Song Zhiyuan

Canine bone marrow mesenchymal stromal cells with lentiviral mHCN4 gene transfer create cardiac pacemakers

BACKGROUND AIMS The study objective was to test the ability of canine mesenchymal stromal cells (cMSC) transfected with the mouse hyperpolarization-activated cyclic nucleotide-gated channel 4 (mHCN4) gene to deliver a biologic pacemaker to the canine heart. METHODS AND RESULTS cMSC that were transfected by lentiviral vector with the cardiac pacemaker gene mHCN4 expressed high levels of Cs(+) -sensitive current (26.4 ± 1.8pA/pF at -140 mV; (n = 17) and were activated in the diastolic potential range with a reversal potential of -29.7 ± 2.5 mV (n = 14), confirming that the expressed current was Funny current (I(f))-like. Next, 3 × 10(6) cMSC transfected with either control plasmid or the mHCN4 gene construct were injected subepicardially into the canine right ventricular wall in situ. During sinus arrest, all control hearts had spontaneous atrioventricular node rhythms [rate = 21 ± 5 beats per minute (b.p.m.)]. In the mHCN4 group, six of eight animals developed spontaneous ventricular rhythms of right-sided origin (rate = 45 ± 9b.p.m.; P < 0.01). Moreover, immunohistochemical analysis of the injected regions demonstrated neither apoptosis nor cellular or humoral rejection at 2 weeks. CONCLUSIONS These results demonstrate that genetically modified cMSC can express functional HCN4 channels in vitro and in vivo and represent a novel delivery system for pacemaker genes into the heart.

mHCN4 genetically modified canine mesenchymal stem cells provide biological pacemaking function in complete dogs with atrioventricular block.

The study was undertaken to assess the properties of mouse HCN4 (mHCN4)‐modified canine mesenchymal stem cells (cMSCs) in dogs with experimentally induced complete atrioventricular (AV) block and electronic pacing.

CRT-P/D治疗射血分数降低的心力衰竭患者超反应发生的相关因素分析

目的 探讨心脏再同步化治疗(cardiac resynchronization therapy with pacemaker function/defibrillation function, CRT-P/D)在射血分数降低的心力衰竭患者中发生超反应的预测因素及不良事件的影响因素。 方法 回顾分析我科2015年1月至2017年1月收治的59例因心力衰竭采用CRT-P/D治疗并随访12个月以上的患者, 采集一般临床资料、心电图、心脏超声、不良事件发生等临床数据。以术后12个月左室射血分数(left ventricular ejection fraction, LVEF)升高幅度分3组:超反应组(幅度≥15%, 14例), 中度反应组(5%≤幅度＜15%, 23例), 轻度/无反应组(幅度＜5%, 22例)。采用方差分析及多因素Logistic回归分析筛选CRT-P/D治疗发生超反应的独立预测因素。采用Kaplan-Meier法进行预后分析, COX回归模型明确影响预后的危险因素。 结果 超反应组术前心房颤动比例、右房内径(right atrial diameter, RA)明显低于中度反应组及轻度/无反应组(P 结论 对于CRT-P/D治疗射血分数降低的心力衰竭患者, 病程＜1.5年、RA＜35 mm, 双心室起搏比率＞96%是发生超反应的重要预测因素, 心房颤动是影响术后不良事件发生的独立因素。

R-V 1 振幅对完全性左束支传导阻滞患者心脏再同步化治疗反应性的影响

目的探讨V 1 导联起始r波振幅(R-V 1 )对真性左束支传导阻滞(t-CLBBB)患者心脏再同步化治疗(cardiac resynchronization therapy，CRT)反应性的影响。 方法从2012年10月至2016年12月在我院接受CRT的108例患者中，遴选出术前心电图为t-CLBBB且资料完整的患者共55例，根据R-V 1 振幅分为研究组(R-V 1 ≥0.1 mV， n =26)和对照组(R-V 1 ＜0.1 mV， n =29)进行回顾性分析。由专人随访并记录心电图与超声心动图，评估CRT患者心功能应用纽约心脏病学会(New York Heart Association，NYHA)分级变化，测量QRS时限，检测各心腔舒张末期内径(左心室、右心室、左心房、右心房)、左心室射血分数(LVEF)、短轴缩短率(FS)等，评估两组患者的CRT反应性及临床获益情况。患者均完成至少6个月随访。 结果CRT前两组间各项资料差异无统计学意义( P >0.05)。CRT后两组患者QRS波时限、LVEF、FS、LVEDD等指标的改善差异均有统计学意义( P P 结论术前R-V 1 ＜0.1 mV的患者CRT反应性明显优于R-V 1 ≥0.1 mV的患者。R-V 1 振幅可作为影响CRT反应性的独立预测因素。

GW24-e2279 Geminin interference facilitate vascular smooth muscle cells proliferation via up-regulation of Cdt1

