Monkeypox Virus Detection in Different Clinical Specimen Types.

A global monkeypox outbreak began in May 2022. Limited data exist on specimen type performance in associated molecular diagnostics. Consequently, a diverse range of specimen sources were collected in the initial weeks of the outbreak in Ontario, Canada. Our clinical evaluation identified skin lesions as the optimal diagnostic specimen source.

microRNA-133a attenuates cardiomyocyte hypertrophy by targeting PKCδ and Gq.

During the past decade, microRNAs have continuously been suggested as a promising therapeutic tool due to their beneficial effects, such as their multi-targets and multi-functions in pathologic conditions. As a pathologic phenotype is generally regulated by multiple signaling pathways, in this study we identified a microRNA regulating multiple target genes within cardiac hypertrophic signaling pathways. microRNA-133a is known to play a crucial role in cardiac hypertrophy. However, the role of microRNA-133a, which may regulate several signaling pathways in norepinephrine-induced cardiac hypertrophy via multi-targeting, has not been investigated. In the current study, we showed that microRNA-133a can protect cardiomyocyte hypertrophy against norepinephrine stimulation in neonatal rat ventricular cardiomyocytes via new targets, PKCδ and Gq, all of which are related to downstream signaling pathways of the α1-adrenergic receptor. Taken together, these results suggest the advantages of the therapeutic use of microRNAs as an effective potential drug regulating multiple signaling pathways under pathologic conditions.

Hot Deformation Behavior of Hot-Extruded AA7175 Through Hot Torsion Tests.

The hot deformation behavior of hot-extruded AA7175 was investigated with flow curves and processing maps through hot torsion tests. The flow curves and the deformed microstructures revealed that dynamic recrystallization (DRX) occurred in the hot-extruded AA7175 during hot working. The failure strain was highest at medium temperature. This was mainly influenced by the dynamic precipitation of fine rod-shaped MgZn2. The processing map determined the optimal deformation condition for the alloy during hot working.

Exogenous miRNA-146a Enhances the Therapeutic Efficacy of Human Mesenchymal Stem Cells by Increasing Vascular Endothelial Growth Factor Secretion in the Ischemia/Reperfusion-Injured Heart.

Adult stem cells have been studied as a promising therapeutic modality for the functional restoration of the damaged heart. In the present study, a strategy for enhancing the angiogenic efficacy of human mesenchymal stem cells (hMSCs) using micro-RNA was examined. We investigated whether micro-RNA-146a (miR-146a) influences the secretion of vascular endothelial growth factor (VEGF) and angiogenesis of MSCs. Our data indicated that miR-146a-transfected hMSCs (hMSCmiR-146a) decreased the expression of neurofibromin 2, an inhibitor of p21-activated kinase-1 (PAK1). miR-146a also increased the expression of Ras-related C3 botulinum toxin substrate 1 and PAK1, which are known to induce VEGF expression, and the formation of vascular branches was increased in hMSCmiR-146a compared to hMSCs treated with VEGF. VEGF and p-Akt were increased in hMSCmiR-146a. Furthermore, injection of hMSCmiR-146a after ischemia/reperfusion (I/R) injury led to a reduction of fibrosis area and increased VEGF expression, confirming the regenerative capacity such as reparative angiogenesis in the infarcted area. Cardiac functions in I/R injury were improved following injection of hMSCmiR-146a compared to the I/R group. Taken together, these data suggest that miR-146 is a novel microRNA that regulates VEGF expression, and its use may be an effective strategy for enhancing the therapeutic efficacy of hMSC transplantation into the I/R-injured heart.

MicroRNA-29b inhibits migration and proliferation of vascular smooth muscle cells in neointimal formation.

