From Cell to Gene: Deciphering the Mechanism of Heart Failure With Single-Cell Sequencing.

Heart failure (HF) is a prevalent cardiovascular disease with significant morbidity and mortality rates worldwide. Due to the intricate structure of the heart, diverse cell types, and the complex pathogenesis of HF, further in-depth investigation into the underlying mechanisms  is required. The elucidation of the heterogeneity of cardiomyocytes and the intercellular communication network is particularly important. Traditional high-throughput sequencing methods provide an average measure of gene expression, failing to capture the "heterogeneity" between cells and impacting the accuracy of gene function knowledge. In contrast, single-cell sequencing techniques allow for the amplification of the entire genome or transcriptome at the individual cell level, facilitating the examination of gene structure and expression with unparalleled precision. This approach offers valuable insights into disease mechanisms, enabling the identification of changes in cellular components and gene expressions during hypertrophy associated with HF. Moreover, it reveals distinct cell populations and their unique roles in the HF microenvironment, providing a comprehensive understanding of the cellular landscape that underpins HF pathogenesis. This review focuses on the insights provided by single-cell sequencing techniques into the mechanisms underlying HF and discusses the challenges encountered in current cardiovascular research.

Empagliflozin rescues pro-arrhythmic and Ca2+ homeostatic effects of transverse aortic constriction in intact murine hearts.

We explored physiological effects of the sodium-glucose co-transporter-2 inhibitor empagliflozin on intact experimentally hypertrophic murine hearts following transverse aortic constriction (TAC). Postoperative drug (2-6 weeks) challenge resulted in reduced late Na+ currents, and increased phosphorylated (p-)CaMK-II and Nav1.5 but not total (t)-CaMK-II, and Na+/Ca2+ exchanger expression, confirming previous cardiomyocyte-level reports. It rescued TAC-induced reductions in echocardiographic ejection fraction and fractional shortening, and diastolic anterior and posterior wall thickening. Dual voltage- and Ca2+-optical mapping of Langendorff-perfused hearts demonstrated that empagliflozin rescued TAC-induced increases in action potential durations at 80% recovery (APD80), Ca2+ transient peak signals and durations at 80% recovery (CaTD80), times to peak Ca2+ (TTP100) and Ca2+ decay constants (Decay30-90) during regular 10-Hz stimulation, and Ca2+ transient alternans with shortening cycle length. Isoproterenol shortened APD80 in sham-operated and TAC-only hearts, shortening CaTD80 and Decay30-90 but sparing TTP100 and Ca2+ transient alternans in all groups. All groups showed similar APD80, and TAC-only hearts showed greater CaTD80, heterogeneities following isoproterenol challenge. Empagliflozin abolished or reduced ventricular tachycardia and premature ventricular contractions and associated re-entrant conduction patterns, in isoproterenol-challenged TAC-operated hearts following successive burst pacing episodes. Empagliflozin thus rescues TAC-induced ventricular hypertrophy and systolic functional, Ca2+ homeostatic, and pro-arrhythmogenic changes in intact hearts.

Dexmedetomidine inhibited arrhythmia susceptibility to adrenergic stress in RyR2R2474S mice through regulating the coupling of membrane potential and intracellular calcium.

BACKGROUND: Dexmedetomidine (DEX), a highly selective α2-adrenoceptor agonist, can decrease the incidence of arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the underlying mechanisms by which DEX affects cardiac electrophysiological function remain unclear. METHODS: Ryanodine receptor (RyR2) heterozygous R2474S mice were used as a model for CPVT. WT and RyR2R2474S/+ mice were treated with isoproterenol (ISO) and DEX, and electrocardiograms were continuously monitored during both in vivo and ex vivo experiments. Dual-dye optical mapping was used to explore the anti-arrhythmic mechanism of DEX. RESULTS: DEX significantly reduced the occurrence and duration of ISO-induced of VT/VF in RyR2R2474S/+ mice in vivo and ex vivo. DEX remarkably prolonged action potential duration (APD80) and calcium transient duration (CaTD80) in both RyR2R2474S/+ and WT hearts, whereas it reduced APD heterogeneity and CaT alternans in RyR2R2474S/+ hearts. DEX inhibited ectopy and reentry formation, and stabilized voltage-calcium latency. CONCLUSION: DEX exhibited an antiarrhythmic effect through stabilizing membrane voltage and intracellular Ca2+. DEX can be used as a beneficial perioperative anesthetic for patients with CPVT or other tachy-arrhythmias.