Objectives Silencing Geminin gene selectively by using RNA interference and investigate the mechanism of the Geminin gene silence regulating VSMCs proliferation. Methods VSMCs proliferation were determined by [3H]-thymidine incorporation, 5-ethynyl-2’-deoxyuridine (EdU) incorporation and flow cytometry; mRNA and Protein expression of Geminin and Cdt1 were determined by real time-PCR, westernblotting, and immunohistochemistry; the interrelationships of Geminin and Cdt1 were examed by immunofluorescence and co-immunoprecipitation (Co-IP). Results After transfected three pairs of siRNA sequences targeting Geminin gene respectively, a significant decrease of Geminin expression was observed in one of them, which acted as a stable model of building lentiviral interference. After Geminin gene silencing, the 3H-thymidine and EdU incorporation of positive interference group was significantly higher than that of two controls. The flow cytometry showed Geminin gene silencing contributed to the proliferation of VSMCs by supporting the G0/G1-S cell-cycle progression, and may be no apoptotic exists in this process. The protein expression and mRNA of Cdt1 in positive group significantly increased, compared with that of two controls. The results of immunofluorescence and Co-IP showed the close interaction between Cdt1 and Geminin gene in the proliferation of the VSMCs. Conclusions Geminin gene silencing contributes to the proliferation of A10 by supporting the G0/G1-S cell-cycle progression has been demonstrated, interestingly, this positive effect is in short term (24-48h) and reversibility. We believed that the results of the study are not caused by our research method, on account of lentivirus-delivered siRNA transfection instead of liposomes. Lentivirus is one kind of reversal viruses that could exhibit the basic feature and structure of reversal. It can integrate into the host genome and demonstrate long-term expression of integrated genes, and causes efficient down-regulation of gene expression, resulting in a functional inactivation of the target genes. In our study, we constructed a lentivirus vector-mediating RNAi targeting of Geminin (RNAi group), and it down-regulated Geminin expression of up to 80-90% efficacy in A10 cells with great specificity. In conclusion, lentivirus-delivered siRNAs are capable of specific, highly stable and functional silencing of Geminin gene expression in our study.

GW24-e3884 Canine bone marrow mesenchymal stromal cells modified with Shox2 gene rebuild biological pacemakers in vitro

Objectives Heart development is a strictly regulated process, and many gene families contribute to regulating genetic network to achieve this program. The homeobox genes are such a family encodes transcription factors, and it contains two closely related members, Shox and Shox2. During the embryonic cardiac development, Shox2 is restrictedly expressed in the sinoatrial node (SAN) and the sinus valves. Analyses of Shox2 knockout mouse models have demonstrated that Shox2 plays a crucial role during the SAN formation and the pacemaking system development. This study objective was to examine the ability of canine mesenchymal stromal cells (cMSCs) transfected with the mouse Shox2 gene to deliver a biological pacemaker. Methods We used lentivirus vectorto integrate the mouse Shox2 gene into the genome of cMSCs and were able to achieve stable expression of the Shox2 gene permanently. The cMSCs were randomised into two groups: the Shox2 group transfected with LV-Shox2- EGFP-cMSCs, and the control group transfected with LV-EGFP-cMSCs. Then all the positively transduced cMSCs were cultured with the new born rat myocardial cell lysate. After co-cultivation for seven days, we investigated the morphological changes of these transfected cMSCs. At the same time, the expression of cardiomyogenic markers Shox2 and HCN4 were determined by quantitative RT-PCR, Western Blot and immunofluorescence staining. In addition, the kinetics characters of If like current channels were detected by whole-cellpatch clamp. Results Shox2 and HCN4 proteins were highly expressed in experimental group compared with the control group. Ahyperpolarization-activated inward current was recorded by whole-cell patchclamp in cMSCs transfected with Shox2 gene. The If current recorded from the experimental group was sensitive to extracellular Cs+. Meanwhile, this current couldn’t be observed in control cells under the same conditions. Conclusions These results demonstrate that cMSCs modified by successful transfection with Shox2 can differentiate so as to develop spontaneous mechanical activity in vitro. Shox2 plays an essential role in the pacemaker development through the promotion of HCN4.

Transcatheter occlusion of pulmonary arteriovenous fistula by closure of the effluent vein with transseptal puncture

We describe a case of a 38-year-old female with pulmonary arteriovenous fistula (PAVF), who underwent successful transcatheter occlusion of the effluent vein via a transpulmonary venous approach with transseptal puncture. The patient presented with cough and shortness of breath from exercise. Cyanosis and clubbed finger were observed. A continuous murmur was heard at the third to fourth intercostal space over the left sternal border. Computer tomography angiography detected a single PAVF with 2 tortuous feeding arteries and 1 straight effluent vein in the left upper lobe. The delivery sheath and guide wire failed to be advanced into the fistula via transpulmonary arterial approach due to the tortuous feeding arteries. The PAVF had a wide and straight effluent vein that drained the fistula. Transseptal puncture in the atrial septum was performed with a transseptal sheath. The PDA occluder was advanced through the stretched flap into the effluent vein via a transpulmonary venous approach. Occluder deployment to occlude the effluent vein increased the arterial partial pressure of oxygen and preceded the disappearance of murmur. We conclude that transcatheter occlusion of the effluent vein is effective for closure of the PAVF with transseptal puncture. The transpulmonary venous approach is a feasible alternative, in cases with tortuous feeding arteries and a straight, dilated effluent vein that only drains the fistula.

