Influence of ECM proteins and their analogs on cells cultured on 2-D hydrogels for cardiac muscle tissue engineering

This study assessed the role of immobilized cell adhesion moieties on controlling the cellular attachment, adhesion and phenotype of cardiac muscle cells towards developing scaffolds for cardiac muscle tissue engineering. Collagen I, laminin and the cell-adhesive oligopeptide, arginine-glycine-aspartic acid (RGD) at concentrations of 0.5 and 5 mM were covalently bound to flexible two-dimensional hydrogels. A robust skeletal myoblast cell line demonstrated good bioactivity for the modified hydrogels, resulting in myoblast attachment and development of an intracellular contractile network after 1 day. Primary neonatal rat ventricular myocytes cultured for up to 7 days, however, were more sensitive to the different modified substrates. Although total cardiomyocyte DNA content did not vary significantly with surface modification, immunostaining for the contractile protein Troponin I and focal adhesion protein vinculin revealed marked improvements in spreading and intracellular contractile protein deposition for cells attached to protein-modified hydrogels over those modified with RGD, regardless of RGD concentration. On the RGD-modified surfaces, cardiomyocytes self-associated, forming aggregates that exhibited a disorganized cytoarchitecture. Cardiomyocyte maturation was assessed through the fetal gene program where expression for atrial natriuretic peptide decreased and sarco(endo)plasmic reticulum Ca²⁺ increased with culture time for the protein-modified surfaces, indicating a trend towards maturation, while the α/β-myosin heavy-chain ratio remained near fetal expression levels for all surfaces. Overall, our findings suggest that whole proteins, collagen and laminin, are effective in promoting cardiomyocyte interaction with hydrogels and cardiomyocyte maturation while RGD does not provide adequate extracellular matrix cues for cardiomyocytes.     

Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro – Correlation between contraction force and electrophysiology

Cardiomyocytes from human pluripotent stem cells (hPSC-CM) have many potential applications in disease modelling and drug target discovery but their phenotypic similarity to early fetal stages of cardiac development limits their applicability. In this study we compared contraction stresses of hPSC-CM to 2nd trimester human fetal derived cardiomyocytes (hFetal-CM) by imaging displacement of fluorescent beads by single contracting hPSC-CM, aligned by microcontact-printing on polyacrylamide gels. hPSC-CM showed distinctly lower contraction stress than cardiomyocytes isolated from hFetal-CM. To improve maturation of hPSC-CM in vitro we made use of commercial media optimized for cardiomyocyte maturation, which promoted significantly higher contraction stress in hPSC-compared with hFetal-CM. Accordingly, other features of cardiomyocyte maturation were observed, most strikingly increased upstroke velocities and action potential amplitudes, lower resting membrane potentials, improved sarcomeric organization and alterations in cardiac-specific gene expression. Performing contraction force and electrophysiology measurements on individual cardiomyocytes revealed strong correlations between an increase in contraction force and a rise of the upstroke velocity and action potential amplitude and with a decrease in the resting membrane potential.We showed that under standard differentiation conditions hPSC-CM display lower contractile force than primary hFetal-CM and identified conditions under which a commercially available culture medium could induce molecular, morphological and functional maturation of hPSC-CM in vitro. These results are an important contribution for full implementation of hPSC-CM in cardiac disease modelling and drug discovery.     

Proteinase-activated receptor agonists stimulate the increase in intracellular Ca<Superscript>2+</Superscript> in cardiomyocytes and proliferation of cardiac fibroblasts from chick embryos

We studied activation of cultured cardiomyocytes and cardiac fibroblasts from chick embryos induced by agonists of PAR1 (thrombin and PAR1 peptide agonist) and PAR2 (trypsin, factor Xa, and peptide SLIGRL) by analyzing changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and cardiac fibroblast proliferation. Exposure of cardiomyocytes with thrombin induced immediate permanent dose-dependent increase in [Ca²⁺]_i. Ca²⁺ response decreased in a calcium-free medium. Peptide agonists of PAR1 and PAR2 also stimulated the increase in [Ca²⁺]_i in cardiomyocytes. Thrombin induced a short-term increase in [Ca²⁺]_i in cardiac fibroblasts and potentiated cell proliferation. PAR2 agonists trypsin and peptide SLIGRL stimulated proliferation of cardiac fibroblasts. Our results indicate that cardiomyocytes and cardiac fibroblasts from chick embryos have at least two types of PAR (types 1 and 2). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 12, pp. 609–612, December, 2007     

