[Expression of MiRNA-19a in Cells of Multiple Myeloma Patients and Its Effect on the Characteristics of Myeloma Cells].

OBJECTIVE: To detect the expression level of miR-19a, one of the oncogenic miR-17-92 cluster, in multiple myeloma cell lines Lp-1 and U266 in vitro and to explore the effects of miR-19a on biological behavior, such as proliferation, migration and apoptosis of Lp-1 and U266 myeloma cells by transfection with miR-19a mimic through LipofectamineTM2000. METHODS: The reverse transcription-PCR was applied to detect the expression level of miR-19a in multiple myeloma cell lines Lp-1 and U266 in vitro. The CCK8 was used to assay the effect of miR-19a on the proliferation of Lp-1 and U266 cells in vitro, the transwell migration test was adopted to determine the effect of up-regulation of miR-19a on the migration of Lp-1 and U266 multiple myeloma cells in vitro. The flow cytometry was used to detect the effect of miR-19a on the apoptosis of Lp-1 and U266 cells in vitro. RESULTS: The miR-19a expression was higher in Lp-1 and U266 multiple myeloma cells; compared with the transfected cells with a specific miR-19a NC, those samples transfected with miR-19a mimic displayed significantly higher expression of miR-19a (P<0.05), indicating a higher transfection efficiency; the miR-19a could promote the proliferation of Lp-1 and U266 multiple myeloma cells in vitro. MiR-19a could promote migration ability of Lp-1 and U266 multiple myeloma cell lines in vitro and could inhibit the apoptosis of Lp-1 and U266 cells. CONCLUSION: miR-19a is overexpressed significantly in Lp-1 and U266 multiple myeloma cells, and promots the proliferation and invasion of the myeloma cells, but inhibits their apoptosis.

Simulated microgravity hampers Notch signaling in the fight against myocardial ischemia­reperfusion injury.

The gravitational field is an important determinant of cardiovascular function. Exposure to microgravity during spaceflight may lead to a series of maladaptive alterations in the cardiovascular system. The authors have previously demonstrated that microgravity can increase the susceptibility to myocardial ischemia­reperfusion (IR) injury under simulated microgravity. Although Notch1 signaling protects against myocardial IR injury, whether Notch1 protects against myocardial IR injury under simulated weightlessness remains unknown. The present study is designed to investigate the role of the Notch1 receptor in myocardial IR injury under simulated weightlessness. The differences in Notch signaling expression and myocardial infarct size following myocardial IR were compared between normal rats and tail­suspended rats that were kept in 30˚ head­down tilt and hindlimb unloading position. The data revealed low expression levels of Notch1 receptor and its endogenous ligand Jagged1 in normal adult rat hearts. However, significantly higher expression of Notch1 was observed in the border zone compared with the infarcted area and the remote zone following myocardial IR. Notch1 expression was notably reduced in the infarcted hearts of tail­suspended rats compared with the control group. Conversely, the myocardial infarct size was significantly increased in tail­suspended rats compared with the control rats. In conclusion, these data suggested that the proper function of Notch signaling may be hampered under simulated microgravity.

[L-VDCC autoregulation abnormality contributes to calcium overload in myocardial ischemia-reperfusion injury].

Calcium overload is a vital mechanism of myocardial ischemia-reperfusion injury, which is a hot therapeutic target in cardiovascular research. It has been well recognized that the dysfunction of calcium relevant proteins, including L-type voltage- dependent calcium channel (L-VDCC), sarco/endoplasmic reticulum ATPase 2a (SERCA2a)/phospholamban (PLB), RyR2, Na+/Ca2+ exchanger, Na+/H+ exchanger, etc. contributes to calcium overload in cardiomyocytes during ischemia-reperfusion injury, in which the diastolic calcium concentration is increased and the amplitude of calcium transients is decreased. There are two phases in calcium increase. The early phase is partially mediated by calcium channels, and the latter one is mainly mediated by Na+/Ca2+ exchanger. L-VDCC, a main subtype of calcium channels in myocardium, is involved in calcium overload, but the underlying molecular mechanism is not well elucidated yet. L-VDCC is regulated by intrinsic and extrinsic pathways. PKG and PKA as extrinsic regulators are not proper candidates to increase L-VDCC activity of cardiomyocyte in vitro, whereas the myocardial ischemia-reperfusion injury is highly possible to enhance L-VDCC activity by delaying calcium-dependent inactivation (CDI), advancing calcium-dependent facilitation (CDF), and weakening distal carboxy terminus (DCT) inhibition. Therefore, it is rational to propose that the L-VDCC autoregulation abnormality may play an important role in calcium overload during myocardial ischemia-reperfusion injury.