A practical model to induce bradycardia by chemical ablation of sinus node and bilateral vagus nerve stimulation in rabbits, which satisfies the requirement for biopacemaking experiment in vivo

Objectives To investigate the feasibility of the bradyarrhythmia rabbit model built by sinus node ablation and bilateral vagus nerve stimulation. Methods 30 two-month-old Japanese white rabbits were randomly and equally divided into three groups: L group, R group and B group. Under 4∼6V stimulating voltage at 2.5 Hz, 5 Hz, 10 Hz, 15 Hz, 20 Hz stimulus frequency (1 Hz=60 beats/min), rabbits in three groups received left, right or bilateral vagus nerve stimulation after chemical ablation, respectively. ECGs were used to detect the change of HR and rhythm after stimulation, time for reaching the stimulus endpoint, recovery time, and HR and rhythm during the stimulus endpoint with continued stimulation. Results Basic heart rate significantly dropped down in those rabbits after chemical ablation with 20% formaldehyde (307±21 beats/min vs 126±28 beats/min, p<0.01). Only one case of sinus arrest occurred under right vagus nerve stimulation at 20 Hz. Heart rate in most rabbits slowed down with the left vagus nerve stimulation. Sinus arrest and junctional escape occurred with the increasing-frequency pulse stimulation. Sinus arrest and ventricular escape beat occurred with the bilateral vagus nerve stimulation at 10 Hz or more and two cases were that 3-degree atrioventricular block. It took less time to reach the stimulus endpoint with the increase of pulse frequency for bilateral vagus nerve stimulation. There was a significant statistical difference in the time in reaching the stimulus endpoint at ≥10 Hz compared with 2.5 Hz (p<0.05). The sinus rhythm of all rabbits restored to normal as before stimulation. But recovery time was needed with the increase of pulse frequency. There was a statistically significant difference in the recovery time at ≥5 Hz compared with 2.5 Hz (p<0.05). Conclusions Combination of sinus node ablation and bilateral vagus nerve stimulation can successfully establish a reliable bradyarrhythmia rabbit model, which satisfies the requirement for experiment in vivo.

CURATIVE EFFECT OF ENDOVASCULAR THERAPY ON TAKAYASU ARTERITIS

Objective To observe the curative effect of endovascular therapy including percutaneous transluminal angioplasty (PTA) and PTA plus stenting on Takayasu arteritis (TA). Methods Forty-eight inpatients with TA (17 males and 31 females) at the age of 19–53 years (mean 27.6±18.1 years), who underwent endovascular therapy (PTA only or PTA plus stenting)in Department of Cardiology, Southwest Hospital, Third Military Medical University (Chongqing, China) from January 2002 to May 2009, were enrolled in this study. Treatment outcome and data including erythrocyte sedimentation rate, C-reactive protein, CTA, MRA, Doppler vascular ultrasound findings obtained during a follow-up period of 42.8 months were analysed. Results A total of 180 lesions were detected in the 48 patients by angiography. Of the 101 lesions that underwent endovascular therapy, 29 were found in subclavian artery and arteria innominata, 28 in carotid, 34 in renal artery, two pulmonary, and eight coronary artery, respectively. Good revascularisation was achieved in all these lesions. No residual stenosis occurred in 76 lesions (75.2%) with only minimal residual stenosis observed in 25 patients (24.8%). Restenosis was observed in three lesions (12.0%) after treatment with PTA only and in five lesions (6.5%) after treatment with PTA plus stenting during the follow-up period of 3–6 months. No significant complication occurred in all recurrent stenoses after endovascular therapy. Conclusions Endovascular therapy including simple PTA or PTA plus stenting is a safe and effective treatment modality for chronic inactive TA.

Endovascular stenting for treatment of pulmonary branch stenosis in adult

Objective To explore the curative effect and safety of endovascular stenting for treatment of pulmonary branch stenosis. Methods Transthoracic echocardiography and right-sided haemodynamics revealed severe pulmonary hypertension in 2 cases (1 male and 1 female). In one case, pulmonary angiography revealed a severe stenosis at origination of right lower lobe branch, haemodynamics showed gradient pressure of 70 mm Hg; In another case, pulmonary branch stenoses at origination of left and right pulmonary artery was revealed, the right pulmonary stenosis was more severe (gradient pressure of 74 mm Hg). According to pulmonary angiography, CP stent (CP8Z34) and BIB balloon (16 mm) was selected in one case, CP stent (CP8Z45) and BIB balloon (16 mm) was selected in another case. Result After CP stent was deployed, the pulmonary branch stenosis disappeared, and descent of the gradient pressure is very obvious (70–10 mm Hg in one case, 74–24 mm Hg in another case). The symptom of dyspnoea was significantly relieved post procedure. At follow-up, the patients remained asymptomatic without complications, Transthoracic echocardiography revealed right ventricle had decreased. Conclusion Endovascular stenting for treatment of pulmonary branch stenosis in adult is effective and safe.