Ca2+-tolerant adult canine myocytes: preparation and response to anoxia/acidosis

A procedure is described for the production of large numbers of Ca2+-tolerant adult canine ventricular myocytes. Gentle tissue dissociation is achieved in Spinner flasks using 320 mosM enzyme buffer containing collagenase hyaluronidase, and trypsin in the presence of millimolar levels of Ca2+. The technique allows for the complete removal of erythrocytes, the gentle removal of closely adhering nonmuscle cells (less than 3% contamination), and the selective removal of damaged from nondamaged myocytes. Total myocyte yields averaged 4-6 x 10(6)/g wet wt; 61.0 +/- 6.5% of the cells have rod-shaped morphology and are "viable" based on trypan blue exclusion. Only 3.9 +/- 1.1% of the rod-shaped cells beat spontaneously when challenged with 2 mM Ca2+. Exposure to 2 mM Ca2+ at 37 degrees C results in minimal loss of viability (2.1 +/- 1.3%/h) based on both trypan blue uptake and creatine kinase release. Simulation of cellular (i.e., membrane) injury in vitro is performed by the separate application of the perturbations of anoxia, acidosis, and low glucose to the canine myocyte suspensions; the data suggest that these myocytes are affected independently by these perturbations and are in good agreement with the results obtained by others using the isolated rat myocyte system.     

Cytoskeletal injury and subsarcolemmal bleb formation in dog heart during in vitro total ischemia.

In previous studies a two-step hypothesis explaining the mechanism of lethal ischemic injury to cardiac myocytes has been advanced. It proposes that damage to the myocyte cytoskeleton precedes, and predisposes the cell to, mechanical injury induced by cell swelling or by ischemic contracture. This study quantitated the prevalence of breakage of the major cytoskeletal attachment between the plasmalemma and peripheral myofibers as a function of the duration (0-180 minutes) of in vitro total ischemia in dog heart papillary muscle. Breakages of Z-band, plasmalemmal attachment complexes were few before 120 minutes of ischemia, but thereafter became more prevalent; the transition between the initial rate of appearance of the breaks and the later fast rates coincided with the appearance of severe cell swelling, ischemic contracture, and ultrastructural criteria of irreversible ischemic injury. Z-band, plasmalemmal attachment complex breakage and cell swelling resulted in formation of subsarcolemmal blebs. Two major bleb types have been discerned on ultrastructural appearance using as the criteria the preservation of integrity of the plasma-lemma and subplasmalemmal leptomeres. The identification of two types of blebs suggests two independent mechanisms of injury, the first directed at Z-band attachments, and the second at the cytoskeletal structures of A- and I-band regions of the plasmalemma.     

Contribution of I Ks to ventricular repolarization in canine myocytes

The role of the slow delayed rectifier K⁺ current (I _Ks) in cardiac repolarization seems to be largely influenced by the experimental conditions including the species and tissue studied. The aim of this study was to determine the contribution of I _Ks to repolarization in canine ventricular myocytes by measuring the frequency dependent action potential lengthening effect of 10 μM chromanol 293B using sharp microelectrodes. Pretreatment with isoproterenol (2 nM), E-4031 (1 μM), and injection of inward current pulses were applied to modify action potential configuration. Chromanol alone caused moderate but statistically significant lengthening of action potentials at cycle lengths longer than 500 ms. The lengthening effect of chromanol, which was strongly enhanced in the presence of either isoproterenol or E-4031, was proportional to the amplitude of plateau, whereas poor correlation was found with action potential duration. Similar results were obtained when action potential configuration was modified by injection of depolarizing current pulses. Computer simulations revealed that activation of I _Ks is a sharp function of the plateau amplitude within the physiological range, while elongation of repolarization may enhance I _Ks only when it is excessive. It was concluded that the effect of I _Ks on ventricular repolarization critically depends on the level of action potential plateau; however, other factors, like action potential duration, cycle length, or suppression of other K⁺ currents can also influence its contribution.     

