Fasting improves tolerance to acute hypoxia in rats.

Acute high-altitude illness seriously threatens the health and lives of people who rapidly ascend to high altitudes, but there is currently no particularly effective method for the prevention or treatment of acute high-altitude illness. In the present study, we found that fasting preconditioning effectively improved the survival rate of rats exposed to a simulated altitude of 7620 m for 24 h, and a novel animal model of rapid adaptation to acute hypoxia was established. Compared with control treatment, fasting preconditioning activated AMPK, induced autophagy, decreased ROS levels, and inhibited NF-κB signaling in the cardiac tissues of rats. Our results suggested that fasting effectively improved the acute hypoxia tolerance of rats, which was gradually enhanced with prolongation of fasting. In addition, the acute hypoxia tolerance of young rats was significantly higher than that of adult rats. These experimental results lay the foundation for achieving rapid adaptation to acute hypoxia in humans.

Elevated ROS depress mitochondrial oxygen utilization efficiency in cardiomyocytes during acute hypoxia.

Mitochondria are important sites for the production of ATP and the generation of ROS in cells. However, whether acute hypoxia increases ROS generation in cells or affects ATP production remains unclear, and therefore, monitoring the changes in ATP and ROS in living cells in real time is important. In this study, cardiomyocytes were transfected with RoGFP for ROS detection and MitGO-Ateam2 for ATP detection, whereby ROS and ATP production in cardiomyocytes were respectively monitored in real time. Furthermore, the oxygen consumption rate (OCR) of cardiomyocytes was measured. Similar results were produced for adult and neonatal rat cardiomyocytes. Hypoxia (1% O2) reduced the basal OCR, ATP-linked OCR, and maximal OCR in cardiomyocytes compared with these OCR levels in the cardiomyocytes in the normoxic group (21% O2). However, ATP-linked OCR, normalized to maximal OCR, was increased during hypoxia, indicating that the electron leakage of complex III exacerbated the increase of ATP-linked oxygen consumption during hypoxia and vice versa. Combined with the result that cardiomyocytes expressing MitGO-Ateam2 showed a significant decrease in ATP production during hypoxia compared with that of normoxic group, acute hypoxia might depress the mitochondrial oxygen utilization efficiency of the cardiomyocytes. Moreover, cardiomyocytes expressing Cyto-RoGFP or IMS-RoGFP showed an increase in ROS generation in the cytosol and the mitochondrial intermembrane space (IMS) during hypoxia. All of these results indicate that acute hypoxia generated more ROS in complex III and increased mitochondrial oxygen consumption, leading to less ATP production. In conclusion, acute hypoxia depresses the mitochondrial oxygen utilization efficiency by decreasing ATP production and increasing oxygen consumption as a result of the enhanced ROS generation at mitochondrial complex III.

Comparison of hypoxic effects induced by chemical and physical hypoxia on cardiomyocytes.

The degree and duration of chemical hypoxia induced by sodium dithionite (Na2S2O4) have not been reported. It is not yet clear how much reduction in the O2 concentration (physical hypoxia) can lead to hypoxia in cultured cardiomyocytes. In this study, oxygen microelectrodes were used to measure changes in the O2 concentration in media containing different concentrations of Na2S2O4. Then, hypoxic effects of 0.8, 1.0, and 2.0 mM Na2S2O4 or 1%, 3%, and 5% O2 in cultured cardiomyocytes from neonatal rats were observed and compared. The results showed that the O2 concentration failed to remain constant by Na2S2O4 treatment during the 180-minute observation period. Only the 2.0 mM Na2S2O4 group significantly increased the expression of hypoxia-inducible factor 1α (HIF-1α) and hypoxic responses. Notably, 3% O2 only significantly increased the expression of HIF-1α in cardiomyocytes, while 1% O2 not only increased the expression of HIF-1α but also increased the apoptotic rate in cardiomyocytes. These results suggest that Na2S2O4 is not suitable for establishing a hypoxic model in cultured neonatal rat cardiomyocytes, and neonatal rat cardiomyocytes cultured at or below 1% O2 induced significant hypoxic effects, which can be used as a starting O2 concentration for establishing a hypoxic cell model.