Hyponatraemia aggravates cardiac susceptibility to ischaemia/reperfusion injury

Hyponatraemia is defined as a serum sodium concentration of <135 mEql/L and is the most common electrolyte disturbance in patients with chronic heart failure. We hypothesize that hyponatraemia may induce Ca²⁺ overload and enhance reactive oxygen species (ROS) production, which will exacerbate myocardial injury more than normonatraemia. We investigated the effect of hyponatraemia on the ability of the heart to recover from ischaemia/reperfusion episodes. Cardiomyocytes were obtained from 1‐ to 3‐day‐old Sprague Dawley rats. After isolation, cardiomyocytes were placed in Dulbecco's modified Eagle's medium (DMEM) containing low sodium concentration (110, 120, or 130 mEq/L) or normal sodium concentration (140 mEq/L) for 72 hours. Exposure of cardiomyocytes to each of the low‐sodium medium significantly increased both ROS and intracellular Ca²⁺ levels compared with the exposure to the normal‐sodium medium. In vivo, 8‐week‐old male Sprague Dawley rats were divided into four groups: control group (Con), furosemide group (Fur), low‐sodium diet group (Lsd) and both furosemide and low‐sodium diet group (Fur + Lsd). The hearts subjected to global ischaemia exhibited considerable decrease in left ventricular developed pressure during reperfusion, and the size of infarcts induced by ischaemia/reperfusion significantly increased in the Fur, Lsd and Fur + Lsd compared with that in the Con. Hyponatraemia aggravates cardiac susceptibility to ischaemia/reperfusion injury by Ca²⁺ overload and increasing in ROS levels.     

Topographical effects on fiber-mediated microRNA delivery to control oligodendroglial precursor cells development

Effective remyelination in the central nervous system (CNS) facilitates the reversal of disability in patients with demyelinating diseases such as multiple sclerosis. Unfortunately until now, effective strategies of controlling oligodendrocyte (OL) differentiation and maturation remain limited. It is well known that topographical and biochemical signals play crucial roles in modulating cell fate commitment. Therefore, in this study, we explored the combined effects of scaffold topography and sustained gene silencing on oligodendroglial precursor cell (OPC) development. Specifically, microRNAs (miRs) were incorporated onto electrospun polycaprolactone (PCL) fiber scaffolds with different fiber diameters and orientations. Regardless of fiber diameter and orientation, efficient knockdown of differentiation inhibitory factors were achieved by either topography alone (up to 70%) or fibers integrated with miR-219 and miR-338 (up to 80%, p < 0.05). Small fiber promoted OPC differentiation by inducing more RIP⁺ cells (p < 0.05) while large fiber promoted OL maturation by inducing more MBP⁺ cells (p < 0.05). Random fiber enhanced more RIP⁺ cells than aligned fibers (p < 0.05), regardless of fiber diameter. Upon miR-219/miR-338 incorporation, 2 μm aligned fibers supported the most MBP⁺ cells (∼17%). These findings indicated that the coupling of substrate topographic cues with efficient gene silencing by sustained microRNA delivery is a promising way for directing OPC maturation in neural tissue engineering and controlling remyelination in the CNS.     

Young developmental age cardiac extracellular matrix promotes the expansion of neonatal cardiomyocytes in vitro

A major limitation to cardiac tissue engineering and regenerative medicine strategies is the lack of proliferation of postnatal cardiomyocytes. The extracellular matrix (ECM) is altered during heart development, and studies suggest that it plays an important role in regulating myocyte proliferation. Here, the effects of fetal, neonatal and adult cardiac ECM on the expansion of neonatal rat ventricular cells in vitro are studied. At 24 h, overall cell attachment was lowest on fetal ECM; however, ∼80% of the cells were cardiomyocytes, while many non-myocytes attached to older ECM and poly-l-lysine controls. After 5 days, the cardiomyocyte population remained highest on fetal ECM, with a 4-fold increase in number. Significantly more cardiomyocytes stained positively for the mitotic marker phospho-histone H3 on fetal ECM compared with other substrates at 5 days, suggesting that proliferation may be a major mechanism of cardiomyocyte expansion on young ECM. Further study of the beneficial properties of early developmental aged cardiac ECM could advance the design of novel biomaterials aimed at promoting cardiac regeneration.     