Dbh+ catecholaminergic cardiomyocytes contribute to the structure and function of the cardiac conduction system in murine heart.

The heterogeneity of functional cardiomyocytes arises during heart development, which is essential to the complex and highly coordinated cardiac physiological function. Yet the biological and physiological identities and the origin of the specialized cardiomyocyte populations have not been fully comprehended. Here we report a previously unrecognised population of cardiomyocytes expressing Dbhgene encoding dopamine beta-hydroxylase in murine heart. We determined how these myocytes are distributed across the heart by utilising advanced single-cell and spatial transcriptomic analyses, genetic fate mapping and molecular imaging with computational reconstruction. We demonstrated that they form the key functional components of the cardiac conduction system by using optogenetic electrophysiology and conditional cardiomyocyte Dbh gene deletion models. We revealed their close relationship with sympathetic innervation during cardiac conduction system formation. Our study thus provides new insights into the development and heterogeneity of the mammalian cardiac conduction system by revealing a new cardiomyocyte population with potential catecholaminergic endocrine function.

The characteristics and molecular targets of antiarrhythmic natural products.

Arrhythmia is one of the most common cardiovascular diseases. The search for new drugs to suppress various types of cardiac arrhythmias has always been the focus of attention. In the past decade, the screening of antiarrhythmic active substances from plants has received extensive attention. These natural compounds have obvious antiarrhythmic effects, and chemical modifications based on natural compounds have greatly increased their pharmacological properties. The chemical modification of botanical antiarrhythmic drugs is closely related to the development of new and promising drugs. Therefore, the structural characteristics and action targets of natural compounds with antiarrhythmic effects are reviewed in this paper, so that pharmacologists can select antiarrhythmic lead compounds from natural compounds based on the disease target - chemical structural characteristics.

Single-cell RNA sequencing of murine hearts for studying the development of the cardiac conduction system.

The development of the cardiac conduction system (CCS) is essential for correct heart function. However, critical details on the cell types populating the CCS in the mammalian heart during the development remain to be resolved. Using single-cell RNA sequencing, we generated a large dataset of transcriptomes of ~0.5 million individual cells isolated from murine hearts at six successive developmental corresponding to the early, middle and late stages of heart development. The dataset provides a powerful library for studying the development of the heart's CCS and other cardiac components. Our initial analysis identified distinct cell types between 20 to 26 cell types across different stages, of which ten are involved in forming the CCS. Our dataset allows researchers to reuse the datasets for data mining and a wide range of analyses. Collectively, our data add valuable transcriptomic resources for further study of cardiac development, such as gene expression, transcriptional regulation and functional gene activity in developing hearts, particularly the CCS.

Novel XIAP mutation with early-onset Crohn's disease complicated with acute heart failure: a case report.

BACKGROUND: The X-linked inhibitor of apoptosis (XIAP) protein is encoded by the XIAP gene and is critical for multiple cell responses and plays a role in preventing cell death. XIAP mutations are associated with several diseases, primarily including hemophagocytic lymphohistiocytosis and inflammatory bowel disease (IBD). We report the clinical features and results associated with hemizygous mutation of the XIAP gene in a young male with Crohn's disease complicated with acute heart failure.This 16-year-old patient ultimately died of heart failure. CASE PRESENTATION: A young male of 16 years of age was initially diagnosed with Crohn's disease based on evidences from endoscopic and histological findings. Although supportive care, anti-infective drugs and biologics were administered consecutively for 11 months, his clinical manifestations and laboratory indices (patient's condition) did not improved. Additionally, the patient exhibited a poor nutritional status and sustained weight loss. Subsequently, acute heart failure led to the exacerbation of the patient's condition. He was diagnosed with wet beriberi according to thiamine deficiency, but the standard medical therapy for heart failure and thiamine supplementation did not reverse the adverse outcomes. Comprehensive genetic analysis of peripheral blood-derived DNA revealed a novel hemizygous mutation of the XIAP gene (c.1259_1262 delACAG), which was inherited from his mother. CONCLUSION: A novel XIAP mutation (c.1259_1262 delACAG) was identified in this study. It may be one of the potential pathogenic factors in Crohn's disease and plays an important role in the progression of heart failure. Additionally, thiamine deficiency triggers a vicious cycle.

