies against myocardial β_1 receptor on acting potential duration and L-type Ca~(2+) currents,under condition of current clamp and potential clamp. And then detected intracellular Ca~(2+) concentration of murine cardiac myocytes by laser confocal technique.
Results Total incidence of myocarditis of BALB/c mice in experiment group was 69.4% at 70 day after cytomegalovirus infection. Total mortality was 11.1% in this group at end of the experiment. There wasn't one murine died in another control group. Microscopical pathology showed that changes of myocardium pathologically were obvious in experiment group from 7day to 14 day after the virus infection. The main pathological changes were that some myocytes were swelling and denaturalization, other single myocyte was necrosis, and inflammatory cells infiltrated in myocardial tissue in a focus and dispersed fashion. Even if at 70 day after the virus infection, inflammatory cells still infiltrated in heart tissue dispersedly. Half quantitative analysis by pathology showed that the myocarditis induced by cytomegalovirus was mild. Result of immunohistochemistry showed that strong expression of proteins of cytokin IL-1β and TNF-α in myocardium at 3-7 day after the virus infection. In experiment group, gene of cytomegalovirus was deteced at 3-7 day after virus infection, and was undetectable from 14 day to 70 day after virus infection. Whereas, gene of cytomegalovirus was undetected at all the time in control group. From 1 to 7 weeks after virus infection, the anti-cardiac β_1 receptor autoantibody titer of sera in control group were zero, but mild elevation from 8 to 10 weeks. The anti-cardiac β_1 receptor autoantibody titer of sera were zero at former 5 weeks after virus infection and were significantly high elevation at latter 5 weeks after virus infection. Total incidences of cardiac arrhythmias were about 50% in A group. Most of cardiac arrhythmias were sinus arrhythmias, atrial arrhythmias and conduction black at the former 5 weeks, and were ventricular arrhythmias and atrial arrhythmias at the latter 5 weeks in A group. After separating ventricular muscle cells of mice, prolongations of acting potential duration of cardiomyocytes were observed when adding anti-cardiac β_1 receptor autoantibody diluted at 1:100 to incubation liquid, but this phenomenon were not found if adding metoprolol to incubation liquid beforehand. Autoantibodies against β_1-adrenoceptor diluted at 1:100 significantly increased the I_(Ca-L) peak current amplitude and elevation of intracellular Ca~(2+) fluorescent intensity. Wheres, blocking of β_1-adrenoceptor by metoprolol beforehand, autoantibodies against β_1-adrenoceptor diluted at 1:100 induced a slight increase of I_(Ca-L) peak current amplitude and mild elevation of intracellular Ca~(2+) fluorescent intensity.
Conclusion Murine cytomegalovirus can cause acute and chronic myocarditis, even more induce various cardiac arrhythmias in BALB/c mice. The pathologicall changes of myocardium and arrhythmias are related with cytomegalovirus infection at early period of the virus infection, but may be related with autoantibodies against β _1-adrenoceptor at chronic period. Autoantibodies against β_1-adrenoceptor may lead ventricular arrhythmias by increasing I_(Ca-L) current and elevation of intracellular Ca~(2+) concentration.
Part II
Correlation between viral infection, idiopathic right ventricular
arrhythmias and its morphological abnormalities of right
ventricle delineated by MRI
Background The etiology of idiopathic right ventricular arrhythmias (IRVA) is
unknown.
Objective To evaluate correlation between viral infection, IRVA and its
morphological abnormalities of right ventricle delineated by magnetic resonance
imaging(MRI) in patients with IRVA.
Methods Case control study was performed. All subjects were divided into four
groups: IRVA group, idiopathic left ventricular tachycardia (ILVT) group, other
cardiovascular diseases control (Heart-Disease-Control) group and healthy control
group. IRVA group had fourty-five patients; fifty subjects were in each of other three groups. All these four groups were age- and sex- matched. Every subject underwent baseline tests, including electrocardiogram, X-ray chest, transthoracic echocardiography, etc, on routine basis. Then all of these subjects went through more tests, including viral serology, MRI, serum cardiac autoantibodies, the mark of damage of myocardium, etc. The subjects in IRVA group, in ILVT group and in Heart-Disease-Control group were follow-up in 6-12 months.