Inadequate cytoplasmatic calcium signals in alveolar macrophages after cardiac surgery

Objective and design  

Patients undergoing cardiac surgery have an elevated risk for pulmonary complications. A dysfunction of alveolar macrophages (AM) might promote postoperative infections. Therefore intracellular calcium [Ca²⁺]_i as an important second messenger in cellular signaling was assessed in AM.     

Differential effects of the formin inhibitor SMIFH2 on contractility and Ca2+ handling in frog and mouse cardiomyocytes

Genetic mutations in actin regulators have been emerging as a cause of cardiomyopathy, although the functional link between actin dynamics and cardiac contraction remains largely unknown. To obtain insight into this issue, we examined the effects of pharmacological inhibition of formins, a major class of actin-assembling proteins. The formin inhibitor SMIFH2 significantly enhanced the cardiac contractility of isolated frog hearts, thereby augmenting cardiac performance. SMIFH2 treatment had no significant effects on the Ca²⁺ sensitivity of frog muscle fibers. Instead, it unexpectedly increased Ca²⁺ concentrations of isolated frog cardiomyocytes, suggesting that the inotropic effect is due to enhanced Ca²⁺ transients. In contrast to frog hearts, the contractility of mouse cardiomyocytes was attenuated by SMIFH2 treatment with decreasing Ca²⁺ transients. Thus, SMIFH2 has opposing effects on the Ca²⁺ transient and contractility between frog and mouse cardiomyocytes. We further found that SMIFH2 suppressed Ca²⁺-release via type 2 ryanodine receptor (RyR2); this inhibitory effect may explain the species differences, since RyR2 is critical for Ca²⁺ transients in mouse myocardium but absent in frog myocardium. Although the mechanisms underlying the enhancement of Ca²⁺ transients in frog cardiomyocytes remain unclear, SMIFH2 differentially affects the cardiac contraction of amphibian and mammalian by differentially modulating their Ca²⁺ handling.     

Chronic CaMKII inhibition blunts the cardiac contractile response to exercise training

Activation of the multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) plays a critical role modulating cardiac function in both health and disease. Here, we determined the effect of chronic CaMKII inhibition during an exercise training program in healthy mice. CaMKII was inhibited by KN-93 injections. Mice were randomized to the following groups: sham sedentary, sham exercise, KN-93 sedentary, and KN-93 exercise. Cardiorespiratory function was evaluated by ergospirometry during treadmill running, echocardiography, and cardiomyocyte fractional shortening and calcium handling. The results revealed that KN-93 alone had no effect on exercise capacity or fractional shortening. In sham animals, exercise training increased maximal oxygen uptake by 8% (p < 0.05) compared to a 22% (p < 0.05) increase after exercise in KN-93 treated mice (group difference p < 0.01). In contrast, in vivo fractional shortening evaluated by echocardiography improved after exercise in sham animals only: from 25 to 32% (p < 0.02). In inactive mice, KN-93 reduced rates of diastolic cardiomyocyte re-lengthening (by 25%, p < 0.05) as well as Ca²⁺ transient decay (by 16%, p < 0.05), whereas no such effect was observed after exercise training. KN-93 blunted exercise training response on cardiomyocyte fractional shortening (63% sham vs. 18% KN-93; p < 0.01 and p < 0.05, respectively). These effects could not be solely explained by the Ca²⁺ transient amplitude, as KN-93 reduced it by 20% (p < 0.05) and response to exercise training was equal (64% sham and 47% KN-93; both p < 0.01). We concluded that chronic CaMKII inhibition increased time to 50% re-lengthening which were recovered by exercise training, but paradoxically led to a greater increase in maximal oxygen uptake compared to sham mice. Thus, the effect of chronic CaMKII inhibition is multifaceted and of a complex nature.     

