Circadian rhythms widely exist in living organisms, and they are regulated by the biological clock. Growing evidence has shown that circadian rhythms are tightly related to the physiological function of the cardiovascular system, including blood pressure, heart rate, metabolism of cardiomyocytes, function of endothelial cells, and vasoconstriction and vasodilation. In addition, disruption of circadian rhythms has been considered as one of the important risk factors for cardiovascular diseases, such as myocardial infarction. This review summarizes the recent research advances in the relationship between circadian clock and cardiovascular diseases, hoping to improve treatment strategies for patients with cardiovascular diseases according to the theory of biological clock.
Blood Pressure ; Cardiovascular Diseases ; physiopathology ; Circadian Clocks ; Circadian Rhythm ; Endothelial Cells ; cytology ; Heart Rate ; Humans ; Myocytes, Cardiac ; metabolism ; Vasoconstriction ; Vasodilation

OBJECTIVE: To investigate the effects of myeloid differentiation-2 (MD2) gene silencing on high glucose-induced proliferation inhibition, apoptosis and inflammation in rat cardiomyocytes. METHODS: The immortalized rat cardiomyocyte cell line H9C2 were transfected with MD2 small interfering RNA (si-MD2) and negative control for 24 h, then stimulated with high glucose (HG) for 48 h. RT-qPCR was performed to detect the mRNA levels of MD2 and inflammatory factors TNF-α, IL-1β and IL-6. MTS and flow cytometry were used to evaluate cell proliferation, cell cycle and apoptosis rate. Western blot was used to detect protein expression levels and phosphorylation levels. RESULTS: The mRNA and protein levels of MD2 in H9C2 cells were dramatically decreased after transfected with si-MD2 (P＜0.01). After stimulation of high glucose, the mRNA levels of inflammatory factors, the cells in G0/G1 phase , the cell apoptosis rate and the protein level of cleaved Caspase-3 were significantly increased, while the cell proliferation ability was decreased (P＜0.01). MD2 gene silencing antagonized the effects of high glucose on cell proliferation, cell cycle, cell apoptosis and the mRNA levels of TNF-α, IL-1β , IL-6(P＜0.05). Western blot analysis showed that the phosphorylation levels of extracellular signal-regulated kinase(ERK1/2), P38 mitogen-activated protein kinase(P38 MAPK) and C-Jun N-terminal kinase(JNK) protein were increased significantly in H9C2 cells treated with high glucose, which could be reversed by silencing of MD2 (P＜0.01). CONCLUSION This study demonstrates that MD2 gene silencing reverses high glucose-induced myocardial inflammation, apoptosis and proliferation inhibition via the mechanisms involving suppression of ERK, P38 MAPK, JNK signaling pathway.
Animals ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Cytokines ; metabolism ; Gene Silencing ; Glucose ; Inflammation ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lymphocyte Antigen 96 ; genetics ; Myocytes, Cardiac ; cytology ; Rats ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVE: To investigate the effects of Notch signal on hypoxic induction factor (HIF-1α) and autophagy-associated genes Beclin1, LC3I, LC3II in oxygen-glucose deprivation (OGD) induced myocardial cell injury. METHODS: The OGD model was established using hypoxic culture box and hypoglycemic DMEM medium. The cells were divided into normal control group, OGD group, OGD + NC siRNA group, OGD + Notch1 siRNA group and OGD + HIF-1α siRNA group. Western blot was used to detect the interference effects of HIF-1α siRNA and Notch1 siRNA. The effects of Notch1 siRNA and HIF-1α siRNA on the activity of myocardial cells in OGD model were detected by the CCK-8 assay. The effects of Notch1 siRNA and HIF-1α siRNA on autophage-associated genes Beclin1, LC3I and LC3II expression were detected by Western blot. RESULTS: The results of Western blot showed that HIF-1α siRNA could effectively knock down the expression of HIF-1α in myocardial cells in OGD model, and Notch1 siRNA could effectively knock down the expression of Notch1 and HIF-1α in myocardial cells in OGD model. The result of CCK-8 assay showed that Notch1 siRNA and HIF-1α siRNA reduced the activity of myocardial cells in OGD model, and there was no statistical difference between the two groups. Western blot results showed that Notch1 siRNA and HIF-1α siRNA could reduce the expressions of the autophagy-associated genes Beclin1, LC3I and LC3II, and reduce the ratio of LC3II to LC3I at mRNA level. CONCLUSION Notch1 plays a role in myocardial protection by regulating the expression of HIF-1α to regulate the autophagy in OGD model cells.
Autophagy ; Beclin-1 ; metabolism ; Cell Hypoxia ; Cells, Cultured ; Glucose ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Myocytes, Cardiac ; cytology ; pathology ; Oxygen ; Receptors, Notch ; metabolism ; Signal Transduction