Results MRI revealed right ventricular wall structural abnormalities more often in
IRVA group (31 [68.9%] of 45) than in ILVT group (3[6%] of 50, p<0.01), in
Heart-Disease-Control group (5[10%] of 50, p<0.01) and in healthy control group
(6[12%] of 50, p<0.01). These structural abnormalities included fixed focal wall
thinning, fatty deposits, small saccular aneurysm, etc. The positive rates of serum IgM
antibodies against Coxsackie B virus were low in each of these four groups (among
the four groups, p>0.05). But the positive rates of serum IgM antibodies against
cytomegalovirus were markedly higher in IRVA group (33[73.3%] of 45) than in other
three groups (6[12.0%] of 50, 4[8.0%] of 50 and 7[14.0%] of 30; IRVA group vs other
three groups, p<0.01). After 6-12 months follow-up, the positive rates of serum IgM
antibodies against cytomegalovirus in IRVA group (30 [66.7%] of 45) kept high, too.
Analysis of correlation found intensities of correlation between Coxsackie B virus
infection, IRVA and its right ventricular wall structural abnormalities were
low(p>0.05), but intensities of correlation between cytomegalovirus infection, IRVA
and its right ventricular wall structural abnormalities were high(p<0.001). Serum
levels of the mark of damage of myocardium (including creatine kinase, creatine
kinase-MB, and cardiac-specific troponin-I) were in normal ranges in all of these
groups(p>0.05). Among four common cardiac autoantibodies, the positive rates of
autoantibody against β_1 adrenergic receptor were higher in IRVA group than in other
three groups(IRVA group vs other three groups, p<0.01). But the positive rates of
other three autoantibodies were low in each of these four groups (among four groups,
p>0.05).
Conclusion The positive rates of cytomegalovirus infection are high in patients with IRVA. Focal structural abnormalities of right ventricular wall occur more often in these patients. Cytomegalovirus infection is correlated with occurrences of IRVA and its right ventricular wall structural abnormalities in IRVA patients. Cytomegalovirus infection may be the cause of IRVA. Effects of cytomegalovirus may be partly mediated by autoantibodies against β_1 adrenergic receptor.
Part III
Effects of radiofrequency ablation on idiopathic right
ventricular arrhythmias accompanied with persistent
cytomegalovirus infection
Background Radiofrequency catheter ablation (RFCA) has been used widely for treatment of tachyarrhythmias for many years.
Objective To explore effects of RFCA on idiopathic right ventricular arrhythmias (IRVA) accompanied with persistent cytomegalovirus infection. Methods Forty-five patients with IRVA were involved in this study. These patients meted with selecting criterion of IRVA. Before ablation, all patients received routine tests, including electrocardiography, X-ray chest, transthoracic echocardiography, etc. Then all of these patients went through more tests, including cytomegalovirus serology, MRI, serum cardiac autoantibodies, the mark of damage of myocardium and intracardiac electrophysiology, etc. All patients were follow-up in 6-12 months, and then received some tests again, including tests of cytomegalovirus serology and serum cardiac autoantibodies.
Results These forty-five patients detected serum IgM antibodies against cytomegalovirus. Among these patients, thirty-three patients (33/45, 73.3%) before ablation and thirty patients (30/45, 66.7%) after ablation 6-12 moonth follow-up were positive of the antibodies in serum. MRI revealed right ventricular wall structural abnormalities found in thirty-one patients (31 [68.9%] of 45). These structural abnormalities included fixed focal wall thinning, fatty deposits, small saccular aneurysm, etc. Positions of the right ventricular structural abnormalities were not related with origins of IRVA. Among four serum autoantibodies, only positives of autoantibodies against β_1 adrenergic receptor were high (62.2%), wheres the positives of other three autoantibodies were low. After follow-up, the positives of autoantibodies against β_1 adrenergic receptor kept high too (64.4%). In patients with positive IgM antibodies against cytomegalovirus, most of them were positive of autoantibodies against β_1 adrenergic receptor (25/33 , 75.8%). Intracardiac electrophysiology showed, thirty-five patients (35/45, 77.8%) had spontaneously IRVA, which configuration of QRS waves were same with that of usually time. Outbreak of IRVA in other nine patients (9/45, 20%) need infusion of epinephrine. IRVA were not induced in one patient (1/45, 2.2%) and ablation was abandoned. Among the forty-four patients received ablation, RFCA was success in forty-three patients (43/44, 97.7%) immediately after RFCA. One patient stoped ablation as strong chest pain. After 6-12 moonth follow-up, there were reoccurrents in five patients (5/44, 11.4%), but second ablation was success. The total rates of success were 95.6% (43/45). In thirty-three patients with positive IgM antibodies against cytomegalovirus, thirty-one (31/33, 93.9%) patients had success of RFCA. Among twenty-eight patients with positive autoantibodies against β_1 adrenergic receptor, RFCA was success in twenty-seven patients (27/28, 96.4%).
