Extensive skinning of cell membrane diminishes the force-inhibiting effect of okadaic acid on smooth muscles of Guinea pig hepatic portal vein

The influences of cell membrane permeabilization (skinning) on the okadaic acid-induced inhibition of vascular smooth muscle contraction were studied in guinea pig hepatic portal vein. Pretreatment by 1 microM okadaic acid in the absence of Ca(2+) suppressed subsequent submaximal Ca(2+)-induced contraction in preparations permeabilized with Staphylococcus aureus alpha-toxin or beta-escin, but not in those treated with saponin or Triton X-100. The SDS-PAGE of elutants from the preparation suggests that the loss of the inhibitory effect of okadaic acid in preparations skinned with saponin or Triton X-100 results from the leakage of some cellular components with a molecular mass of 67 to 200 kDa.     

Effects of Ca antagonist on the contractile force in glycerinated dog heart muscles

The effect of Ca antagonist on the contractile apparatus was investigated in glycerinated cardiac muscle preparations obtained from canine hearts. Each muscle preparation had three consecutive isometric contractions. The 1st and 3rd contractions were produced with a control contraction solution, and compared with the 2nd contraction which was induced with a contraction solution containing verapamil. The results showed that maximal developed tension (Po) was enhanced significantly by 1.02×10^–2 mM of verapamil, and the augmentation of contractility was dependent on the concentrations of verapamil. Thus, not only Po, but also dT/dt increased tremendously at 1.02 mM of verapamil. Such contractile potentiation by verapamil was also ascertained by another Ca antagonist, Diltiazem hydrochloride. The developed tension was maximum at pCa 4.0, and no developed tension was found at pCa 8.0. The relationship between pCa and tension with verapamil shifted to the left from that without verapamil, showing higher sensitivity to Ca²⁺. From these results, it was strongly indicated that Ca antagonist is a potentiating agent of the contractile force.     

A study of the short-term effects of glucose 6-phosphate on the contractile activation properties of skinned single muscle fibres of the rat. Implications for solution design

The present study highlights possible problems that can arise from the incorrect preparation of control and test solutions for use in Ca2+-activation experiments using single skinned skeletal muscle fibres and EGTA-based Ca2+ buffers. We show here, using glucose 6-phosphate (G6-P) as our "test" compound, that the Ca2+-activation properties of skinned single fibre segments from the extensor digitorum longus muscle of the rat are highly dependent on the form in which the G6-P is added and on the correct balancing of an appropriate anion in control solutions. Test solutions prepared by the direct addition of 10 mM monosodium G6-P salt to a set of control solutions of defined pCa resulted in significantly greater submaximal force responses than the corresponding controls. This is equivalent to an increase in the sensitivity of the contractile-regulatory system to Ca2+ (pCa50=-log10[Ca2+] that produces 50% of maximum force) by 0.19+/-0.01 pCa units. In contrast, addition of disodium G6-P to control solutions caused a slight reduction in the apparent sensitivity of the contractile apparatus to Ca2+ by 0.04+/-0.01 pCa units (P<0.01). Rather than being indicative of the effects of G6-P on the contractile apparatus, these opposing effects are due to differences between test and control solutions with respect to pH and Na+ concentration brought about by the G6-P salts. When all ionic species were carefully balanced, 10 mM G6-P was found to have only a small sensitizing effect on Ca2+-activation properties compared to control, without affecting the maximum Ca2+-activated force response. Our findings highlight the often-overlooked need for careful balancing of the ionic composition in control and test solutions when examining the true effects of different compounds on the Ca2+-activation characteristics of single skinned muscle fibre preparations.     

Contractile properties of skinned preparations from ischaemic canine myocardium and coronary arteries