Circuit-Specific Control of Blood Pressure by PNMT-Expressing Nucleus Tractus Solitarii Neurons.

The nucleus tractus solitarii (NTS) is one of the morphologically and functionally defined centers that engage in the autonomic regulation of cardiovascular activity. Phenotypically-characterized NTS neurons have been implicated in the differential regulation of blood pressure (BP). Here, we investigated whether phenylethanolamine N-methyltransferase (PNMT)-expressing NTS (NTSPNMT) neurons contribute to the control of BP. We demonstrate that photostimulation of NTSPNMT neurons has variable effects on BP. A depressor response was produced during optogenetic stimulation of NTSPNMT neurons projecting to the paraventricular nucleus of the hypothalamus, lateral parabrachial nucleus, and caudal ventrolateral medulla. Conversely, photostimulation of NTSPNMT neurons projecting to the rostral ventrolateral medulla produced a robust pressor response and bradycardia. In addition, genetic ablation of both NTSPNMT neurons and those projecting to the rostral ventrolateral medulla impaired the arterial baroreflex. Overall, we revealed the neuronal phenotype- and circuit-specific mechanisms underlying the contribution of NTSPNMT neurons to the regulation of BP.

Dual-Dye Optical Mapping of Hearts from RyR2 R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia.

The pro-arrhythmic cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia episodes following physical activity, stress, or catecholamine challenge, which can deteriorate into potentially fatal ventricular fibrillation. The mouse heart is a widespread species for modeling inherited cardiac arrhythmic diseases, including CPVT. Simultaneous optical mapping of transmembrane potential (Vm) and calcium transients (CaT) from Langendorff-perfused mouse hearts has the potential to elucidate mechanisms underlying arrhythmogenesis. Compared with the cellular level investigation, the optical mapping technique can test some electrophysiological parameters, such as the determination of activation, conduction velocity, action potential duration, and CaT duration. This paper presents the instrumentation setup and experimental procedure for high-throughput optical mapping of CaT and Vm in murine wild-type and heterozygous RyR2-R2474S/+ hearts, combined with programmed electrical pacing before and during the isoproterenol challenge. This approach has demonstrated a feasible and reliable method for mechanistically studying CPVT disease in an ex vivo mouse heart preparation.

Ventricular SK2 upregulation following angiotensin II challenge: Modulation by p21-activated kinase-1.

Effects of hypertrophic challenge on small-conductance, Ca2+-activated K+(SK2) channel expression were explored in intact murine hearts, isolated ventricular myocytes and neonatal rat cardiomyocytes (NRCMs). An established experimental platform applied angiotensin II (Ang II) challenge in the presence and absence of reduced p21-activated kinase (PAK1) (PAK1cko vs. PAK1f/f, or shRNA-PAK1 interference) expression. SK2 current contributions were detected through their sensitivity to apamin block. Ang II treatment increased such SK2 contributions to optically mapped action potential durations (APD80) and their heterogeneity, and to patch-clamp currents. Such changes were accentuated in PAK1cko compared to PAK1f/f, intact hearts and isolated cardiomyocytes. They paralleled increased histological and echocardiographic hypertrophic indices, reduced cardiac contractility, and increased SK2 protein expression, changes similarly greater with PAK1cko than PAK1f/f. In NRCMs, Ang II challenge replicated such increases in apamin-sensitive SK patch clamp currents as well as in real-time PCR and western blot measures of SK2 mRNA and protein expression and cell hypertrophy. Furthermore, the latter were enhanced by shRNA-PAK1 interference and mitigated by the PAK1 agonist FTY720. Increased CaMKII and CREB phosphorylation accompanied these effects. These were rescued by both FTY720 as well as the CaMKII inhibitor KN93, but not its inactive analogue KN92. Such CREB then specifically bound to the KCNN2 promoter sequence in luciferase assays. These findings associate Ang II induced hypertrophy with increased SK2 expression brought about by a CaMKII/CREB signaling convergent with the PAK1 pathway thence upregulating the KCNN2 promoter activity. SK2 may then influence cardiac electrophysiology under conditions of cardiac hypertrophy and failure.

A dataset of dual calcium and voltage optical mapping in healthy and hypertrophied murine hearts.