Conclusion RFCA is effect on IRVA in IRVA patients accompanied with persistent cytomegalovirus infection and positive serum autoantibodies against β_1 adrenergic receptor.
背景鼠巨细胞病毒(MCMV)感染诱导小鼠心律失常的研究尚属空白。
目的探讨MCMV感染诱导BALB/c小鼠心肌炎室性心律失常及其可能机理。
方法60只雄性4周龄BALB/c小鼠随机分成二组：实验组(A组，36只，MCMV腹腔注射感染小鼠)和对照组(B组，24只，3T3细胞裂解液腹腔注射小鼠)。于病毒接种后3、7、14、21、28、35、42、49、56、63和70天，眼内眦静脉取血提取血清测定血清抗心肌β_1-受体抗体，记录心电图，用断颈法分批处死小鼠，摘取心脏，提取少量心肌组织检测MCMV基因表达，并进行病理学和炎性因子免疫组化检查。分离BALB/c小鼠心室肌细胞，在电流钳和电压钳模式下，观察抗β1-受体抗体对心肌细胞动作电位及I_(Ca-L)电流的影响；运用激光共聚焦技术，观察抗β1-受体抗体对心肌细胞胞浆游离Ca~(2+)浓度([Ca~(2+)]_I)的影响。
结果A组36只小鼠70天心肌炎累积发病25只(25/36，69．4％)；累积死亡4只(4/36，11.1％)；B组无1只死亡。显微病理显示，A组接种病毒后7-14天，心肌病理改变最明显，表现为心肌细胞肿胀变性，单个心肌细胞核固缩、小灶性坏死，心肌组织局灶性或弥散性炎性细胞浸润；即使病毒感染后70天，心肌间质仍有散在炎性细胞浸润。病理学半定量分析发现，A组心肌组织在病毒感染后7-14天炎性细胞浸润和心肌小灶性坏死达高峰；心肌病理积分均在2分以下，属轻度病毒性心肌炎。免疫组化结果显示，感染病毒后3天，心肌组织炎性因子IL-1β和TNF-α蛋白表达呈阳性；而感染病毒后7天，心肌IL-1β和TNF-α蛋白呈强阳性表达。运用PCR技术，A组接种病毒后第3天和第7天心肌组织可检测到MCMV特异基因片段表达；但在病毒接种后第14天至第70天，心肌组织却不能检测到MCMV特异基因片段的表达；B组心肌始终不能检测到MCMV表达。B组小鼠前7周血清抗β1受体抗体滴度为0，而在第8-10周滴度轻微升高；A组小鼠前5周血清抗β1抗体滴度为0，但从第6周开始升高直至第10周；接种病毒后6-10周，二组血清抗β1受体抗体滴度比较，具有极显著性差异，P＜0.001。A组小鼠心电图异常累计发生率达50％；感染病毒后3天即可记录到心律失常；前5周心律失常主要是以窦性心律失常、房性心律失常和传导阻滞为主；而从第6周至第10周，虽然心肌组织不能检测到MCMV基因片段，但仍有心律失常发生，主要表现为室性心律失常和房性心律失常，此时血清抗β1受体抗体滴度明显升高。分离小鼠心室肌细胞，在电流钳模式下可见，1：100抗β1-受体抗体可明显延长动作电位平台期，预先给予美托洛尔阻断β1-受体，1：100抗β1-受体抗体基本没有延长动作电位平台期的作用；在电压钳模式下，1：100抗β1受体抗体可显著增加I_(Ca-L)电流，预先加入美托洛尔，则I_(Ca-L)基本不变。运用激光共聚焦技术可见，1：100抗β1受体抗体可显著增加心肌细胞胞内钙荧光强度，预先加入美托洛尔，抗β1-受体抗体仅能轻微增加胞内钙荧光强度。
结论MCMV感染可引起小鼠急慢性心肌炎，导致各种类型心律失常；感染早期的心肌病理改变及心律失常发生可能与MCMV感染直接损伤心肌有关，而慢性期的心肌病理改变和心律失常发生，很可能与MCMV诱导产生的自身抗体-抗β1受体抗体的作用相关；抗β1受体抗体可能通过增加I_(Ca-L)电流，增加心肌胞内钙浓度，诱发早期后除极，导致室性心律失常的发生。
第二部分巨细胞病毒感染与特发性右室心律失常及其右室壁异常相关性的研究
背景特发性右室心律失常(IRVA)的病因不明。
目的探讨巨细胞病毒(CMV)感染与IRVA发生及其MRI右室影像异常的相关性。
