Effects of tamoxifen on CD147 glycosylation and MMPs in the diabetic rat myocardium / 中国应用生理学杂志

<p><b>OBJECTIVE</b>Over the last few decades, diabetic cardiomyopathy has been identified as a significant contributor in cardiac morbidity. However, the mechanisms of diabetic cardiomyopathy have not been clarified.</p><p><b>METHODS</b>In the present study, a diabetic rat model was induced by the intraperitoneal injection of streptozotocin. The myocardial CD147 expression and extent of glycosylation, as well as thematrixmetalloproteinases(MMPs) expression and activity, were observed in the diabetic and synchronous rats.</p><p><b>RESULTS</b>The results showed that CD147 located on sarcolemma of cardiomyocytes. The myocardial CD147 expression and glycosylation were significantly increased in the diabetic rats as compared with the control. Expression of MMP-2 protein, MMP-2 and MMP-9 activity were also increased in left ventricular myocardium in the diabetic rats. Tamoxifen only inhibited the enhanced expression of myocardial CD147 in the diabetic rats, but not in synchronous control rats. Tamoxifen inhibited glycosylation of myocardial CD147 in both diabetic and control rats. The inhibition of tamoxifen on CD147 glycosylation was stronger than on the expression in the myocardium. The extent of myocardial CD147glycosylation was positively related toMMP-2 and MMP-9 activity. Tamoxifen induced an inhibition of myocardial MMP-2 and MMP-9 activity in the control and diabetic rats.</p><p><b>CONCLUSION</b>These results indicate that myocardial CD147 expression, especially the extent of glycosylation, regulates MMP-2 and MMP-9 activity, then accelerates cardiac pathological remodeling inducing diabetic cardiomyopathy. Tamoxifen inhibits myocardial CD147 glycosylation and further depress the activity of MMPs. Therefore, tamoxifen may protect the diabetic rats against diabetic myocardium.</p>

Dynamic assembly of intercalated disc during postnatal development in the rat myocardium / 生理学报

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 / 生理学报

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.

Four-week simulated weightlessness increases the expression of atrial natriuretic peptide in the myocardium / 生理学报

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.

Autophagic flux of cardiomyocytes from 20-week transverse abdominal aortic constriction rats / 生理学报

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.

Depressed cardiac output at higher pacing rate in isolated working heart of rat / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the regulation of heart rate to cardiac pump function in the phase of negative force-frequency relationship and their possible mechanisms.</p><p><b>METHODS</b>The left ventricular pressure, aortic pressure, and cardiac output were measured in isolated working heart of rat from 240 to 300 beats/min of pacing rate.</p><p><b>RESULTS</b>Cardiac output of isolated working heart was decreased by a proximally 20% (P < 0.01) with the increase in the pacing rate from 240 to 300 beats/min. Left ventricular end-systolic pressure (LVESP) was declined by 4.8% (P < 0.05), but left ventricular end-diastolic pressure (LVEDP) was elevated by 139% (P < 0.01) with an increase in the pacing rate. Left atrium was enlarged at 300 beats/min of pacing rate. The time from peak to 75% relaxation in left ventricular pressure was shortened with the increased pacing rate. Pressure at aortic valve close was raised (P < 0.01) and ejection duration was shortened with the increased pacing rate (P < 0.01).</p><p><b>CONCLUSION</b>Those above results suggest that there are different mechanisms between the depressed cardiac output at higher heart rate and negative force-frequency relationship. The frequency-dependent acceleration of relaxation facilitates the decline of left ventricular pressure, and then may elevate the pressure of aortic valve close in the condition that the shape of aortic pressure curve stays the same. Therefore, the ejection duration is shortened at higher pacing rate. The shortened ejection duration may induce a decrease in stroke volume of the left ventricle. The increment of heart rate is not enough to compensate the decreased stroke volume. Finally, cardiac output shows a decrease at higher heart rate.</p>

In vitro heart models simulating in vivo cardiac ischemia-reperfusion injury / 生理学报

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.