Glycosylated CD147 reduces myocardial collagen cross-linking in cardiac hypertrophy.

The mechanism of transition from chronic pressure overload-induced cardiac hypertrophy to heart failure is still unclear. Angiotensin II (Ang II) may be an important factor that mediates the transition in the end-stage of cardiac hypertrophy. In the present study, Goldblatt two-kidney one-clip (2K1C) rat model was used to simulate Ang II-induced hypertension. The elevated Ang II not only induced the concentric hypertrophy of left ventricle and cardiac fibrosis, but also increased the expression and glycosylation of CD147 in 2K1C rats. The left ventricular structure and function detected by echocardiogram showed a sign of the transition from cardiac hypertrophy to heart failure in 16 weeks of 2K1C rats. Ang II can activate N-acetylglucosamine transferase V (GnT-V), a key enzyme for CD147 glycosylation. Retinoic acid, an agonist of GnT-V, further increased glycosylated CD147, and activated matrix metalloproteinase-2/-9 (MMP-2 and MMP-9) in the hypertrophied left ventricle of 2K1C rat. Meanwhile, collagen cross-linking in the hypertrophied left ventricle significantly reduced in 2K1C rats. On the contrary, tunicamycin, an inhibitor of N-glycan biosynthesis, inhibited glycosylation of CD147 and activity of MMP-2 and MMP-9, and then maintained a stable of collagen cross-linking in the 2K1C rat hearts. The above results suggested that Ang II increased glycosylated CD147 which activated MMP-2 and MMP-9. Collagens were degraded by the activated MMPs and then reduced collagen cross-linking. Finally, the hypertrophied left ventricle was progressively dilated in chronic pressure overload due to losing the limitation of collagen cross-linking. Therefore, the compensated hypertrophy of left ventricle gradually transited to congestive heart failure.

Simulated microgravity increases myocardial susceptibility to ischemia-reperfusion injury via a deficiency of AMP-activated protein kinase.

Gravitation is an important factor in maintaining cardiac contractility. Our study investigated whether simulated microgravity increases myocardial susceptibility to ischemia-reperfusion (IR) injury. Using the Langendorff-perfused heart model with 300 beats/min pacing, 4-week tail suspension (SUS) and control (CON) male Sprague-Dawley rats (n = 10 rats/group) were subjected to 60 min of left anterior descending coronary artery (LAD) occlusion followed by 120 min of reperfusion. Left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), creatine kinase (CK) and lactate dehydrogenase (LDH) activity, and infarct size were assessed. Data demonstrated that there were significantly increased LVEDP, CK, LDH, and infarct size in SUS compared with CON (P < 0.05), accompanied by decreased LVESP (P < 0.05). Furthermore, TUNEL-positive cardiomyocytes were higher in SUS than that in CON (P < 0.01), and AMP-activated protein kinase (AMPK) phosphorylation and Bcl-2/Bax in SUS were less compared with CON (P < 0.05). Similarly, isolated hearts pre-treated with A-769662 exhibited better recovery of cardiac function, increased AMPK phosphorylation, and reduced necrosis and apoptosis. Furthermore, AMPKα protein showed a significant suppression in 4-week hindlimb unweighting rats. These results suggest that AMPK deficiency increases myocardial susceptibility to IR injury in rats subjected to simulated microgravity.

Nuclear Translocation of Calpain-2 Mediates Apoptosis of Hypertrophied Cardiomyocytes in Transverse Aortic Constriction Rat.

