低压恒流灌流分离成年大鼠心肌细胞

目的:探寻合适的初始灌流压力与定量确定酶消化终止点,以提高恒流灌注分离成年大鼠心肌细胞的产量与质量.方法:采用Langendorff装置,行主动脉插管逆向灌流,0.08%胶原酶Ⅰ消化大鼠心脏,监测灌流压力的变化.结果:调节灌流流速以15 kPa初始压力恒流灌流时(n=4),灌流压力明显升高(压力峰值＞25 kPa),造成左心室在松弛状态下明显扩张,使分离的心肌细胞存活率降低,且收缩功能显著降低.以10 kPa初始压力行恒流灌流时,酶消化引起的压力升高均低于18.75 kPa,左心室扩张不明显;当酶消化过程中压力降至10 kPa(n=3)或5kPa(n=4)时终止消化,心肌细胞分别呈消化不足或过度的形态,且心肌细胞存活率均低于10%,恢复正常细胞外液钙离子浓度后,大部分心肌细胞死亡.当酶消化时灌流压降至7.5 kPa时终止消化(n=15),分离即刻心肌细胞存活率为82.6%±4.8%,复钙后心肌细胞存活率为30.4%±4.5%,复钙后4 h的存活率仍为24.8%±5.4%.细胞形态完好,边缘锐利、横纹清晰,无明显博动,收缩功能正常.结论:为获得存活率较高的成年大鼠心肌细胞,宜采用低压恒流灌流分离方法,即初始灌流压力保持在10 kPa,胶原酶Ⅰ消化的终止压力为7.5 kPa.     

犬心肌细胞分离方法的改良

目的:建立高效可靠的成年犬心肌细胞分离方法,获得高产量与高质量的心肌细胞,以便进行犬心肌细胞收缩功能的研究.方法:采用改良的Langendorff灌流胶原酶消化法分离得到左心室心肌细胞.荧光显微镜观察细胞生长状态和形态并利用单细胞收缩动态边缘检测系统测定心肌细胞收缩功能的改变.结果:结果显示,与传统方法相比,即刻分离的心肌细胞状态良好,复钙后的心肌细胞成活率达到70％-80％,转染重组腺病毒β2-EGFP培养48 h,心肌细胞成活率为60％-70％.给予持续电场刺激,心肌细胞可以保持收缩节律和幅度稳定30 min以上.结论:该方法操作简单,分离的活细胞产量高,质量好,节约实验成本和时间,为心血管相关研究提供良好的细胞模型.     

成年小鼠心肌细胞的分离培养及鉴定

目的探讨成年小鼠心肌细胞分离培养的方法,评价心肌细胞的存活状况。方法应用改良Langendorff方法进行成年小鼠心脏灌流,分离并培养心肌细胞,评价杆状心肌细胞的比例变化。台盼蓝染色判定心肌细胞活力,应用毒胡萝卜素(thapsigargin)治疗心肌细胞24 h后,WST方法测定细胞生存率。结果在每个成年小鼠心脏分离了40~60万的心肌细胞,台盼蓝染色结果68%为存活心肌细胞。心肌细胞培养1 h2、4 h及48 h后,杆状心肌细胞的比例分别为70%、50%及30%。心肌细胞生存率随着毒胡萝卜素浓度的增加逐渐减低,其中1、5及10μmol/L毒胡萝卜素的生存率明显低于对照组(P<0.05,P<0.01)。结论应用改良Langendorff方法及严格控制心脏灌流培养的条件,可以成功地分离培养成年小鼠心肌细胞,有助于进行细胞分子水平的研究。     

成年大鼠心肌细胞分离培养及兴奋-收缩耦联表征

目的：比较两种细胞分离液分离成年大鼠心肌细胞,进一步表征成年大鼠心室肌细胞兴奋-收缩耦联。方法 Langendorff装置进行主动脉逆流灌流,分别用两种细胞分离液分离成年大鼠心肌细胞,无血清培养并进行腺病毒感染。显微镜下观察单个心肌细胞的形态学特点,荧光显微镜下检测病毒感染。采用IonOptix仪器检测心肌细胞肌节收缩-舒张指标以及心肌细胞钙离子摄入-排出指标。结果两种分离液均可获得70％横纹清晰的长杆状心肌细胞,培养可存活7 d以上。腺病毒感染48 h,绿色荧光蛋白持续表达7 d以上。分离液一获得的心肌细胞不能很好地随电场刺激产生收缩,分离液二获得的细胞可用于检测兴奋-收缩耦联特性,心肌细胞肌节缩短分数为11.61％±2.15％,舒张时间为（0.177±0.031） s,钙瞬变幅度为30.79％±9.74％,钙瞬变衰减时间为（0.300±0.074） s。结论两种分离液均可用于分离和培养成年大鼠心肌细胞,并用于腺病毒转染等长时程研究。分离液二更适用于检测成年大鼠心肌细胞的兴奋-收缩耦联特性。     