Tetanic contraction induces enhancement of fatigability and sarcomeric damage in atrophic skeletal muscle and its underlying molecular mechanisms / 中国应用生理学杂志

Muscle unloading due to long-term exposure of weightlessness or simulated weightlessness causes atrophy, loss of functional capacity, impaired locomotor coordination, and decreased resistance to fatigue in the antigravity muscles of the lower limbs. Besides reducing astronauts' mobility in space and on returning to a gravity environment, the molecular mechanisms for the adaptation of skeletal muscle to unloading also play an important medical role in conditions such as disuse and paralysis. The tail-suspended rat model was used to simulate the effects of weightlessness on skeletal muscles and to induce muscle unloading in the rat hindlimb. Our series studies have shown that the maximum of twitch tension and the twitch duration decreased significantly in the atrophic soleus muscles, the maximal tension of high-frequency tetanic contraction was significantly reduced in 2-week unloaded soleus muscles, however, the fatigability of high-frequency tetanic contraction increased after one week of unloading. The maximal isometric tension of intermittent tetanic contraction at optimal stimulating frequency did not alter in 1- and 2-week unloaded soleus, but significantly decreased in 4-week unloaded soleus. The 1-week unloaded soleus, but not extensor digitorum longus (EDL), was more susceptible to fatigue during intermittent tetanic contraction than the synchronous controls. The changes in K+ channel characteristics may increase the fatigability during high-frequency tetanic contraction in atrophic soleus muscles. High fatigability of intermittent tetanic contraction may be involved in enhanced activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and switching from slow to fast isoform of myosin heavy chain, tropomyosin, troponin I and T subunit in atrophic soleus muscles. Unloaded soleus muscle also showed a decreased protein level of neuronal nitric oxide synthase (nNOS), and the reduction in nNOS-derived NO increased frequency of calcium sparks and elevated intracellular resting Ca2+ concentration ([Ca2+]i) in unloaded soleus muscles. High [Ca2+]i activated calpain-1 which induced a higher degradation of desmin. Desmin degradation may loose connections between adjacent myofibrils and further misaligned Z-disc during repeated tetanic contractions. Passive stretch in unloaded muscle could preserve the stability of sarcoplasmic reticulum Ca2+ release channels by means of keeping nNOS activity, and decrease the enhanced protein level and activity of calpain to control levels in unloaded soleus muscles. Therefore, passive stretch restored normal appearance of Z-disc and resisted in part atrophy of unloaded soleus muscles. The above results indicate that enhanced fatigability of high-frequency tetanic contraction is associated to the alteration in K+ channel characteristics, and elevated SERCA activity and slow to fast transition of myosin heavy chain (MHC) isoforms increases fatigability of intermittent tetanic contraction in atrophic soleus muscle. The sarcomeric damage induced by tetanic contraction can be retarded by stretch in atrophic soleus muscles.

Cardioprotection by the inhibitory effect of nitric oxide / 生理学报

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.

Remodeling of cardiac gap junctions and arrhythmias / 生理学报

In the heart, gap junctions mediate electrical and chemical coupling between adjacent cardiomyocytes, forming the cell-to-cell pathways for orderly spread of the wave of electrical excitation responsible for a functional syncytium. Three principal connexins are expressed in cardiomyocytes, connexin 43 (CX43), CX40, and CX45. CX43 predominates in ventricular muscle cells. Most of the gap junctions, assembled from CX43, are located at the intercalated discs, often with larger junctional plaques at the disc periphery. The gap junctions are rarely distributed to the sides of the cardiomyocyte. The ischemia-reperfusion, cardiac hypertrophy, heart failure, hypercholesterolemia, and diabetes mellitus induce gap junction remodeling. The gap junction remodeling induced by above-mentioned diseases shows similar characteristics, including down-regulation of CX43, reduction in gap junction plaque size, increased heterogeneity and lateralization of gap junction distribution, and dephosphorylation of CX43. The elevated angiotensin II concentration in local myocardium may play an important role in the gap junction remodeling. The down-regulation of CX43 and lateralization of gap junction distribution alter anisotropic spread of the impulse of ventricular myocardium. The dephosphorylation of CX43 not only reduces electrical conductance, but also decreases permeability of chemicals between cardiomyocytes. The lateralization of gap junctions may increase the number of hemichannels formed by CX43. The opening of hemichannels induces ATP efflux and Na(+) influx, which forms a delayed after-depolarization. The gap junction remodeling in pathological condition produces arrhythmia substrate in the ventricles. In this review, the current knowledge on the relationship between the remodeling of cardiac gap junctions and arrhythmias were summarized.

