心脏收缩力调节对慢性心力衰竭兔心肌自噬的影响及可能机制

doi:10.3969/j.issn.1004-583X.2024.11.011

摘要/Abstract

摘要：

目的研究心脏收缩力调节(CCM)干预对慢性心力衰竭(CHF)兔心肌自噬的影响及可能机制。方法将30只体重2.5~3.5 kg的健康大白兔随机分成假手术组(Sham组)、HF组和CCM干预组(CCM组),每组各10只。HF组通过升主动脉根部套扎法建立慢性心力衰竭模型,而CCM组在成功建立HF模型后进行为期4周的CCM干预。在实验的第12周和第16周,对各组进行心脏功能的超声心动图检测,利用免疫荧光染色技术检测自噬过程中一个关键的标记蛋白微管相关蛋白1轻链3(microtuble-associated protein 1 light chain 3, LC3)的表达情况,此外,运用western blot蛋白印迹技术,观察P62蛋白、Beclin1蛋白和LC3B(II/I)蛋白等表达水平,并测定各组动物心肌组织中蛋白激酶B(AKT)的三个亚型AKT1、AKT2、AKT3,磷脂酰肌醇-3-激酶(PI3K)的亚型PI3K(α110)、PI3K(α85)以及哺乳动物雷帕霉素靶蛋白(mTOR)的表达情况。结果实验第12周末,HF组和CCM组左室舒张末期内径(LVEDD)和收缩末期内径(LVESD)相较于假手术组增加,而左心室射血分数(LVEF)和左室短轴缩短率(LVFS)相比假手术组明显下降(P<0.05);实验第16周末,CCM组LVEF、LVFS、LVESD、LVEDD指标相较于HF组有明显改善(P<0.05)。与HF组相比,经CCM干预后的兔的心肌组织P62蛋白表达水平显著升高,LC3免疫荧光染色阳性染色减少,Beclin1蛋白和LC3B蛋白表达水平显著下降,LC3B(II/I)比值显著下降(P<0.05)。此外,经CCM干预后的心肌组织内AKT1、AKT2、AKT3、mTOR蛋白表达水平显著减少(P<0.05),而PI3K(α110)、PI3K(α85)蛋白的表达则增加(P<0.05), 与HF组相比差异有统计学意义。结论 CCM干预可通过调节PI3K/AKT信号通路,改善HF兔心肌自噬相关蛋白的异常表达,从而减轻心肌自噬活动,并改善心肌的舒缩功能。

关键词: 心力衰竭, 自噬, 心肌收缩, 自噬相关蛋白, PI3K/AKT, 兔

Abstract:

Objective To explore the effect of cardiac contractility modulation (CCM) on the autophagy of cardiomyocytes in rabbits with chronic heart failure (CHF) and the underlying mechanisms. Methods Thirty healthy New Zealand White rabbits weighing 2.5-3.5 kg were randomly assigned to the sham operation group, HF group, and CCM group, with 10 rabbits in each group. A CHF model was established by the ascending aortic constriction method, followed by CCM for 4 weeks. At 12 and 16 weeks, echocardiography was employed to assess the cardiac function. Expression level of the microtuble-associated protein 1 light chain 3 (LC3) was detected by immunofluorescence staining. Protein levels of P62, Beclin1, light chain 3B II/I (LC3B II/I), protein kinase B (AKT)1, AKT2, AKT3, phosphatidylinositol 3-kinase (PI3K) α110, PI3K α85 and mammalian target of rapamycin (mTOR) in myocardial tissue were detected by Western blot.Results At 12 weeks, rabbits in the HF and CCM groups had significantly larger left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD), and lower left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) than those of sham operation group (P<0.05). At 16 weeks, significant improvements in LVEF, LVFS, LVESD and LVEDD were observed in the CCM group than HF group (P<0.05). Compared with those of the HF group, rabbits in the CCM group had significantly upregulated p62, PI3K α110 and PI3K α85, less LC3-positive immunofluorescence staining, downregulated Beclin1, LC3B, AKT1-3 and mTOR in myocardium and lower LC3B(II/I) ratio (all P<0.05). Conclusion CCM can improve the abnormal expression of LC3B (II/I), Beclin1 and p62 protein in myocardium of CHF rabbits, reduce the activity of autophagy in rabbit myocardium, and improve myocardial contractile and diastolic function by regulating the PI3K/AKT signaling pathway.

Key words: heart failure, autophagy, myocardial contraction, autophagy related protein, Pi3k/akt, rabbit

中图分类号:

R541.6

郝清卿, 吕仕林, 张静, 张立涛, 张飞飞. 心脏收缩力调节对慢性心力衰竭兔心肌自噬的影响及可能机制[J]. 临床荟萃, 2024, 39(11): 1026-1034.