The influence of prolonged ischaemia on the regulation of contraction in the myocardium and in the smooth muscle of coronary arteries was investigated. Chemically skinned preparations were used which enabled the contraction to be studied with the environment of the contractile filaments controlled. Myocardial ischaemia was produced in anaesthetized adult beagle dogs by occlusion of the left anterior descending artery for 3 h and followed by 30 min reperfusion. Myocardial tissue and segments from coronary arteries were obtained from the ischaemic infarcted wall region (in vivo ischaemic) and compared with control preparations from perfused coronary arteries and from the free wall of the left ventricle. Coronary and myocardial preparations were also obtained from the heart after a 3 h period in vitro under anoxic conditions at 37°C (in vitro ischaemic) simulating a state of extreme ischaemia. Control myocardial fibres were fully relaxed at pCa (-log-[Ca²⁺]) 9 and developed 24±5% (n=7) of maximum force at intermediate calcium concentration (pCa 5.5). In contrast, the in vivo and in vitro ischaemic preparations produced force at pCa 9 (28±13 and 39±8%, respectively, n=5 and 7) and showed an increased force development at pCa 5.5 (53±11 and 75±5%). The in vivo and in vitro ischaemic coronary arteries relaxed more slowly following calcium removal than control vessels. The in vitro ischaemic vascular preparations developed active force at pCa 9 and showed increased levels of myosin light chain phosphorylation and reduced phosphatase activity. This suggests a reduced rate of dephosphorylation as a cause for the changes in contracile behaviour of the smooth muscle. In conclusion, extreme ischaemia in vitro is associated with a loss of calcium regulation and an increased calcium sensitivity of the contractile system in myocardium and changes in the phosphorylation/dephosphorylation reactions of coronary arteries. The changes in myocardium appear to occur also during ischaemia in vivo, and might contribute to contracture development in cells under conditions when adenosine triphosphate synthesis is reestablished after reperfusion.     

Myocardial cation contents during induction of calcium paradox

Myocardial cation contents were measured in isolated rat hearts perfused under various conditions. Reperfusion of Ca2+-deprived hearts produced marked increases in myocardial Ca2+ and Na+ and decreases in Mg2+ and K+ contents. These changes were dependent on the Ca2+ concentration and duration of perfusion during the periods of Ca2+ deprivation and reperfusion. The loss of Ca2+ and K+ contents normally seen after Ca2+-free exposure as well as the reperfusion-induced changes were prevented if the Ca2+-free medium contained low (35 mM) Na+ or was cooled to 21 degrees C. Reperfusion with normal Ca2+, low Na+ medium augmented the increase in myocardial Ca2+ content, while reducing K+ or Mg2+ or increasing Mg2+ in the reperfusion medium had no effect. Addition of verapamil, D600, or propranolol to the reperfusion solution did not alter the reperfusion-induced cation changes observed using control medium. These data suggest that during Ca2+ depletion, the mechanisms responsible for regulating calcium influx are either lost or inactivated, so that reperfusion-induced changes are governed solely by the driving force favoring calcium influx. The occurrence of Ca2+ overload under this condition has been implicated in the irreversible damage to myocardium and contractile failure.     

Adenosine affects the release of Ca2+ from the sarcoplasmic reticulum via A2A receptors in ferret skinned cardiac fibres

In this study, it was shown that adenosine potentiates caffeine-induced Ca2+ release. It was then proposed that the enhancement of the caffeine-induced Ca2+ release might occur by a direct effect on the ryanodine Ca2+ release channel or on other Ca2+ regulation mechanisms. Furthermore, A2A receptors may be functional on the ferret cardiac sarcoplasmic reticulum. Using chemically skinned fibres, experiments were conducted on ferret cardiac muscle to find out whether adenosine and the A1 and A2A adenosine receptor agonists (CCPA and CGS 21680) and antagonists (DPCPX and ZM 241385) affected caffeine-induced Ca2+ release and the Ca2+ sensitivity of contractile proteins. Changes in the caffeine-induced contracture brought about by adenosine and by adenosine-receptor agonists and antagonists were recorded in saponin-skinned fibres (50 microg ml(-1)). Tension-pCa relationships were then obtained by exposing Triton X-100-skinned fibres (1% v/v) sequentially to solutions of decreasing pCa. Adenosine (1-100 nm) and the specific A2A receptor agonist CGS 21680 (1-50 nm) produced a concentration-dependant potentiation of the caffeine-induced Ca2+ release from saponin-skinned fibres. The data plotted versus adenosine and CGS 21680 concentrations displayed sigmoid relationships (Hill relationship), with potentiation of Ca2+ release by 22.2 +/- 1.6 (n = 6) and 10.9 +/- 0.4% (n = 6), respectively. In addition, the potentiation of caffeine-induced Ca2+ release by adenosine (50 nm; 15.3 +/- 1.0%; n = 6) and by CGS 21680 (50 nm; 11.2 +/- 0.4%; n = 6) was reduced by the specific A2A receptor antagonist ZM 241385 (50 nm) to 8.0 +/- 1.4 (n = 4) and 5.4 +/- 1.2% (n = 4), respectively. The A1 receptor agonist CCPA (1-50 nm) and antagonist DPCPX (50 nm) had no significant effects on caffeine responses. In Triton X-100-skinned fibres, the maximal Ca(2+)-activated tension of the contractile proteins (41.3 +/- 4.1 mN mm(-2); n = 8), the Hill coefficient (nH = 2.2 +/- 0.1; n = 8) and the pCa50 (6.15 +/- 0.05; n = 8) were not significantly modified by adenosine (100 nm) or by CGS 21680 (50 nm).     