方法进行病例对照研究。分为四组，即IRVA组(45例)、特发性左室心动过速(ILVT)组(50例)、其他心脏病平行对照组(Heart-Disease-Control，50例)和健康对照组(Healthy-Control，50例)，每组性别年龄匹配。接受常规检查(心电图、X线胸片、心脏超声等)后，进行病毒血清学、心脏磁共振(MRI)、血清抗心肌自身抗体和心肌损伤标志物检查，随访6-12个月。
结果IRVA组中，单纯右室频发早搏12例、右室频发早搏伴反复性阵发性右室心动过速33例；根据心电图判断，心律失常起源于右室流出道37例、右室游离壁或心尖8例。ILVT组中，起源于左室间隔45例、左室心尖3例、左室游离壁2例。MRI结果显示，IRVA组所有患者左室均未发现异常，其中31例(31/45，68.9％)存在右室壁局灶性和散在性病变；ILVT组中2例(2/50，4％)左室间隔部存在微小室壁瘤，3例(3/50，6％)右室游离壁近间隔处变薄；Heart-Disease-Control组中5例(5/50，10％)右室壁存在局灶性病变；Healthy-Control组中6例(6/50，12％)右室存在上述局灶性病变；IRVA组MRI右室异常阳性率显著高于其它三组，差异具有极显著性(P＜0.01)。四组血清柯萨奇B组病毒(CVB)IgM抗体阳性率均低，组间比较无显著性差异。但IRVA组血清巨细胞病毒(CMV)IgM抗体阳性率(73.3％)显著高于其他三组(P＜0.01)；即使随访6-12个月，IRVA组CMV IgM阳性率仍然持续增高(66.7％)。四组血清CK、CK-MB、cTNI的测值结果均在正常范围，组间比较无显著性差异(P＞0.05)。相关性分析发现，CVB感染与IRVA发生及其MRI右室壁异常的关联强度较低(P＞0.05)，而CMV感染与IRVA发生及其MRI右室壁异常的关联强度较高(P＜0.001)。比较四组血清抗心肌自身抗体可见，四组的抗ANT抗体、抗M_2抗体及抗MHC抗体阳性率均低，组间差异无显著性(P＞0.05)；但抗β1受体抗体阳性率在四组间却存在显著性差异，表现为IRVA组的抗β1受体抗体阳性率显著高于其他三组(P＜0.01)，即使随访6-12个月，IRVA组的抗β1受体抗体阳性率依然增高。
结论IRVA患者CMV感染阳性率高，多数右室壁存在局限性病变；CMV感染与IRVA的发生及其右室壁异常相关，可能是引起IRVA并导致右室病变的病因；CMV感染引起IRVA可能与自身抗体-抗β1受体抗体作用有关。
第三部分巨细胞病毒持续感染状态特发性右室心律失常的射频消融疗效观察
背景射频消融(RFCA)广泛地应用于治疗快速性心律失常。
目的探讨RFCA对于伴有巨细胞病毒(CMV)持续感染状态的特发性右室心律失常(IRVA)的治疗效果。
方法符合入选标准的IRVA患者45例，其中男17例、女28例，年龄21-53(37.5±17)岁；IRVA病史3-25(9.7±8)年；IRVA表现为单纯右室频发早搏12例、右室频发早搏伴反复性阵发性右室心动过速33例；根据心电图判断，心律失常起源于右室流出道37例、右室游离壁或心尖8例。术前接受常规检查(心电图、X线胸片、心脏超声等)，并进行CMV病毒血清学、心脏磁共振(MRI)、血清抗心肌自身抗体、血清心肌损伤标志物以及心内电生理等项目检查。所有患者射频消融术后随访6-12个月，并再次接受CMV病毒血清学及血清抗心肌自身抗体检测。
结果45例IRVA患者术前均检测了血清CMV IgM抗体，33例(33/45，73.3％)阳性，随访6-12个月，仍有30例CMV IgM抗体阳性(30/45，66.7％)，提示多数IRVA患者处于CMV病毒持续感染状态。术前MRI影像显示，31例(31/45，68.9％)右室壁存在局灶性和散在性病变，其中右室游离壁变薄27例(27/45，60％)、右室流出道壁变薄9例(9/45，20％)、局限性脂肪沉积25例(25/45，55.6％)、右室游离壁微小膨胀瘤1例(1/45，2.2％)；MRI右室病变部位与IRVA部位不一致。术前4种血清抗心肌自身抗体中，只有抗心肌β_1受体抗体阳性率较高(62.2％)，而其他3种抗心肌自身抗体的阳性率较低；随访以后，抗心肌β_1受体抗体阳性率仍然持续增高(64.4％)，提示多数IRVA患者血清持续存在抗心肌β_1受体抗体。