Hao Qingqing, Lv Shilin, Zhang Jing, Zhang Litao, Zhang Feifei. Effects of cardiac contractility modulation on the autophagy of cardiomyocytes in rabbits with chronic heart failure and the underlying mechanisms[J]. Clinical Focus, 2024, 39(11): 1026-1034.

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://huicui.hebmu.edu.cn/CN/10.3969/j.issn.1004-583X.2024.11.011

https://huicui.hebmu.edu.cn/CN/Y2024/V39/I11/1026

图/表 11

表1 开胸术前3组超声心动图结果

Tab.1 Echocardiographic results in three groups before thoracotomy

组别	例数	IVSd(mm)	LVPWd(mm)	LVESD(mm)	LVEDD(mm)	LVEF (%)	LVFS (%)
Sham组	9	2.04±0.18	1.98±0.15	9.07±0.57	11.31±0.26	67.32±3.62	34.39±3.04
HF组	9	2.01±0.20	2.00±0.15	9.20±0.44	11.17±0.47	66.94±3.49	34.40±3.35
CCM组	9	203.00±0.20	1.98±0.15	9.08±0.57	10.98±0.51	68.33±2.51	35.54±2.43
$F$ 值		0.06	0.04	0.17	0.34	0.44	0.45
P值		0.942	0.963	0.841	0.280	0.649	0.642

表1 开胸术前3组超声心动图结果

Tab.1 Echocardiographic results in three groups before thoracotomy

组别	例数	IVSd(mm)	LVPWd(mm)	LVESD(mm)	LVEDD(mm)	LVEF (%)	LVFS (%)
Sham组	9	2.04±0.18	1.98±0.15	9.07±0.57	11.31±0.26	67.32±3.62	34.39±3.04
HF组	9	2.01±0.20	2.00±0.15	9.20±0.44	11.17±0.47	66.94±3.49	34.40±3.35
CCM组	9	203.00±0.20	1.98±0.15	9.08±0.57	10.98±0.51	68.33±2.51	35.54±2.43
$F$ 值		0.06	0.04	0.17	0.34	0.44	0.45
P值		0.942	0.963	0.841	0.280	0.649	0.642

表2 手术后12周3组超声心动图结果

Tab.2 Echocardiographic results of three groups at 12 weeks postoperatively

组别	例数	IVSd(mm)	LVPWd(mm)	LVESD(mm)	LVEDD(mm)	LVEF (%)	LVFS (%)
Sham组	9	2.09±0.23	2.05±0.19	9.07±0.32	11.18±0.56	67.74±4.81	34.18±3.47
HF组	9	2.01±0.24	2.09±0.15	12.13±0.52^*	14.70±0.^*	38.22±1.99^*	19.58±1.82^*
CCM组	9	2.05±0.14	2.13±0.18	11.98±0.54^*	14.48±0.78^*	38.11±1.62^*	19.64±1.43^*
$F$ 值		0.35	0.40	120.66	68.55	265.39	109.87
P值		0.708	0.678	0.000	0.000	0.000	0.000

表2 手术后12周3组超声心动图结果

Tab.2 Echocardiographic results of three groups at 12 weeks postoperatively

组别	例数	IVSd(mm)	LVPWd(mm)	LVESD(mm)	LVEDD(mm)	LVEF (%)	LVFS (%)
Sham组	9	2.09±0.23	2.05±0.19	9.07±0.32	11.18±0.56	67.74±4.81	34.18±3.47
HF组	9	2.01±0.24	2.09±0.15	12.13±0.52^*	14.70±0.^*	38.22±1.99^*	19.58±1.82^*
CCM组	9	2.05±0.14	2.13±0.18	11.98±0.54^*	14.48±0.78^*	38.11±1.62^*	19.64±1.43^*
$F$ 值		0.35	0.40	120.66	68.55	265.39	109.87
P值		0.708	0.678	0.000	0.000	0.000	0.000

表3 手术后16周3组超声心动图结果

Tab.3 Echocardiographic results of three groups at 16 weeks postoperatively

组别	例数	IVSd(mm)	LVPWd(mm)	LVESD(mm)	LVEDD(mm)	LVEF (%)	LVFS (%)
Sham组	9	2.06±0.20	2.03±0.19	9.40±0.44	11.29±0.42	68.00±4.47	34.31±3.66
HF组	9	2.05±0.25	2.11±0.20	11.82±0.36^*	14.40±0.38^*	37.11±1.69^*	18.76±1.18^*
CCM组	9	2.03±0.17	2.13±0.21	10.01±0.28^*#	12.94±0.48^*#	45.22±1.86^*#	22.72±1.15^*#
$F$ 值		0.04	0.63	106.52	116.98	263.75	109.73
P值		0.965	0.544	0.000	0.000	0.000	0.000