Pathological hypertrophy underlies sudden cardiac death due to its high incidence of occurrence of ventricular arrhythmias. The alteration of transmural electrophysiological properties in hypertrophic cardiac murine tissue has never been explored previously. In this dataset, we have for the first time conducted high-throughput simultaneous optical imaging of transmembrane potential and calcium transients (CaT) throughout the entire hypertrophic murine hearts at high temporal and spatial resolution. Using ElectroMap, we have conducted multiple parameters analysis including action potential duration/calcium transient duration, conduction velocity, alternans and diastolic interval. Voltage-calcium latency was measured as time difference between action potential and CaT peak. The dataset therefore provides the first high spatial resolution transmural electrophysiological profiling of the murine heart, allowing interrogation of mechanisms driving ventricular arrhythmias associated with pathological hypertrophy. The dataset allows for further reuse and detailed analyses of geometrical, topological and functional analyses and reconstruction of 2-dimensional and 3-dimentional models.

ACE2 contributes to the maintenance of mouse epithelial barrier function.

BACKGROUND: The whole world was hit hard by the coronavirus disease-19 (COVID-19). Given that angiotensin I converting enzyme 2 (ACE2) is the viral entry molecule, understanding ACE2 has become a major focus of current COVID-19 research. ACE2 is highly expressed in the gut, but its role has not been fully understood and thus COVID-19 treatments intending to downregulate ACE2 level may cause untoward side effects. Gaining insight into the functions of ACE2 in gut homeostasis therefore merits closer examination, and is beneficial to find potential therapeutic alternatives for COVID-19. METHODS: We took advantage of Ace2 knockout out mice and isolated intestinal organoids to examine the role of ACE2 in intestinal stemness. Inflammatory bowel disease (IBD) mouse model was established by 4% dextran sodium sulfate. LGR5 and KI67 levels were quantitated to reflect the virtue of intestinal stem cells (ISCs). FITC-dextran 4 (FD-4) assay was used to assess intestinal barrier function. RESULTS: Western blotting identified the expression of ACE2 in colon, which was consistent with the results of immunofluorescence and RT-PCR. Moreover, Ace2-/- organoids showed decreased LRG5 and KI67 levels, and elevated calcium concentration. Furthermore, the permeability of ace2-/- organoids was markedly increased compared with ace2+/+ organoids. Collectively, ace2-/- mice were more susceptible than ace2+/+ mice to IBD, including earlier bloody stool, undermined intestinal architecture and more pronounced weight loss. CONCLUSIONS: Our data reveal that ACE2 contributes to the proliferation of intestinal stem cells and hence orchestrates the mucosal homeostasis.

A Protocol for Dual Calcium-Voltage Optical Mapping in Murine Sinoatrial Preparation With Optogenetic Pacing.

Among the animal models for studying the molecular basis of atrial and sinoatrial node (SAN) biology and disease, the mouse is a widely used species due to its feasibility for genetic modifications in genes encoding ion channels or calcium handling and signaling proteins in the heart. It is therefore highly valuable to develop robust methodologies for studying SAN and atrial electrophysiological function in this species. Here, we describe a protocol for performing dual calcium-voltage optical mapping on mouse sinoatrial preparation (SAP), in combination with an optogenetic approach, for studying SAP membrane potential, intracellular Ca2+ transients, and pacemaker activity. The protocol includes the details for preparing the intact SAP, robust tissue dual-dye loading, light-programmed pacing, and high-resolution optical mapping. Our protocol provides an example of use of the combination of optogenetic and optical mapping techniques for investigating SAP membrane potential and intracellular Ca2+ transients and pacemaker activity with high temporal and spatial resolution in specific cardiac tissues. Thus, our protocol provides a useful tool for studying SAP physiology and pathophysiology in mice.

Pnmt-Derived Cardiomyocytes: Anatomical Localization, Function and Future Perspectives.

In this mini-review, we provide an overview of phenylethanolamine-N-methyl transferase (Pnmt)-derived cardiomyocytes (PdCMs), a recently discovered cardiomyocyte subpopulation. We discuss their anatomical localization, physiological characteristics, possible function, and future perspectives. Their unique distribution in the heart, electrical activity, Ca2+ transient properties, and potential role in localized adrenergic signaling are discussed.

Cardiac Optogenetics and Optical Mapping - Overcoming Spectral Congestion in All-Optical Cardiac Electrophysiology.

