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.

Correlative analysis of different HBV genotypes and autoantibodies in hepatitis B patients / 中华肝脏病杂志

To study the genotypes of hepatitis B virus (HBV) in patients from Chongqing district and determine the prevalence of autoantibodies in these patients. HBV genotyping was carried out by restriction fragment length polymorphism analysis of venous blood serum samples from 252 chronic hepatitis B (CHB) patients and 25 healthy controls. Indirect immunofluorescent assay was used to detect autoantibodies, including antinuclear antibody, anti-mitochondrial antibody, anti-smooth muscle (SM) antibody, and anti-liver and kidney microsomal antibody. Immunospot assay was used to detect anti-ribonucleoprotein/anti-SM antibodies, anti-SS-A antibody, anti-SS-B antibody, anti-scl-70 antibody, and anti-Jo-1 antibody. Correlations between the production of autoantibodies and patient age and sex and various genetypes of HBV were analyzed by the Chi-squared test. The most frequent HBV genotype in CHB patients was B (67.3%), followed by genotype C (32.7%). Genotypes A, D, E, F, G and H were not detected in any of the CHB patients. The positive rate of autoantibodies was higher in the CHB patients than in the healthy group (76.98% vs. 12.00%, X2 = 44.60, P less than 0.05). There was no significant differences in the autoantibodies profiles of CHB patients carrying the B or C genotypes ( X2 = 0.0016, P more than 0.05). The main HBV genotypes in CHB patients in the Chongqing district are B and C. Autoantibodies are prevalent among these CHB patients, and are correlated with patient age and course of liver disease but not HBV genotype or patient sex.

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.

The relationship between the gene polymorphism of TGF-beta1 and early renal injury in patients with essential hypertension, and the effect of the gene polymorphism of TGF- beta1 on the individual treatment with valsartan / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the association of transforming growth factor-beta1(TGF-beta1) gene +869T/C polymorphism and the serum level of TGF-beta1 in patients with early essential hypertension and early renal injury. To study the effect of the gene polymorphism of TGF-beta1 on the individual treatment with valsartan.</p><p><b>METHODS</b>Eighty patients with early essential hypertension and early renal injury were treated with valsartan, and their polymorphism of TGF-beta1 gene +869T/C was analyzed by sequence specific primers-polymerase chain reaction method. Before valsartan treatment and 8 weeks after the treatment, their urinary microamount albumin (MA) levels were determined by using radioimmunity method, and their serum levels of TGF-beta1 were determined by enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>The blood pressure level of patients with TGF-beta 1 gene +869 CC genotype, TT genotype, TC genotype showed no significant difference (P> 0.05) before treatment. The urinary MA level of the three genotypes was CC genotype's > TC genetype's > TT genotype's in sequence, and the urinary MA level of CC genotype was significantly higher than that of TC genotype and of TT genotype (P< 0.01). The serum levels of TGF-beta1 in the three genotypes was CC genotype's > TC genotype's > TT genotype's in sequence (P< 0.01). There was statistically significant positive correlation between the urinary MA level and the serum level of TGF-beta1(P< 0.05). After 8 weeks treatment with valsartan, there was significant decrease of the blood pressure, the urinary MA level, and the serum level of TGF-beta1. However, the blood pressure reduction of the three types had no statistical significant difference in sequence. The absolute value of urinary MA reduction was CC genotype's > TC genotype's > TT genotype's in sequence, the reduction absolute value of urinary MA in CC genotype was higher than that of TC genotype and of TT genotype (P< 0.05), but the reduction percentage of the urinary MA in the three genotypes showed no statistically significant difference (P> 0.05). The reduction of serum levels of TGF-beta1 was CC genotype's > TC genotype's > TT genotype's in sequence, there was significant difference between each other (P< 0.01). However, the reduction percentage of the serum level of TGF-beta 1 in the three types showed no statistically significant difference (P> 0.05).</p><p><b>CONCLUSION</b>(1) The renal injury of CC genotype in patients with early essential hypertension and early renal injury is more serious than that of TC genotype and of TT genotype. (2)The serum level of TGF-beta 1 can accurately indicate the severity of the renal injury and the protective effect on kidney with valsartan in patients with essential hypertension and early renal injury. (3)The treatment effect of valsartan in the patients with different TGF-beta 1 +869T/C genotype has no difference, i.e., besides effectively decreasing the blood pressure and protecting the renal function, valsartan can reduce the serum level of TGF-beta1 without the influence of the gene polymorphism of TGF-beta1.</p>

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.

Correlation between Level of Nitric Oxide/Nitricoxide Synthase and Ultra-Structure Changes of Placenta Exposured in Lead Gestation Period in Rats / 实用儿科临床杂志

Objective To study the correlation between level of nitric oxide/ nitricoxide synthase(NO/NOS) on placenta homogenate and ultra-structure changes of placenta in pregnancy lead exposure in rats.Methods Seventeen normal pregnant rats and 46 rats of exposured in lead which were divided into A,B,C groups were studied.The level of NO/NOS of placenta were measured by nitrate reductase and NOS kit.Placentas were randomly selected from each group to detect ultra-structure by electron-microscope.Results There were significant difference among A,B and control groups on level of NO/NOS(all P0.05).Compensation hyperplasy or minor injury were observed in lead exposure of stage groups.Lead exposure during whole gestation period,the lead level was maxmum,and decompensation were observed on placental construction.Conclusions There is a close correlations between level of lead,NO/NOS and pathological change of placental tissue,and both of them may play an important role in the pathogenesis of peripartum lead exposure.