表3 手术后16周3组超声心动图结果

Tab.3 Echocardiographic results of three groups at 16 weeks postoperatively

组别	例数	IVSd(mm)	LVPWd(mm)	LVESD(mm)	LVEDD(mm)	LVEF (%)	LVFS (%)
Sham组	9	2.06±0.20	2.03±0.19	9.40±0.44	11.29±0.42	68.00±4.47	34.31±3.66
HF组	9	2.05±0.25	2.11±0.20	11.82±0.36^*	14.40±0.38^*	37.11±1.69^*	18.76±1.18^*
CCM组	9	2.03±0.17	2.13±0.21	10.01±0.28^*#	12.94±0.48^*#	45.22±1.86^*#	22.72±1.15^*#
$F$ 值		0.04	0.63	106.52	116.98	263.75	109.73
P值		0.965	0.544	0.000	0.000	0.000	0.000

图1 HF兔的典型心脏超声成像和3组心脏超声指标比较 a~b.HF兔典型心脏超声成像;c~h.三组心脏超声指标比较;注:与sham组比较,*P<0.05;与HF组比较,#P<0.05

Fig.1 The typical echocardiographic imaging of HF rabbits and the echocardiographic parameters of the three groups a-b. Typical echocardiographic imaging of HF rabbits; c-h. Comparison of echocardiographic parameters among the three groups

图2 3组心肌组织LC3免疫荧光染色比较(×400)

Fig.2 LC3 immunofluorescence staining of myocardial tissue among three groups (×400)

表4 各组心肌组织Beclin1、P62、LC3B(II/I)蛋白表达水平比较

Tab.4 Beclin1, P62 and LC3B(II/I) protein expression levels of myocardial tissue among three groups

组别	例数	Beclin1	P62	LC3B(II/I)
Sham组	9	0.34±0.15	0.50±0.08	1.00±0.10
HF组	9	0.85±0.06^*	0.18±0.07^*	1.47±0.15^*
CCM组	9	0.60±0.11^*#	0.28±0.06^*#	1.13±0.12^#

图3 3组心肌组织Beclin1、P62、LC3B(II/I)蛋白条带及表达水平比较 a.3组Beclin1、P62、LC3B(II/I)蛋白的Western blot蛋白条带结果;b.Beclin1蛋白表达情况;c.P62蛋白表达情况;d.LC3B(II/I)表达情况;注:与sham组比较,*P<0.05;与HF组比较,#P<0.05,柱状图和误差条表示平均值±标准差

Fig.3 The protein bands and expression levels of Beclin1, P62 and LC3B(II/I) of myocardial tissue among the three groups a. Western blot results of Beclin1, P62 and LC3B(II/I) proteins in the three groups; b. Beclin1 protein expression; c.P62 protein expression; d. LC3B (II/I) expression

图4 3三组心肌组织中AKT1、AKT2、AKT3、PI3K(α110) 、PI3K (α85)蛋白条带及表达水平比较 a.3组AKT1、AKT2、AKT3、PI3K(α110)、PI3K (α85)的Western blot蛋白条带结果;b.AKT1表达情况;c.AKT2表达情况;d.AKT3表达情况;e.PI3K(α110)蛋白表达情况;f.PI3K(α85)蛋白表达情况;注:与sham组比较,*P<0.05;与HF组比较,#P<0.05;柱状图和误差条表示平均值±标准差

Fig.4 The protein bands and expression levels of AKT1, AKT2, AKT3, PI3K(α110) and PI3K(α85) of myocardial tissue among the three groups a. Western blot results of AKT1, AKT2, AKT3, PI3K(α110) and PI3K(α85) in the three groups; b.AKT1 expression; c. AKT2 expression; d.AKT3 expression; e.PI3K(α110) protein expression; f.PI3K (α85) protein expression

表5 三组心肌组织AKT1, AKT2, AKT3, PI3K (α110), PI3K (α85)蛋白表达水平比较

Tab.5 AKT1, AKT2, AKT3, PI3K (α110), and PI3K (α85) protein expression levels of myocardial tissue among three groups

组别	例数	AKT1	AKT2	AKT3	PI3K (α110)	PI3K (α85)
Sham组	9	0.10±0.03	0.24±0.07	0.19±0.05	0.40±0.04	0.62±0.10
HF组	9	0.52±0.06^*	0.58±0.07^*	0.50±0.06^*	0.15±0.05^*	0.24±0.04^*
CCM组	9	0.22±0.04^*#	0.42±0.06^*#	0.29±0.06^*#	0.26±0.08^*#	0.45±0.05^*#

