Regulation of cardiomyocyte proliferation during development and regeneration

The regulation of cardiomyocyte proliferation is important for heart development and regeneration. The proliferation patterns of cardiomyocytes are closely related to heart morphogenesis, size, and functions. The proliferation levels are high during early embryogenesis; however, mammalian cardiomyocytes exit the cell cycle irreversibly soon after birth. The cell cycle exit inhibits cardiac regeneration in mammals. On the other hand, cardiomyocytes of adult zebrafish and probably newts can proliferate after cardiac injury, and the hearts can be regenerated. Therefore, the ability to reproliferate determines regenerative ability. As in other cells, the relationship between proliferation and differentiation is very interesting, and is closely related to cardiac development, regeneration and homeostasis. In this review, these topics are discussed.     

Electron microscopy and 3D reconstruction of F-actin decorated with cardiac myosin-binding protein C (cMyBP-C)

Myosin-binding protein C (MyBP-C) is an ∼ 130-kDa rod-shaped protein of the thick (myosin containing) filaments of vertebrate striated muscle. It is composed of 10 or 11 globular 10-kDa domains from the immunoglobulin and fibronectin type III families and an additional MyBP-C-specific motif. The cardiac isoform cMyBP-C plays a key role in the phosphorylation-dependent enhancement of cardiac function that occurs upon β-adrenergic stimulation, and mutations in MyBP-C cause skeletal muscle and heart diseases. In addition to binding to myosin, MyBP-C can also bind to actin via its N-terminal end, potentially modulating contraction in a novel way via this thick-thin filament bridge. To understand the structural basis of actin binding, we have used negative stain electron microscopy and three-dimensional reconstruction to study the structure of F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions varying from 25 to 180 mM KCl concentration. Three-dimensional helical reconstructions, carried out at the 180-mM KCl level to minimize nonspecific binding, showed MyBP-C density over a broad portion of the periphery of subdomain 1 of actin and extending tangentially from its surface in the direction of actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it could modulate tropomyosin position and would interfere with myosin head binding to actin.     

Cardiospheres and tissue engineering for myocardial regeneration: potential for clinical application

Tissue engineering is an increasingly expanding area of research in the cardiovascular field that involves engineering, chemistry, biology and medicine. Cardiac tissue engineering (CTE) aims to regenerate myocardial damage by combining cells, matrix, biological active molecules and physiological stimuli. The rationale behind CTE applications is that in order to regenerate the ventricular wall after a myocardial infarction it is necessary to combine procedures that regenerate both cardiomyocytes and the extracellular matrix. The application of (stem) cells together with a matrix could represent an environment protected from the inflammatory and pro-apoptotic signals, a stemness/survival reservoir slowly releasing cells and factors promoting tissue regeneration and angiogenesis. This review will focus on the applications and advantages that CTE application could offer compared to conventional cell therapy.     

用心脏舒张期与收缩期时限比值评估青少年心脏功能

目的：观察青少年在安静状态及最大运动量状态下心脏舒张期时限与收缩期时限比值（D/S）的变化情况,并运用该指标对青少年心脏功能的评估进行探讨。方法：选取重庆市主城区中学309名（男159名,女150名）青少年为研究对象,受试者完成最大运动量的台阶运动,在安静状态和运动后即刻进行D/S比值的测试。根据安静状态D/S值的大小范围将受试者心脏功能分级为优（ D/S≥2）、良（1.5≤D/S<2）、中（1≤D/S<1.5）、差（D/S<1）。结果：安静状态下男生心脏功能评定为优、良、中、差的占比分别为： 7.5%、49.1%、 40.9%、2.5%;女生分别为： 5.3%、48.0%; 44.7%、2.0%。运动后男、女青少年D/S值都明显下降,但心脏功能评定等级高的受试者运动后D/S仍高于等级低的受试者。结论：本次研究的青少年虽有过半心脏功能处于健康状态,但处于亚健康状态的比例较高,有少部分学生心脏功能较差,其心脏健康应得到高度重视;心脏功能等级越高的学生运动后心脏供血时间越长,其心脏功能更好。应用D/S值可以有效评估青少年心脏供血时限的相关情况;对青少年心脏功能进行分级可以针对不同分级群体制定有针对性的运动训练,同时预防心脏安全事故的出现。该评估方法操作简单、客观,可作为一种日常的心脏监测手段在学校普及。     

Cathepsin K knockout alleviates aging‐induced cardiac dysfunction

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload‐induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age‐dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca²⁺ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura‐2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24‐month‐old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross‐sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca²⁺ release compared to young (6‐month‐old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged‐cathepsin K knockout mice compared to their wild‐type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age‐induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis‐inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin‐induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age‐related decline in cardiac function via suppressing caspase‐dependent and caspase‐independent apoptosis.     