Nitric Oxide Protects L-Type Calcium Channel of Cardiomyocyte during Long-Term Isoproterenol Stimulation in Tail-Suspended Rats.

The aim of this study was to investigate the effects of nitric oxide (NO) and reactive oxygen species (ROS) on L-type calcium channel (LTCC) gating properties of cardiomyocytes during long-term isoproterenol (ISO) stimulation. Expression and activity of nNOS as well as S-nitrosylation of LTCC α1C subunit significantly decreased in the myocardium of SUS rats. Long-term ISO stimulation increased ROS in cardiomyocytes of SUS rats. ISO-enhanced calcium current (I Ca,L) in the SUS group was less than that in the CON group. The maximal I Ca,L decreased to about 80% or 60% of initial value at the 50th minute of ISO treatment in CON or SUS group, respectively. Specific inhibitor NAAN of nNOS reduced maximal I Ca,L to 50% of initial value in the CON group; in contrast, NO donor SNAP maintained maximal I Ca,L in SUS group to similar extent of CON group after 50 min of ISO treatment. Long-term ISO stimulation also changed steady-state activation (P < 0.01), inactivation (P < 0.01), and recovery (P < 0.05) characteristics of LTCC in SUS group. In conclusion, NO-induced S-nitrosylation of LTCC α1C subunit may competitively prevent oxidation from ROS at the same sites. Furthermore, LTCC can be protected by NO during long-term ISO stimulation.

ROS-Induced Nuclear Translocation of Calpain-2 Facilitates Cardiomyocyte Apoptosis in Tail-Suspended Rats.

Isoproterenol (ISO) induced nuclear translocation of calpain-2 which further increased susceptibility of cardiomyocyte apoptosis in tail-suspended rats. The underlying mechanisms remain elusive. In the present study, the results showed that ISO (10 nM) significantly elevated NADPH oxidases (NOXs) activity and NOXs-derived ROS productions which induced nuclear translocation of calpain-2 in cardiomyocytes of tail-suspended rats. In contrast, the inhibition of NADPH oxidase or cleavage of ROS not only reduced ROS productions, but also resisted nuclear translocation of calpain-2 and decreased ISO-induced apoptosis of cardiomyocyte in tail-suspended rats. ISO also increased the constitutive binding between calpain-2 and Ca(2+)/calmodulin-dependent protein kinase II δB (CaMK II δB) in nuclei, concomitant with the promotion of CaMK II δB degradation and subsequent down-regulation of Bcl-2 mRNA expression and the ratio of Bcl-2 to Bax protein in tail-suspended rat cardiomyocytes. These effects of ISO on cardiomyocytes were abolished by a calpain inhibitor PD150606. Inhibition of calpain significantly reduced ISO-induced loss of the mitochondrial membrane potential, cytochrome c release into the cytoplasm, as well as the activation of caspase-3 and caspase-9 in mitochondrial apoptotic pathway. In summary, the above results suggest that ISO increased NOXs-derived ROS which activated nuclear translocation of calpain-2, subsequently nuclear calpain-2 degraded CaMK II δB which reduced the ratio of Bcl-2 to Bax, and finally the mitochondria apoptosis pathway was triggered in tail-suspended rat cardiomyocytes. Therefore, calpain-2 may represent a potentially therapeutic target for prevention of oxidative stress-associated cardiomyocyte apoptosis.

[In vitro heart models simulating in vivo cardiac ischemia-reperfusion injury].