Apoptosis of cardiomyocytes plays an important role in the transition from cardiac hypertrophy to heart failure. Hypertrophied cardiomyocytes show enhanced susceptibility to apoptosis. Therefore, the aim of this study was to determine the susceptibility to apoptosis and its mechanism in hypertrophied cardiomyocytes using a rat model of transverse abdominal aortic constriction (TAC). Sixteen weeks of TAC showed compensatory and pathological hypertrophy in the left ventricle. TUNEL-positive nuclei were significantly increased in TAC with angiotensin II (Ang II) treatment. Calpain inhibitor, PD150606, effectively inhibited Ang II-induced apoptosis of hypertrophied cardiomyocytes. Ang II increased nuclear translocation of intracellular Ca(2+) activated calpain-2 in hypertrophied cardiomyocytes. Ang II enhanced the interaction between activated calpain-2 and Ca(2+)/calmodulin-dependent protein kinase II δB (CaMKIIδB), and promoted the degradation of CaMKIIδB by calpain-2 in the nuclei of hypertrophied cardiomyocytes. Consequently, the depressed CaMKIIδB downregulated the expression of antiapoptotic Bcl-2 leading to mitochondrial depolarization and release of cytochrome c led to apoptosis of hypertrophied cardiomyocytes. In conclusion, hypertrophied cardiomyocytes show increased susceptibility to apoptosis during Ang II stimulation via nuclear calpain-2 and CaMKIIδB pathway.

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.

Thymosin alpha 1 suppresses proliferation and induces apoptosis in breast cancer cells through PTEN-mediated inhibition of PI3K/Akt/mTOR signaling pathway.

Thymosin alpha 1 (Tα1), an immunoactive peptide, has been shown to inhibit cell proliferation and induce apoptosis in human leukemia, non-small cell lung cancer, melanoma, and other human cancers. However, the response and molecular mechanism of breast cancer cells exposed to Tα1 remain unclear. PTEN, a tumor suppressor gene, is frequently mutated in a variety of human cancers. In the present study, we aimed to investigate the biological roles of PTEN in the growth inhibition of human breast cancer cells exposed to Tα1. Using wild-type and mutant PTEN-expressing cells, we found a strong correlation between PTEN status and Tα1-mediated growth inhibition of breast cancer cells. The growth inhibition effect was more pronounced in breast cancer cells in which Tα1 enhanced PTEN expression, whereas endogenous PTEN knockdown reversed the growth inhibition effect of Tα1 in breast cancer cells. Further investigation revealed that PTEN up-regulation, which was induced by Tα1, can inhibit the activation of the PI3K/Akt/mTOR signaling pathway, leading to the growth inhibition of breast cancer cells. The addition of the synergy between Tα1 and the inhibition of PI3K/Akt/mTOR activation could strongly block cell viability in PTEN down-regulated breast cancer cells. PTEN-overexpressing cells not only up-regulated Bax and cleaved caspase-3/9 and PARP expression but also down-regulated Bcl-2 compared to the treatment with Tα1 alone. Together these findings suggest that PTEN mediates Tα1-induced apoptosis through the mitochondrial death cascade and inhibition of the PI3K/Akt/mTOR signaling pathway in breast cancer cells.

Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-κB essential modulator.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to the swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that infection with the highly pathogenic PRRSV strain JXwn06 antagonized type I IFN expression induced by poly(I·C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(I·C)-induced IFN-ß production by PRRSV was dependent on the blocking of NF-κB signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFN-ß expression. Moreover, we verified that nsp4 inhibited NF-κB activation induced by signaling molecules, including RIG-I, VISA, TRIF, and IKKß. nsp4 was shown to target the NF-κB essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFN-ß production. These findings might have implications for our understanding of PRRSV pathogenesis and its mechanisms for evading the host immune response. IMPORTANCE: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs. Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of the NF-κB signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism used by nsp4 to suppress NF-κB-mediated IFN-ß production. We found that nsp4 interfered with the NF-κB signaling pathway through the cleavage of NEMO (a key regulator of NF-κB signaling) at the E349-S350 site, leading to the downregulation of IFN-ß production induced by poly(I·C). The data presented here may help us to better understand PRRSV pathogenesis.