The influence of contraction modes on the phosphorylation of p38/Akt / 中国应用生理学杂志

<p><b>OBJECTIVE</b>Muscle contraction may prompt glucose uptake through non-insulin-dependent ways, and it may be due to the enhanced activation of key proteins known to regulate glucose metabolism, like p38 and Akt. Our experiment focused on the impact of different contraction modes on the phosphorylation of the molecules, thus to explore effective ways to lower blood glucose.</p><p><b>METHODS</b>Isolated muscle strips perfusion technique and Western blot analysis were employed to investigate the influence of different modes of contraction on the activation of the molecules.</p><p><b>RESULTS</b>Muscle contraction led to an increase in p38 phosphorylation, with the greatest effect observed after 5 minutes of 10% DC (duty cycle) contraction and 5 minutes of 1% DC contraction. However, phosphorylation of Akt were not altered by the two contraction modes.</p><p><b>CONCLUSION</b>The level of phosphorylation of p38 was higher at the optimal contraction modes, but these modes could not increase the level of phosphorlation of Akt.</p>

Morphological signs of survival cultured adult rat cardiomyocytes / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To clarify the key morphological signs for the survival of adult rat cardiomyocytes in primary culture.</p><p><b>METHODS</b>The adult rat hearts were retrogradely superfused by Langendorff apparatus. Cardiomyocytes were digested by collagenase I and cultured in three groups: (1) Serum free medium + BA (Bongkrekic acid, apoptotic inhibitor), (2) 5% serum medium, and (3) 5% serum medium + BA. The morphological alterations were observed and the percentage of rod-shaped cardiomyocytes, the apoptotic rate of cells, the rate of pseudopodium formation and the nuclear distances of cardiomyocytes were detected during culture.</p><p><b>RESULTS</b>(1) The percentage of rod-shaped cardiomyocytes decreased gradually in the first 3 days of cell culture. The percentage of rod-shaped cardiomyocytes cultured without fetal bovine serum (FBS) decreased more rapidly than those cultured with FBS. No differences were noticed between with and without the addition of apoptotic inhibitor BA. The apoptotic rate of cardiomyocytes increased in the first 3 days of cell culture, and the apoptotic rate of cells cultured without FBS increased more than that cultured with FBS. Also BA had no effect on apoptotic rate. (2) Cardiomyocytes cultured with FBS spread from the intercalated disk and extended pseudopodium on the second or third day of cell culture. Cardiomyocytes with thin membranous pseudopodium developed would survive and spread laterally at the 6th day of culture. Cells with the elongated morphology gradually spread extensively and took on a spheroidal shape. Myofibrils gradually lost their parallel. Cells cultured without FBS had no pseudopodium formation. The intercalated disk of cells gradually changed blunt. There was no effect on the rate of pseudopodium formation when added with apoptotic inhibitor BA. (3) Cytoskeletal remodeling occurred in survived cardiomyocytes. After 6 days of culture, cardiomyocytes exhibited characteristic of redifferentiation. (4) The distance between nuclei decreased in a single cardiomyocyte cultured with FBS for the cytoskeletal reconstruction, whereas it remained unchanged in cardiomyocytes cultured without FBS.</p><p><b>CONCLUSION</b>We clarify the pseudopodium developed on the second or third day of cell culture will be the critical morphological signs of survival cultured adult rat cardiomyocytes. It is necessary to add FBS for the formation of pseudopodium.</p>

Calpain mediates cardiac troponin I degradation in tail-suspended rats / 生理学报

The aim of the present study was to investigate the expressions of calpain and calpastatin in the myocardium of simulated weightlessness rats, and to elucidate the underlying mechanism of cardiac troponin I (cTnI) degradations. Tail-suspended (SUS) rats were used as a simulated weightlessness model on the ground. The myocardium of rats was homogenized, and the expressions of calpain-1, calpain-2, calpastatin and cTnI were analyzed by Western blotting technique. Calpastatin expression was significantly decreased in 2- and 4-week SUS groups compared with that in the synchronous controls (P<0.05). Calpain-2 expression was slightly decreased, whereas calpain-1 expression was unaltered in SUS groups. However, calpain-1/calpastatin and calpain-2/calpastatin ratios were increased after tail-suspension, being significantly higher in 2- and 4-week SUS groups than those in the synchronous controls (P<0.05, P<0.01). Cardiac TnI degradation was significantly increased after tail-suspension (P<0.01), but cTnI degradation in both SUS and control groups was significantly inhibited by a non-specific inhibitor of calpain, PD150606 (P<0.01). These results suggest that an increase in calpain activity may enhance cTnI degradation in the myocardium of tail-suspended rats.