Alterations in behavior in adult offspring mice following maternal inflammation during pregnancy

Maternal intrauterine inflammation during pregnancy poses a major threat of neurodevelopmental brain damage in offspring and may cause poor cognitive and perceptual outcomes. In mice, we have previously shown that maternal inflammation induced by lipopolysaccharide (LPS) at gestation day 17th increased the levels of the pro-inflammatory cytokine IL-6 in the fetal brain. In this study, we used the same system and examined the effect of short, systemic maternal inflammation on anxiety and social behavior of the offspring. Adult offspring from the maternal inflammation group showed increased anxiety, as indicated by the elevated plus maze. Social interaction among offspring from the test groups was examined when two unfamiliar mice from different litters were introduced into a new home-cage. Offspring from the maternal inflammation group showed reduced activity, indicating increased fear. In addition, offspring from the maternal inflammation group were less aggressive towards their cagemates and they spent a significantly longer time trimming the whiskers of their cagemates during the first 30 min of their interaction, compared to offspring from the control group. Our data suggest that short systemic maternal inflammation have long-lasting consequences on the adult mouse stress and social behavior.     

Calcium channel heterogeneity in canine left ventricular myocytes

Regional variations in the electrophysiological properties of myocytes across the left ventricular wall play an important role in both the normal physiology of the heart and the genesis of arrhythmias. To investigate the possible contributions of calcium channels to transmural electrical heterogeneity, whole-cell patch-clamp recordings were made from isolated canine epicardial and endocardial left ventricular myocytes. Two major differences in Ca²⁺ channel properties were found between epi- and endocardial cells. First, the L-type Ca²⁺ current was larger in endocardial than in epicardial myocytes. The average peak current density at +10 mV in endocardial myocytes was  3.4 ± 0.2 pA pF⁻¹  , and was 45 % higher than that in epicardium (  2.3 ± 0.1 pA pF⁻¹  ). The kinetic properties of the L-type current in epi- and endocardial cells were not significantly different. Second, a low-threshold, rapidly activating and inactivating Ca²⁺ current that resembled the T-type current was present in all endocardial myocytes but was small or absent in epicardial myocytes. This T-like current had an average peak density of  0.5 pA pF⁻¹  at −40 mV in endocardial cells. In most endocardial cells the T-like Ca²⁺ current comprised two components: a Ni²⁺-sensitive T-type current and a tetrodotoxin-sensitive Ca²⁺ current. We conclude that there are considerable regional variations in the density and properties of Ca²⁺ channels across the canine left ventricular wall. These variations may contribute to the overall transmural electrical heterogeneity.     

降钙素基因相关肽和心房肽对心肌缺血犬冠脉循环及离体冠脉的影响

本实验利用冠脉内给药和离体血管灌流等方法，观察并比较了降钙素基因相关肽（ＣＧＲＰ）和心房肽（ＡＮＰ）对犬冠脉循环的影响及心肌缺血后冠脉对二者反应性的变化。结果表明，冠脉内注射ＣＧＲＰ或ＡＮＰ均能明显逆转急性心肌缺血所致冠脉血流量的显著减少和大小冠脉阻力的显著增加，其中对远端小冠脉阻力降低的程度明显大于犬冠脉，而且ＣＧＲＰ的上述作用显著强于ＡＮＰ。ＣＧＲＰ和ＡＮＰ均可呈剂量依赖性舒张正常及心肌缺血后的大、小冠脉血管；二者对大冠脉的舒张作用显著弱于小冠脉，而ＣＧＲＰ的舒血管效应又强于ＡＮＰ。心肌缺血后大冠脉对ＣＧＲＰ和ＡＮＰ反应性显著降低，而小冠脉无此现象。结果提示，ＣＧＲＰ是一种强有力的内源性扩血管物质，对缺血性心脏病的治疗有益。     

Occurence of lipid bodies in canine type II pneumocytes during hypothermic lung ischemia