Ⅱ型胶原酶加压灌流提高成年大鼠心肌细胞分离效率

目的探讨效率更高的成熟心肌细胞分离方法。方法采用Ⅱ型胶原酶升主动脉逆行灌流法分离成年大鼠心肌细胞。对照组采用Langendorff装置灌流;实验组在Langendorff装置基础上加压匀速灌流。在倒置显微镜下观察细胞形态,并计算杆状细胞比率及产量;通过台盼蓝染色和局部场剌激评价心肌细胞活性。结果分离即刻,实验组杆状细胞比率显著高于对照组【(90.3±4.4)%νs.(53.4±5.2)%,P<0.01】;实验组活细胞产量也显著高于对照组[(3.6±0.7)×107νs.(1.9±0.6)×107,P<0.01]。复钙过程中,实验组发生自发性收缩的细胞比率及变圆细胞比率均明显低于对照组【(10.4±2.1)%νs.(18.9±4.5)%,P<0.01;(5.3±1.3)%νs.(8.6±1.7)%,P<0.05】。实验组台盼蓝染色阴性的杆状细胞比率和局部场剌激条件下收缩细胞比率明显高于对照组【(95.3±8.2)%νs.(90.6±9.8)%,P<0.05;(92.2±7.6)%νs.(85±6.4)%,P<0.01】。结论Ⅱ型胶原酶加压灌流可获得高产量、高活性的心肌细胞,提高了成熟心肌细胞的分离效率。     

模拟失重降低大鼠心肌细胞对异丙肾上腺素的反应性

本文旨在研究模拟失重对大鼠单个心肌细胞无负荷收缩功能的影响以及对异丙肾上腺素(isoproterenol,ISO)反应性的变化.采用人鼠尾部悬吊法在地面模拟失重状态,4周后以胶原酶I消化分离心肌细胞,分别对左、右两心室心肌细胞进行收缩功能测量.结果显示,悬吊4周大鼠(悬吊组)左,右心室心肌细胞的长度和宽度与正常大鼠(对照组)相比均无显著差异.随刺激频率增加,对照组与悬吊组大鼠心肌细胞缩短幅值均逐步增加.在1.0、2.0与4.0 Hz刺激下,对照组大鼠左心室心肌细胞缩短幅值分别为(8.50±1.26)%、(9.00±1.38)%与(9.23±1.83)%,右心室心肌细胞缩短幅值分别为(9.80±2.48)%、(10.03±2.48)%与(10.28±2.27)%;与对照组大鼠相比,在1.0与2.0Hz刺激下,悬吊组大鼠左心室心肌细胞无负荷缩短幅值分别降低12.2%、10.9%(P《0.05),右心室则分别降低16.5%、16.3%(P《0.05);但是在4.0 Hz刺激下却无显著性改变.与同一频率刺激下的对照组大鼠相比,悬吊组大鼠左、右心室心肌细胞达到缩短峰值的时程(time to peak shortening,TPS)明显缩短(P《0.05);而从缩短峰值至75%舒张的时程(TR75)则明显延长(P《0.05).在各刺激频率下,悬吊组大鼠左、右心室心肌细胞缩短(+dL/dtmax)与舒张(-dL/dtmax)速度均未发生明显改变.用1、5、10 nmol/L ISO灌流达稳态水平后,对照组大鼠心肌细胞缩短幅值分别增加了(10.63±0.83)%、(35.06±5.22)%和(71.64±6.83)%;而悬吊组大鼠心肌细胞缩短幅值仅增加(5.75±0.76)%、(23.97±4.50)%和(26.38±8.13)%,均有显著性差异(P《0.05,P《0.01).用10、50、100 nmol/L forskolin 灌流达稳定水平后,对照组大鼠心肌细胞缩短幅值分别增加了(3.04±0.27)%、(9.81±2.66)%、(20.20±3.47)%;而悬吊组大鼠心肌细胞缩短幅值仅增加了(1.42±0.53)%、(3.83±1.71)%、(5.49±4.08)%,均有显著性差异(P《0.05).以上结果表明,模拟失重4周降低人鼠心肌细胞无负荷缩短幅值以及对ISO的反应性.     