Cardiac hypertrophy and changes in contractile function of cardiomyocyte / 生理学报

To investigate the cellular mechanisms of pressure-overload cardiac hypertrophy transition to heart failure, we observed time course of changes in morphology and contractile function of cardiomyocytes in transverse abdominal aortic constriction (TAC) rats. Since TAC rats suffered higher stress, body weight had a slower growth rate compared with that of synchronous control rats. Therefore, the left ventricular to body weight ratio produced experimental bias to evaluate the degree of cardiac hypertrophy. Length and width of collagenase-isolated cardiomyocyte were directly measured. Length, width and calculated surface area of cardiomyocyte showed a progressive increase in 8-, 16-, and 20-week TAC rats. The increasing rate of surface area in cardiomyocytes was higher at the middle stage of TAC (from the eighth to sixteenth week). Due to the constraint of fibrosis formation, the increasing rate of surface area in cardiomyocytes was slower at the late stage of TAC (from the sixteenth to twentieth week). The sarcomere length of cardiomyocytes was unchanged, whereas sarcomere numbers were significantly increased in 8-, 16-, and 20-week TAC rats. Shortening amplitude of unloaded contraction in single cardiomyocyte was significantly enhanced in 1-week TAC rats, but not altered in 8-week TAC rats compared with that in the synchronous control rats. On the contrary, unloaded shortening amplitude of single cardiomyocyte was significantly reduced in 16- and 20-week TAC rats. The above results suggest that the reduced shortening amplitude may be associated with intrinsic molecular alterations in hypertrophied cardiomyocytes.

Calcium leak of sarcoplasmic reticulum induces degradation of troponin I in skeletal muscle fibers / 生理学报

The troponin I subunit (TnI) was used as a molecular marker to explore the relationship between the resting intracellular Ca(2+) concentration and myofibril degradation in muscle fibers. The isolated soleus muscle strips of rats were treated by caffeine and H2O2. Caffeine is an opener to increase the calcium release channel open probability of sarcoplasmic reticulum (SR) in contraction phase. H2O2 induces a calcium leak of SR calcium release channel in relaxation phase. The expression and degradation of TnI were detected by Western blot. The resting tension of tetanic contraction and expression of TnI were not changed, but the developed tension was lowered in isolated soleus muscle strips during 40 min of calcium-free Krebs perfusion. Low concentrations of caffeine (1 and 5 mmol/L) perfusion induced a transient increase in resting tension during fatigue period, but did not alter the extent of fatigue, recovery rate after fatigue and expression of TnI in muscle strips. High concentration of caffeine (10 mmol/L) perfusion induced a progressive increase in resting tension, a higher rate of fatigue and a decrease in recovery rate after fatigue in muscle strips. There was a detectable degradation of TnI in soleus after 10 mmol/L caffeine treatment. H2O2 perfusion facilitated a progressive increase in resting tension in a dose-dependent manner, but did not influence the fatigue rate of tetanic contraction. The recovery rate after fatigue showed a quick resumption before decline during H2O2 perfusion. Degradation of TnI occurred in 5 and 10 mmol/L H2O2-treated soleus muscles. Since resting tension is dependent on intracellular Ca(2+) concentration, the above-mentioned results suggest that SR Ca(2+) leakage in relaxation phase may induce a degradation of TnI in skeletal muscle fibers.

Protein kinase C regulates unloading contraction of cardiomyocytes in hypertrophic heart of spontaneously hypertensive rat / 生理学报