Type II pneumocytes defend the pulmonary alveolus by synthesis and secretion of surfactant and by contributing to alveolar epithelial regeneration. Lipid bodies are regarded as intracellular domains for the synthesis of eicosanoid mediators that can be induced by inflammatory stimuli. The aim of the present study was to establish whether hypothermic ischemic lung storage without further preservation measures leads to an induction of lipid body formation in canine type II pneumocytes. The lungs of 18 dogs were fixed for transmission electron microscopy (TEM) immediately after cardiac arrest (six double lungs) and after ischemic storage in Tutofusin solution at 4 degrees C for 20 min, 4 hr, 8 hr, and 12 hr (six single lungs, respectively). Type II pneumocytes were analyzed qualitatively by conventional TEM (CTEM) and quantitatively by stereology. The relative phosphorus content of surfactant containing lamellar bodies, lipid bodies, and intermediate forms was investigated by energy-filtering TEM (EFTEM). By CTEM, lipid bodies as well as forms intermediate between lipid bodies and lamellar bodies were already noted in the control group but were more pronounced in the ischemia groups. Beginning at 20 min of ischemic storage, a significant increase in the volume density of lipid bodies was noted in the ischemic groups as compared to the control group. By EFTEM, the highest intracellular phosphorus signals were recorded over lamellar bodies and lamellar areas of intermediate forms in all experimental groups, while lipid bodies and homogeneous areas of intermediate forms did not show a clear phosphorus signal. These results indicate that the formation of lipid bodies in canine type II pneumocytes is induced early during ischemic lung storage.     

Restoration of myocardial contractility during gradual reperfusion after total ischemia

Institute of Experimental Cardiology, All-Union Cardiological Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. N. Smirnov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 4, pp. 407–409, April, 1989.     

成年比格犬骨髓间质干细胞体外软骨方向分化的实验研究

目的：体外诱导成年比格犬骨髓间质干细胞（BMSCs）定向分化为软骨细胞，探讨体外诱导成软骨的方法和条件。方法:比格犬股骨取骨髓10 mL，体外行原代和传代培养扩增，加入转化生长因子（TGF-β1），以高密度细胞团块培养，诱导BMSCs分化为软骨细胞。甲苯胺蓝染色检测软骨基质的分泌，免疫组织化学染色检测软骨特异性Ⅱ型胶原表达。结果:诱导的软骨样组织甲苯胺蓝染色阳性;Ⅱ型胶原免疫组织化学检测阳性。结论： 应用含TGF-β1的诱导液在体外可以诱导比格犬BMSC分化为软骨细胞，诱导的软骨细胞可作为软骨组织工程较理想的种子细胞。     

Alterations in immunological reactivity during pregnancy in mice determined in vitro by lymphoproliferation tests

Primiparous and multiparous mice, either syngeneically or allogeneically pregnant, were sacrificed at various stages of pregnancy, and the immune reactivity of cells from different lymphatic organs was analyzed by mixed lymphocyte reaction (MLR) and concanavalin A (Con A)-induced lymphocyte proliferation. In the MLR, spleen cells and cells from the axillary lymph nodes of pregnant animals showed similar changes in their response to allogeneic cells during the course of gestation. In comparison to age-matched virgin controls they had an increased reactivity between the 7th and 11th day of pregnancy. During the preimplantation period and the last week of pregnancy, their alloreactivity was comparable to that of the controls. Cells from the para-aortic lymph nodes, which drain the uterus, also showed an increased reactivity at mid-gestation, but in the preimplantation period and third week of pregnancy their alloreactivity was even decreased in comparison to the control animals. When T cell immunocompetence was measured by means of lymphocyte transformation induced with Con A, the pattern of reactivity was completely different. Lymphoid cells from all the above-mentioned tissues showed the highest response to Con A during the preimplantation period, constantly weakening towards term.     

Role of α2-adrenoceptors in brain resistance to total ischemiaadrenoceptors in brain resistance to total ischemia

Neuroprotective effect of epinephrine is revealed against the background of prazosin, β-antagonists and their combinations protect against cerebral ischemia. α₂-Antagonists (but not α₁) block the neuroprotective effect of these combinations, methyldopa, guanabenz, clonidine, and oxymetazoline and decrease brain resistance to ischemia. α₂-Adrenoreceptors participate in the endogenic mechanism of brain resistance to ischemia and in the neuroprotective effect. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 10, pp. 413–416, October, 1997     

Irreversible injury of isolated adult rat myocytes. Osmotic fragility during metabolic inhibition.