Reduced length-dependent cross-bridge recruitment in skinned fiber preparations of human failing myocardium

A depressed activity of myosin ATPase has been described in human failing myocardium. Since alterations in cross-bridge kinetics may affect both systolic and diastolic cardiac function, the present study simultaneously investigated Ca²⁺-dependent tension and actomyosin ATPase activity (MYO) in triton X-skinned fiber preparations of human non-failing (donor hearts, n=8) and failing (dilated cardiomyopathy, n=11) left ventricular myocardium at increasing sarcomeric length (1.9 and 2.1 μm, α-actinin staining). The MYO/tension ratio was analyzed as a parameter characterizing myofibrillar energetics. At a sarcomere length of 1.9 μm, the Ca²⁺ sensitivity of tension was significantly increased in human failing compared to non-failing myocardium. In human non-failing myocardium, maximal Ca²⁺-activated tension [1.9 μm vs. 2.1 μm, 23.7 (1.9) vs. 28.3 (1.9) mN/mm²] and the Ca²⁺ sensitivity of tension [EC₅₀Ca²⁺ (pCa): 5.67 (0.06) vs. 7.07 (0.11)] were increased by increasing sarcomere length. This was accompanied by an enhancement in Ca²⁺-dependent MYO [+72 (11) vs. +101 (9) μM ADP/s] as well as an increase in the Ca²⁺-sensitivity of MYO [EC₅₀Ca²⁺ (pCa): 5.84 (0.08) vs. 6.86 (0.08)]. In human failing myocardium, only Ca²⁺ sensitivity of tension (but not of MYO) increased. Tension cost was increased in failing vs. non-failing tissue [1.9 μm: 4.18 (0.06) vs. 3.53 (0.06) (mN·s)/(mm²·μM ADP); 2.1 μm: 4.28 (0.13) vs. 3.52 (0.05) (mN·s)/(mm²·μM ADP)]. We concluded that, in human failing myocardium, the length-dependent force generation may be blunted due to an already increased Ca²⁺ affinity of troponin C as well as an impairment of length-dependent cross-bridge recruitment. Electronic Publication     

The effects of tetrandrine on the contractile function and microvascular permeability in the stunned myocardium of rats

The effects of tetrandrine (Tet) on the contractile function and microvascular permeability in stunned rat myocardium in vivo were studied. Stunned myocardium was induced by 15 (MS(15) group) or 20 (MS(20) group) min of myocardial ischemia plus 60 min of reperfusion. The following was shown. (1) FITC-BSA concentration was 166.0 +/- 7. 9 microg/g myocardium in the control group. The concentrations in ischemic myocardium increased by 35.4 and 45.6% in MS(15) and MS(20) groups respectively (p<0.05). (2) Administration of Tet (64.2 and 96. 3 micromol/kg, I.P.) 20 min before ischemia not only ameliorated the contractile function, but also reduced the FITC-BSA concentrations in ischemic myocardium. At 60 min after reperfusion, the contractile function parameters in Tet-treated groups were significantly superior to those in corresponding stunning groups. FITC-BSA concentrations in Tet-treated groups were lower than those in stunning groups. Then, there was already no significant difference in FITC-BSA concentrations between Tet-treated groups and the control group. The FITC-BSA concentrations at the end of experiments were correlated negatively with dp/dt(max) (r = -0.83, p<0.01). (3) Tet inhibited KCl-induced calcium influx in isolated cardiomyocytes. The results suggest that Tet given before ischemia may be involved in the reduction of microvascular permeability in stunned myocardium, which might be associated with its calcium channel blocking effect.     

Molecular analysis of non-specific supersensitivity induced by AF64A in rat iris smooth muscle.