Optogenetic control of the heart is an emergent technology that offers unparalleled spatio-temporal control of cardiac dynamics via light-sensitive ion pumps and channels (opsins). This fast-evolving technique holds broad scope in both clinical and basic research setting. Combination of optogenetics with optical mapping of voltage or calcium fluorescent probes facilitates 'all-optical' electrophysiology, allowing precise optogenetic actuation of cardiac tissue with high spatio-temporal resolution imaging of action potential and calcium transient morphology and conduction patterns. In this review, we provide a synopsis of optogenetics and discuss in detail its use and compatibility with optical interrogation of cardiac electrophysiology. We briefly discuss the benefits of all-optical cardiac control and electrophysiological interrogation compared to traditional techniques, and describe mechanisms, unique features and limitations of optically induced cardiac control. In particular, we focus on state-of-the-art setup design, challenges in light delivery and filtering, and compatibility of opsins with fluorescent reporters used in optical mapping. The interaction of cardiac tissue with light, and physical and computational approaches to overcome the 'spectral congestion' that arises from the combination of optogenetics and optical mapping are discussed. Finally, we summarize recent preclinical work applications of combined cardiac optogenetics and optical mapping approach.

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Increases Small-Conductance Ca2+-Activated K+ Current in Patients with Chronic Atrial Fibrillation.

BACKGROUND Increased small-conductance Ca2+-activated K+ current (SK), abnormal intracellular Ca2+ handling, and enhanced expression and activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been found in clinical and/or experimental models of atrial fibrillation (AF), but the cumulative effect of these phenomena and their mechanisms in AF are still unclear. This study aimed to test the hypothesis that CaMKII increases SK current in human chronic AF. MATERIAL AND METHODS Right atrial appendage tissues from patients with either sinus rhythm (SR) or AF and neonatal rat atrial myocytes were used. Patch clamp, qRT-PCR, and Western blotting techniques were used to perform the study. RESULTS Compared to SR, the apamin-sensitive SK current (IKAS) was significantly increased, but the mRNA and protein levels of SK1, SK2, and SK3 were significantly decreased. In AF, the steady-state Ca2+ response curve of [i]IKAS[/i] was shifted leftward and the [Ca2+]i level was significantly increased. CaMKII inhibitors (KN-93 or autocamtide-2-related inhibitory peptide (AIP)) reduced the IKAS in both AF and SR. The inhibitory effect of KN-93 or AIP on [i]IKAS[/i] was greater in AF than in SR. The expression levels of calmodulin, CaMKII, and autophosphorylated CaMKII at Thr287 (but not at Thr286) were significantly increased in AF. Furthermore, KN-93 inhibited the expression of (Thr287)p-CaMKII and SK2 in neonatal rat atrial myocytes. CONCLUSIONS SK current is increased via the enhanced activation of CaMKII in patients with AF. This finding may explain the difference between SK current and channels expression in AF, and thus may provide a therapeutic target for AF.

Atrial-selective block of sodium channels by acehytisine in rabbit myocardium.

Acehytisine, a multi-ion channel blocker, can markedly inhibit INa, ICa, IKur, If at various concentrations and effectively terminate and prevent atrial fibrillation (AF) in patients and animal models, but the molecular mechanism underlying its blockage remains elusive. In this study, we investigated the effects of acehytisine on action potentials and sodium channels of atrial and ventricular myocytes isolated from rabbit, using whole-cell recording system. We found that acehytisine exerted stronger blocking effects on sodium channels in atria than in ventricles, especially at depolarization (IC50: 48.48 ± 7.75 µmol/L in atria vs. 560.17 ± 63.98 µmol/L in ventricles). It also significantly shifted steady state inactivation curves toward negative potentials in atrial myocytes, without affecting the recovery kinetics from inactivation of sodium channels in the same cells. In addition, acehytisine inhibited INa in a use-dependent manner and regulated slow inactivation kinetics by different gating configurations. These findings indicate that acehytisine selectively blocks atrial sodium channels and possesses affinity to sodium channel in certain states, which provides additional evidence for the anti-AF of acehytisine.

Myricetin inhibits Kv1.5 channels in HEK293 cells.