在CMVIgM抗体阳性患者中，多数抗心肌β_1受体抗体为阳性(25/33，75.8％)。所有患者术前均检测了血清心肌损伤标志物，测值结果在正常范围。心内电生理检查显示，35例(35/45，77.7％)有自发IRVA，QRS波形态与平时发作时一致；9例(9/45，20％)静脉滴注异丙肾上腺素方可诱发出与平素发作ORS波形态一致的IRVA；1例(1/45，2.2％)不能诱发IRVA并放弃RFCA。在44例接受RFCA中，43例(43/44，97.7％)术后即刻成功，1例放电时出现剧烈胸痛终止手术；随访6-12个月，5例(5/44，11.6％)复发，再次消融成功；RFCA总成功率为95.6％(43/45)。33例CMV IgM抗体阳性病例中，31例(31/33，93.9％)消融成功；28例抗心肌β_1受体抗体阳性病例中，27例(27/28，96.4％)消融成功。
结论对于伴有CMV持续感染状态及血清抗β_1受体抗体阳性的IRVA患者，射频消融仍然是治疗IRVA的有效方法。
Part I
Evaluation of myocarditis and cardiac arrhythmias induced by murine cytomegalovirus and their mechanisms
Bacground Lack of study of murine cardiac arrhythmias induced by cytomegalovirus Objective To evaluate the myocarditis and the cardiac arrhythmias induced by murine cytomegalovirus and to investigate their mechanisms.
Methods sixty male four weeks Balb/c mice were random divided into two groups: one was experiment group (A group, abdominal injection of murine cytomegalovirus) and control group (B group, abdominal injection of 3T3 liquid). At 3,7,14,21,28,35,42,49,56,63 and 70 day after cytomegalovirus infection, blood taken from canthus vessels was used to detect serum antibodies against myocardial β_1 receptor. Meantime, electrocardiographies of mice were recorded. After killing mice, getting out their hearts, tested gene express of murine cytomegalovirus, took pathological examination and immunohistochemical examination. After separating ventricular muscle cells of mice, tested effects of above antibodies against myocardial β_1 receptor on acting potential duration and L-type Ca~(2+) currents,under condition of current clamp and potential clamp. And then detected intracellular Ca~(2+) concentration of murine cardiac myocytes by laser confocal technique.