Role of Ca(2+) in the rapid cooling-induced Ca(2+) release from sarcoplasmic reticulum in ferret cardiac muscles

Rapid lowering of the solution temperature (rapid cooling, RC) from 24 to 3°C within 3 s releases considerable amounts of Ca²⁺ from the sarcoplasmic reticulum (SR) in mammalian cardiac muscles. In this study, we investigated the intracellular mechanism of RC-induced Ca²⁺ release, especially the role of Ca²⁺, in ferret ventricular muscle. Saponin-treated skinned trabeculae were placed in a glass capillary, and the amount of Ca²⁺ released from the SR by RC and caffeine (50 mM) was measured with fluo-3. It was estimated that in the presence of ATP about 45% of the Ca²⁺ content in the SR was released by RC. The amount of SR Ca²⁺ released by RC was unchanged by the replacement of ATP by AMP-PCP (a non-hydrolysable ATP analogue and agonist for the ryanodine receptor but not for the Ca²⁺ pump of SR), suggesting that the suppression of the Ca²⁺ pump of SR at low temperature might not be a major mechanism in RC-induced Ca²⁺ release. The free Ca²⁺ concentration of the solution used for triggering RC-induced Ca²⁺ release was estimated to be only about 20 nM with fluo-3 or aequorin. When this solution was applied to the preparation at 3°C, only a small amount of Ca²⁺ was released from SR presumably by the Ca²⁺-induced Ca²⁺ release (CICR) mechanism. Thus, in mammalian cardiac muscles, RC releases a part of the (<50%) stored Ca²⁺ contained in the SR, and the mechanism of RC-induced Ca²⁺ release may differ from that of CICR, which is thought to play a role in frog skeletal muscle fibres that express ryanodine receptors of different types.     

Micropattern width dependent sarcomere development in human ESC-derived cardiomyocytes

In this study, human embryonic stem cell-derived cardiomyocytes were seeded onto controlled two-dimensional micropatterned features, and an improvement in sarcomere formation and cell alignment was observed in specific feature geometries. High-resolution photolithography techniques and microcontact printing were utilized to produce features of various rectangular geometries, with areas ranging from 2500 μm² to 160,000 μm². The microcontact printing method was used to pattern non-adherent poly(ethylene glycol) regions on gold coated glass slides. Matrigel and fibronectin extracellular matrix (ECM) proteins were layered onto the gold-coated glass slides, providing a controlled geometry for cell adhesion. We used small molecule-based differentiation and an antibiotic purification step to produce a pure population of immature cardiomyocytes from H9 human embryonic stem cells (hESCs). We then seeded this pure population of human cardiomyocytes onto the micropatterned features of various sizes and observed how the cardiomyocytes remodeled their myofilament structure in response to the feature geometries. Immunofluorescence was used to measure α-actinin expression, and phalloidin stains were used to detect actin presence in the patterned cells. Analysis of nuclear alignment was also used to determine how cell direction was influenced by the features. The seeded cells showed clear alignment with the features, dependent on the width rather than the overall aspect ratio of the features. It was determined that features with widths between 30 μm and 80 μm promoted highly aligned cardiomyocytes with a dramatic increase in sarcomere alignment relative to the long axis of the pattern. This creation of highly-aligned cell aggregates with robust sarcomere structures holds great potential in advancing cell-based pharmacological studies, and will help researchers to understand the means by which ECM geometries can affect myofilament structure and maturation in hESC-derived cardiomyocytes.     

Functional characterization of cardiac progenitor cells and their derivatives in the embryonic heart post‐chamber formation

There is scant information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Here we simultaneously tracked three lineage‐specific markers (Nkx2.5, MLC2v, and ANF) and confirmed that CPCs with an Nkx2.5⁺MLC2v^?ANF^? phenotype are present in the embryonic (E) day 11.5 mouse ventricular myocardium. We demonstrated that these CPCs could give rise to working cardiomyocytes and conduction system cells. Using a two‐photon imaging analysis, we found that the majority of CPCs are not capable of developing Ca²⁺ transients in response to β‐adrenergic receptor stimulation. In contrast, Nkx2.5⁺ cells expressing MLC2v but not ANF are capable of developing functional Ca²⁺ transients. We showed that Ca²⁺ transients could be invoked in Nkx2.5⁺MLC2v⁺ANF⁺ cells only upon inhibition of Gi, muscarinic receptors, or nitric oxide synthase (NOS) signaling pathways. Our data suggest that these inhibitory pathways may delay functional specification in a subset of developing ventricular cells. Developmental Dynamics 238:2787–2799, 2009. © 2009 Wiley‐Liss, Inc.     