Multisite phosphorylation of Bcl-2 via protein kinase Cδ facilitates apoptosis of hypertrophic cardiomyocytes.

Activated protein kinase Cδ (PKCδ) associated with cardiac hypertrophy moves from the cytoplasm to the mitochondria and subsequently triggers the apoptotic signalling pathway. The underlying mechanisms remain unknown. The aim of the present study was to investigate whether mitochondrial translocation of PKCδ phosphorylates multiple sites of Bcl-2, resulting in an imbalance between Bcl-2 and Bak or Bax, thus enhancing the susceptibility of hypertrophic cardiomyocytes to angiotensin II (AngII)-induced apoptosis. Chronic pressure overload was induced by transverse aortic constriction (TAC) in rats. The apoptotic rate increased in hypertrophied cardiomyocytes. In AngII-treated hearts (10 nmol/L, 60 min), there was an increase in the number of TERMINAL deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL)-positive cells; PKCδ inhibition with 500 nmol/L δV1-1 for 60 min prevented the AngII-induced increase in apoptosis. In the hypertrophied myocardium, PKCδ expression increased, whereas that of Bcl-2 decreased compared with the synchronous control. Treatment of hearts with 10 nmol/L AngII for 60 min activated PKCδ and induced translocation of PKCδ to the mitochondria, where activated PKCδ facilitated the phosphorylation of Bcl-2 at serine-87 and serine-70 sites. The multisite phosphorylated Bcl-2 was released from the mitochondria, and exhibited reduced affinity for Bak and Bax. The imbalance between Bcl-2 and Bak/Bax induced the release of mitochondrial cytochrome c and then activated the caspase 3 apoptotic pathway during AngII stimulation (10 nmol/L, 60 min) of hypertrophied cardiomyocytes. Inhibition of PKCδ reduced these effects of AngII. The results suggest that PKCδ can counteract the anti-apoptotic effect of Bcl-2 and may promote cardiomyocyte apoptosis through multisite phosphorylation of Bcl-2 in hypertrophied cardiomyocytes.

[Dynamic assembly of intercalated disc during postnatal development in the rat myocardium].

The intercalated disc (ICD) complex of cardiomyocyte consists of fascia adherens, desmosomes and gap junctions which are mainly constructed by their transmembrane proteins: N-cadherin (N-cad), desmoglein-2 (DSG2) and connexin 43 (Cx43), respectively. The aim of this study was to observe the dynamic changes in colocalization of N-cad, DSG2 and Cx43 with each other in the rat left ventricular myocardium at 1, 7, 14, 28 and 90 day(s) after birth (P1, P7, P14, P28 and P90) using immunofluorescent staining. The results showed that, N-cad, DSG2 and Cx43 located all around the plasma membrane at the P1. These proteins accumulated to the long ends of cardiomyocytes, indicating preliminary formation of the ICD at the P7. The localization of three proteins at the ICD increased progressively, but their lateral localization showed an inverse trend from the P14 to P90. However, Cx43 still kept a certain amount of lateral localization in cardiomyocytes even at the P90 as compared with N-cad and DSG2. Quantitative colocalization of proteins was analyzed by the stereological method. Total percentage of colocalization of N-cad with DSG2 was 33.5% at the P1, and increased to 38.6% at the P7, 9.4% in ICD and 29.2% in lateral side. The total percentage of colocalization of N-cad with DSG2 increased to 65.7% at the P90, ICD colocalization increasing to 60.5% and lateral colocalization decreasing to 5.2%. Total percentage of colocalization of N-cad with Cx43 increased from 10.3% at the P1 to 37.1% at the P90, and only ICD colocalization increased, but lateral colocalization kept about 5%. The colocalization pattern of DSG2 with Cx43 was similar to that of N-cad with Cx43. Total percentage of colocalization of N-cad with DSG2 was higher than those of N-cad or DSG2 with Cx43. The above results suggest that the formation of mechanical junctions at the ICD of cardiomyocyte is prior to that of electrochemistry junctions during postnatal development. In other words, cardiomyocyte growth needs a stable mechanical environment at first.