Results Total incidence of myocarditis of BALB/c mice in experiment group was 69.4% at 70 day after cytomegalovirus infection. Total mortality was 11.1% in this group at end of the experiment. There wasn't one murine died in another control group. Microscopical pathology showed that changes of myocardium pathologically were obvious in experiment group from 7day to 14 day after the virus infection. The main pathological changes were that some myocytes were swelling and denaturalization, other single myocyte was necrosis, and inflammatory cells infiltrated in myocardial tissue in a focus and dispersed fashion. Even if at 70 day after the virus infection, inflammatory cells still infiltrated in heart tissue dispersedly. Half quantitative analysis by pathology showed that the myocarditis induced by cytomegalovirus was mild. Result of immunohistochemistry showed that strong expression of proteins of cytokin IL-1β and TNF-α in myocardium at 3-7 day after the virus infection. In experiment group, gene of cytomegalovirus was deteced at 3-7 day after virus infection, and was undetectable from 14 day to 70 day after virus infection. Whereas, gene of cytomegalovirus was undetected at all the time in control group. From 1 to 7 weeks after virus infection, the anti-cardiac β_1 receptor autoantibody titer of sera in control group were zero, but mild elevation from 8 to 10 weeks. The anti-cardiac β_1 receptor autoantibody titer of sera were zero at former 5 weeks after virus infection and were significantly high elevation at latter 5 weeks after virus infection. Total incidences of cardiac arrhythmias were about 50% in A group. Most of cardiac arrhythmias were sinus arrhythmias, atrial arrhythmias and conduction black at the former 5 weeks, and were ventricular arrhythmias and atrial arrhythmias at the latter 5 weeks in A group. After separating ventricular muscle cells of mice, prolongations of acting potential duration of cardiomyocytes were observed when adding anti-cardiac β_1 receptor autoantibody diluted at 1:100 to incubation liquid, but this phenomenon were not found if adding metoprolol to incubation liquid beforehand. Autoantibodies against β_1-adrenoceptor diluted at 1:100 significantly increased the I_(Ca-L) peak current amplitude and elevation of intracellular Ca~(2+) fluorescent intensity. Wheres, blocking of β_1-adrenoceptor by metoprolol beforehand, autoantibodies against β_1-adrenoceptor diluted at 1:100 induced a slight increase of I_(Ca-L) peak current amplitude and mild elevation of intracellular Ca~(2+) fluorescent intensity.
Conclusion Murine cytomegalovirus can cause acute and chronic myocarditis, even more induce various cardiac arrhythmias in BALB/c mice. The pathologicall changes of myocardium and arrhythmias are related with cytomegalovirus infection at early period of the virus infection, but may be related with autoantibodies against β _1-adrenoceptor at chronic period. Autoantibodies against β_1-adrenoceptor may lead ventricular arrhythmias by increasing I_(Ca-L) current and elevation of intracellular Ca~(2+) concentration.
Part II
Correlation between viral infection, idiopathic right ventricular
arrhythmias and its morphological abnormalities of right
ventricle delineated by MRI
Background The etiology of idiopathic right ventricular arrhythmias (IRVA) is
unknown.
Objective To evaluate correlation between viral infection, IRVA and its
morphological abnormalities of right ventricle delineated by magnetic resonance
imaging(MRI) in patients with IRVA.
Methods Case control study was performed. All subjects were divided into four
groups: IRVA group, idiopathic left ventricular tachycardia (ILVT) group, other
cardiovascular diseases control (Heart-Disease-Control) group and healthy control
group. IRVA group had fourty-five patients; fifty subjects were in each of other three groups. All these four groups were age- and sex- matched. Every subject underwent baseline tests, including electrocardiogram, X-ray chest, transthoracic echocardiography, etc, on routine basis. Then all of these subjects went through more tests, including viral serology, MRI, serum cardiac autoantibodies, the mark of damage of myocardium, etc. The subjects in IRVA group, in ILVT group and in Heart-Disease-Control group were follow-up in 6-12 months.
Results MRI revealed right ventricular wall structural abnormalities more often in
IRVA group (31 [68.9%] of 45) than in ILVT group (3[6%] of 50, p<0.01), in
Heart-Disease-Control group (5[10%] of 50, p<0.01) and in healthy control group
(6[12%] of 50, p<0.01). These structural abnormalities included fixed focal wall
thinning, fatty deposits, small saccular aneurysm, etc. The positive rates of serum IgM
antibodies against Coxsackie B virus were low in each of these four groups (among
the four groups, p>0.05). But the positive rates of serum IgM antibodies against
cytomegalovirus were markedly higher in IRVA group (33[73.3%] of 45) than in other
three groups (6[12.0%] of 50, 4[8.0%] of 50 and 7[14.0%] of 30; IRVA group vs other
three groups, p<0.01). After 6-12 months follow-up, the positive rates of serum IgM
antibodies against cytomegalovirus in IRVA group (30 [66.7%] of 45) kept high, too.
Analysis of correlation found intensities of correlation between Coxsackie B virus
infection, IRVA and its right ventricular wall structural abnormalities were
low(p>0.05), but intensities of correlation between cytomegalovirus infection, IRVA
and its right ventricular wall structural abnormalities were high(p<0.001). Serum
levels of the mark of damage of myocardium (including creatine kinase, creatine
kinase-MB, and cardiac-specific troponin-I) were in normal ranges in all of these
groups(p>0.05). Among four common cardiac autoantibodies, the positive rates of
autoantibody against β_1 adrenergic receptor were higher in IRVA group than in other
three groups(IRVA group vs other three groups, p<0.01). But the positive rates of
other three autoantibodies were low in each of these four groups (among four groups,
p>0.05).
