Cardiomyocyte apoptosis in the failing heart — A critical review from definition and classification of cell death

It has been suggested that apoptosis may be responsible for a significant amount of the cardiomyocyte death that contributes to the development and progression of heart failure. However, studies of actual heart disease and in vivo experimental models have provided little or no direct morphological evidence that cardiomyocyte apoptosis occurs at any stage of heart failure, despite the availability of much indirect evidence that includes detection of DNA fragmentation and apoptosis-related factors. The Nomenclature Committee on Cell Death (NCCD), an international organization consulting on cell death, proposed an international standard for the definition and classification of cell death, in which cell death was defined based purely on morphological criteria. This is because there is no clear-cut equivalence between ultrastructural alterations and biochemical cell death characteristics. This review will first introduce the NCCD definition and classification of cell death and, based on this classification, survey the available data from both animals and humans to critically assess the impact of cardiomyocyte apoptosis during the progression of heart failure of various etiologies. Particularly noteworthy is the wide variation in the reported rates of apoptosis – e.g., the difference was > 1000-fold in one heart failure model – but even more importantly, no morphological (ultrastructural) data has ever been shown definitively demonstrating apoptosis of a cardiomyocyte. We conclude from our survey that even the existence of cardiomyocyte apoptosis in heart failure remains controversial.     

Inhibition of p38 MAP kinase improves survival of cardiac myocytes with hypoxia and burn serum challenge

This study was aimed to investigate the effects of SB203580, the specific p38 mitogen-activated protein (MAP) kinase inhibitor, on cardiac myocyte survival and secretion of cytokines in an in vitro model of hypoxia and burn serum challenge. Results demonstrated that hypoxia and burn serum induced a persistent activation of p38 MAP kinase in primary cultured neonatal rat cardiomyocytes during the 12h period of stimulation, concomitant with a time-dependent increased expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide (iNOS), a progressively developed oxidative stress reflected by malondialdehyde (MDA) production, and myocytes injury evidenced by the increased levels of released lactate dehydrogenase (LDH) and the decreased myocyte viability. Furthermore, hypoxia and burn serum resulted in a significant increase in myocyte apoptosis, which may account for the impairment of myocyte viability as observed. SB203580 abolished p38 MAP kinase activation, blunted the upregulation of TNF-alpha, iNOS and the subsequent nitric oxide (NO) production, reduced oxidative stress, and alleviated hypoxia and burn serum-induced myocytes injury or apoptosis. These results demonstrated for the first time that inhibition of p38 MAP kinase improves survival of cardiac myocytes with hypoxia and burn serum challenge possibly via reducing the production of cytokines, such as TNF-alpha and NO, and the subsequent oxidative stress, providing strong evidence that the excessive inflammatory cytokines produced by cardiomyocytes themselves may be sufficient to cause myocardial injury after burn.     

Production of arrays of cardiac and skeletal muscle myofibers by micropatterning techniques on a soft substrate

Micropatterning and microfabrication techniques have been widely used to pattern cells on surfaces and to have a deeper insight into many processes in cell biology such as cell adhesion and interactions with the surrounding environment. The aim of this study was the development of an easy and versatile technique for the in vitro production of arrays of functional cardiac and skeletal muscle myofibers using micropatterning techniques on soft substrates. Cardiomyocytes were used for the production of oriented cardiac myofibers whereas mouse muscle satellite cells for that of differentiated parallel myotubes. We performed micro-contact printing of extracellular matrix proteins on soft polyacrylamide-based hydrogels photopolymerized onto functionalized glass slides. Our methods proved to be simple, repeatable and effective in obtaining an extremely selective adhesion of both cardiomyocytes and satellite cells onto patterned soft hydrogel surfaces. Cardiomyocytes resulted in aligned cardiac myofibers able to exhibit a synchronous contractile activity after 2 days of culture. We demonstrated for the first time that murine satellite cells, cultured on a soft hydrogel substrate, fuse and form aligned myotubes after 7 days of culture. Immunofluorescence analyses confirmed correct expression of cell phenotype, differentiation markers and sarcomeric organization. These results were obtained in myotubes derived from satellite cells from both wild type and MDX mice which are research models for the study of muscle dystrophy. These arrays of both cardiac and skeletal muscle myofibers could be used as in vitro models for pharmacological screening tests or biological studies at the single fiber level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