Characteristics of supersensitivity induced by the pretreatment with AF64A, an inhibitor of choline uptake at parasympathetic nerve endings, were examined in rat iris sphincter. In preparations isolated and skinned by beta-escin after the micro injection of AF64A to eyes in vivo, the amplitude of maximum contraction in pCa 4.5 solution was increased by 180% of the control from the contralateral eyes. The Ca2+ sensitivity of the contractile system was slightly but significantly increased by AF64A injection; the half maximum contraction was obtained at pCa 5.87 and 6.05 in the control and AF64A-injected eyes, respectively. The increase in maximum contraction in AF64A injected ones was neither affected by the addition of calmodulin, GTPgammaS nor H-7. The increase in Ca2+ sensitivity by AF64A injection was not affected by calmodulin, enhanced by GTPgammaS and abolished by H-7. AF64A injection increased the total protein content only by 30% of the control. The contents of contractile proteins per iris were quantified using Western blotting with monoclonal antibodies. The contents of actin and calponin were increased by AF64A, whereas those of myosin, calmodulin and caldesmon were not affected. The results indicate that AF64A-induced enhancement of the maximum contraction is not mainly due to the increase in the contents of major contractile proteins and that the increase in Ca2+ sensitivity could be due to the mechanism in which changes in protein kinase C and/or GTP binding protein activity are involved.     

非创伤性预处理在离体心脏抗再灌注损伤中作用机制的探讨

目的:观察非创伤性肢体缺血预处理对大鼠离体再灌注心肌是否有保护作用。方法:实验采用体重(250±30)gSD雄性大鼠25只随机分成3组,在Langendorff装置上对大鼠离体心脏进行灌流。对照组(C,n=8):在灌注全程均用富氧K-H液(充以95%O₂+5%CO₂),在恒压(8.33kPa)、恒温(37℃)条件下灌注;缺氧/复氧组(A,n=8):预灌15min后,灌注心脏先全心缺血缺氧15min,随后15min复氧再灌注(37℃);非创伤性肢体缺血预处理组(N-WIP,n=9):先将大鼠双后肢捆绑5min,松开5min,反复4次后,随后的方法同R组。在相应时点分别测定冠脉流出液和心肌匀浆中超氧化物歧化酶(SOD)活性,Ca²⁺-Mg²⁺-ATP酶活性和丙二醛(MDA)含量,同时记录心肌细胞的单相动作电位(MAP)和心肌收缩张力曲线。结果:非创伤性肢体缺血预处理能使再灌注心律失常发生率显著低于A组;心肌组织中MDA含量显著低于A组,心肌组织中SOD活性显著高于A组,心肌细胞的膜电位、Ca²⁺-Mg²⁺-ATP酶活性及肌张力较稳定。结论:非创伤性肢体缺血预处理对大鼠离体再灌注心肌有明显的保护作用,可能是通过增强心肌的抗氧化能力、稳定心肌Ca²⁺-Mg²⁺-ATP酶活性和膜相结构等途径,提高心肌细胞对再灌注损伤的抵抗力。     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

Protein phosphatase 2A affects myofilament contractility in non-failing but not in failing human myocardium

Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation-contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (ΔpCa50=0.05±0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ΔpCa50=0.10±0.03) and dilated (DCM, ΔpCa50=0.06±0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56α was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts.     

Phosphorylation status of regulatory proteins and functional characteristics in myocardium of dilated cardiomyopathy of Syrian hamsters

To understand the pathophysiology of hereditary cardiomyopathy, we measured the phosphorylation status of regulatory proteins, troponin I (TnI), troponin T (TnT), myosin light chain 2 (MLC2), and myosin-binding protein C (MyBP-C), and the Ca2+-dependence of tension development and ATPase activity in skinned right ventricular trabeculae obtained from cardiomyopathic (TO-2 strain, n = 8) and control (F1B strain, n = 8) hamsters. The Ca2+ sensitivities of tension development and ATPase activity (mean +/- SD) were significantly (P < 0.0001) higher in the TO-2 strain (pCa50 5.64 +/- 0.04 in tension and 5.65 +/- 0.04 in ATPase activity) than in the F1B strain (pCa50 5.48 +/- 0.03 in tension and 5.51 +/- 0.03 in ATPase activity). No significant differences in their maximum values were observed between TO-2 (40.8 +/- 7.4 mN/mm2 in tension and 0.52 +/- 0.15 micromol/l/s in ATP consumption) and F1B (42.3 +/- 8.5 mN/mm2 in tension and 0.58 +/- 0.41 micromol/l/s in ATP consumption) preparations, indicating that the tension cost (ATPase activity/tension development) in TO-2 was quite similar to that in F1B. The phosphorylation levels of MLC2 and TnI were significantly (P < 0.01) lower in TO-2 than in F1B. These results suggest that the increase in the Ca2+ sensitivities of tension development and the ATPase activity in TO-2 hearts result from the decreased basal level of TnI phosphorylation, and these features can be considered to produce the incomplete diastolic relaxation and partly improve the systolic function in TO-2 hearts.     