Conclusion The positive rates of cytomegalovirus infection are high in patients with IRVA. Focal structural abnormalities of right ventricular wall occur more often in these patients. Cytomegalovirus infection is correlated with occurrences of IRVA and its right ventricular wall structural abnormalities in IRVA patients. Cytomegalovirus infection may be the cause of IRVA. Effects of cytomegalovirus may be partly mediated by autoantibodies against β_1 adrenergic receptor.
Part III
Effects of radiofrequency ablation on idiopathic right
ventricular arrhythmias accompanied with persistent
cytomegalovirus infection
Background Radiofrequency catheter ablation (RFCA) has been used widely for treatment of tachyarrhythmias for many years.
Objective To explore effects of RFCA on idiopathic right ventricular arrhythmias (IRVA) accompanied with persistent cytomegalovirus infection. Methods Forty-five patients with IRVA were involved in this study. These patients meted with selecting criterion of IRVA. Before ablation, all patients received routine tests, including electrocardiography, X-ray chest, transthoracic echocardiography, etc. Then all of these patients went through more tests, including cytomegalovirus serology, MRI, serum cardiac autoantibodies, the mark of damage of myocardium and intracardiac electrophysiology, etc. All patients were follow-up in 6-12 months, and then received some tests again, including tests of cytomegalovirus serology and serum cardiac autoantibodies.
Results These forty-five patients detected serum IgM antibodies against cytomegalovirus. Among these patients, thirty-three patients (33/45, 73.3%) before ablation and thirty patients (30/45, 66.7%) after ablation 6-12 moonth follow-up were positive of the antibodies in serum. MRI revealed right ventricular wall structural abnormalities found in thirty-one patients (31 [68.9%] of 45). These structural abnormalities included fixed focal wall thinning, fatty deposits, small saccular aneurysm, etc. Positions of the right ventricular structural abnormalities were not related with origins of IRVA. Among four serum autoantibodies, only positives of autoantibodies against β_1 adrenergic receptor were high (62.2%), wheres the positives of other three autoantibodies were low. After follow-up, the positives of autoantibodies against β_1 adrenergic receptor kept high too (64.4%). In patients with positive IgM antibodies against cytomegalovirus, most of them were positive of autoantibodies against β_1 adrenergic receptor (25/33 , 75.8%). Intracardiac electrophysiology showed, thirty-five patients (35/45, 77.8%) had spontaneously IRVA, which configuration of QRS waves were same with that of usually time. Outbreak of IRVA in other nine patients (9/45, 20%) need infusion of epinephrine. IRVA were not induced in one patient (1/45, 2.2%) and ablation was abandoned. Among the forty-four patients received ablation, RFCA was success in forty-three patients (43/44, 97.7%) immediately after RFCA. One patient stoped ablation as strong chest pain. After 6-12 moonth follow-up, there were reoccurrents in five patients (5/44, 11.4%), but second ablation was success. The total rates of success were 95.6% (43/45). In thirty-three patients with positive IgM antibodies against cytomegalovirus, thirty-one (31/33, 93.9%) patients had success of RFCA. Among twenty-eight patients with positive autoantibodies against β_1 adrenergic receptor, RFCA was success in twenty-seven patients (27/28, 96.4%).
Conclusion RFCA is effect on IRVA in IRVA patients accompanied with persistent cytomegalovirus infection and positive serum autoantibodies against β_1 adrenergic receptor.
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17．沈关心．抗体的制备技术：抗体的纯化．见：沈关心，周汝麟．主编．现代免疫学实验技术．第1版．武汉：湖北科学技术出版社，1998；33-34．
18．邢斌，招明高，王海芳等．成年小鼠心肌细胞的分离及电生理特性．心脏杂志．2002；14(3)：210-212．
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8．李国光．病例对照研究．聂绍发主编．临床流行病学．武汉：湖北科技出版社．2003，1st，P55-71．
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27．郭继鸿 主编．心电图学．第一版．北京：人民卫生出版社，2002，401-437，545-594．
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16．程丽、余宏．第八章 疱疹病毒．杨占秋 余宏 主编．临床病毒学．中国医药科技出版社．2000年，第一版．P101-141．
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