蛋白激酶C对自发性高血压大鼠肥大心肌细胞无负荷收缩功能的调节

在慢性压力超负荷引起心肌肥大过程中,蛋白激酶C(protein kinase C,PKC)的激活起关键性作用,激活的PKC也能调节心肌收缩性能.本文旨在研究自发性高血压大(spontaneously hypertensive rat,SHR)心肌肥大的不同阶段PKC调节心肌收缩性能的特征.采用胶原酶法分离4月龄与10月龄Wistar-Kyoto(WKY)、SHR大鼠的心肌细胞,观测单个心肌细胞无负荷缩短幅值以及在PKC激动剂与抑制剂作用下心肌收缩性能的变化.结果表明:刺激频率从1 Hz增至3 Hz,WKY大鼠心肌细胞无负荷缩短幅值逐渐增加,呈正阶梯效应;4月龄SHR大鼠心肌细胞的缩短幅值较WKY大鼠增强,但在各刺激频率下其缩短幅值基本保持不变;10月龄SHR大鼠心肌细胞的缩短幅值在1 Hz刺激条件下与WKY大鼠无差别,随刺激频率增加,缩短幅值降低,呈负阶梯效应.在PKC激动剂PMA灌流条件下,50、100与200 nmol/L的PMA分别降低WKY大鼠心肌细胞缩短幅值至(69.8±1.9)%、(58.2 2.2)%与(22.7±2.5)%(均P<0.01),呈浓度依赖关系;PMA对4月龄SHR大鼠心肌细胞缩短幅值的降低更明显,分别降至(6.1±0.7)%、(2.4±0.2)%与(12.5±2.6)%(均P<0.01);PMA降低10月龄SHR大鼠心肌细胞缩短幅值至(65.7±1.6)%、(53.9±4.0)%与(16.3±2.0)%(均P<0.01),小于对4月龄SHR大鼠心肌细胞缩短幅值的作用.PKC抑制剂staurosporine增加WKY大鼠心肌细胞缩短幅值,在200 nmol/L的staurosporine灌流条件下,WKY大鼠、4月龄SHR大鼠、10月龄SHR大鼠心肌细胞缩短幅值分别增JJH(63.63±4.53)%、(80.82±4.61)%、(80.97±4.59)%(均P<0.05).结果提示,在SHR大鼠心肌肥大初期,具有负性肌力作用的PKC异构体可能被激活,并参与对心肌收缩性能的调节;而心肌肥大稳定阶段,这些PKC活性可能恢复至正常水平.     

δ-阿片受体在缺血后处理心肌保护中的作用

目的：探讨δ-阿片受体是否参与缺血后处理对抗心肌缺血/复灌（I/R）损伤和心肌细胞低氧/复氧（H/R）损伤作用及其机制。方法：采用离体大鼠心脏Langendorff灌流模型,全心停灌30 min、复灌120 min复制I/R模型。测定心室力学指标和复灌时冠脉流出液中乳酸脱氢酶（lactate dehydrogenase,LDH）活性,实验结束测定心肌组织formazan含量。酶解分离的心肌细胞采用低氧60min、复氧60min复制H/R模型,测定心肌细胞存活率。结果：在离体心脏模型上,与I/R组相比,缺血后处理组（停灌后复灌即刻立即给予6次全心停灌/复灌循环）心肌组织的formazan含量明显增高,复灌期间冠脉流出液中LDH明显降低,同时缺血后处理明显改善心室力学指标,缓解冠脉流量的减少 在分离心肌细胞模型上,低氧后处理明显提高心肌细胞存活率。δ-阿片受体阻断剂naltrindole（NTI）和线粒体钙激活钾通道（KCa）阻断剂paxilline（Pax）在离体大鼠心脏模型和分离心肌细胞模型上均能明显减弱缺血后处理的作用。在心肌细胞模型上,与H/R组相比,δ-阿片受体激动剂DADLE明显提高心肌细胞存活率,其作用可被paxilline所阻断。结论：缺血后处理具有抗心肌缺血/复灌损伤的作用,这种保护作用可能与其激活δ-阿片受体和开放KCa有关。     