[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.

[Autophagic flux of cardiomyocytes from 20-week transverse abdominal aortic constriction rats].

Cardiac autophagy dramatically increases in heart failure induced by sustained pressure overload. However, it has not yet been addressed if enhanced autophagy plays a role in protecting myocardium or mediating progression from compensative hypertrophy to heart failure. The aim of the present study was to detect autophagic flux of cardiomyocytes from 20-week transverse abdominal aortic constriction (TAC) rats. Fasting rats were used as the positive control for detecting cardiac autophagy. Echocardiography was applied to find the changes of cardiac structure and function. Immunofluorescent histochemistry and Western blot were used to analyze the related biomolecular indexes reflecting cardiac autophagic flux. After the previous methods for detecting cardiac autophagy were confirmed, the autophagic flux in cardiomyocytes of rats subjected to 20-week TAC was examined. The results showed that fasting had no obvious influence on parameters of cardiac structure in rats, including interventricular septal wall thickness and left ventricle posterior wall thickness, but heart rate, diastolic left ventricle internal dimension, fractional shortening of left ventricle dimension, ejection fraction and mitral inflow velocity decreased in rats after fasting for 3 d. Meanwhile, positively stained particles of LC3 and cathepsin D, but not ubiquitin and complement 9, distributed within cardiomyocytes of 3-day fasting rats, indicating augmented autophagic flux. Compared with sham rats, 20-week TAC rats did not show any changes of LC3, cathepsin D, ubiquitin and complement 9 in myocardium detected by immunofluorescent histochemistry. In addition, protein levels of LC3, cathepsin D and p62 in myocardium of TAC rats did not changed. These results reveal the unchanged autophagic flux in cardiomyocytes at middle or late phase of cardiac hypertrophy in TAC rats, implying a balance between inhibition of hypertrophy and activation of pressure load stress on autophagy.

[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.

[Four-week simulated weightlessness increases the expression of atrial natriuretic peptide in the myocardium].

One of the major circulatory changes that occur in human during space flight and simulated weightlessness is a cerebral redistribution of body fluids, which is accompanied by an increase of blood volume in the upper body. Therefore, atrial myocardium should increase the secretion of atrial natriuretic peptide (ANP), but the researches lack common conclusion until now. The present study was to investigate the expression level of ANP in simulated weightlessness rats, and to confirm the changes of ANP by observing the associated proteins of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The tail-suspended rat model was used to simulate weightlessness. Western blots were carried out to examine the expression levels of ANP and SNARE proteins in atrial and left ventricular myocardium. The results showed that ANP expression in atrial myocardium showed an increase in 4-week tail-suspended rats (SUS) compared with that in the synchronous control rats (CON). We only detected a trace amount of ANP in the left ventricular myocardium of the CON, but found an enhanced expression of ANP in left ventricular myocardium of the SUS. Expression of VAMP-1/2 (vesicle associated SNARE) increased significantly in both atrial and left ventricular myocardium in the SUS compared with that in the CON. There was no difference of the expression of syntaxin-4 (target compartment associated SNARE) between the CON and SUS, but the expression of SNAP-23 showed an increase in atrial myocardium of the SUS compared with that in the CON. Synip and Munc-18c as regulators of SNAREs did not show significant difference between the CON and SUS. These results suggest that the expression of ANP shows an increase in atrial and left ventricular myocardium of 4-week tail-suspended rats. Enhanced expression of VAMP-1/2 associated with ANP vesicles confirms the increased expression of ANP in atrial and left ventricular myocardium.

Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling.