The proliferation and migration of smooth muscle cells (SMCs) are considered to be key steps in the progression of atherosclerosis and restenosis. Certain stimuli, such as, interleukin-3 (IL-3) are known to stimulate proliferation and migration in vascular diseases. Meanwhile, microRNAs (miRs) have been revealed as critical modulators of various diseases in which miR-29b is known to regulate cell growth by targeting Mcl-1 and MMP2. However, roles of miR-29b in vascular smooth muscle cells remain almost unknown. We hypothesized that miR-29b may control the proliferation and migration processes induced by IL-3 stimulation by inhibiting its own specific targets in SMCs. MiR-29b significantly suppressed the proliferation and migration of SMCs through the inhibition of the signaling pathway related to Mcl-1 and MMP2. We also found that miR-29b expression levels significantly declined in balloon-injured rat carotid arteries and that the overexpression of miR-29b by local oligonucleotide delivery can inhibit neointimal formation. Consistent with the critical role of miR-29b in vitro, we observed down-regulated expression levels of Mcl-1 and MMP2 from the neointimal region. These results indicate that miR-29b suppressed the proliferation and migration of SMCs, possibly through the inhibition of Mcl-1 and MMP2, and suggest that miR-29b may serve as a useful therapeutic tool to treat cardiovascular diseases such as, atherosclerosis and restenosis.

Regulation of mitochondrial morphology by positive feedback interaction between PKCδ and Drp1 in vascular smooth muscle cell.

Dynamin-related protein-1 (Drp1) plays a critical role in mitochondrial fission which allows cell proliferation and Mdivi-1, a specific small molecule Drp1 inhibitor, is revealed to attenuate proliferation. However, few molecular mechanisms-related to Drp1 under stimulus for restenosis or atherosclerosis have been investigated in vascular smooth muscle cells (vSMCs). Therefore, we hypothesized that Drp1 inhibition can prevent vascular restenosis and investigated its regulatory mechanism. Angiotensin II (Ang II) or hydrogen peroxide (H2 O2 )-induced proliferation and migration in SMCs were attenuated by down-regulation of Drp1 Ser 616 phosphorylation, which was demonstrated by in vitro assays for migration and proliferation. Excessive amounts of ROS production and changes in mitochondrial membrane potential were prevented by Drp1 inhibition under Ang II and H2 O2 . Under the Ang II stimulation, activated Drp1 interacted with PKCδ and then activated MEK1/2-ERK1/2 signaling cascade and MMP2, but not MMP9. Furthermore, in ex vivo aortic ring assay, inhibition of the Drp1 had significant anti-proliferative and -migration effects for vSMCs. A formation of vascular neointima in response to a rat carotid artery balloon injury was prevented by Drp1 inhibition, which shows a beneficial effect of Drp1 regulation in the pathologic vascular condition. Drp1-mediated SMC proliferation and migration can be prevented by mitochondrial division inhibitor (Mdivi-1) in in vitro, ex vivo and in vivo, and these results suggest the possibility that Drp1 can be a new therapeutic target for restenosis or atherosclerosis.

MicroRNA-17-mediated down-regulation of apoptotic protease activating factor 1 attenuates apoptosome formation and subsequent apoptosis of cardiomyocytes.

Heart diseases such as myocardial infarction (MI) can damage individual cardiomyocytes, leading to the activation of cell death programs. The most scrutinized type of cell death in the heart is apoptosis, and one of the key events during the propagation of apoptotic signaling is the formation of apoptosomes, which relay apoptotic signals by activating caspase-9. As one of the major components of apoptosomes, apoptotic protease activating factor 1 (Apaf-1) facilitates the formation of apoptosomes containing cytochrome c (Cyto-c) and deoxyadenosine triphosphate (dATP). Thus, it may be possible to suppress the activation of the apoptotic program by down-regulating the expression of Apaf-1 using miRNAs. To validate this hypothesis, we selected a number of candidate miRNAs that were expected to target Apaf-1 based on miRNA target prediction databases. Among these candidate miRNAs, we empirically identified miR-17 as a novel Apaf-1-targeting miRNA. The delivery of exogenous miR-17 suppressed Apaf-1 expression and consequently attenuated formation of the apoptosome complex containing caspase-9, as demonstrated by co-immunoprecipitation and immunocytochemistry. Furthermore, miR-17 suppressed the cleavage of procaspase-9 and the subsequent activation of caspase-3, which is downstream of activated caspase-9. Cell viability tests also indicated that miR-17 pretreatment significantly prevented the norepinephrine-induced apoptosis of cardiomyocytes, suggesting that down-regulation of apoptosome formation may be an effective strategy to prevent cellular apoptosis. These results demonstrate the potential of miR-17 as an effective anti-apoptotic agent.