小鼠胚胎心肌细胞的分离及电生理特性

本文旨在探索小鼠胚胎心肌细胞的分离方法并观察其电生理特性。应用胶原酶B消化法获得不同时期单个小鼠胚胎心肌细胞;利用全细胞膜片钳技术,记录胚胎心肌细胞的超极化激活的非选择性内向阳离子电流(If)和L-钙电流(ICa-L),并用电流钳记录其自发性动作电位。胚胎心肌细胞通过相差显微镜依据其形态和自发性收缩进行鉴定。本法分离所获得的胚胎心肌细胞容易进行全细胞膜片钳记录,可用于记录If,ICa-L．电流和自发性动作电位,己证实胚胎心肌细胞If和Ica-L的电生理特性与成年起搏细胞或心肌细胞相似。本实验建立的分离方法简单、稳定、有效、可靠,最早可获得8．5d的胚胎心肌细胞。胚胎心肌细胞的电生理记录为探索胚胎心肌细胞的电生理特性提供了一个可用的模型,并可能为某些心脏疾病产生的机制提供实验依据。     

可视化动缘探测系统检测心肌细胞舒缩功能

目的:分离成年大鼠心室肌细胞,利用可视化动缘探测系统检测其收缩/舒张功能.方法:常规酶解法分离制备钙耐受心肌细胞;用Ca2 荧光探剂fura-2/AM(0.5μmol/L)负载心肌细胞30 min(25℃)后,以含氧台氏液灌流并予电场刺激(0.5 Hz,5 ms),采用动缘探测系统检测心肌细胞收缩/舒张功能及细胞内荧光信号变化.结果:可视化动缘探测系统可实时同步观察和记录心肌细胞机械功能和细胞内钙瞬变变化,并可计算得到细胞收缩/舒张和钙瞬变的变化幅度、时程及速率等一系列指标,从而直接反映细胞水平的心肌功能改变.结论:可视化动缘探测系统为人们提供了一个在单心肌细胞水平探讨心肌肌源性功能及其改变的新方法,是当今心血管研究的一项重要技术.     

Variation of mechanical properties along the length of the aorta in C57bl/6 mice

The objective of the present study is to obtain a systematic set of data on the mechanical properties along the entire length of the mouse aorta. The ascending aorta of seven mice was cannulated near the aortic valve, and the aorta was preconditioned with several cyclic changes in pressure. The perfusion pressure was then increased in 30-mmHg increments from 0 to 150 mmHg. Cab-O-Sil, colloidal silica, was mixed into the perfusate to prevent flow through the microvessels and hence attain zero-flow distensions. Our results show that the residual circumferential strain leads to a uniformity of transmural strain of the aorta in the loaded state along the entire length of the aorta. This uniformity is attained in the range of 60-120 mmHg. At pressures <60 mmHg, the outer strain is greater than the inner strain, whereas at pressures >120 mmHg, the converse is true. Furthermore, we found that the circumferential and longitudinal stress-strain relationships are linear in the pressure range of 30-120 mmHg. Finally, the circumferential modulus is greatest (most rigid) near the diaphragm, and the majority of volume compliance (85%) is in the thoracic compared with the abdominal aorta. These findings are important for an understanding of the hemodynamics of the cardiovascular system of the normal mouse and will serve as a reference state for the study of various diseases in knock-in and knock-out models of this species.     

Mechanosensitive cardiac C-fiber response to changes in left ventricular filling, coronary perfusion pressure, hemorrhage, and volume expansion in rats