图5 3组心肌组织中mTOR蛋白条带及表达水平比较注:与sham组比较,*P<0.05;与HF组比较,#P<0.05

Fig.5 The protein bands and expression levels of mTOR of myocardial tissue among the three groups

表6 三组心肌组织mTOR蛋白表达水平比较

Tab.6 The mTOR protein expression levels of myocardial tissue among three groups

组别	例数	mTOR
Sham组	9	0.35±0.14
HF组	9	0.75±0.22^*
CCM组	9	0.50±0.14^#

参考文献 18

[1]	郑博, 刘耀琨, 霍勇. 2019 年中国心血管领域主要临床研究回顾[J]. 中华心血管病杂志(网络版), 2020, 3(1): 1-9.
[2]	Zhang F, Liu L, Xie Y, et al. Cardiac contractility modulation ameliorates myocardial metabolic remodeling in a rabbit model of chronic heart failure through activation of AMPK and PPAR-α pathway[J]. Open Med (Wars), 2022, 17(1):365-374.
[3]	Abraham WT, Fisher WG, Smith AL, et al. Cardiac resynchronization in chronic heart failure[J]. N Engl J Med, 2002, 346(24):1845-1853.
[4]	Tsukada M, Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae[J]. FEBS Lett, 1993, 333(1-2):169-174. doi: 10.1016/0014-5793(93)80398-e pmid: 8224160
[5]	He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy[J]. Annu Rev Genet, 2009, 43(1):67-93.
[6]	Kuschyk J, Kloppe A, Schmidt-Schweda S, et al. Cardiac contractility modulation: A technical guide for device implantation[J]. Rev Cardiovasc Med, 2017, 18(1):1-13.
[7]	Kleemann T. Cardiac contractil ty modulation. A new form of therapy for patients with heart failure and narrow QRS complex?[J]. Herz, 2015, 40(7):945-951. doi: 10.1007/s00059-015-4362-8 pmid: 26446549
[8]	Ha T, Li Y, Gao X, et al. Attenuation of cardiac hypertrophy by inhibiting both mTOR and NFκB activation in vivo[J]. Free Radic Biol Med, 2005, 39(12):1570-1580.
[9]	McMullen JR, Sherwood MC, Tarnavski O, et al. Inhibition of mTOR signaling with rapamycin regresses established cardiac hypertrophy induced by pressure overload[J]. Circulation, 2004, 109(24):3050-3055. doi: 10.1161/01.CIR.0000130641.08705.45 pmid: 15184287
[10]	Singh SR, Zech AT, Geertz B, et al. Activation of autophagy ameliorates cardiomyopathy in Mybpc3-targeted knockin mice[J]. Circ Heart Fail, 2017, 10(10):e004140.
[11]	Song L, Su M, Wang S, et al. MiR-451 is decreased in hypertrophic cardiomyopathy and regulates autophagy by targeting TSC 1[J]. J Cell Mol Med, 2014, 18(11):2266-2274. doi: 10.1111/jcmm.12380 pmid: 25209900
[12]	Bhuiyan MS, Pattison JS, Osinska H, et al. Enhanced autophagy ameliorates cardiac proteinopathy[J]. J Clin Invest, 2013, 123(12):5284-5297. doi: 10.1172/JCI70877 pmid: 24177425
[13]	Schlossarek S, Englmann DR, Sultan KR, et al. Defective proteolytic systems in Mybpc3-targeted mice with cardiac hypertrophy[J]. Basic Res Cardiol, 2012, 107(1):235. doi: 10.1007/s00395-011-0235-3 pmid: 22189562
[14]	Xie Z, Lau K, Eby B, et al. Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice[J]. Diabetes, 2011, 60(6):1770-1778. doi: 10.2337/db10-0351 pmid: 21562078
[15]	Hill SM, Wrobel L, Ashkenazi A, et al. VCP/p97 regulates Beclin-1-dependent autophagy initiation[J]. Nat Chem Biol, 2021, 17(4): 448-455. doi: 10.1038/s41589-020-00726-x pmid: 33510452
[16]	Yang Z, Su W, Zhang Y, et al. Selective inhibition of PKCβ2 improves Caveolin-3/ eNOS signaling and attenuates lipopolysaccha-ride-induced injury by inhibiting autophagy in H9C2 cardiomyocytes[J]. J Mol Histol, 2021, 52(4): 705-715.
[17]	Lamark T, Svenning S, Johansen T. Regulation of selective autophagy:The p62/SQSTM1 paradigm[J]. Essays Biochem, 2017, 61(6):609-624. doi: 10.1042/EBC20170035 pmid: 29233872
[18]	Zhang DM, Liu JS, Deng LJ, et al. Arenobufagin, a natural bufadienolide from toad venom, induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/mTOR pathway[J]. Carcinogenesis, 2013, 34(6):1331-1342.