Innervating sympathetic neurons regulate heart size and the timing of cardiomyocyte cell cycle withdrawal

AbstractSympathetic drive to the heart is a key modulator of cardiac function and interactions between heart tissue and innervating sympathetic fibres are established early in development. Significant innervation takes place during postnatal heart development, a period when cardiomyocytes undergo a rapid transition from proliferative to hypertrophic growth. The question of whether these innervating sympathetic fibres play a role in regulating the modes of cardiomyocyte growth was investigated using 6‐hydroxydopamine (6‐OHDA) to abolish early sympathetic innervation of the heart. Postnatal chemical sympathectomy resulted in rats with smaller hearts, indicating that heart growth is regulated by innervating sympathetic fibres during the postnatal period. In vitro experiments showed that sympathetic interactions resulted in delays in markers of cardiomyocyte maturation, suggesting that changes in the timing of the transition from hyperplastic to hypertrophic growth of cardiomyocytes could underlie changes in heart size in the sympathectomized animals. There was also an increase in the expression of Meis1, which has been linked to cardiomyocyte cell cycle withdrawal, suggesting that sympathetic signalling suppresses cell cycle withdrawal. This signalling involves β‐adrenergic activation, which was necessary for sympathetic regulation of cardiomyocyte proliferation and hypertrophy. The effect of β‐adrenergic signalling on cardiomyocyte hypertrophy underwent a developmental transition. While young postnatal cardiomyocytes responded to isoproterenol (isoprenaline) with a decrease in cell size, mature cardiomyocytes showed an increase in cell size in response to the drug. Together, these results suggest that early sympathetic effects on proliferation modulate a key transition between proliferative and hypertrophic growth of the heart and contribute to the sympathetic regulation of adult heart size.

Abbreviations

DIV

days in vitro

6‐OHDA

6‐hydroxydopamine

P

postnatal day

PH3

phospho‐histone H3

WGA

wheat germ agglutinin

     

Salusins对大鼠离体心脏及心肌细胞的生物学效应的研究

目的:研究新发现的心血管活性肽salusin的心脏及心肌细胞的生物学效应。方法:利用Langendorff装置灌流的成年大鼠离体心脏及原代培养的乳鼠心肌细胞,测定心功能和心肌细胞[⁴⁵Ca²⁺]摄入及[³H]-亮氨酸([³H]-Leu)掺入量。结果:10^-12-10^-7mol/L salusin-α及salusin-β对成年大鼠离体心脏功能无明显影响;但10^-10-10^-6mol/L salusin-α及salusin-β均呈浓度依赖性地促进心肌细胞[⁴⁵Ca²⁺]摄入及[³H]-Leu掺入,其效应在10^-8mol/L时达峰值;salusin-α及salusin-β对心肌细胞[⁴⁵Ca²⁺]摄入的效应可被钙通道阻断剂尼卡地平(nicardipine)所拮抗,且与内皮素有协同效应。 Salusin-α及salusin-β对心肌细胞[³H]-Leu掺入的效应可被尼卡地平、钙调磷酸酶抑制剂FK506、丝裂原活化蛋白激酶(MAPK)阻断剂PD₉₈₀₅₉及蛋白激酶C(PKC)阻断剂chelerthine chloride等不同程度抑制。Salusin-β对心肌细胞 摄入的效应强于salusin-α,但[³H]-Leu掺入的效应两者之间无显著性差异。结论:Salusin-α及salusin-β对成年大鼠离体心脏功能无直接效应,但能促进乳鼠心肌细胞钙摄入及蛋白合成,其效应可能与钙通道、钙调神经磷酸酶、MAPK和PKC等信号转导途径有关。Salusin可能具有心肌生长、肥大的调节的作用。     

HGF percutaneous endocardial injection induces cardiomyocyte proliferation and rescues cardiac function in pigs

Objective

To investigate the effect of cardiomyocyte proliferation induced by human hepatocyte growth factor (HGF) in pigs with chronic myocardial infarction (CMI).