Adult rat cardiomyocytes isolated by constant flow perfusion at low pressure / 中国应用生理学杂志

<p><b>AIM</b>In order to get high-yield and high-quality cardiomyocytes from adult rat. We needed to clarify the optimal perfusion pressure in a constant flow condition and stopping digestion pressure.</p><p><b>METHODS</b>The rat was injected with heparin (1 000 IU/kg, i. p.) 20 minutes prior to the experimental protocol. The heart was excised and the aorta was cannulated rapidly. The cannulated heart was mounted on a Langendorff perfusion apparatus with constant flow and perfusion pressure was monitored. The initial perfusion pressure was maintained at 15 kPa or 10 kPa by regulating the flow rate. The heart was digested by 0.08 % collagenase I at 37 degrees C and the enzymatic digestion was terminated immediately when the perfusion pressure was lowered to 10 kPa, 7.5 kPa or 5 kPa.</p><p><b>RESULTS</b>While the initial perfusion pressure was 15 kPa (n = 4), enzymatic digestion induced great extent increment in perfusion pressure (maximal pressure > 25 kPa). The left ventricle was enlarged significantly and the contractility of myocytes was decreased. While the initial perfusion pressure was kept at 10 kPa, the digestion only resulted in less extent increment in perfusion pressure (maximal pressure < 18.75 kPa). The left ventricle was enlarged slightly. In the same initial perfusion pressure, if the digestion was stopped at 10 kPa (n = 3), or 5 kPa (n = 4), myocytes appeared over much or insufficiency digestion, respectively. The viability of freshly isolated cardiomyocytes was less than 10%. The most of myocytes died after restoration of extracellular Ca2+ concentration. If the digestion was stopped at 7.5 kPa (n = 15). The viability of cardiomyocytes was 82.6% +/- 4.8% in fresh isolation, 30.4% +/- 4.5% after restoration of normal extracellular Ca2+ concentration, or 24.8% +/- 5.4% after 4 h standing. The most of rod-shaped cardiomyocytes were quiescent and had visible cross striations, sharp edges, and normal contractility.</p><p><b>CONCLUSION</b>When the initial perfusion pressure is kept at 10 kPa (low pressure) and the enzymatic digestion is stopped at 7.5 kPa, high yield and high quality cardiomyocytes can be obtained.</p>

Interleukin 10 gene -627 polymorphisms in Chinese patients with early-onset coronary heart disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between interleukin 10 (IL10) gene -627 polymorphisms and serum IL10 level and early-onset coronary heart disease (CHD).</p><p><b>METHODS</b>The genotype and allele frequency of IL10 gene -627 site was assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). DNA samples were obtained from 163 patients with CHD and 112 controls. Serum IL10 level was detected by ELISA.</p><p><b>RESULTS</b>No significant difference was found in the distribution of IL10 genotype and allele frequency between the healthy controls and the patients with CHD; Chi-square values were 1.9324 and 1.5703 respectively, P > 0.05. Stratification analyses based on different sex still found no significant difference in the distribution of IL10 genotype and allele frequency between the healthy controls and the CHD patients; the Chi-square values in male groups were 1.2708 versus 0.8595, and in female groups were 0.8254 versus 0.7127, P > 0.05. Serum IL10 level showed significant differences among AA genotype, AC genotype and CC genotype, but no significant difference was noted between healthy controls and CHD patients.</p><p><b>CONCLUSION</b>These results suggest that IL10 gene -627 polymorphisms are not associated with an increased risk of CHD, but it might assume a role in IL10 gene expression.</p>

Isolation of cardiomyocytes from the adult mouse heart / 生理学报

In order to culture cardiomyocytes or to observe the contractile function of adult mouse cardiomyocytes, it is necessary to isolate high-yield and high-quality cardiomyocytes at first. The mouse was injected with heparin (5,000 IU/kg, i.p.) 20 min prior to the experimental protocol, then was sacrificed by cervical dislocation. The heart was excised and the aorta was cannulated rapidly. The cannulated heart was mounted on a Langendorff perfusion apparatus with constant flow and perfusion pressure was monitored. The initial perfusion pressure was maintained at 40 mmHg by regulating the flow rate. The heart was digested by 0.05 % crude collagenase I at 37 degrees C and the enzymatic digestion was terminated immediately when the perfusion pressure was lowered to 28 mmHg. The heart was then cut off the cannula and the atria and aorta dissected away. The ventricular tissue was chopped and the single myocyte was dispersed gently by a wide tipped pipette. The viability of freshly isolated cardiomyocytes was more than 70 %. The cardiomyocytes were kept in Joklik's minimum essential medium containing 1 % BSA and 10 mmol/L BDM, then extracellular calcium was restored step-wise to a final concentration of 1.25 mmol/L. The viability of cardiomyocytes reduced to (40-50) % after 4 h standing. More than 90 % of rod-shaped cardiomyocytes were quiescent and had visible cross striations and sharp edges. The amplitude of unloaded shortening in cardiomyocytes was (9.72+/-0.43) % during 1.0 Hz stimulation, (11.28+/-0.43) % at 2.0 Hz and (11.40+/-0.45) % at 5.0 Hz. These results indicate that high yield and high quality cardiomyocytes can be obtained. In addition, the standards of identifying cardiomyocyte quality are concise and are suitable to culture the cardiomyocytes or to study the physiological function of cardiomyocytes.