The effect of 2,3-butanedione 2-monoxime (BDM) on ventricular trabeculae from the avian heart

Summary 2,3-butanedione 2-monoxime (BDM, 3–30 mm) decreased twitch force of intact ventricular trabeculae isolated from 19-day embryonic chick hearts in a dose-dependent manner. The responses to BDM were rapid and reversible. In an attempt to determine the cellular basis for the inhibitory effect of BDM, experiments were carried out on skinned muscle fibres and isolated myocytes. In trabeculae skinned with Triton X-100, BDM depressed maximum calcium activated force (F_max) with an IC₅₀ of 14 mm. At 3 mm BDM, the proportional decrease in twitch force in intact tissue was similar to that of F_max in skinned tissue. At higher BDM concentrations (10 and 30 mm), however, the proportional decrease in twitch force was greater than that of F_max. BDM (up to 10 mm) had no effect on the normalized force-pCa relationship. In saponin-skinned preparations, BDM (3 and 30 mm) released calcium from the fully loaded sarcoplasmic reticulum to a slightly greater extent in the absence of calcium (pCa 8.5) than in the presence of a fixed level of free calcium (pCa 5.5). Whole cell patch clamping of freshly isolated chick myocytes demonstrated that BDM caused a dose-dependent decrease in the T-and L-type calcium current. Therefore, at low BDM concentrations (3 mm), the decrease in twitch force can be ascribed predominantly to depression of the contractile apparatus while, at higher concentrations of BDM, there is an additional inhibitory effect of BDM on excitation-contraction coupling.     

Nandrolone decanoate treatment induces changes in contractile responses of rat untrained fast-twitch skeletal muscle

This investigation was designed to examine whether short-term administration of anabolic-androgenic steroids (AAS) (nandrolone decanoate) could produce changes in contractile responses of untrained rat fast- (edl) and slow- (soleus) twitch skeletal muscle. Twenty male rats were divided into two groups, one group received weekly (for 6 weeks) an intramuscular injection of AAS, nandrolone decanoate (15 mg kg(-1)) and the second group received weekly the similar doses of vehicle (sterile peanut oil). In edl intact isolated small bundles (two to four cells), it was found that nandrolone decanoate treatment increases the K+ contracture tension (146 mM) relative to maximum tension by 56%, whereas no change was observed in the time to peak tension and in the time constant of relaxation. By contrast, in treated soleus muscle, compared with control, no significant modification was found in the K+ contracture characteristics. The change in edl contractile responses was associated with a shift to more negative potential of the voltage-dependence activation and the steady-state inactivation curves which also shifted leftward in treated soleus fibres. Furthermore, in edl skinned Triton X-100 fibres, the Ca2+ sensitivity of contractile proteins (pCa50) was increased, while electrophoresis analysis indicates no significant effect of nandrolone decanoate treatment on myosin heavy chain (MHC) isoforms. The present results show that nandrolone decanoate treatment produces more pronounced changes in untrained fast muscle function rather than soleus by acting at different levels of the excitation-contraction coupling mechanism without changes in the MHC isoforms and that contractile responses became similar to those found in soleus muscle.     

Force-velocity relation in chemically skinned rat portal vein

Rat portal veins were chemically skinned using Triton X-100 and mounted for isometric and quick release experiments at 20°C. The skinned preparations were activated by Ca²⁺ (EGTA-buffered) in solutions containing 2 mM free-Mg²⁺ and 1 M calmodulin. Half maximal isometric force was obtained at pCa=6.2. Maximal force of the skinned preparations, at pCa=4.5, was 8.2±0.8 mN/mm² (n=6). Force-velocity relations were determined at varied Ca²⁺-concentrations. Maximal shortening velocity (V _max) was 0.10±0.01 lengths/s at pCa=4.5. At decreasing Ca²⁺-levelsV _max decreased (at pCa=6.25,V _max=0.05 l/s). At pCa =9 an increased level of free-Mg²⁺ (15mM) induces a slow and submaximal increase in tension. Force velocity relations of Mg²⁺-induced contractures were not different from those of Ca²⁺-contractures of similar magnitude (pCa=6.3). The results indicate that the degree of activation of the contractile system, as regulated by Ca²⁺ and Mg²⁺, influences the kinetic properties of the actomyosin interaction as well as the force development.     