ROS-mediated bidirectional regulation of miRNA results in distinct pathologic heart conditions.

Under distinct pathological heart conditions, the expression of a single miRNA can display completely opposite patterns. However, the mechanism underlying the bidirectional regulation of a single miRNA and the clinical implications of this regulation remain largely unknown. To address this issue, we examined the regulation of miR-1, one of the most abundant miRNAs in the heart, during cardiac hypertrophy and ischemia/reperfusion (I/R). Our data indicated that different magnitudes and chronicities of ROS levels in cardiomyocytes resulted in differential expression of miR-1, subsequently altering the expression of myocardin. In animal models, the administration of a miR-1 mimic attenuated cardiac hypertrophy by suppressing the transverse aortic constriction-induced increase in myocardin expression, whereas the administration of anti-miR-1 ameliorated I/R-induced cardiac apoptosis and deterioration of heart function. Our findings indicated that a pathologic stimulus such as ROS can bidirectionally alter the expression of miRNA to contribute to the development of pathological conditions exhibiting distinct phenotypes and that the meticulous adjustment of the pathological miRNA levels is required to improve clinical outcomes.

PLCδ1 protein rescues ischemia-reperfused heart by the regulation of calcium homeostasis.

Myocardial Ca(2+) overload induced by ischemia/reperfusion (I/R) is a major element of myocardial dysfunction in heart failure. Phospholipase C (PLC) plays important roles in the regulation of the phosphoinositol pathway and Ca(2+) homeostasis in various types of cells. Here, we investigated the protective role of PLCδ1 against myocardial I/R injury through the regulation of Ca(2+) homeostasis. To investigate its role, PLCδ1 was fused to Hph1, a cell-permeable protein transduction domain (PTD), and treated into rat neonatal cardiomyocytes and rat hearts under respective hypoxia-reoxygenation (H/R) and ischemia-reperfusion conditions. Treatment with Hph1-PLCδ1 significantly inhibited intracellular Ca(2+) overload, reactive oxygen species generation, mitochondrial permeability transition pore opening, and mitochondrial membrane potential elevation in H/R neonatal cardiomyocytes, resulting in the inhibition of apoptosis. Intravenous injections of Hph1-PLCδ1 in rats with I/R-injured myocardium caused significant reductions in infarct size and apoptosis and also improved systolic and diastolic cardiac functioning. Furthermore, a small ions profile obtained using time-of-flight secondary ion mass spectrometry showed that treatment with Hph1-PLCδ1 leads to significant recovery of calcium-related ions toward normal levels in I/R-injured myocardium. These results suggest that Hph1-PLCδ1 may manifest as a promising cardioprotective drug due to its inhibition of the mitochondrial apoptotic pathway in cells suffering from I/R injury.

Suppression of miR-181a attenuates H2O2-induced death of mesenchymal stem cells by maintaining hexokinase II expression.

BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.

MicroRNA-365 inhibits the proliferation of vascular smooth muscle cells by targeting cyclin D1.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of disease progression in atherosclerosis. Cell proliferation is regulated by cell cycle regulatory proteins. MicroRNAs (miR) have been reported to act as important gene regulators and play essential roles in the proliferation and migration of VSMCs in a cardiovascular disease. However, the roles and mechanisms of miRs in VSMCs and neointimal formation are far from being fully understood. In this study, cell cycle-specific cyclin D1 was found to be a potential target of miR-365 by direct binding. Through an in vitro experiment, we showed that exogenous miR-365 overexpression reduced VSMC proliferation and proliferating cell nuclear antigen (PCNA) expression, while miR-365 was observed to block G1/S transition in platelet-derived growth factor-bb (PDGF-bb)-induced VSMCs. In addition, the proliferation of VSMCs by various stimuli, including PDGF-bb, angiotensin II (Ang II), and serum, led to the downregulation of miR-365 expression levels. The expression of miR-365 was confirmed in balloon-injured carotid arteries. Taken together, our results suggest an anti-proliferative role for miR-365 in VSMC proliferation, at least partly via modulating the expression of cyclin D1. Therefore, miR-365 may influence neointimal formation in atherosclerosis patients.