The activation of protein kinase C (PKC) is not only a pivotal node during cardiac hypertrophy in chronic pressure-overloaded heart, but also involved in the regulation of cardiac contractility. The aim of this paper was to observe PKC modulation in cardiac contractility at different stages of cardiac hypertrophy in spontaneously hypertensive rat (SHR). The cardiomyocytes were isolated from 4- and 10-month-old normotensive Wistar-Kyoto (WKY) and SHR rat hearts. The shortening amplitude of unloading contraction in cardiomyocytes was observed by an Edge Detector system. The shortening amplitude in WKY rat cardiomyocytes increased gradually as the stimulating frequency increased from 1 to 3 Hz. The shortening amplitude was positively correlated with stimulating frequency. The shortening amplitude in 4-month-old SHR group was enhanced as compared with that in WKY group at the same stimulating frequency. When the stimulating frequency increased, the shortening amplitude did not increase in 4-month-old SHR group. There was no difference in shortening amplitude between 10-month-old SHR and WKY groups at 1-Hz stimulating frequency. But the shortening amplitude in 10-month-old SHR group decreased when the stimulating frequency increased to 3 Hz. The perfusion of 50, 100 or 200 nmol/L PMA (a non-specific agonist of PKCs) significantly reduced the shortening amplitude in WKY and SHR groups. The shortening amplitude was reduced to (69.8±1.9)%, (58.2±2.2)% and (22.7±2.5)% (all P<0.01), respectively, in WKY group, as compared with that in HEPES buffer perfusion (100%). It was reduced to (6.1±0.7)%, (2.4±0.2)% and (12.5±2.6)% (all P<0.01) in 4-month-old SHR group, and (65.7±1.6)%, (53.9±4.0)% and (16.3±2.0)% (all P<0.01) in 10-month-old SHR group. The decreases in shortening amplitude in 4-month-old SHR group were more significant than those in 10-month-old SHR and WKY groups. On the other hand, 200 nmol/L of staurosporine, an inhibitor of PKC, significantly increased the shortening amplitude of cardiomyocytes in WKY, 4-month-old SHR, and 10-month-old SHR groups by (63.63±4.53)%, (80.82±4.61)% and (80.97±4.59)%, respectively (all P<0.05). The results suggest that the PKC isoforms inducing negative inotropic effect may be activated at the early stage of cardiac hypertrophy in SHR rats, and are possibly involved in the modulation of cardiac contractility. The activated PKC isoforms return to their normal activity at the stable stage of cardiac hypertrophy in SHR rats.

Increased activity of sarcoplasmic reticulum Ca(2+)-ATPase in soleus of hyperthyroid rat accelerates fatigue during intermittent tetanic contraction / 生理学报

The elevated plasma level of thyroxin and/or triiodothyronine in hyperthyroidism not only induces a transition from the innervated slow-twitch muscle fibers to fast-twitch fibers, but also changes the contractile function in transition muscle fibers. So the muscle weakness of thyrotoxic myopathy would relate to alteration in fatigability of tetanic contraction in muscles, especially in slow-twitch fibers. The aim of the present study was to observe the extent of fatigue of soleus in 4-week hyperthyroid rats and elucidate its underlying mechanism. The isolated soleus muscle strips were perfused in Krebs-Henseleit solution with or without an inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), cyclopiazonic acid (CPA). The contractile function of soleus was observed in twitch and intermittent tetanic contraction. The body weight in 4-week hyperthyroid rats decreased as compared with that in the control group [(292±13) g vs (354±10) g], but there was no difference between hyperthyroid and control groups in the wet weight of soleus [(107.3±8.6) mg vs (115.1±6.9) mg]. The time to peak tension (TPT) and time from peak tension to 75% relaxation (TR(75)) in twitch contraction were shortened in the soleus of hyperthyroid rats, and the TR(75) of tetanic contraction was also shortened as compared with that in the control group [(102.8±4.1) ms vs (178.8±15.8) ms]. The optimal stimulation frequency at which a maximal tension of tetanic contraction happened was shifted from 100 Hz in the control group to 140 Hz in hyperthyroid group. The soleus of hyperthyroid rat was easier to fatigue than that of the control rat during intermittent tetanic contraction. The SERCA activity also increased in soleus of hyperthyroid rat. The TR(75) in tetanic contraction was prolonged and showed an increased fatigue resistance in the soleus of control and hyperthyroid groups treated with 1.0 μmol/L CPA. The fatigue resistance of tetanic contraction in the soleus of hyperthyroid rat increased further with 5.0 μmol/L CPA treatment, but the resting tension kept rising. The 10 μmol/L CPA reduced the fatigue resistance of tetanic contraction in the soleus of hyperthyroid rat. The above results demonstrate that the SERCA activity in soleus can also influence the relaxation duration of twitch contraction like that in the myocardium. The SERCA activity in slow-twitch fibers is possibly involved in the regulation of fatigue resistance of intermittent tetanic contraction.

Chemical constituents from the tuber of Cremastra appendiculata / 药学学报

To study the chemical constituents of "Shan-Ci-Gu" (the tuber of Cremastra appendiculata (D. Don) Makino), the compounds were isolated with silica gel and reverse phase silica gel as well as Sephadex column chromatographic method. Their structures were elucidated on the basis of modern spectra technology. Seven compounds were isolated and identified as 5-methoxybibenzyl-3, 3'-di-O-beta-D-glucopyranoside (1), militarine (2), loroglossin (3), protocatechuic acid (4), succinic acid (5), gastrodin (6), and daucosterol (7). Compound 1 is a new compound. Compounds 2 -6 were isolated from this plant for the first time.