葱白提取物预处理对心肌缺血再灌注损伤及相关生化因素的影响

目的:观察葱白提取物(FOB)预处理对大鼠离体心脏缺血再灌注损伤(MIRI)及相关生化指标的影响,探讨FOB抗MIRI的机制。方法:利用Langendorff建立大鼠离体心脏缺血再灌注(I/R)模型,观察左心室发展压(LVDP)恢复和再灌注痉挛度。用生化技术检测冠脉流出液中乳酸脱氢酶(LDH)、磷酸肌酸激酶(CK)活性和心肌细胞线粒体超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、ATP酶活性以及丙二醛(MDA)含量。结果:(1)FOB(25g/L、50g/L、100g/L)预处理对心肌I/R损伤后的LVDP恢复和再灌注痉挛度有明显改善作用,并随浓度增加,对心脏保护作用逐渐增强而加强,100g/LFOB预处理后的LVDP恢复和痉挛度的改善均接近正常组值(分别与I/R组和25g/LFOB组相比,均P<0.01;与50g/LFOB相比,均P<0.05),心脏恢复跳动更迅速,收缩和舒张规则有力;(2)FOB(25g/L、50g/L、100g/L)预处理明显降低LDH、CK活性和MDA含量,显著增加SOD、GSHPx、ATP酶活性,且随FOB浓度增高,上述作用更加明显,100g/LFOB预处理组各项生化指标与Control组无明显差异。结论:FOB预处理能有效保护MIRI,其机制与FOB减少心脏I/R时心肌细胞内Ca2+超载和抗脂质过氧化有密切关系。     

骨形态蛋白2体外诱导大鼠骨髓间充质干细胞分化为心肌样细胞

目的 应用骨形态蛋白2(BMP-2)体外诱导骨髓间充质干细胞(MSCs)向心肌样细胞分化,探索MSCs向心肌细胞分化的诱导方法.方法 取SD大鼠四肢骨骨髓,分离培养MSCs,应用BMP-2定向诱导,相差显微镜观察细胞形态学变化,应用免疫细胞化学、激光扫描共焦显微镜技术检测结蛋白(desmin)、α-横纹肌肌动蛋白(α-sareomeric actin)、心肌特异性肌钙蛋白(C-TnT)的表达,透射电镜鉴定.在诱导后7d、21d和28d 3个时间点以半定量RT-PCR方法检测细胞心肌早期转录因子(GATA4)和心肌特异性α-肌凝蛋白重链(α-MHC)的表达.结果 BMP-2诱导后的MSCs细胞伸出伪足,排列方向渐趋一致.MSCs体外经BMP-2诱导后分化的细胞结蛋白、α-横纹肌肌动蛋白、C-TnT均表达阳性,结蛋白、α-横级肌肌动蛋白阳性率较高,分别为37.28%和63.94%,而C-TnT阳性率较低为34.66%.透射电镜下可见到平行排列的肌丝,大量的粗面内质网和线粒体,富含糖原和核糖体.RT-PCR结果显示,GATA4于诱导后7d弱表达,21d表达增强,28d表达减弱.α-MHC在诱导后7d不表达,21d弱表达,28d表达明显.结论骨髓间充质干细胞在BMP-2诱导下可定向分化为心肌样细胞,是自体心肌细胞的一种良好供体来源.     

灯盏花素对大鼠心室肌细胞钠通道的影响

目的： 研究灯盏花素对大鼠心室肌细胞膜钠通道的影响,在离子通道水平探讨灯盏花素的抗心律失常作用机制。方法： 用急性酶解法获得单个大鼠心室肌细胞,标准的全细胞膜片钳技术记录钠通道电流(INa)。结果： (1) 灯盏花素呈浓度依赖性抑制INa,在-30 mV时,含1、3、30、100 mg·L-1灯盏花素的细胞外液分别灌流细胞3 min,分别阻断INa峰电流(7.98±0.60)%、(37.73±2.31)%、(65.58±2.90)% 和(88.09±5.60)%。INa激活电位为-70 mV,最大峰电位为-30 mV,翻转电位为5 mV,激活和失活过程呈电压和时间依赖性。30 mg·L-1灯盏花素使电流电压曲线明显上移,峰值电流从（13.49±1.25）pA/pF 减少至（4.78±0.85）pA/pF,n=8,P<0.05,冲洗后可以不完全恢复。（2）灯盏花素能使钠电流失活曲线明显左移。（3）灯盏花素使钠电流激活曲线明显右移。（4）灯盏花素使钠电流复活明显减慢。结论： 灯盏花素能够抑制心肌细胞钠通道电流,并呈浓度依赖性。     