Cardiomyocytes from phorbol myristate acetate-activated mesenchymal stem cells restore electromechanical function in infarcted rat hearts.

Despite the safety and feasibility of mesenchymal stem cell (MSC) therapy, an optimal cell type has not yet emerged in terms of electromechanical integration in infarcted myocardium. We found that poor to moderate survival benefits of MSC-implanted rats were caused by incomplete electromechanical integration induced by tissue heterogeneity between myocytes and engrafted MSCs in the infarcted myocardium. Here, we report the development of cardiogenic cells from rat MSCs activated by phorbol myristate acetate, a PKC activator, that exhibited high expressions of cardiac-specific markers and Ca(2+) homeostasis-related proteins and showed adrenergic receptor signaling by norepinephrine. Histological analysis showed high connexin 43 coupling, few inflammatory cells, and low fibrotic markers in myocardium implanted with these phorbol myristate acetate-activated MSCs. Infarct hearts implanted with these cells exhibited restoration of conduction velocity through decreased tissue heterogeneity and improved myocardial contractility. These findings have major implications for the development of better cell types for electromechanical integration of cell-based treatment for infarcted myocardium.

MicroRNA-145 suppresses ROS-induced Ca2+ overload of cardiomyocytes by targeting CaMKIIδ.

A change in intracellular free calcium (Ca(2+)) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca(2+) signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca(2+)-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H2O2-mediated Ca(2+) overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca(2+) overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca(2+)-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca(2+) overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

Prions in Microbes: The Least in the Most.

Prions are infectious proteins that mostly replicate in self-propagating amyloid conformations (filamentous protein polymers) and consist of structurally altered normal soluble proteins. Prions can arise spontaneously in the cell without any clear reason and are generally considered fatal disease-causing agents that are only present in mammals. However, after the seminal discovery of two prions, [PSI+] and [URE3], in the eukaryotic model microorganism Saccharomyces cerevisiae, at least ten more prions have been discovered, and their biological and pathological effects on the host, molecular structure, and the relationship between prions and cellular components have been studied. In a filamentous fungus model, Podospora anserina, a vegetative incomparability-related [Het-s] prion that directly triggers cell death during anastomosis (hyphal fusion) was discovered. These prions in eukaryotic microbes have extended our understanding to overcome most fatal human prion/amyloid diseases. A prokaryotic microorganism (Clostridium botulinum) was reported to have a prion analog. The transcriptional regulators of C. botulinum-Rho can be converted into the self-replicating prion form ([RHO-X-C+]), which may affect global transcription. Here, we outline the major issues with prions in microbes and the lessons learned from the relatively uncovered microbial prion world.

Evaluation of Actual Ventilation Rates and Efficiency in Research-Scale Pig Houses Based on Ventilation Configurations.

Accurate ventilation control is crucial for maintaining a healthy and productive environment in research-specialized pig facilities. This study aimed to evaluate actual ventilation rates and ventilation efficiency by investigating different inlet and exhaust configurations. The research was conducted in two pig rooms, namely pig room A and pig room B, in the absence of animals and workers to focus solely on evaluating the ventilation system's performance. Actual ventilation rates were measured using hood-type anemometers, and the local air change per hour was analyzed at various measurement points via tracer gas decay experiments. The results demonstrated that specific inlet and exhaust combinations, such as side inlet/chimney outlet and ceiling inlet/side outlet, exhibited higher ventilation rates. However, the measured ventilation rates were much lower than the manufacturer's specifications. The side exhaust fan closer to the pig activity space demonstrated better ventilation effectiveness for the animals than the chimney exhaust fan. Additionally, the ceiling inlet exhibited superior air distribution and uniformity. Lower ventilation rates and higher infiltration ratios resulted in reduced ventilation efficiency, with the difference between pig and worker activity spaces being pronounced. This study emphasizes the importance of selecting optimal inlet and exhaust configurations to achieve efficient ventilation and create a healthy environment for both pigs and workers.