Contractile activity and reperfusion-induced calcium gain after ischemia in the isolated rat heart

Reperfusion of cardiac muscle after an ischemic episode results in the cells becoming overloaded with Ca2+. Gross ultrastructural changes, including the formation of contraction bands, also occur. The present study investigates the relationship, if any, between contractile activity during reperfusion and Ca2+ gain. Contractile activity was inhibited with 2,3-butanedione monoxime (BDM). Isolated perfused rat hearts were subjected to 30 minutes ischemia before reperfusion in the presence or absence of BDM. BDM (10 MIN) significantly reduced the Ca2+ gained during reperfusion. It also enhanced the ATP and creatine phosphate supplies. Ultrastructural examination of cells from hearts reperfused in the presence of BDM for 30 minutes revealed cells with relaxed myofibrils, some glycogen and intact sarcolemmal membranes, compared with cells from hearts reperfused in the absence of BDM which showed contraction bands, sarcolemmal discontinuities and swollen mitochondria. The 'protection' afforded by BDM did not result in a restoration of the cells to their normal state. Removal of BDM and continued reperfusion with Krebs-Henseleit buffer resulted in a gain in Ca2+ and ultrastructural damage, including contraction band formation. These findings suggest a role for contractile activity in the Ca2+ gain. However, preventing the damage which occurs as a result of contractile activity is not sufficient to restore the cells to their preischemic state. This suggests that the damage caused as a result of contractile activity is secondary to some other primary deleterious event.     

顿抑心肌的微循环障碍机制研究

目的 :探讨顿抑心肌微循环改变及机制。方法 :制备左前降支冠脉(LAD)阻断不同时间 ( 15和 60min)后再灌注 2h犬心肌顿抑模型 ,在不同观察时间点行静脉心肌声学造影 (MCE) ,计算心肌视频密度峰值(PVI)、MCE曲线上升斜率 (α)和曲线早期下降斜率 ( β) ,分别代表心肌血流灌注量、灌注速度和排空速度。测定相应时间点冠状静脉窦血乳酸浓度。结果 :⑴心肌顿抑早期PVI显著增高 ,1h后恢复至结扎前水平 ;⑵再灌注期顿抑区与正常区PVI比值、α比值、β比值显著高于LAD结扎前 ,随着再灌注时间的延长比值逐渐回降 ;⑶再灌注期冠状静脉窦血乳酸浓度明显增高。结论 :心肌顿抑早期心肌微循环处于“高动力”状态 ,血流灌注增加与排空加快并存 ;顿抑心肌缺氧代谢加强 ;心肌内微循环短路可能是心肌顿抑微循环障碍的机制。     

The length dependency of calcium activated contractions in the femoral artery smooth muscle studied with different methods of skinning

The relationship between the calcium concentration and the isometric tension obtained with different techniques of skinning provides information on the biochemical events of contraction in vascular smooth muscle. Muscle preparations of the rabbit femoral artery were skinned with triton X-100, saponin, -escin and -toxin and the relationship between the calcium concentration and isometric tension was determined at different preparation lengths. We determined the calcium sensitivity as a function of muscle length with different techniques of skinning. At a pCa of 6.0, triton X-100 skinned smooth muscle of the femoral artery generated 50% of the maximal tension. In -toxin skinned preparations, this calcium sensitivity was shifted to a pCa of 5.6. The sensitivity of the saponin and -escin skinned preparations were in between those of the triton X-100 and the -toxin skinned preparations. The cooperativity of the regulation of contraction varied among the differently skinned preparations between 3 (-toxin) and 6 (triton X-100). The relationships between the calcium concentration and the isometric tension of the differently skinned preparations up to the optimal length for tension generation did not exhibit any length dependency. The length tension relationship, obtained from the maximal response at the highest calcium concentration is in line with that from other studies. The presence of intracellular proteins and membranes affects the regulation of contraction in smooth muscle of the femoral artery.