Isolated myocytes can be established as a valid model for studying changes in cytoskeletal proteins during the development of irreversible injury only if isolated cells develop lesions similar to those that occur during irreversible injury to intact hearts, specifically osmotic fragility and subsarcolemmal blebs. In the first experiment, isolated cells were irreversibly injured by metabolic inhibition with 5 mM Iodoacetic acid (IAA) and 6 mM amobarbital (Amy). Osmotic fragility of control and injured cells was determined by comparing the rates of development of trypan blue permeability during 60 minutes of isotonic or hypotonic (50% reduction in osmolality) incubations. Cell morphology was monitored by light and electron microscopy. Control cells remained elongated and excluded trypan blue. Metabolically inhibited cells rapidly contracted to a nearly square shape. The inhibited squared cells initially excluded trypan blue, but during 60 minutes of incubation became permeable to trypan blue. Cells in hypotonic buffer developed blue staining at a more rapid rate than cells in isotonic buffer, indicating increased osmotic fragility. In a second experiment, control and inhibited cells were first incubated for 25 minutes in isotonic buffer and then in either isotonic or hypotonic buffer. In this experiment, inhibited cells also developed more extensive and rapid permeability increases when transferred to the hypotonic buffer than cells maintained in the isotonic buffer. In both experiments, increased permeability of cells to trypan blue was accompanied by formation of subsarcolemmal blebs along the lateral cell border and at the intercalated disks. The results show that metabolically inhibited, isolated myocytes do exhibit morphologic lesions and increased osmotic fragility properties similar to those reported during anoxic or ischemic injury to intact hearts. Therefore, isolated myocytes may be a useful model with which to study cytoskeletal-sarcolemmal membrane changes during development of irreversible injury.     

Electron microscopic cytochemical studies on cardiac myocytes in acute ischemia

In acute myocardial ischemia induced by coronary occlusion in the canine heart, mild to moderate ultrastructural changes such as reduction in glycogen granules appeared around 20 min after coronary occlusion. Severe, irreversible alterations, including a mitochondrial dense deposit, appeared after 60 min. With cytochemical study, ATPase activity decreased at 30 min in the sarcoplasmic reticulum (SR) and on the myofibril preceded by activation of acid phosphatase activity in lysosomes and SR. Permeation of lanthanum ion from glycocalyx into the cytosol was observed 30 min after occlusion. These results indicate that activation of acid hydrolases and an inflow of excess Ca⁺⁺ are likely to be two main causes of ischemic necrosis.     

Transformation of adult rat cardiac myocytes in primary culture

We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long-term cell culture. Morphometric parameters, sarcomere length, T-tubule density, cell capacitance, L-type calcium current (I(Ca,L)), inward rectifier potassium current (I(K1)), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 +/- 70 microm(2) on day 0 to 1432 +/- 50 microm(2) on day 5), T-tubule density, systolic shortening (from 0.11 +/- 0.02 to 0.05 +/- 0.01 microm) and amplitude of calcium transients (from 1.54 +/- 0.19 to 0.67 +/- 0.19) over 5 days of culture. The negative force-frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 +/- 8 to 105 +/- 11 pF) and membrane currents were also reduced (I(Ca,L), from 3.98 +/- 0.39 to 2.12 +/- 0.37 pA pF; and I(K1), from 34.34p +/- 2.31 to 18.00 +/- 5.97 pA pF(-1)). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 +/- 2.5 to 34.2 +/- 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.     

Protective effect of intracerebroventricular injection of adenosine agonists during total cerebral ischemia

Adenosine and its analogs N⁶-cyclohexyladenosine (CHA) and N-ethylcarboxamideadenosine (NECA) are highly effective when injected into the lateral ventricle in very low doses (by 1–2 orders lower than subcutaneously). The protective effect of A-agonists is considered to be mediated by a central mechanism and realized via A-receptors of the brain: the effects of low doses of A-agonists are mediated by A₁-receptors, whereas higher doses exert an extra A₂-effect. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 6, pp. 622–624, June, 1994 Presented by P. V. Sergeev, Member of the Russian Academy of Medical Sciences