Left ventricular (LV) end-diastolic pressure (LVEDP) increase due to volume expansion (VExp) enhances mechanosensitive vagal cardiac afferent C-fiber activity (CNFA), thus decreasing renal sympathetic nerve activity (RSNA). Hypotensive hemorrhage (hHem) attenuates RSNA despite decreased LVEDP. We hypothesized that CNFA increases with any change in LVEDP. Coronary perfusion pressure (CPP), supposedly affected in both conditions, might also be a stimulus of CNFA. VExp and hHem were performed in anesthetized male Sprague-Dawley rats while blood pressure, heart rate, and RSNA were measured. Cervical vagotomy abolished RSNA response in both reflex responses. Single-unit CNFA was recorded while LVEDP was changed. Rapid changes (+/- 4, +/-6, +/-8 mmHg) were obtained by graded occlusion of the caval vein and descending aorta. Prolonged changes were obtained by VExp and hHem. Furthermore, CNFA was recorded in a modified Langendorff heart while CPP was changed (70, 100, 40 mmHg). Rapid LVEDP changes increased CNFA [caval vein occlusion: +16 +/- 3 Hz (approximately +602%); aortic occlusion: +15 +/- 3 Hz (approximately +553%); 70 units; P < 0.05]. VExp and hHem (n = 6) increased CNFA [VExp: +10 +/- 4 Hz (approximately +1,033%); hHem: +10 +/- 2 Hz (approximately +1,225%); P < 0.05]. An increase in CPP increased CNFA [+2 +/- 1 Hz (approximately +225%); P < 0.05], whereas a decrease in CPP decreased CNFA [-0.8 +/- 0.4 Hz (approximately -50%); P < 0.05]. All C fibers recorded originated from the LV. CNFA increased with any LVEDP change but changed equidirectionally with CPP. Thus neither LVEDP nor CPP fully accounts directly for afferent C-fiber and reflex sympathetic responses. The intrinsic afferent stimuli and receptive fields accounting for reflex sympathoinhibition still remain cryptic.     

Less afterload sensitivity in short-term hibernating than in acutely ischemic and stunned myocardium

Short-term hibernating myocardium is characterized by reduced contractile function during persistent moderate ischemia, the recovery of metabolic parameters, and the absence of necrosis. To study the afterload dependence of regional wall excursion in short-term hibernating myocardium, in 11 enflurane-anesthetized swine the left anterior descending coronary artery was cannulated and hypoperfused for 90 min to reduce anterior systolic wall thickening (WT, sonomicrometry) by 60%. Under control conditions, at 5 and 90 min ischemia the descending thoracic aorta was acutely constricted to increase left ventricular (LV) pressure by 30 mmHg. Under control conditions, increased LV pressure resulted in decreased WT [i.e., a negative slope of the relationship between WT and LV end-systolic pressure: -11.2 +/- 4.2 (SD) microm/mmHg]. This slope was further significantly decreased at 5 min ischemia (-26.5 +/- 8.8 microm/mmHg) but returned toward control values in short-term hibernating myocardium at 90 min ischemia (-17.2 +/- 6.6 microm/mmHg). At 30 min reperfusion, the slope was once more significantly decreased (-27.8 +/- 8.1 microm/mmHg). In conclusion, WT in short-term hibernating myocardium is less afterload dependent than in acutely ischemic and reperfused myocardium.     

Mechanical function and fatty acid oxidation in the neonatal pig heart with ischemia and reperfusion

We investigated mechanical function and exogenous fatty acid oxidation in neonatal pig hearts subjected to ischemia, followed by reperfusion. Isolated, isovolumically-beating hearts, from pigs 12 h to 2 days of age, were perfused with an erythrocyte-enriched (hematocrit approximately 15%) solution (37 degrees C). All hearts were studied for 30 min. with a perfusion pressure of 60 mmHg (pre-ischemia). One group of hearts (low-flow ischemia, N = 12) was then perfused for 30 min. with a perfusion pressure of approximately 12 mmHg. In the other group (no-flow ischemic arrest, N = 9), the perfusion pressure was zero for 30 min. Following ischemia in both groups, the perfusion pressure was restored to 60 mmHg for 40 min. (reperfusion). Pre-ischemia parameters for all hearts averaged: left ventricular peak systolic pressure, 99.0 +/- 2.0 mmHg; end diastolic pressure, 1.9 +/- 0.2 mmHg; coronary flow, 3.4 +/- 0.1 ml/min per g; myocardial oxygen consumption, 56.6 +/- 1.6 microliter/min per g and fatty acid oxidation, 33.4 +/- 1.4 nmol/min per g. During low-flow ischemia, hearts released lactate, and the corresponding parameters decreased to: 30.7 +/- 0.9 mmHg; 1.2 +/- 0.3 mmHg; 0.8 +/- 0.1 ml/min per g; 26.6 +/- 2.3 microliters/min per g and 12.9 +/- 1.1 nmol/min per g, respectively. Early in reperfusion in both groups, all parameters, except for fatty acid oxidation, exceeded pre-ischemia values, before recovering to near pre-ischemia values. Late in reperfusion, however, rates of fatty acid oxidation exceeded pre-ischemia rates by approximately 60%. Thus, the neonatal pig heart demonstrated similar recovery following 30 min of low-flow ischemia or no-flow ischemic arrest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circulating catecholamines and cardiorespiratory responses in hypoxic lungfish (Protopterus dolloi): a comparison of aquatic and aerial hypoxia

Circulating catecholamine levels and a variety of cardiorespiratory variables were monitored in cannulated bimodally breathing African lungfish (Protopterus dolloi) exposed to aquatic or aerial hypoxia. Owing to the purported absence of external branchial chemoreceptors in lungfish and the minor role played by the gill in O2 uptake, it was hypothesized that plasma catecholamine levels would increase only during exposure of fish to aerial hypoxia. The rapid induction of aquatic hypoxia (final PWo2 = 25.9+/-1.6 mmHg) did not affect the levels of adrenaline (A) or noradrenaline (NA) within the plasma. Similarly, none of the measured cardiorespiratory variables--including heart rate (fH), blood pressure, air-breathing frequency (fV), O2 consumption (Mo2), CO2 excretion (Mco2), or blood gases--were influenced by acute aquatic hypoxia. In contrast, however, the rapid induction of aerial hypoxia (inspired Po2=46.6+/-3.3 mmHg) caused a marked increase in the circulating levels of A (from 7.9+/-2.0 to 18.8+/-6.1 nmol L(-1)) and NA (from 7.7+/-2.2 to 19.7+/-6.3 nmol L(-1)) that was accompanied by significant decreases in Mo2, arterial Po2 (Pao2), and arterial O2 concentration (Cao2). Air-breathing frequency was increased (by approximately five breaths per hour) during aerial hypoxia and presumably contributed to the observed doubling of pulmonary Mco2 (from 0.25+/-0.04 to 0.49+/-0.07 mmol kg(-1) h(-1)); fH and blood pressure were unaffected by aerial hypoxia. An in situ perfused heart preparation was used to test the possibility that catecholamine secretion from cardiac chromaffin cells was being activated by a direct localized effect of hypoxia. Catecholamine secretion from the chromaffin cells of the heart, while clearly responsive to a depolarizing concentration of KCl (60 mmol L(-1)), was unaffected by the O2 status of the perfusion fluid. The results of this study demonstrate that P. dolloi is able to mobilize stored catecholamines and increase f(V) during exposure to aerial hypoxia while remaining unresponsive to aquatic hypoxia. Thus, unlike in exclusively water-breathing teleosts, P. dolloi would appear to rely solely on internal/airway O2 chemoreceptors for initiating catecholamine secretion and cardiorespiratory responses.     

Cardiac performance of an isolated eel heart: effects of hypoxia and responses to coronary artery perfusion.

The pericardial sac containing the heart was removed from large (2.7-6.3 kg) long-finned eels (Anguilla dieffenbachii). Coronary arteries were cannulated in preparation for perfusion with eel Ringer or red cell suspensions. The hearts were maintained by Ringer perfusion while the performance of the heart was assessed. Responses of the hearts to increases in filling pressure and output pressure were recorded. Maximum cardiac output was 22.3 +/- 1.4 ml/min/kg body mass (mean +/- 1 SEM; N = 9). The highest cardiac power output was measured at maximum cardiac output and was 3.39 +/- 0.32 mW/g ventricle mass (mean +/- 1 SEM; N = 9). Eel hearts could sustain output pressures near 80 cm H2O, but cardiac output was reduced and cardiac power output was 1.89 +/- 0.24 mW/g ventricular mass (mean +/- 1 SEM; N = 9). Maximum cardiac output decreased by 14% when hearts pumped hypoxic Ringer with a PO2 of 11.5 torr. At high input pressures concomitant with high output pressures (80 cm H2O), cardiac power output decreased by 38% upon exposure to hypoxic Ringer. Coronary perfusion of hypoxic hearts with red cell suspensions (mean hematocrit 10.4%) at a rate of 2% of control cardiac output (0.20 ml/min/kg body mass) had no effect on maximum cardiac output. However, coronary perfusion enabled hypoxic hearts to maintain cardiac output when output pressure was raised to 80 cm H2O. Under conditions of high input pressure and high output pressure, power output increased by 20% compared to hypoxic hearts without coronary perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes’ terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.     

Relationships between cardiac hyperactivity, oxygen tension, and release of vasodilator material in coronary autoregulation of guinea-pig hearts

Increases in cardiac activity induce autoregulatory coronary vasodilation. The intermediate steps which trigger this process are thought to be myocardial hypoxia which induces the release of vasodilator mediator(s). The present study examines the relationships between mechanical activity, oxygen tension, and release of vasodilator material in isolated perfused hearts. Guinea-pig isolated hearts were perfused in series, the effluent from donor hearts being regassed prior to entry to recipient hearts. Histamine (1 microgram) and isoproterenol (10 ng) increased the rate and tension of donor hearts and produced predominant coronary vasodilator responses which were followed by the appearance of vasodilator material in the recipient (falls in perfusion pressure, 9.8 +/- 1.1 and 9.1 +/- 2.5 mmHg) (1 mmHg = 133.322 Pa). Exposure of donor hearts to hypoxia also caused vasodilatation and release of vasodilator material (fall in pressure, 11.4 +/- 1.6 mmHg). Pacing-induced tachycardia (6 Hz) of donor hearts promoted the release of vasodilator material, the fall in recipient heart pressure being 11.5 +/- 1.8 mmHg. This was abolished by beta-adrenoceptor blockade and when donor hearts were from reserpine-pretreated guinea pigs. In was concluded that pacing released endogenous catecholamines which in turn released the vasodilator material. Pacing per se did not cause vasodilatation or release of the vasodilator. The Po2 of perfusates from donor hearts was reduced by pacing at 5 Hz (25.7 +/- 5.2 mmHg) and by isoproterenol (10 ng, 32.0 +/- 3.7 mmHg), indicative of an elevated oxygen extraction. The isoproterenol-induced falls in Po2 were abolished by beta-adrenoceptor blockade. However, the pacing-induced falls in Po2 persisted, the values occurring before (25.7 +/- 5.2 mmHg) and after propranolol (45.7 +/- 4.5 mmHg) and before (32.1 +/- 1.1 mmHg) and after practolol (27.3 +/- 4.1 mmHg) not differing significantly (p greater than 0.05). These falls in perfusate Po2 were not accompanied by coronary vasodilatation or release of vasoactive material. Perfusate Po2 changes could therefore be dissociated from the coronary vasodilatation and vasoactive material release, suggesting that hypoxia may not be a prerequisite for the metabolic autoregulatory vasodilatation in response to myocardial hyperactivity induced by cardiac stimulants.     

Moderate glucose deprivation preconditions myocardium against infarction.

Glucose-free perfusion preconditions myocardium against the consequences of subsequent ischemia. We investigated whether mitochondrial ATP-sensitive potassium (mK (ATP)) channels are involved in preconditioning by glucose deprivation, and whether moderate glucose deprivation also preconditions myocardium. Isolated rat hearts underwent 30 min of no-flow ischemia followed by 1 h reperfusion. Controls were not further treated. Three groups were preconditioned by perfusion with 0, 40 or 80 mg/dl (0, 2.22, 4.44 mmol/l) glucose (correction of osmotic pressure by addition of urea) for 10 min followed by 10 min perfusion with normal buffer (150 mg/dl, or 8.33 mmol/l glucose) before the ischemia reperfusion protocol. In one group, 100 micromol/l of the mK (ATP) channel blocker 5-HD was added to the glucose-free perfusate. Two groups were treated with 5-HD or urea before ischemia without preconditioning. Left ventricular developed pressure and maximum ischemic contracture (82 +/- 21 mmHg) were similar in all groups. Mean left ventricular developed pressure was 100 +/- 16 mm Hg under baseline conditions, and poorly recovered to 8 +/- 11 mm Hg during reperfusion. Preconditioning with 0 and 40 mg/dl glucose containing buffer reduced infarct size from 41 +/- 10% (control) to 23 +/- 12% (p = 0.02) and 26 +/- 8% (p = 0.011). The 5-HD blocked preconditioning by glucose deprivation (38 +/- 9%, p = 0.04) while 80 mg/dl glucose, 5-HD and urea had no effect on infarct size (39 +/- 9%; 38 +/- 13%; 37 +/- 8%; p = 1.0 each). We conclude that transient severe glucose deprivation and moderate glucose deprivation preconditions the isolated rat heart. Preconditioning by complete glucose deprivation depends on the opening of mK (ATP) channels.