Our previous in vivo and ex vivo studies suggested that coexistence of two or more troponin T (TnT) isoforms in adult cardiac muscle decreased cardiac function and efficiency (Huang QQ, Feng HZ, Liu J, Du J, Stull LB, Moravec CS, Huang X, Jin JP, Am J Physiol Cell Physiol 294: C213-C22, 2008; Feng HZ, Jin JP, Am J Physiol Heart Circ Physiol 299: H97-H105, 2010). Here we characterized Ca(2+)-regulated contractility of isolated adult cardiomyocytes from transgenic mice coexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT. Without the influence of extracellular matrix, coexistence of the two TnT isoforms resulted in lower shortening amplitude, slower shortening and relengthening velocities, and longer relengthening time. The level of resting cytosolic Ca(2+) was unchanged, but the peak Ca(2+) transient was lowered and the durations of Ca(2+) rising and decaying were longer in the transgenic mouse cardiomyocytes vs. the wild-type controls. Isoproterenol treatment diminished the differences in shortening amplitude and shortening and relengthening velocities, whereas the prolonged durations of relengthening and Ca(2+) transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca(2+), resting sarcomere length of the transgenic cardiomyocytes became shorter than that in wild-type cells. Inhibition of myosin motor diminished this effect of TnT function on cross bridges. The length but not width of transgenic cardiomyocytes was significantly increased compared with the wild-type controls, corresponding to longitudinal addition of sarcomeres and dilatative remodeling at the cellular level. These dominantly negative effects of normal fast TnT demonstrated that chronic coexistence of functionally distinct variants of TnT in adult cardiomyocytes reduces contractile performance with pathological consequences.

Passive stretch reduces calpain activity through nitric oxide pathway in unloaded soleus muscles.

Unloading in spaceflight or long-term bed rest induces to pronounced atrophy of anti-gravity skeletal muscles. Passive stretch partially resists unloading-induced atrophy of skeletal muscle, but the mechanism remains elusive. The aims of this study were to investigate the hypotheses that stretch tension might increase protein level of neuronal nitric oxide synthase (nNOS) in unloaded skeletal muscle, and then nNOS-derived NO alleviated atrophy of skeletal muscle by inhibiting calpain activity. The tail-suspended rats were used to unload rat hindlimbs for 2 weeks, at the same time, left soleus muscle was stretched by applying a plaster cast to fix the ankle at 35° dorsiflexion. Stretch partially resisted atrophy and inhibited the decreased protein level and activity of nNOS in unloaded soleus muscles. Unloading increased frequency of calcium sparks and elevated intracellular resting and caffeine-induced Ca(2+) concentration ([Ca(2+)]i) in unloaded soleus muscle fibers. Stretch reduced frequency of calcium sparks and restored intracellular resting and caffeine-induced Ca(2+) concentration to control levels in unloaded soleus muscle fibers. The increased protein level and activity of calpain as well as the higher degradation of desmin induced by unloading were inhibited by stretch in soleus muscles. In conclusion, these results suggest that stretch can preserve the stability of sarcoplasmic reticulum Ca(2+) release channels which prevents the elevated [Ca(2+)]i by means of keeping nNOS activity, and then the enhanced protein level and activity of calpain return to control levels in unloaded soleus muscles. Therefore, stretch can resist in part atrophy of unloaded soleus muscles.

Analysis of international competitiveness of the China's licorice industry from the perspective of global trade.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a multi-purpose plant raw material, which is widely used in the pharmaceutical industry and food industry, cosmetic industry, etc. It has a wide application in various countries and regions around the world. AIM OF STUDY: This paper studied the trade situation of licorice-related products among major countries and regions in the world, providing a practical reference for the sustainable development of the global licorice industry. MATERIALS AND METHODS: The licorice trade data of licorice-related products came from the United Nations Commodity Trade Database and China Customs data. We analyzed the world's major trading countries by using international market share (IMS), trade competitiveness index (TC), average export price (AEP) and average import price (AIP), and analyzed global trade flows with chord diameter. RESULTS: Uzbekistan, Kazakhstan and Iran mainly export licorice raw materials and low value-added products. China is both a producer and a consumer of licorice raw materials and licorice products. The processing trade of the licorice industry in China has advantages, and the structure of import and export trade has been continuously improved. The United States, France, Germany and other developed countries are still important consumers who rely on the intellectual property rights and brand advantages of licorice products, which have stronger global trade radiation capacity. CONCLUSIONS: China's trade structure has been optimized and its industrial competitiveness has been enhanced. China's experience can be used for reference by other countries, especially those with rich licorice resources among the SCO members.