在生理学研究中如何正确把握试验设计三要素

目的：为从事生理学研究的科研工作者制订科学完善的试验设计方案提供一些有效的技术方法。方法：通过正面阐述三要素和正确把握每个要素的关键点;再通过分析已发表的学术论文中未妥善把握三要素的案例,为生理学研究工作者在制订课题设计方案时能正确把握三要素提供具有可操作性的方法。结果：呈现出试验设计三要素的具体内容,即"受试对象"、"影响因素"和"试验效应",并且,为如何正确把握每个要素提供具体措施。结论：试验设计的三要素是从事试验研究的"物质基础",正确把握它们是制订出科学完善试验设计方案的重要环节之一。     

慢性缺氧对大鼠心室功能和ATP酶活性的影响

将大鼠置于不同模拟海拔高度低压舱内4d,观察其左、右心室功能代偿与失代偿的某些生物化学基础。结果表明,5000m中度缺氧4d使左、右心室功能、重量、心肌蛋白含量及Ca~(2 )-ATP酶活性均有不同程度的增高。提示机体在整体、心脏器官及心肌细胞分子各个水平的代偿机制均有加强。8000m重度缺氧4d后,左室重量增加,dp/dt_(max)与蛋白含量均下降,肌原纤维ATP酶活性则保持中度缺氧的代偿水平,提示左心功能似已受到损害。与此同时,右室蛋白含量虽也明显减少,但其ATP酶活性则继续增高,dp/dt_(max)未出现下降,表明右心功能仍具有相当的代偿能力。从而支持我们关于在短期内因供氧严重不足而造成的左室心肌的直接损伤作用大于右室心肌的推论。     

Turning regenerative technologies into treatment to repair myocardial injuries

Regenerative therapies including stem cell treatments hold promise to allow curing patients affected by severe cardiac muscle diseases. However, the clinical efficacy of stem cell therapy remains elusive, so far. The two key roadblocks that still need to be overcome are the poor cell engraftment into the injured myocardium and the limited knowledge of the ideal mixture of bioactive factors to be locally delivered for restoring heart function. Thus, therapeutic strategies for cardiac repair are directed to increase the retention and functional integration of transplanted cells in the damaged myocardium or to enhance the endogenous repair mechanisms through cell-free therapies. In this context, biomaterial-based technologies and tissue engineering approaches have the potential to dramatically impact cardiac translational medicine. This review intends to offer some consideration on the cell-based and cell-free cardiac therapies, their limitations and the possible future developments.     

Effects of cannabinoid receptor type 2 on endogenous myocardial regeneration by activating cardiac progenitor cells in mouse infarcted heart

Cannabinoid receptor type 2(CB2)activation is recently reported to promote proliferation of some types of resident stem cells(e.g.,hematopoietic stem/progenitor cell or neural progenitor cell).Resident cardiac progenitor cell(CPC)activation and proliferation are crucial for endogenous cardiac regeneration and cardiac repair after myocardial infarction(MI).This study aims to explore the role and possible mechanisms of CB2receptor activation in enhancing myocardial repair.Our results revealed that CB2receptor agonist AM1241 can significantly increase CPCs by c-kit and Runx1 staining in ischemic myocardium as well as improve cardiomyocyte proliferation.AM1241 also decreased serum levels of MDA,TNF-αand IL-6 after MI.In addition,AM1241 can ameliorate left ventricular ejection fraction and fractional shortening,and reduce fibrosis.Moreover,AM1241 treatment markedly increased p-Akt and HO-1 expression,and promoted Nrf-2 nuclear translocation.However,PI3K inhibitor wortmannin eliminated these cardioprotective roles of AM1241.In conclusion,AM1241 could induce myocardial regeneration and improve cardiac function,which might be associated with PI3K/Akt/Nrf2 signaling pathway activation.Our findings may provide a promising strategy for cardiac endogenous regeneration after MI.     