Ammonia and Particulate Matter Emissions at a Korean Commercial Pig Farm and Influencing Factors.

Quantifying emission factors of ammonia and particulate matter (PM) in livestock production systems is crucial for assessing and mitigating the environmental impact of animal production and for ensuring industry sustainability. This study aimed to determine emission factors of ammonia, total suspended particles (TSPs), PM10, and PM2.5 for piglets and growing-finishing pigs at a commercial pig farm in Korea. It also sought to identify factors influencing these emission factors. The research found that the emission factors measured were generally lower than those currently used in Korea, but were consistent with findings from individual research studies in the literature. Seasonal variations were observed, with ammonia emissions peaking in spring and autumn, and PM emissions rising in summer. Correlation analyses indicated that the number of animals and their average age correlated positively with both ammonia and PM emission factors. Ventilation rate was also positively correlated with PM emissions. Future extended field measurements across diverse pig farms will offer deeper insights into the emission factors of pig farms in Korea, guiding the development of sustainable livestock management practices.

Phorbol myristate acetate differentiates human adipose-derived mesenchymal stem cells into functional cardiogenic cells.

To achieve effective regeneration of injured myocardium, it is important to find physiological way of improving the cardiogenic differentiation of stem cells. Previous studies demonstrated that cardiomyocytes from bone marrow-derived mesenchymal stem cells (BMSCs) activated with phorbolmyristate acetate (PMA), a protein kinase C (PKC) activator, restore electromechanical function in infarcted rat hearts. In this study, we investigated the effect of PMA on cardiogenic differentiation of adipose-derived MSCs (ASCs) for clinical applications. To confirm the effect of PMA, ASCs treated with 1µM PMA were grown for nine days. The expression of cardiac-specific markers (cardiac troponin T, myosin light chain, myosin heavy chain) in PMA-treated MSCs was demonstrated by immunocytochemistry. Alhough few α(1A) receptors exist in ASCs, α(1)-adrenergic receptor subtypes were preferentially expressed in PMA-treated ASCs. Moreover, expression of the ß-adrenergic and muscarinic receptors increased in PMA-treated ASCs compared to normal cells. The mRNA levels of Ca(2+)-related factors (SERCA 2a; sarcoplasmic reticulum Ca(2+)-ATPase, LTCC; L-type Ca(2+) channel) in treated ASCs were similar to the levels in cardiomyocytes. Following the transplantation of chemically activated cardiogenic ASCs into infarcted myocardium, histological analysis showed that infarct size, interstitial fibrosis, and apoptotic index were markedly decreased and cardiac function was restored. In conclusion, PMA might induce the cardiogenic differentiation of human ASCs as well as BMSCs. This result suggests successful use of human ASCs in cardiac regeneration therapy.

Up-regulation of miR-26a promotes apoptosis of hypoxic rat neonatal cardiomyocytes by repressing GSK-3ß protein expression.