High glucose alters apoptosis and proliferation in HEK293 cells by inhibition of cloned BK Ca channel.

It has been reported that diabetic vascular dysfunction is associated with impaired function of large conductance Ca(2+) -activated K(+) (BK(Ca) ) channels. However, it is unclear whether impaired BK(Ca) channel directly participates in regulating diabetic vascular remodeling by altering cell growth in response to hyperglycemia. In the present study, we investigated the specific role of BK(Ca) channel in controlling apoptosis and proliferation under high glucose concentration (25 mM). The cDNA encoding the α+ß1 subunit of BK(Ca) channel, hSloα+ß1, was transiently transfected into human embryonic kidney 293 (HEK293) cells. Cloned BK(Ca) currents were recorded by both whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry and analysis of fragmented DNA by agarose gel electrophoresis. Cell proliferation was investigated by flow cytometry assays, MTT test, and immunocytochemistry. In addition, the expression of anti-apoptotic protein Bcl-2, intracellular Ca(2+) , and mitochondrial membrane potential (Δψm) were also examined to investigate the possible mechanisms. Our results indicate that inhibition of cloned BK(Ca) channels might be responsible for hyperglycemia-altered apoptosis and proliferation in HEK-hSloα+ß1 cells. However, activation of BK(Ca) channel by NS1619 or Tamoxifen significantly induced apoptosis and suppressed proliferation in HEK-hSloα+ß1 cells under hyperglycemia condition. When rat cerebral smooth muscle cells were cultured in hyperglycemia, similar findings were observed. Moreover, the possible mechanisms underlying the activation of BK(Ca) channel were associated with decreased expression of Bcl-2, elevation of intracellular Ca(2+) , and a concomitant depolarization of Δψm in HEK-hSloα+ß1 cells. In conclusion, cloned BK(Ca) channel directly regulated apoptosis and proliferation of HEK293 cell under hyperglycemia condition.

Nuclear translocation of calpain-2 regulates propensity toward apoptosis in cardiomyocytes of tail-suspended rats.

The compensatory increase in catecholamine release does not reverse orthostatic intolerance after returning from a long-term spaceflight, but it is unclear whether high dose of catecholamine induces cardiac damage. The tail-suspended rat model was used to simulate the effects of weightlessness on the heart. Apoptotic rates in the left ventricular myocardium did not increase in 4-week of tail-suspended rats compared with the synchronous control. On the contrary, isoproterenol (intraperitoneal injection) and 1-day recovery from the 4-week tail-suspension increased apoptotic rates in the myocardium. Propranolol and PD150606 inhibited cardiomyocyte apoptosis in the recovery group. PD150606 and calpain-2 knockdown also blocked isoproterenol-induced cardiomyocyte apoptosis in tail-suspended rats. The activity and nuclear translocation of calpain-2 increased, but the expression of calpain-1, calpain-2, and calpastatin was unchanged in the myocardium of tail-suspended rats. The Ser-16-phosphorylated phospholamban of the nuclear envelope was higher in tail-suspended rats than in the control rats under isoproterenol stimulation. Isoproterenol treatment also induced a large intranuclear Ca(2+) transient of cardiomyocytes in tail-suspended rats. These results suggest that high-dose isoproterenol phosphorylates phospholamban of the nuclear envelope and increases intranuclear Ca(2+) transient. Larger intranuclear Ca(2+) further activates nuclear calpain-2 and hence induces cardiomyocyte apoptosis.