N-乙酰半胱氨酸对大鼠心脏成纤维细胞增殖和胶原合成的影响

目的：通过观察N-乙酰半胱氨酸（NAC）对大鼠心脏成纤维细胞（CFs）增殖和胶原合成的影响,探讨NAC对心脏重构的作用。方法：以培养的新生SD大鼠CFs为实验对象,给予不同浓度的NAC进行干预,48小时后用MTT比色法检测CFs增殖水平,用3H脯氨酸掺入法测定总胶原合成。结果：与对照组相比,不同浓度NAC作用下的CFs增殖水平和3H脯氨酸掺入量均比对照组低,且具有浓度依赖性（p〈0.05）。结论：NAC能够抑制SD大鼠CFs增殖,并降低其胶原合成,因此NAC对心脏的病理性重构可能具有保护作用。     

Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides

It is well-known that micromolar to millimolar concentrations of cardiac glycosides inhibit Na/K pump activity, however, some early reports suggested nanomolar concentrations of these glycosides stimulate activity. These early reports were based on indirect measurements in multicellular preparations, hence, there was some uncertainty whether ion accumulation/depletion rather than pump stimulation caused the observations. Here, we utilize the whole-cell patch-clamp technique on isolated cardiac myocytes to directly measure Na/K pump current (I(P)) in conditions that minimize the possibility of ion accumulation/depletion causing the observed effects. In guinea pig ventricular myocytes, nanomolar concentrations of dihydro-ouabain (DHO) caused an outward current that appeared to be due to stimulation of I(P) because of the following: (1) it was absent in 0 mM [K(+)](o), as was I(P); (2) it was absent in 0 mM [Na(+)](i), as was I(P); (3) at reduced [Na(+)](i), the outward current was reduced in proportion to the reduction in I(P); (4) it was eliminated by intracellular vanadate, as was I(P). Our previous work suggested guinea pig ventricular myocytes coexpress the alpha(1)- and alpha(2)-isoforms of the Na/K pumps. The stimulation of I(P) appears to be through stimulation of the high glycoside affinity alpha(2)-isoform and not the alpha(1)-isoform because of the following: (1) regulatory signals that specifically increased activity of the alpha(2)-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the alpha(1)-isoform did not affect the stimulation; (3) changes in [K(+)](o) that affected activity of the alpha(1)-isoform, but not the alpha(2)-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the alpha(1)-isoform but not the alpha(2)-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total I(P) increased by 35 +/- 10% (mean +/- SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the alpha(2)-isoform, then activity of the alpha(2)-isoform increased by 107 +/- 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the alpha(2)-isoform, but both the stimulatory and inhibitory concentrations of ouabain were approximately 10-fold lower than those for DHO. Stimulation of I(P) by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the alpha(1)- and alpha(3)-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the alpha(3)-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of I(P) that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of I(P), and where the contributions of the high glycoside affinity alpha(2)- and alpha(3)-isoforms could be separated from that of the alpha(1)-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I(P) in heart by nanomolar concentrations of endogenous ouabain-like molecules.     

急性低氧下心泵“力效应”与心电、心肌乳酸的变化

用“心力环”表达心泵的“力效应”,以侧位肢体导联心电图来检测心脏传导系统的反应,在10条麻醉开胸狗上,观察了急性低氧条件下心泵的力学变化和心电活动演变过程的联系。在急性低氧早期,“心力环”即开始增大和“心力效率”(SV/FL_o)降低时,心电传导时间和心室复极化过程均没有明显改变(P>0.05)。直到以“心力环”环体减小及“心力效率”增加为特征的心泵衰竭出现时,才呈现心室复极化过程的明显缺氧性改变:ST_(Ⅱ)段抬高(P<0.05),T_(Ⅱ)波振幅显著增加(P<0.01);并伴以心率明显地降低(P<0.05)。在10条狗中有4条发生室性早搏或室颤等明显心率紊乱,它们均出现于“心力环”环体显著减小的严重泵衰竭时。另有8条狗在相似的实验条件下的结果表明,心泵衰竭时心肌乳酸含量明显高于对照组(P<0.05)。本研究结果提示,心泵“力效应”变化在急性低氧过程中发生得较早。这样,在评价心泵对急性低氧的耐受性上,心泵“力效应”比心脏电活动更为敏感。     