Methods

A steerable, deflectable 7F catheter incorporating a 27-guage needle was advanced percutaneously to the left ventricular myocardium of 18 pigs with CMI. Pigs were randomized (1:1:1) to receive adenoviral vector HGF (total dose, 1×10¹⁰ genome copies), which was administered as five injections into the infarcted myocardium (total, 1.0 mL), or saline, or Ad-null (control groups). Injections were guided by Ensite NavX left ventricular electroanatomical mapping. HGF and cyclin proteins were detected by western blot and immunoprecipitation analysis. Histological and immunohistochemical analysis determined proliferating cardiomyocytes. Myocardial perfusion and cardiac function were estimated by Gated-Single Photon Emission Computed Tomography (G-SPECT).

Results

Western blot analyses showed that HGF were predominantly expressed in the infarct core and border in the myocardium of the infarcted heart. G-SPECT analysis indicated that the HGF group had better cardiac function and myocardial perfusion four weeks after the injection of Ad-HGF than before the injection of Ad-HGF. After treatment there were more proliferating cardiomyocytes in the HGF group compared to either of the control groups. Furthermore, the HGF group myocardial samples expressed higher levels of p-Akt, cyclin A, cyclin E, cyclin D1, cdk2, cdk4 than those in the control groups.

Conclusion

The over-expression of HGF activates pro-survival pathways, induces cardiomyocyte proliferation, and improves the perfusion and function of the porcine CMI heart.     

The effect of cyclic stretch on maturation and 3D tissue formation of human embryonic stem cell-derived cardiomyocytes

The goal of cardiac tissue engineering is to restore function to the damaged myocardium with regenerative constructs. Human embryonic stem cell–derived cardiomyocytes (hESC-CMs) can produce viable, contractile, three-dimensional grafts that function in vivo. We sought to enhance the viability and functional maturation of cardiac tissue constructs by cyclical stretch. hESC-CMs seeded onto gelatin-based scaffolds underwent cyclical stretching. Histological analysis demonstrated a greater proportion of cardiac troponin T–expressing cells in stretched than non-stretched constructs, and flow sorting demonstrated a higher proportion of cardiomyocytes. Ultrastructural assessment showed that cells in stretched constructs had a more mature phenotype, characterized by greater cell elongation, increased gap junction expression, and better contractile elements. Real-time PCR revealed enhanced mRNA expression of genes associated with cardiac maturation as well as genes encoding cardiac ion channels. Calcium imaging confirmed that stretched constructs contracted more frequently, with shorter calcium cycle duration. Epicardial implantation of constructs onto ischemic rat hearts demonstrated the feasibility of this platform, with enhanced survival and engraftment of transplanted cells in the stretched constructs. This uniaxial stretching system may serve as a platform for the production of cardiac tissue-engineered constructs for translational applications.     

钙调神经磷酸酶在血管紧张素Ⅱ刺激的大鼠心肌细胞肥大中的作用及其活性调节

目的:观察钙调神经磷酸酶(CaN)在血管紧张素Ⅱ(AngⅡ)刺激的大鼠心肌细胞肥大中的作用及其活性调节。方法:建立AngⅡ诱导的大鼠心肌细胞肥大模型,观察CaN抑制剂对AngⅡ刺激的心肌细胞 [³H]-亮氨酸掺入的影响,以及各种因素对心肌细胞CaN酶活性的影响。结果:10、 100、 1000 nmol·L^-1的AngⅡ作用12 h分别使心肌细胞的CaN活性增加了13%、 57%(P＜0.05)、 228%(P＜0.01)。AngⅡ(10 nmol·L^-1)刺激心肌细胞2 h内,CaN活性与对照组无明显差异(P＜0.05);AngⅡ刺激心肌细胞12 h以上,CaN活性才明显增高(P＜0.05)。Losartan(50 μmol·L^-1)、H₇(50 μmol·L^-1)及Fura-2/AM(4 μmol·L^-1)可明显抑制AngⅡ刺激的心肌细胞CaN活性;而PD98059(50 μmol·L^-1)对AngⅡ刺激的心肌细胞CaN活性无明显影响。AngⅡ(10^-7mol/L)刺激的大鼠心肌细胞 [³H]-亮氨酸掺入明显高于对照组(P＜0.01),而CaN特异性抑制剂-环孢素A(0.5~5 μg/mL)可以明显抑制AngⅡ刺激的心肌细胞 [³H]-亮氨酸掺入。结论:依赖Ca²⁺/CaM活化的CaN可能在AngⅡ刺激的心肌细胞肥大中起重要作用;CaN的活化可能有赖于胞内Ca²⁺水平的持续升高,另外,CaN的活性还可能受到蛋白激酶C等信号分子的磷酸化调节。     