Myocardial ischemia is the major cause of morbidity and mortality due to cardiovascular diseases. This disease is a severe stress condition that causes extensive biochemical changes which trigger cardiac cell death. Stress conditions such as deprivation of glucose and oxygen activate the endoplasmic reticulum in the cytoplasm of cells, including cardiomyocytes, to generate and propagate apoptotic signals in response to these conditions. microRNAs (miRNAs) are a class of small non-coding RNAs that mediate posttranscriptional gene silencing. The miRNAs play important roles in regulating cardiac physiological and pathological events such as hypertrophy, apoptosis, and heart failure. However, the roles of miRNAs in reactive oxygen species (ROS)-mediated injury on cardiomyocytes are uncertain. In this study, we identified at the apoptotic concentration of H(2)O(2), miR-26a expression was increased. To determine the potential roles of miR-26a in H(2)O(2)-mediated cardiac apoptosis, miR-26a expression was regulated by a miR-26a or an anti-miR-26a. Overexpression of miR-26a increased apoptosis as determined by upregulation of Annexin V/PI positive cell population, caspase-3 activity and expression of pro-apoptotic signal molecules, whereas inhibition of miR-26a reduced apoptosis. We identified GSK3B as a direct downstream target of miR-26a. Furthermore, miR-26a attenuated viability and increased caspase-3 activity in normal cardiomyocytes. This study demonstrates that miR-26a promotes ROS-induced apoptosis in cardiomyocytes. Thus, miR-26a affects ROS-mediated gene regulation and cellular injury response.

Anti-proliferative effect of rosiglitazone on angiotensin II-induced vascular smooth muscle cell proliferation is mediated by the mTOR pathway.

VSMC (vascular smooth muscle cell) proliferation contributes significantly to intimal thickening in atherosclerosis, restenosis and venous bypass graft diseases. Ang II (angiotensin II) has been implicated in VSMC proliferation though the activation of multiple growth-promoting signals. Although TZDs (thiazolidinediones) can inhibit VSMC proliferation and reduce Ang II-induced fibrosis, the mechanism underlying the inhibition of VSMC proliferation and fibrosis needs elucidation. We have used primary cultured rat aortic VSMCs and specific antibodies to investigate the inhibitory mechanism of rosiglitazone on Ang II-induced VSMC proliferation. Rosiglitazone treatment significantly inhibited Ang II-induced rat aortic VSMC proliferation in a dose-dependent manner. Western blot analysis showed that rosiglitazone significantly lowered phosphorylated ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt (also known as protein kinase B), mTOR (mammalian target of rapamycin), p70S6K (70 kDa S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E-binding protein) levels in Ang II-treated VSMCs. In addition, PPAR-γ (peroxisome-proliferator-activated receptor γ) mRNA increased significantly and CTGF (connective tissue growth factor), Fn (fibronectin) and Col III (collagen III) levels decreased significantly. The results demonstrate that the rosiglitazone directly inhibits the pro-atherosclerotic effect of Ang II on rat aortic VSMCs. It also attenuates Ang II-induced ECM (extracellular matrix) molecules and CTGF production in rat aortic VSMCs, reducing fibrosis. Importantly, PPAR-γ activation mediates these effects, in part, through the mTOR-p70S6K and -4EBP1 system.

Differentially regulated functional gene clusters identified in early hypoxic cardiomyocytes.

Pathological stress including myocardial infarction and hypertension causes a negative effect on calcium regulation and homeostasis. Nevertheless, few studies reveal that Ca(2+) regulatory genes are related to pathological status in cardiomyocytes under early hypoxia. To determine the alteration of Ca(2+)-related gene in hypoxic myocytes, primary neonatal rat ventricular cardiomyocytes (NRVCMs) was isolated. Survival of hypoxic NRVCMs was significantly decreased in 6 h. We confirmed an increase of reactive oxygen species (ROS) generation and Ca(2+) overload in hypoxic NRVCMs by using 2',7'-dichlorodihydro-fluorescein diacetate (H2DCFDA) and FACS analysis. Furthermore, survival/apoptotic signals were also regulated in same condition. The expression profiles of more than 30,000 genes from NRVCMs that were subjected to early hypoxia revealed 630 genes that were differentially regulated. The intracellular Na(+) overload and Ca(2+) handling genes with at least two-fold changes were confirmed. The levels of Ca(2+)-handling proteins (calsequestrin, calmodulin, and calreticulin), ion channels (NCX, Na(+)-K(+)-ATPase, SERCA2a, and PLB), and stress markers (RyR2, ANP, and BNP) were significantly altered in early hypoxia. These results demonstrate that early hypoxia alters Ca(2+)-related gene expression in NRVCMs, leading to pathological status.