OBJECTIVE: To observe whether necroptosis was happened in high glucose (HG) - induced primary cardiomyocytes injury and to investigate the likely mechanism. METHODS: The primary cultured cardiomyocytes were divided into 4 groups (n=9): control group (the cardiomyocytes were incubated with 5.5 mmol/L glucose for 48 h), HG group (the cardiomyocytes were incubated with 30 mmol/L glucose for 48 h), HG + necrostatin-1 (Nec-1) group (the cardiomyocytes was co-incubated with necroptosis inhibitor Nec-1 at 100 μmol/L and HG for 48 h) and hypertonic pressure group (HPG, the cardiomyocytes was co-incubated with 5.5 mmol/L glucose and 24.5 mmol/L mannitol for 48 h). Cell viability was measured by MTT method, reactive oxygen species (ROS) generation was measured by DHE staining. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were tested by ELISA method. The mRNA and protein expressions of necroptosis related genes receptor interacting serine/threonine protein kinase 1 (RIP1), RIP3, mixed lineage kinase domain-like protein (MLKL) were tested by quantitative real-time PCR and Western blot. RESULTS: The results showed HG intervention decreased cardiomyocytes viability, increased ROS generation, up-regulated the levels of TNF-α, IL-6 and IL-1β, increased RIP1, RIP3, MLKL expressions at mRNA and protein levels. Nec-1 treatment attenuated HG-induced increased cardiomyocytes viability, reduced ROS generation, down-regulated the levels of TNF-α, IL-6 and IL-1β, decreased RIP1, RIP3, MLKL expressions at mRNA and protein levels. CONCLUSION Necroptosis was happened in high glucose-induced primary cardiomyocytes injury. Inhibition of necroptosis can reduce high glucose-induced cardiomyocytes damage, may be related to inhibition of oxidative stress and depression of inflammative factors releasing.
Apoptosis ; Cells, Cultured ; Cytokines ; metabolism ; Glucose ; adverse effects ; Humans ; Myocytes, Cardiac ; cytology ; pathology ; Necrosis ; Oxidative Stress ; Reactive Oxygen Species ; metabolism

BackgroundMicroRNA-24 (miR-24) plays an important role in heart failure by reducing the efficiency of myocardial excitation-contraction coupling. Prolonged cardiac hypertrophy may lead to heart failure, but little is known about the role of miR-24 in cardiac hypertrophy. This study aimed to preliminarily investigate the function of miR-24 and its mechanisms in cardiac hypertrophy.

MethodsTwelve Sprague-Dawley rats with a body weight of 50 ± 5 g were recruited and randomly divided into two groups: a transverse aortic constriction (TAC) group and a sham surgery group. Hypertrophy index was measured and calculated by echocardiography and hematoxylin and eosin staining. TargetScans algorithm-based prediction was used to search for the targets of miR-24, which was subsequently confirmed by a real-time polymerase chain reaction and luciferase assay. Immunofluorescence labeling was used to measure the cell surface area, and H-leucine incorporation was used to detect the synthesis of total protein in neonatal rat cardiac myocytes (NRCMs) with the overexpression of miR-24. In addition, flow cytometry was performed to observe the alteration in the cell cycle. Statistical analysis was carried out with GraphPad Prism v5.0 and SPSS 19.0. A two-sided P < 0.05 was considered as the threshold for significance.

ResultsThe expression of miR-24 was abnormally increased in TAC rat cardiac tissue (t = -2.938, P < 0.05). TargetScans algorithm-based prediction demonstrated that CDKN1B (p27, Kip1), a cell cycle regulator, was a putative target of miR-24, and was confirmed by luciferase assay. The expression of p27 was decreased in TAC rat cardiac tissue (t = 2.896, P < 0.05). The overexpression of miR-24 in NRCMs led to the decreased expression of p27 (t = 4.400, P < 0.01), and decreased G0/G1 arrest in cell cycle and cardiomyocyte hypertrophy.

ConclusionMiR-24 promotes cardiac hypertrophy partly by affecting the cell cycle through down-regulation of p27 expression.

Animals ; Cardiomegaly ; genetics ; pathology ; Cell Cycle ; genetics ; physiology ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Male ; MicroRNAs ; genetics ; Myocardium ; metabolism ; Myocytes, Cardiac ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To detect the levels of miR-1, miR-21 and their targeted proteins in hearts of mice after different exercise training, and discuss potential molecular mechanism. METHODS: Male C57BL/6 mice were randomly divided to 3 groups:sedentary (SE), exercise training 1(ET1) and exercise training 2 (ET2). SE did not do any exercise; ET1 undertook swimming training for 8 weeks, once a day, 5 days/week. Swimming 30 min in the 1 week, and the duration was increased 10 min per week to 90 min and maintained in the 7 and 8 week. ET2 performed the same work as ET1 and switched to twice a day by the end of the 5th week. TUNEL assay was applied to test myocardial apoptosis. Western blot and RT-PCR were used to detect proteins and miRs levels respectively. RESULTS: Compared with SE, in ET1, myocardial apoptosis and miR-1 level did not change, but its targeted protein Bcl-2 increased significantly(<0.01). miR-21 and its targeted protein PDCD4 did not change significantly. In ET2, myocardial apoptosis and miR-1 level were decreased significantly(<0.05). Bcl-2 was increased significantly(<0.01). miR-21 also increased significantly (<0.05), but PDCD4 did not decrease significantly. CONCLUSIONS Exercise training in ET2 other than ET1 could down-regulate myocardial apoptosis. Alterations of miR-1 and Bcl-2 may be responsible for this cardioprotection. PDCD4 is not sensitive to exercise training, it is likely that miR-21 and other targeted proteins participate in exercise-regulative apoptosis.
Animals ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; MicroRNAs ; metabolism ; Myocardium ; metabolism ; pathology ; Myocytes, Cardiac ; cytology ; metabolism ; Physical Conditioning, Animal ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA-Binding Proteins ; metabolism ; Random Allocation