The role of Ca2+/calmodulin-dependent protein kinase II and calcineurin in TNF-α-induced myocardial hypertrophy

We investigated whether Ca²⁺/calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN) are involved in myocardial hypertrophy induced by tumor necrosis factor α (TNF-α). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-α (100 µg/L), and Ca²⁺ signal transduction was blocked by several antagonists, including BAPTA (4 µM), KN-93 (0.2 µM) and cyclosporin A (CsA, 0.2 µM). Protein content, protein synthesis, cardiomyocyte volumes, [Ca²⁺]_i transients, CaMKIIδ_B and CaN were evaluated by the Lowry method, [³H]-leucine incorporation, a computerized image analysis system, a Till imaging system, and Western blot analysis, respectively. TNF-α induced a significant increase in protein content in a dose-dependent manner from 10 µg/L (53.56 µg protein/well) to 100 µg/L (72.18 µg protein/well), and in a time-dependent manner from 12 h (37.42 µg protein/well) to 72 h (42.81 µg protein/well). TNF-α (100 µg/L) significantly increased the amplitude of spontaneous [Ca²⁺]_i transients, the total protein content, cell size, and [³H]-leucine incorporation in cultured cardiomyocytes, which was abolished by 4 µM BAPTA, an intracellular Ca²⁺ chelator. The increases in protein content, cell size and [³H]-leucine incorporation were abolished by 0.2 µM KN-93 or 0.2 µM CsA. TNF-α increased the expression of CaMKIIδ_B by 35.21% and that of CaN by 22.22% compared to control. These effects were abolished by 4 µM BAPTA, which itself had no effect. These results suggest that TNF-α induces increases in [Ca²⁺]_i, CaMKIIδ_B and CaN and promotes cardiac hypertrophy. Therefore, we hypothesize that the Ca²⁺/CaMKII- and CaN-dependent signaling pathways are involved in myocardial hypertrophy induced by TNF-α.     

Developmental changes in Ca2+ currents from newborn rat cardiomyocytes in primary culture

Electrophysiological characteristics of neonatal rat ventricular cardiomyocytes in primary culture were studied using the whole-cell patch-clamp recording technique. Cell size, estimated by measurement of membrane capacitance, was significantly increased throughout the culture from 22.4±5.4 pF at day 2 to 55.0±16.1 pF at day 7, reflecting the hypertrophic process which characterises postnatal cell development. The Ca²⁺ current was investigated at day 2 and 7 of the culture which constituted the early postnatal and maximally developed stages, respectively, of isolated cells in our experimental conditions. At 2 days of culture, two types of Ca²⁺ current could be distinguished, as also observed in freshly dissociated newborn ventricular cells. From their potential dependence and pharmacological characteristics, they could be attributed to the T- (I _Ca-T) and L-type (I _Ca-L) Ca²⁺ current components. After 7 days of culture, only the latterI _Ca-L was present and its density was significantly increased when compared to the density in 2-day-old cells, but lower than that obtained in freshly dissociated adult cells. As the age of the culture progressed, the steady-state inactivation curve was shifted toward negative potentials, in the direction of the inactivation curve obtained for adult cells. Compared to the serum-free control conditions, the density ofI _Ca-L was significantly increased in the presence of fetal calf serum throughout the culture. Consequently, the density ofI _Ca-L obtained in 7-day-old cells was similar to the density ofI _Ca-L obtained in freshly dissociated adult cardiac cells. These results show that in rat neonatal ventricular cardiomyocytes, the changes in Ca²⁺ current during development in primary culture can be compared to that observed in vivo during the first weeks of the postnatal period. The data suggest that the composition of the culture medium is a conditioning factor in the development of cardiac cells in culture. However, the determination and the role of specific factors contained in the serum need to be investigated. The data are also discussed in terms of a possible correlation between the expression and maturation of the Ca²⁺ current components and the capabilities of the neonatal cardiac cells to proliferate and/or to hypertrophy. For these reasons primary cultures of neonatal rat cardiac cells could constitute a valuable in vitro model for studies of postnatal development.