Depressed responsiveness of cardiomyocytes to isoproterenol in simulated weightlessness rats / 生理学报

The present study aimed to observe the changes of contractile function and responsiveness to isoproterenol (ISO) in tail-suspended rat cardiomyocytes under simulated weightlessness condition. Tail-suspended rat model was used to simulate weightlessness on the ground. Twenty-four male Sprague-Dawley rats were randomly divided into the control and tail-suspended groups. After 4 weeks of suspension, the rats were injected with heparin (100 IU/100 g body weight, i.p.) and anesthetized with pentobarbital sodium (40 mg/kg body weight). The hearts were removed and the aortas were cannulated rapidly. The cannulated hearts were mounted on a Langendorff perfusion apparatus and perfused with constant flow. The perfusion pressure was monitored. The hearts were digested by 0.08% collagenase I at 37 degrees C. The ventricular tissues were chopped and the single myocytes were dispersed gently by a wide-tipped pipette. The contractile function was measured in the Edge Detector system within 6 h after isolation. The length and width of cardiomyocytes were measured without electric stimulation. Contractile curves of the single cardiomyocytes were recorded at stimulation frequency of 1.0, 2.0 and 4.0 Hz. To observe the responsiveness of cardiomyocytes to ISO, 1, 5 and 10 nmol/L ISO in Kreb's solution was perfused at a stimulation frequency of 2.0 Hz. The length and width of the left and right ventricular cardiomyocytes in tail-suspended group had little difference from that in the control group. The unloaded shortening amplitude increased as stimulation frequency elevated in both the control and tail-suspended groups. It was increased by (8.50±1.26)%, (9.00±1.38)%, (9.23±1.83)% in the left ventricular cardiomyocytes, and (9.80±2.48)%, (10.03±2.48)%, (10.28±2.27)% in the right ventricular cardiomyocytes in the control group at stimulation frequency of 1.0, 2.0 and 4.0 Hz. Compared with that in the control group, the unloaded shortening amplitude decreased by 12.2% and 10.9% in the left ventricular cardiomycytes (P<0.05), and 16.5% and 16.3% in the right ventricular cardiomyocytes (P<0.05) at stimulation frequency of 1.0 and 2.0 Hz in tail-suspended group. There was no significant difference in unloaded shortening amplitude at stimulation frequency of 4.0 Hz between the control and tail-suspended groups. Time to peak shortening (TPS) in tail-suspended group significantly reduced in both the left and right ventricular cardiomyocytes (P<0.05). Time from peak to 75% relaxation (TR(75)) in tail-suspended group significantly prolonged in both the left and right ventricular cardiomyocytes (P<0.05). No significant differences in shortening and relaxation rate (±dL/dt(max)) were observed between the control and tail-suspended groups. The unloaded shortening amplitude increased by (10.63±0.83)%, (35.06±5.22)% and (71.64±6.83)% in the control cardiomyocytes, but increased by (5.75±0.76)%, (23.97±4.50)% and (26.38±8.13)% in tail-suspended group during perfusion with 1, 5 and 10 nmol/L ISO (P<0.05, P<0.01). The unloaded shortening amplitude increased by (3.04±0.27)%, (9.81±2.66)% and (20.20±3.47)% in the control cardiomyocytes, but increased by (1.42±0.53)%, (3.83±1.71)% and (5.49±4.08)% in tail-suspended group during perfusion with 10, 50 and 100 nmol/L forskolin (P<0.05). The results obtained suggest that the unloaded shortening amplitude and responsiveness to ISO decrease in rat cardiomyocytes after 4-week tail-suspension.

Studies on flavones in of Lavandula augustifolia / 中国中药杂志

<p><b>OBJECTIVE</b>To study on the chemical constituents of Lavandula augustifolia.</p><p><b>METHOD</b>The compounds were extracted with 95% ethyl alcohol, isolated by various column chromatography and identified by spectroscopic methods.</p><p><b>RESULT</b>Seven flavanoids were isolated and identified as apigenin (1), ladanein (2), apigenin-7-O-beta-D-(6'-p-hydoxy-cinnamoyloxy) -mannoside (3), luteolin (4), apigenin-7-O-beta-D-glucoside (5), luteolin-7-O-beta-D-glucoside (6), 5, 4'-dihydroxy flavonoid-7-O-beta-D-pyranglycuronate buthyl ester (7).</p><p><b>CONCLUSION</b>All of these seven compounds were obtained from this plant for the first time.</p>