Previous studies have indicated that the Ilex genus exhibits antioxidant, neuroprotective, hepatoprotective, and anti-inflammatory activities. However, the pharmacologic action and mechanisms of Ilex cornuta against cardiac diseases have not yet been explored. The present study was designed to investigate the antioxidant and cardioprotective effects of Ilex cornuta root with in vitro and in vivo models. The anti-oxidative effects of the extract of Ilex cornuta root (ICR) were measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging and MTT assays as well as immunoassay. Furthermore, a rat model of myocardial ischemia was established to investigate the cardioprotective effect of ICR in vivo. Eight compounds were isolated and identified from ICR and exhibited DPPH free-radical scavenging activities. They also could increase cell viability and inhibit morphological changes induced by HO or NaSO in H9c2 cardiomyocytes, followed by increasing the SOD activities and decreasing the MDA and ROS levels. In addition, it could suppress the apoptosis of cardiomyocytes. In the rat model of myocardial ischemia, ICR decreased myocardial infarct size and suppressed the activities of LDH and CK. Furthermore, ICR attenuated histopathological alterations of heart tissues and the MDA levels, while increasing SOD activities in serum. In conclusion, these results suggest that ICR has cardioprotective activity and could be developed as a new food supplement for the prevention of ischemic heart disease.
Animals ; Antioxidants ; metabolism ; pharmacology ; therapeutic use ; Apoptosis ; Cardiovascular Agents ; pharmacology ; therapeutic use ; Cell Survival ; drug effects ; Hydrogen Peroxide ; metabolism ; Ilex ; Malondialdehyde ; metabolism ; Myocardial Infarction ; Myocardial Ischemia ; drug therapy ; metabolism ; pathology ; Myocardium ; cytology ; pathology ; Myocytes, Cardiac ; drug effects ; Oxidative Stress ; drug effects ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Plant Roots ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism

In the present study, three new triterpenoids, 23-hydroxyurs-12, 18-dien-28-oic acid 3β-O-α-L-arabinopyranoside (1), 23-hydroxyurs-12, 18-dien-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-methyl ester (2), and urs-12, 18-dien-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-methyl ester (3), and a known triterpenoid, 3β-hydroxy-urs-2, 18-dien-28-oic acid (4, randialic acid B), were isolated from the aerial parts of Ilex cornuta. Their structures were identified by the spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, and 1D and 2D NMR) and chemical reactions. Compound 4 showed significant cell-protective effects against HO-induced H9c2 cardiomyocyte injury. Compounds 1-4 did not show any significant DPPH radical scavenging activity.
Animals ; Biphenyl Compounds ; metabolism ; Cardiovascular Agents ; chemistry ; isolation & purification ; pharmacology ; Hydrogen Peroxide ; metabolism ; Ilex ; chemistry ; Molecular Structure ; Myocardium ; cytology ; pathology ; Myocytes, Cardiac ; drug effects ; Picrates ; metabolism ; Plant Components, Aerial ; chemistry ; Plant Extracts ; chemistry ; pharmacology ; Rats ; Triterpenes ; chemistry ; isolation & purification ; pharmacology

Objective To study the effect of endometrial stem cells (EnSCs) derived cytokine cocktail (EdCC) on myocardial ischemic reperfusion injury (I/R) in a mouse model. Methods EdCC was concentrated from the culture medium of EnSCs with Millipore ultra-filtration technology and was administrated to a myocardial I/R mouse models through tail vein injection. The infarct area was determined by TTC/Evans Blue staining. The apoptotic cells were counted by TUNEL assay and the protein level of cleaved caspase 3 was evaluated by Western blotting. Results The EdCC extraction efficiency was (222.4±29.3) Μg/10(6) cells in every 24 h,but the protein gradually degraded under-80 ℃ storage. As compared with I/R group,100 Μg fresh EdCC decreased infarct area (P=0.001),reduced apoptotic nuclei in the infarct border (P=0.019),and inhibited cleaved caspase 3 expression (P=0.002). Increasing EdCC dosage did not further reduce the infarct area. The myocardial protective effect of EdCC diminished after 90 days' storage under-80 ℃. Conclusion EdCC reduces myocardial I/R injury through protecting cardiomyocytes from apoptosis within 30 days storage under-80 ℃.
Animals ; Apoptosis ; Caspase 3 ; metabolism ; Cells, Cultured ; Cytokines ; therapeutic use ; Disease Models, Animal ; Endometrium ; cytology ; Female ; Mice ; Myocardial Reperfusion Injury ; therapy ; Myocytes, Cardiac ; drug effects ; Stem Cells ; cytology

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism