大网膜联合组织工程心肌移植改善大鼠心肌梗死后移植细胞的存活

目的 观察大网膜增加组织工程心肌(EHT)血供,改善微环境后对大鼠心肌梗死模型上移植细胞存活的影响.方法 结扎雌性Sprague-Dawley(SD)大鼠左冠状动脉,制作心肌梗死模型.以雄性大鼠骨髓间充质干细胞(MSCs)为种子细胞,种植于共聚乙交酯-丙交酯(poly lactic acid-CO-glycolic ac-id,PLGA)补片上构建EHT.符合心肌梗死标准的动物随机分成网膜EHT组、单纯EHT组、心肌梗死组和假手术组,每组6只鼠.组织工程心肌植入后4周,用TUNEL法检测移植细胞凋亡,标本取材做病理学检查,免疫组化方法检测内皮细胞标志物vWF表达.结果 TONEL检测发现EHT植入后1周网膜EHT组移植细胞凋亡少于单纯EHT组(P<0.05),2周、4周时两组间凋亡无明显差异(P>0.05).vWF染色发现EHT植入后1周、2周、4周网膜EHT组微血管密度均高于单纯EHT组(P<0.05).结论 网膜包裹MSCs-PLGA构建的EHT覆盖于心肌梗死表面后能够通过增加微血管密度,改善局部微环境,减少移植细胞的早期凋亡,促进移植细胞的存活.     

胸段硬膜外阻滞对兔心肌梗死后左心室间质胶原代谢的影响

目的 胸段硬膜外阻滞(TEB)对兔心肌梗死(MI)后左室间质早期重构的影响.方法 成年健康新西兰兔随机均分为三组.各组均建立TEB治疗模型:心肌梗死组(MI组)及胸段硬膜外治疗组(TEB组)均结扎左冠状动脉(LAD)制备心肌梗死模型;假手术组(S组)和MI组于硬膜外推注生理盐水,TEB组于硬膜外推注0.1%罗哌卡因0.5 mg/kg.每日2次.4周后处死动物,保存左室组织,制备VG染色心肌组织切片观察间质胶原变化,用病理图像分析软件测量间质胶原含量,计算胶原容积分数.提取左室前壁心肌组织总RNA,用RT-PCR法检测心肌组织基质金属蛋白酶-2(MMP-2)mRNA表达水平,用明胶酶谱法检测MMP-2酶比活性.结果 TEB组间质胶原生成较MI组大为减少,胶原容积分数(ICVF)TEB组在非梗死区及梗死灶交界区均较MI组明显降低(P<0.01),其中TEB组非梗死区ICVF与S组比较差异无统计学意义;MMP-2 mRNA表达水平TEB组明显低于MI组(P<0.01),TEB组基质金属蛋白酶-2活性形式(AMMP-2)和基质金属蛋白酶-2原形(LMMP-2)比活性较MI组降低(P<0.05).结论 TEB通过影响MI后MMP-2表达及酶活性改变,抑制MI后心肌间质胶原代谢,可能阻止心肌梗死后左心室间质重构.     

心室辅助前后心肌间质改变与预后

目的 探讨心室辅助前、后心肌胶原含量改变与预后的关系.方法 依据心室辅助后病人是否心脏移植分为移植组和撤离组,取左心室心肌行心肌胶原(Sircol法及羟脯氨酸定量)和蛋白测定,比较心室辅助前、后胶原及蛋白含量的差异与不同预后病人的关系.结果 心室辅助前,除总羟脯氨酸外,两种预后病人的胶原和蛋白质含量的差别均有统计学意义(P<0.05),撤离组明显高于移植组;撤离组蛋白质含量甚至高于移植组的辅助后测定值(P<0.05).移植组心室辅助后盐溶胶原及蛋白质和总羟脯氨酸含量较辅助前增加(P<0.05).结论 扩张性心肌病病人心肌间质重塑的含义不同,部分胶原及蛋白质含量增加可能与良好预后有关;心室辅助改善心功能的机制可能与间质胶原改变有关.     

骨髓间质干细胞移植对香猪急性心肌梗死后心肌结构和心功能的影响

目的 移植自体骨髓间质干细胞(BMSCs)到香猪急性心肌梗死区内,研究移植BMSCs对心肌结构和心功能的影响. 方法将24只贵州香猪采用计算器随机法分为实验组(n＝12)和对照组(n＝12),抽取香猪自体骨髓,经体外分离出BMSCs并培养和经5-氮胞苷(5-azacytidine)转化,利用结扎左前降支(LAD)的方法建立急性心肌梗死动物模型,经LAD和梗死区多点注射的方法将实验组香猪注射BMSCs(细胞总数2×106个),对照组注射等量的细胞培养液.3周和6周后,用超声心动图(UCG)观察两组移植后心肌结构和心功能改变的情况. 结果实验组左心室射血分数、左心室短轴缩短率和室壁增厚率明显高于对照组;左心室室壁、室间隔厚度和心室腔的大小在两组之间也存在明显差别,实验组室壁和室间隔厚度明显大于对照组,而心室腔小于对照组. 结论 BMSCs梗死区心肌移植后可减轻心室重构的进程,减轻心肌的变薄程度,使心室腔未明显扩大.BMSCs移植还可增加心肌的收缩力,改善心功能.     

磁性靶向材料介导骨髓间充质干细胞经静脉移植修复梗死心肌

目的探讨用磁性靶向材料介导骨髓间充质干细胞（MSCs）经静脉移植时到达心肌梗死部位的程度以及对心肌梗死修复的影响。方法取体外扩增的第4代MSCs，先测定MSCs的表面标记，用含10μmol的5-氮杂胞苷诱导后，将MSCs细胞核DAPI（4，6-联脒-2-苯基吲哚）染色后备用移植。将28只SD大鼠分为3组，A组：10只，磁性靶向材料和MSCs接触结合后经大鼠尾静脉移植，将磁石接触心肌梗死部位皮肤表面30min后继续饲养；B组：9只，未与磁性靶向材料结合的MSCs经大鼠尾静脉移植；C组：9只，将MScs直接移植心肌梗死部位。于移植2d后检查MSCs在梗死部位聚集的情况，30d后检查心肌梗死部位的功能及形态的改变。结果在透射电子显微镜下观察可见3～5个磁性靶向材料分子和MSCs的细胞膜结合。A组MSCs归巢率为38％，B组6％，C组53％，A组和C组聚集MSC数目明显多于B组（P〈0．01）。A组和C组移植后左心室射血分数（LVEF）、左心室短轴缩短率（LVFS）较移植前明显改善（LVEF46％±6％VS．38％±8％，51％±5％ vs．35％±4％；LVFS28％±6％vs．20％±7％，32％±4％vs．20％±5％，P〈0．05）；光学显微镜下观察梗死心肌内均可找到标记DAPI的移植细胞。B组移植后左心室收缩功能各项指标无明显改善，光学显微镜下在梗死心肌内未找到标记了DAPI的移植细胞（MSCs归巢率为38％）。C组与A组结果比较差异无统计学意义，但实验过程中死亡率较高。结论磁性靶向材料介导MSCs经静脉移植方法能聚集更多的MSCs于梗死心肌部位，减小梗死区面积，有效改善心肌梗死后的心功能。     

骨髓间充质干细胞自体移植治疗心肌梗死的实验研究

目的探讨兔骨髓间充质干细胞(MSCs)移植至缺血心肌后的增殖分化情况,对缺血心肌细胞的修复重建能力及心功能改善情况。方法将20只新西兰白兔随机分为骨髓间充质干细胞移植组(MSCs组,n=10)和对照组(n=10),采用结扎冠状动脉左前降支(LAD)制备心肌梗死模型,2周后分别将Dil标记的1×106个细胞悬液400μl或等量L-DMEM培养基用微量注射器注入梗死灶边缘,于建模前、建模后2周、细胞移植后2、4周采用多普勒超声心动图检测左心室收缩期末内径(LVESD)、左心室舒张期末内径(LVEDD),计算左心室射血分数(LVEF)、左心室短轴缩短率(LVFS)评价心脏收缩功能,同时进行心肌声学造影评价心肌组织的血流灌注情况。细胞移植后8周处死所有动物,病理学检查移植细胞在梗死区的生长状况。结果多普勒超声心动图检测结果显示:两组动物建模前、建模后2周LVEF、LVFS差异无统计学意义(0.72±0.08vs.0.71±0.04,0.56±0.11vs.0.55±0.09;0.35±0.06vs.0.35±0.04,0.24±0.08vs.0.23±0.03,P>0.05),细胞移植后2、4周MSCs组LVEF、LVFS值均明显高于对照组(0.71±0.05vs.0.60±0.05,0.72±0.07vs.0.62±0.08;0.34±0.03vs.0.29±0.01,0.35±0.06vs.0.27±0.05,P<0.05);病理学检查见自体MSCs移植8周后存活于梗死心肌中,表达肌细胞特异性标志,并且能显著增加瘢痕区毛细血管密度(38.6±7.6/mm2vs.21.4±3.9/mm2,P<0.05),心肌声学造影亦显示梗死局部血流灌注MSCs组较对照组明显改善。结论自体MSCs移植缺血心肌中可向心肌细胞分化,增加心肌血流灌注,改善心脏收缩功能。     

单宁酸缓释微球延缓大鼠心室重构的实验研究

目的探讨单宁酸(tannic acid,TA)缓释微球心肌注射对急性心肌梗死(acute myocardial infarction,AMI)后心室重构的影响。方法制备TA缓释微球,检测其体外释放药物参数。制备大鼠心肌梗死模型,将80只大鼠按随机数字表法分为空白微球组[聚乳酸-羟基乙酸共聚物(PLGA)组,n=24]、TA缓释微球(PLGA-TA)组(n=24)、TA组(n=16)和生理盐水(NS)组(n=16)。术后用超声心动图评价心功能;术后4周,观察心肌梗死边缘组织的心肌细胞外基质(ECM)排列;术后2周和4周,测定心肌梗死区胶原蛋白含量。结果 TA持续从微球载体中释放1个月。术后2周PLGA-TA组、TA组的左心室射血分数(LVEF)、左心室短轴缩短率(LVFS)、左心室舒张期末内径(LVEDD)和左心室收缩期末内径(LVESD)指标均明显优于其它两组(P<0.05);术后4周,PLGA-TA组的LVEF、LVFS、LVEDD、LVESD指标均明显优于其它3组(P<0.05)。术后4周PLGA-TA组心肌ECM排列较TA组更为有序整齐。术后4周PLGA-TA组的心肌梗死区胶原蛋白含量高于TA组[(88.88±7.28)μg/mg心肌干重vs.(72.43±9.02)μg/mg心肌干重]、PLGA组[(88.88±7.28)μg/mg心肌干重vs(.71.97±6.06)μg/mg心肌干重]和NS组[(88.88±7.28)μg/mg心肌干重vs.(68.86±7.55)μg/mg心肌干重],差异有统计学意义(F=7.162,P=0.003);而TA组、PLGA组、NS组之间比较差异无统计学意义(P>0.05)。结论 TA缓释微球心肌注射可较长时间遏制AMI后ECM的降解,有效延缓心室重构的发生。     

速度向量成像技术对梗死心肌和缺血心肌纵向运动的研究

目的 应用速度向量成像(velocity vector imaging,VVI)技术分析梗死心肌和缺血心肌纵向运动特点,评价VVI技术测定心肌运动功能的价值.方法选择2007年12月-2008年1月行超声心动图检查者,其中梗死组6例,均为急性前肇心肌梗死;缺血组9例,前降支狭窄＞70%,有心绞痛症状;正常对照组16例.应用Sequoia 512超声成像仪在VVI技术模式下采集心尖左心室长轴和左心室两腔心切面动态图像.采用Syngo US workplace工作站对图像进行脱机分析,定量分析左心室前壁和前间隔梗死节段、缺血节段和正常节段的纵向速度、位移、应变和应变率. 结果 正常对照组左心室前壁和前间隔纵向收缩期峰值速度(Vs)和峰值位移(D)自基底段向心尖段递减,差异有统计学意义(P＜0.05):峰值应变(S)和收缩期峰值应变率(SRs)在基底段、中间段和心尖段上差异无统计学意义(P＞0.05).梗死组心肌各项指标均较正常对照组和缺血组减低,S和SRs减低更明显,差异均有统计学意义(P＜0.05).缺血组心肌仅前壁基底段和中间段的S显著低于正常对照组(P＜0.05).以任意节段S＜-6.94%作为诊断急性心肌梗死的临界值,敏感度和特异度均为100%;以任意节段SRs＜-0.81%作为诊断急性心肌梗死的临界值,敏感度为100%,特异度为80%;以任意节段SRs＜-0.46%作为诊断急性心肌梗死的临界值,其敏感度为83%,特异度为100%. 结论 VVI技术具有准确定量评价急性心肌梗死的临床应用价值,尤其足应变和应变率能提供更多有价值的信息.     

阿托伐他汀对心肌梗死后大鼠基质金属蛋白酶-2及微小RNA-21的作用

目的 观察阿托伐他汀对心肌梗死后大鼠基质金属蛋白酶(MMP)-2、微小RNA(miRNA,miR)-21表达的影响.方法 选取140只Wistar大鼠建立急性心肌梗死模型,分为假手术组、对照组、阿托伐他汀低剂量组和高剂量组,进行左室重量指数、心肌胶原容积分数、miRNA-21及MMP-2 mRNA表达测定.结果 阿托伐他汀治疗组左室重量指数、心肌胶原容积分数、梗死周边区miR-21 、MMP-2 mRNA的表达均降低(P＜0.05);各组梗死周边区miR-21表达量与MMP-2 mRNA水平呈正相关(r对照组=0.611,P＜0.05;r阿托伐他汀低剂量组=0.502,P＜0.05;r阿托伐他汀低剂量组=0.541,P＜0.05).结论 阿托伐他汀能降低梗死周边区MMP-2和miR-21的表达,发挥减轻心室重构的作用.     

一氧化氮在异丙酚减轻大鼠离体心脏缺血再灌注损伤中的作用

目的 探讨一氧化氮在异丙酚减轻大鼠离体心脏缺血再灌注损伤中的作用.方法 成年雄性SD大鼠,体重220～330 g,采用Langendorff装置建立大鼠离体心脏灌注模型,选取符合实验标准的心脏模型18个,随机分为3组(n=6):K-H液灌注组(A组)、异丙酚灌注组(B组)和异丙酚+L-NAME灌注组(C组).各组用相应的K-H液灌注15 min,常温全心停灌20 min,然后用相应的K-H液复灌60 min.采用电化学微传感器法测定心肌一氧化氮(NO)含量,免疫组化法测定心肌一氧化氮合酶(NOS)含量,分光光度计测定心肌NOS活性,测定心率、左心室舒张末压、左心室发展压、左心室压变化速率最大值和左心室压变化速率最小值,定时收集右心室流出液以测定冠脉流量.结果 与A组相比,B组和C组复灌后各时点心功能改善(P＜0.05);与C组相比,B组复灌后各时点心功能改善(P＜0.05);B组心肌NO含量和NOS活性较A组升高(P＜0.05),但2组心肌NOS含量差异无统计学意义(P＞0.05).结论 缺血再灌注导致心肌内源性NO的含量降低.异丙酚减轻大鼠离体心脏缺血再灌注损伤可能是通过增加心肌内源性NO生成实现的.     

超声评价骨髓间充质干细胞移植治疗心肌梗死

心肌梗死是冠心病患者主要死亡原因之一。骨髓间充质干细胞（MSCs）移植治疗心肌梗死具有良好的前景。作为重要的检查方法 ,超声检查已应用于MSCs移植治疗心肌梗死的动物实验及临床研究中。本文对超声检查在心肌梗死MSCs移植治疗中的研究进展作一综述。     

骨髓间充质干细胞分化为心肌细胞的实验研究

目的 探讨骨髓间充质干细胞(MsCs)定向分化为心肌细胞的可行性，建立一种以干细胞移植为主治疗心肌梗死的治疗策略。方法利用Percoll分离骨髓细胞培养获得MsCs；用5-氮杂胞苷(0．1、1、5、10、50和100μmol/L)定向诱导24h，分别在诱导培养的第14d和21d，检测细胞中的α-横纹肌肌动蛋白表达。将培养的MSCs，移植于急性梗死心肌组织内，4周后进行组织学和免疫组织化学染色，并用超声检测室壁运动和心功能改变。结果骨髓间充质干细胞经5-氮杂胞苷诱导，培养21d后，5和10μmol/L 5-氮杂胞苷诱导后的MSCs有一部分细胞表达α-横纹肌肌动蛋白，并一些细胞自发性跳动；细胞移植4周后，在缺血心肌有一部分MSCs与宿主心肌细胞形成连接并分化为具有典型的肌小节和表达α-横纹肌肌动蛋白阳性的心肌细胞。结论MsCs可以分化成心肌细胞。     

Mobilization and homing of bone marrow stromal cells in myocardial infarction.

OBJECTIVE: Marrow stromal cells (MSCs) contain multipotent cells, which may participate in the repair of damaged organs. We tested the hypothesis that MSCs are recruited to the heart upon myocardial infarction (MI), and play pathophysiological roles in the healing and adaptation process. METHODS: Donor MSCs from isogenic Lewis rats were harvested, multiplied and labeled with Lac Z reporter gene. Ten million labeled cells were injected intravenously into the recipient rats (n=30). One week later, 10 rats were killed to examine the distribution of the labeled MSCs. Other rats underwent either coronary artery ligations (n=14) or sham operations (n=6). The hearts were removed at various time points (1-8 weeks) and stained for beta-galactosidase activity. Phenotypes of labeled cells were identified with immunohistochemical stains. RESULTS: In rats killed at 1 week, labeled cells had homed into the bone marrow of the recipients, and none found in their hearts. In the coronary ligated hearts, labeled cells were seen in and near the infarct at all time points studied (14/14), but none in the sham operated hearts (6/6). There was evidence for myogenic differentiation. Some of these labeled cells showed positive staining for cardiomyocyte specific troponin I-c at 4 weeks, while others appeared in the vascular walls expressing smooth muscle alpha-actin. CONCLUSIONS: Following myocardial infarction, MSC's are signaled and recruited to the injured heart, where they undergo differentiation, and may participate in the pathophysiology of post-infarct remodeling, angiogenesis, and maturation of the scar. Therapeutic implantation of MSCs thus may further enhance such effects.     

心肌梗死后移植骨髓间充质干细胞后全身细胞的分布

目的 观察经心肌梗死边缘区移植骨髓间充质干细胞(MSCs)24 h和2周后的全身分布.方法 雄性SD大鼠的MSCs用溴脱氧尿苷(BrdU)标记.将雌性SD大鼠经心梗3周后随机分为2组.第1组为标记细胞(3×10,50μl)经心肌注入心梗边缘区(n=12).第2组为等体积的磷酸盐缓冲液(PBS)经心肌注入心梗边缘区(n=8).移植细胞24 h及2周后,体内细胞的分布情况通过实时定量逆转录-聚合酶链反应(RT-PCR)检测及病理切片免疫组织化学染色来评价.结果 细胞经心梗边缘区移植后可以分布在心脏以及心外脏器脾脏、肺脏和肝脏中.移植24 h后细胞主要在心脏中分布(467 467±191 387),其他脏器中的分布较少,而在2周后移植细胞在包括心脏(112 388±43 751)在内的各个脏器中的分布都很少,病理切片免疫组织化学染色结果显示与RT-PCR结果相符.结论 MSCs经心梗边缘区移植后主要在心脏中分布,同时脾脏、肺脏、肝脏中也有分布,但其分布数量随着移植时间锐减.     

Human Mesenchymal Stromal Cells Improve Cardiac Perfusion in an Ovine Immunocompetent Animal Model

Background: Mesenchymal stromal cells (MSCs) hold considerable promise in the treatment of ischemic heart disease. Most preclinical studies of MSCs for acute myocardial infarction (AMI) have been performed either in syngeneic animal models or with human cells in xenogeneic immunodeficient animals. A preferable pre-clinical model, however, would involve human MSCs in an immunocompetent animal. Methods: AMI was generated in adult sheep by inducing ischemia reperfusion of the second diagonal branch. Sheep (n = 10) were randomized to receive an intravenous injection of human MSCs (1 × 10⁶ cells/kg) or phosphate buffered saline. Cardiac function and remodeling were evaluated with echocardiography. Perfusion scintigraphy was used to identify sustained myocardial ischemia. Interaction between human MSCs and ovine lymphocytes was assessed by a mixed lymphocyte response (MLR). Results: Sheep receiving human MSCs showed significant improvement in myocardial perfusion at 1 month compared with baseline measurements. There was no change in ventricular dimensions in either group after 1 month of AMI. No adverse events or symptoms were observed in the sheep receiving human MSCs. The MLR was negative. Conclusion: The immunocompetent ovine AMI model demonstrates the clinical safety and efficacy of human MSCs. The human cells do not appear to be immunogenic, further suggesting that immunocompetent sheep may serve as a suitable pre-clinical large animal model for testing human MSCs.     

基因修饰的骨髓间充质干细胞移植于慢性心肌梗死模型的实验研究

目的 研究血管内皮生长因子（VEGF）165基因修饰的骨髓间充质干细胞（MSCs）移植于慢性心肌梗死模型后的血管新生及对心功能的作用。方法 同源重组法构建含有VEGF。基因的重组腺病毒载体（rAd—VEGF165）;密度梯度离心法分离骨髓单个核细胞,贴壁法培养兔MSCs;rAd—VFGF165转染MSCs,并用4,6-联脒-2-苯基吲哚（DAPI）标记MSCs;结扎前降支法建立兔心肌梗死模型,存活6周的实验动物36只随机分为3组：移植rAd—VFGF165转染的MSCs组（Ⅰ组）12只、单纯移植MSCs组（Ⅱ组）12只和只注射无血清培养基的对照组（Ⅲ组）12只。移植术后4周,超声法检测心脏功能,并同术前比较;荧光显微镜下观察MSCs分布情况;免疫组化法检测梗死区微血管密度。结果 移植4周后Ⅰ 组的MSCs存活率大于其他2组;Ⅰ组的左室射血分数、E／A比值和梗死区微血管密度与其他2组比较差异有统计学意义（P〈0．05）。结论 VEGF165基因和MSCs联合策略是治疗心肌梗死的有效方法,所产生的协同治疗效果大于单纯的MSCs移植。     

Isolation of Mesenchymal Stromal Cells From Peripheral Blood of ST Elevation Myocardial Infarction Patients

Bone marrow mesenchymal stromal cells (MSCs) have shown therapeutic potential in the treatment of myocardial infarction patients. However, bone marrow requires invasive harvesting techniques. Therefore, the aim was to carry out a feasibility study of using autologous peripheral blood (PB) as a source for MSCs and platelet lysate (PL), a potential novel therapeutic intervention in acute ST elevation myocardial infarction (STEMI) patients. Autologous PL and MSCs were prepared from STEMI patient and healthy control blood. MSCs were analyzed by trilineage differentiation and flow cytometry. PB MSCs were isolated from 83% of patients (n = 6) but not from controls. The use of PL was feasible in the first passage but not in subsequent ones due to volume. To conclude, PB is a promising alternative to bone marrow. It negates the need for invasive harvesting techniques, and reduces hemorrhagic risk in this patient population routinely managed with anticoagulant and antiplatelet agents.     

Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects

Background. A novel therapeutic option for the treatment of acute myocardial infarction involves the use of mesenchymal stem cells (MSCs). The purpose of this study was to investigate whether implantation of autologous MSCs results in sustained engraftment, myogenic differentiation, and improved cardiac function in a swine myocardial infarct model.

Methods. MSCs were isolated and expanded from bone marrow aspirates of 14 domestic swine. A 60-minute left anterior descending artery occlusion was used to produce anterior wall infarction. Piezoelectric crystals were placed within the ischemic region for measurement of regional wall thickness and contractile function. Two weeks later animals autologous, Di-I–labeled MSCs (6 × 10⁷) were implanted into the infarct by direct injection. Hemodynamic and functional measurements were obtained weekly until the time of sacrifice. Immunohistochemistry was used to assess MSC engraftment and myogenic differentiation.

Results. Microscopic analysis showed robust engraftment of MSCs in all treated animals. Expression of muscle-specific proteins was seen as early as 2 weeks and could be identified in all animals at sacrifice. The degree of contractile dysfunction was significantly attenuated at 4 weeks in animals implanted with MSCs (5.4% ± 2.2% versus −3.37% ± 2.7% in control). In addition, the extent of wall thinning after myocardial infarction was markedly reduced in treated animals.

Conclusions. Mesenchymal stem cells are capable of engraftment in host myocardium, demonstrate expression of muscle specific proteins, and may attenuate contractile dysfunction and pathologic thinning in this model of left ventricular wall infarction. MSC cardiomyoplasty may have significant clinical potential in attenuating the pathology associated with myocardial infarction.