成熟心肌细胞诱导胚胎干细胞定向分化为心肌样细胞

目的： 拟证实成熟心肌细胞对胚胎干细胞（ESCs）定向分化的诱导作用。方法：分离培养SD大鼠乳鼠心肌细胞,以DAPI（4’6-联眯-2-苯基吲哚）染核作为细胞标记。取昆明小鼠3.5-4 d的囊胚培养后分离出ESCs,以1和（或）2代的小鼠ESCs细胞团与心肌细胞共培养。录像动态观察ESCs向心肌细胞分化的情况;分别于共培养后3、7、14 d对ESCs行肌钙蛋白T（cTnT）免疫荧光染色检测。结果：1代或2代的ESCs和/或细胞团与心肌细胞共培养约7 d左右出现成节律搏动的心肌样细胞;最好实验批次,可计数到1/4以上的ESCs和/或ESCs细胞团出现节律性搏动。心肌细胞共培养体系中添加0.6% DMSO时,节律搏动的心肌样细胞分化率未见提高。心肌细胞诱导组,记录已搏动和未搏动ESCs诱导分化后心肌样细胞cTnT蛋白荧光染色阳性;ESCs与心肌成纤维细胞共培养诱导组,分化的细胞cTnT蛋白荧光染阴性;0.6% DMSO诱导组约3％的细胞cTnT蛋白荧光染色阳性;DMSO联合心肌细胞诱导组,结果与单纯心肌细胞诱导组相似。结论：未经建系操作的ESCs可直接诱导分化为节律搏动的心肌细胞;成熟心肌细胞与ESCs共培养状态下,成熟心肌细胞是ESCs定向分化较强的诱导因素。     

Activation of the cardiac Na-Ca exchanger by sorcin via the interaction of the respective Ca-binding domains

Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca²⁺ binding. The sarcolemmal Na⁺/Ca²⁺ exchanger (NCX1) may be an important sorcin target in cardiac muscle. In this study, RNAi knockdown of sorcin, purified sorcin or sorcin variants was employed in parallel measurements of: (i) NCX activity in isolated rabbit cardiomyocytes using electrophysiological techniques and (ii) sorcin binding to the NCX1 calcium binding domains (CBD1 and (iii) using surface plasmon resonance and gel overlay techniques. Sorcin is activated by Ca²⁺ binding to the EF3 and EF2 regions, which are connected by the D helix. To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca²⁺ due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3 μM) increased NCX activity in isolated cardiomyocytes. The relative stimulatory effects of the sorcin variants were: W105G > wt sorcin > Sorcin Calcium Binding Domain (SCBD) > W99G > E124A. Sorcin binding to both CBD1 and 2 was observed. In the presence of 50 µM Ca²⁺, the interaction with CBD1 followed the order W105G > SCBD > wt sorcin > W99G > E124A. In sorcin, the interacting surface can be mapped on the C-terminal Ca²⁺-binding domain in the D helix region comprising W99. The fast association/dissociation rates that characterize the interaction of sorcin with CBD1 and 2 may permit complex formation/dissociation during an excitation/contraction cycle.     

银杏叶提取物对家兔心室肌细胞瞬时外向钾电流和动作电位的影响

目的:探讨银杏叶提取物(Egb761)对家兔心室肌细胞瞬时外向钾电流(Ito)和动作电位的作用,揭示其抗心肌缺血及缺血引起的心律失常的离子机制。方法:酶解法分离家兔的心室肌细胞。全细胞膜片钳技术记录心肌细胞的Ito和动作电位及其被Egb761作用后的变化。结果:①在电压钳制方式下,60μg/L Egb761作用心室肌细胞5 min后,各个钳制电位下的Ito均明显增大,在钳制电位为+50 mV时,Egb761使Ito的电流密度由对照组的(7.59±0.19)pA/pF增加到(11.18±0.89)pA/pF(P<0.01,n=8),Egb761还使Ito的I-V曲线比对照组Ito的I-V明显抬高,但I-V曲线方向没有发生改变,表明Egb761引起了心肌细胞Ito的明显外流。②在电流钳制下,对照组心室肌细胞动作电位都具有从0期到4期的动作电位形态,60μg/L Egb761使心肌细胞动作电位形态呈三角形尖锥锋形,动作电位时程(APD)明显缩短,其复极化50%时程(APD50)和复极化90%时程(APD90)分别由(83.6±4.3)ms缩短为(51.3±3.2)ms和由(168.7±4.1)ms缩短为(93.8±4.4)ms(分别与对照组相比,P<0.01,n=8),尽管Egb761使动作电位幅度(APA)和静息电位(RP)降低,但与对照组相比,没有显著性差异(P>0.05)。结论:Egb761可使心室肌细胞Ito显著增加和APD明显缩短,从而减轻心肌缺血时细胞内阳离子超载对心肌造成的损伤和心肌缺血引起的心律失常的发生,以及增加心脏泵血功能。