犬双心室多点组合同步起搏的心肌力学效应研究

目的 :探讨多点组合同步心室起搏对犬心肌收缩 /舒张力学效应和心脏作功的影响。方法 :12只犬 ,随机进行 5种组合模式的双心室同步起搏 ,并以自身窦性心律状态 (SNR )作为对照。记录各起搏状态下 :左室内压上升和下降最大数率 (±dp/dtmax)、左室松弛时间常数 (τ)、左 /右室游离壁室壁肌张力 (L/RV tensileforce ,L/RV TF)、每搏量 (SV )、左室每搏功 (LVSW )和右室每搏功 (RVSW )等心肌收缩 /舒张力学和心脏作功参数。结果 :双室cHisB LVPL起搏和RVA LVPL起搏的心肌收缩力学参数 +dp/dtmax和L/RV TF较右室双点cHisB RVA起搏增加 ,前两组的心肌舒张力学参数 dp/dtmax也较cHisB RVA起搏增加 ,而τ值较后者缩短。双室三点cHisB RVA LVPL起搏和cHisB RVA LVA起搏的上述各参数均优于双室cHisB LVPL起搏和RVA LVPL起搏。而cHisB RVA LVPL起搏的 +dp/dtmax和L/RV TF均较cHisB RVA LVA起搏增加。cHisB RVA LVPL起搏 dp/dtmax较cHisB RVA LVA起搏提高 6.0 % ,τ值缩短 3 .7%。cHisB LVPL起搏和RVA LVPL起搏的SV、LVSW和RVSW等心室作功参数均较cHisB RVA起搏增加 ,而HisB RVA LVPL起搏的上述心脏作功各参数 ,亦分别较cHisB RVA LVA起搏和cHisB LVPL起搏有不同程度的增加。结论 :双室三点cHisB RVA LVPL组合同     

运动性和高血压性心肌肥大重塑时心肌初级和次级应答基因表达的差异

我们前期研究表明运动性和高血压性心肌肥大细胞表型变化在结构、功能和代谢方面均表现不同,但两者基因表达的不同特征尚不清楚。本实验采用Ｎｏｒｔｈｅｒｎ分子杂交方法对游泳运动１２周大鼠和自发性高血压大鼠（ＳＨＲ）肥大心脏心肌初级和次级应答基因表达进行比较研究。结果表明,游泳大鼠心系数比对照大鼠提高２６％（Ｐ〈０．０１）,心肌ｃ－ｆｏｓ和心房钠尿肽（ＡＮＦ）基因表达在最后一次运动后即刻明显增强,在运动后２     

Intramyocardial transplantation of cardiac telocytes decreases myocardial infarction and improves post‐infarcted cardiac function in rats

The midterm effects of cardiac telocytes (CTs) transplantation on myocardial infarction (MI) and the cellular mechanisms involved in the beneficial effects of CTs transplantation are not understood. In the present study, we have revealed that transplantation of CTs was able to significantly decrease the infarct size and improved cardiac function 14 weeks after MI. It has established that CT transplantation exerted a protective effect on the myocardium and this was maintained for at least 14 weeks. The cellular mechanism behind this beneficial effect on MI was partially attributed to increased cardiac angiogenesis, improved reconstruction of the CT network and decreased myocardial fibrosis. These combined effects decreased the infarct size, improved the reconstruction of the LV and enhanced myocardial function in MI. Our findings suggest that CTs could be considered as a potential cell source for therapeutic use to improve cardiac repair and function following MI, used either alone or in tandem with stem cells.     

N-乙酰半胱氨酸对大鼠心脏成纤维细胞增殖和胶原合成的影响

目的:通过观察N-乙酰半胱氨酸(NAC)对大鼠心脏成纤维细胞(CFs)增殖和胶原合成的影响,探讨NAC对心脏重构的作用。方法:以培养的新生SD大鼠CFs为实验对象,给予不同浓度的NAC进行干预,48小时后用MTT比色法检测CFs增殖水平,用3H脯氨酸掺入法测定总胶原合成。结果:与对照组相比,不同浓度NAC作用下的CFs增殖水平和3H脯氨酸掺入量均比对照组低,且具有浓度依赖性(p<0.05)。结论:NAC能够抑制SD大鼠CFs增殖,并降低其胶原合成,因此NAC对心脏的病理性重构可能具有保护作用。     

Neuregulin‐1 is essential for nerve plexus formation during cardiac maturation

The Neuregulin‐1 (Nrg1)/ErbB pathway plays multiple, critical roles in early cardiac and nervous system development and has been implicated in both heart and nerve repair processes. However, the early embryonic lethality of mouse Nrg1 mutants precludes an analysis of Nrg1's function in later cardiac development and homeostasis. In this study, we generated a novel nrg1 null allele targeting all known isoforms of nrg1 in zebrafish and examined cardiac structural and functional parameters throughout development. We found that zebrafish nrg1 mutants instead survived until young adult stages when they exhibited reduced survivorship. This coincided with structural and functional defects in the developing juvenile and young adult hearts, as demonstrated by reduced intracardiac myocardial density, cardiomyocyte cell number, swimming performance and dysregulated heartbeat. Interestingly, nrg1 mutant hearts were missing long axons on the ventricle surface by standard length (SL) 5 mm, which preceded juvenile and adult cardiac defects. Given that the autonomic nervous system normally exerts fine control of cardiac output through this nerve plexus, these data suggest that Nrg1 may play a critical role in establishing the cardiac nerve plexus such that inadequate innervation leads to deficits in cardiac maturation, function and survival.