The aim of this study was to compare in vivo and several in vitro cardiac ischemia-reperfusion (I-R) myocardial injury models, and choose a superior in vitro cardiac I-R model. Sprague-Dawley (SD) rats were randomly grouped into in vivo, Langendorff, Langendorff + pacing, and working heart groups. Left anterior descending (LAD) coronary artery was ligated for 60 min and then reperfused for 120 min in in vivo and in vitro rat hearts. Cardiac function and myocardial infarct size were measured by using pressure transducer and TTC/Evans blue double staining, respectively. The results showed that heart rate was greater in in vivo model than those in the three in vitro models. Coronary flows were dropped after LAD ligation and could recover at early phase of releasing LAD ligation in I-R models of the isolated working heart, Langendorff and Langendorff with 300 beats/min of electrical stimulation. Left ventricular end-systolic pressure (LVESP) decreased during ischemia, and partially restored during reperfusion in the three in vitro models. Left ventricular end-diastolic pressure (LVEDP) increased during ischemia in the three in vitro models. LVEDP was significantly higher in the isolated working heart than those in Langendorff models during ischemia, whereafter decreased slowly during reperfusion. LVEDP elevated further in the initiation of reperfusion period and then decreased, but did not recover to normal levels during reperfusion in Langendorff and Langendorff + pacing groups. Left ventricular myocardial infarct size was (60.4 ± 5.4)% in in vivo I-R model, which was significantly higher than that in Langendorff model and the isolated working heart. Notably, there was no significant difference in myocardial infarct size between in vivo model and Langendorff model with electrical stimulation. These results suggest that Langendorff I-R model with 300 beats/min of electrical stimulation can simulate the in vivo I-R myocardial injury.

[Metabolites of long-time preserved-acutely isolated rat cardiomyocytes affect L-type Ca(2+) channel current].

The variability of peak current of L-type calcium channel (I(Ca,L)) shows an increase in cardiomyocytes after 6 h of preservation when the acutely isolated cardiomyocytes are preserved in a small volume buffer solution. The mechanism of the increased variability of I(Ca,L) is not clear. In order to obtain more accurately and stably experimental data of I(Ca,L), the aim of this study was to observe the pH changes of preservation buffer solution with acutely isolated rat cardiomyocytes, and the effects of pH changes on the shape of cardiomyocytes, the function of mitochondria and the gating property of L-type calcium channel. The results indicated that the pH was kept stable in 100 mL buffer solution, but was decreased from 7.20 to 6.95 in 20 mL buffer solution during 10 h of cardiomyocyte preservation. Therefore, 100 mL or 20 mL preservation solution was used as a normal control or acidotic group, respectively. The ratio of abnormal to normal rod-shaped cardiomyocytes increased in the acidotic group after 6 h of preservation. The acidosis induced a reduction in mitochondrial membrane potential indicated by JC-1 fluorescent probe after 8 h of cardiomyocyte preservation. The acidosis also shifted the autofluorescence of NADPH from blue to green after 8 h of cardiomyocyte preservation. The above changes in mitochondrial function induced a significant decrease in the peak I(Ca,L) and a shift in the clamped voltage at peak I(Ca,L) from +10 mV to 0 mV, after 10 h of cardiomyocyte preservation. These results suggest that the best way to preserve acutely isolated cardiomyocytes is to use a larger volume buffer system. In order to get stable peak I(Ca,L), we need to not only select a normal shape of cardiomyocyte at a bright field but also a blue fluorescent myocyte at an ultraviolet excitation.

[Cardioprotection by the inhibitory effect of nitric oxide].

Endothelial and neuronal nitric oxide synthases (eNOS and nNOS) are constitutively expressed in cardiomyocytes under the physiological condition, while inducible nitric oxide synthase (iNOS) is only expressed in cell stress. Nitric oxide (NO) derived from the constitutive isoforms of eNOS and nNOS plays four kinds of inhibitory effects on the myocardium: reducing the contractile frequency of cardiomyocyte, slightly attenuating cardiac contractility, accelerating relaxation and increasing distensibility of cardiomyocyte, and slightly inhibiting mitochondrial respiration and improving the efficiency of myocardial oxygen consumption. In conditions of enhanced cardiac reserve and cardiac hypertrophy, NO derived from eNOS, which forms a complex with a certain kind of receptor on the sarcolemma, modulates receptor-mediated signaling and generates an "accentuated antagonism" by moderate inhibition of cardiac contractility. NO derived from the complex of nNOS-ryanodine receptor (RyR) stabilizes RyR calcium release and increases the efficiency of Ca(2+) cycling in sarcoplasmic reticulum by the inhibitory effects. However, besides the above-mentioned inhibitions of NO derived from eNOS and nNOS, NO derived from iNOS generally prevents mitochondrial permeability transition pore opening by inhibiting mitochondrial respiration under the conditions of the myocardial ischemia-reperfusion injury and heart failure. Therefore, both in the physiological condition and in the pathological condition, NO exhibits a moderate inhibition in cardiac function, and eventually produces cardioprotection.