Myricetin (Myr) is a flavonoid that exerts anti-arrhythmic effects. However, its potential effects on ion channels have remained elusive. The aim of the present study was to investigate the effects of Myr on Kv1.5 channels in HEK293 cells. The current of Kv1.5 channels (Ikur) in HEK293 cells was recorded using the whole-cell patch-clamp technique and the expression of the Kv1.5 protein was measured using western blot analysis 24 h after treatment with Myr. The results showed that 5 µM Myr significantly reduced Ikur from 215.04 ± 40.59 to 77.72 ± 17.94 pA/pF (P<0.05; n=5). Myr increased the current suppression from 0 to 0.31 ± 0.12 and 0.55 ± 0.11 over 5 or 20 min, respectively. In addition, Ikur decreased from 376.23 ± 1.30 to 270.19 ± 4.28 pA/pF when the frequency was increased from 0.5 to 4 Hz in HEK293 cells treated with 10 µM Myr for 5 min. Furthermore, Myr reduced hKv1.5 protein expression in a dose-dependent manner. These results demonstrated that Myr inhibited Ikur and the expression of hKv1.5 in HEK293 cells in a dose-, time- and frequency-dependent manner. These observations partly explained the mechanisms by which Myr exerts anti-arrhythmic effect.

Remodeling of Kv1.5 channel in right atria from Han Chinese patients with atrial fibrillation.

BACKGROUND: The incidence of atrial fibrillation (AF) in rheumatic heart diseases (RHD) is very high and increases with age. Occurrence and maintenance of AF are very complicated process accompanied by many different mechanisms. Ion-channel remodeling, including the voltage-gated potassium channel Kv1.5, plays an important role in the pathophysiology of AF. However, the changes of Kv1.5 channel expression in Han Chinese patients with RHD and AF remain poorly understood. The aim of the present study was to investigate whether the Kv1.5 channels of the right atria may be altered with RHD, age, and sex to contribute to AF. MATERIAL/METHODS: Right atrial appendages were obtained from 20 patients with normal cardiac functions who had undergone surgery, and 26 patients with AF. Subjects were picked from 4 groups: adult and aged patients in normal sinus rhythm (SR) and AF. Patients were divided into non-RHD and RHD groups or men and women groups in normal SR and AF, respectively. The expression of Kv1.5 protein and messenger RNA (mRNA) were measured using Western blotting and polymerase chain reaction (PCR) method, respectively. RESULTS: Compared with the SR group, the expression of Kv1.5 protein decreased significantly in the AF group. However, neither Kv1.5 protein nor KCNA5 mRNA had significant differences in adult and aged groups, non-RHD and RHD group, and men and women group of AF. CONCLUSIONS: The expression of Kv1.5 channel protein changes with AF but not with age, RHD, and sex in AF.

Nitidine chloride induces apoptosis in human hepatocellular carcinoma cells through a pathway involving p53, p21, Bax and Bcl-2.

Nitidine chloride (NC), a novel benzo[c]phenanthridine alkaloid, induces the growth inhibition of cancer cells. Previously it was demonstrated that SMMC-7721 human hepatocellular carcinoma (HCC) cells are highly susceptible to the antiproliferative effects of NC. However, the specific mechanisms remained unclear. In the present study the pathways of growth inhibition induced by NC in SMMC-7721 cells were investigated. The effects of NC on SMMC-7721 cell proliferation were characterized by MTT and colony formation assays. Additionally, BALB/c nude mice were transplanted with SMMC-7721 cells to verify the inhibition of HCC by NC in vivo. The results showed that NC inhibited the proliferation of SMMC-7721 cells in vitro in a time- and dose-dependent manner and identified efficacy in vivo in a mouse model of HCC. Acridine orange (AO) staining, transmission electron microscopy, Annexin V/PI staining, TUNEL assay and caspase-3 activation assays were used to investigate apoptosis and the cell cycle distribution. Inhibition was mediated in part by cell cycle arrest in G2/M, leading to chromatin condensation, DNA fragmentation and the formation of apoptotic bodies. Apoptosis was also verified by Annexin V/PI staining, TUNEL assay and caspase-3 activation. To assess the levels of the cell cycle and apoptotic regulators, immunohistochemical staining, ELISA, real-time PCR and RNA interference (RNAi) were employed. The apoptotic process triggered by NC involved the upregulation of p53, p21 and Bax, and the downregulation of Bcl-2. These data elucidate a pathway of apoptosis in SMMC­7721 cells that involves G2/M arrest, upregulation of p53, Bax, caspase-3 and p21, and downregulation of Bcl-2.