Pre-treatment of synthetic elastomeric scaffolds by cardiac fibroblasts improves engineered heart tissue

Native myocardium consists of several cell types, of which approximately one-third are myocytes and most of the nonmyocytes are fibroblasts. By analogy with monolayer culture in which fibroblasts were removed to prevent overgrowth, early attempts to engineer myocardium utilized cell populations enriched for cardiac myocytes (CMs; approximately 80-90% of total cells). We hypothesized that the pre-treatment of synthetic elastomeric scaffolds with cardiac fibroblasts (CFs) will enhance the functional assembly of the engineered cardiac constructs by creating an environment supportive of cardiomyocyte attachment and function. Cells isolated from neonatal rat ventricles were prepared to form three distinct populations: rapidly plating cells identified as CFs, slowly plating cells identified as CMs, and unseparated initial population of cells (US). The cell fractions (3 x 10(6) cells total) were seeded into poly(glycerol sebacate) scaffolds (highly porous discs, 5 mm in diameter x 2-mm thick) using Matrigeltrade mark, either separately (CM or CF), concurrently (US), or sequentially (CF pre-treatment followed by CM culture, CF + CM), and cultured in spinner flasks. The CF + CM group had the highest amplitude of contraction and the lowest excitation threshold, superior DNA content, and higher glucose consumption rate. The CF + CM group exhibited compact 100- to 200-mum thick layers of elongated myocytes aligned in parallel over layers of collagen-producing fibroblasts, while US and CM groups exhibited scattered and poorly elongated myocytes. The sequential co-culture of CF and CM on a synthetic elastomer scaffold thus created an environment supportive of cardiomyocyte attachment, differentiation, and contractile function, presumably due to scaffold conditioning by cultured fibroblasts. When implanted over the infarcted myocardium in a nude rat model, cell-free poly(glycerol sebacate) remained at the ventricular wall after 2 weeks of in vivo, and was vascularized.     

Electrolyte and Morphologic Alterations of Myocardium in Adriamycin-Treated Rabbits

Rabbits receiving adriamycin (ADR) on a chronic schedule developed significant histopathologic, ultrastructural and tissue electrolyte alterations of the ventricular myocardium. Rabbits that developed clinicopathologic evidence of cardiomyopathy with ADR had histologic lesions of the myocardium, including perivascular fibrosis, interstitial fibrosis and edema and myocytolysis. Ultrastructurally, large vacuoles resembling distended sarcoplasmic reticulum displaced the contractile elements and mitochondria, which were diminished in number within affected myocytes. Frequently, mitochondria appeared as electron-dense structures surrounded by layers of membranes resembling myelin figures. In addition, rabbits with cardiomyopathy had marked elevations in ventricular Ca, Na and H₂O concentrations. Serum electrolytes were not significantly elevated, but lactic dehydrogenase (LDH) and creatine phosphokinase (CPK) were significantly increased, indicative of a cardiomyopathy. Rabbits receiving ADR but not developing clinicopathologic evidence of heart failure also had significant elevations in ventricular Ca, Na and H₂O. Rabbits with no cardiomyopathy had no increases either in serum electrolyte concentrations or CPK and LDH levels. These studies indicate that marked increases in ventricular tissue Ca precede and accompany morphologic evidence of chronic myocardial degeneration and may be instrumental in the development of the ADR-induced cardiomyopathy.     

Repair of infarcted myocardium using mesenchymal stem cell seeded small intestinal submucosa in rabbits

Myocardial infarction (MI) remains a common and deadly disease. Using tissue-engineered cardiac grafts to repair infarcted myocrdium is considered to be a therapeutic approach. This study tested the feasibility of using MSCs-seeded SIS to repair chronic myocardial infarction in a rabbit model. MI in rabbits was created by ligation of the left anterior descending artery. BrdU-labeled mesenchymal stem cells (MSCs) were seeded on the small intestinal submucosa and cultured for 5–7 days prior to implantation. Four weeks after myocardial infarction, cardiac grafts were implanted onto the epicardial surface of infarcted myocardium. Four weeks after implantation of the membranes, a serial of tests including echocardiography, hemodynamics, histology and immunohistochemistry were undertaken to evaluate the effect of the implanted grafts on recovery of the infarcted myocardium. It was shown that left ventricular contractile function and dimension, the capillary density of the infarcted region, and myocardial pathological changes were significantly improved in rabbits implanted either SIS or MSCs-seeded SIS. But the MSCs-seeded SIS was more effective. Immunofluorescence staining demonstrated the migration of Brdu-labeled MSCs from the membrane into the infarcted area and their differentiation to cardiomyocytes and smooth muscle cells. Taken together, these results suggest that MSCs-seeded SIS can be used to repair chronic myocardial infarction, which enhances myocardial regeneration.     

Hibernation and stunning as manifestations of ischemic dysfunction of the myocardium

Current information on hybernation and stunning which are the basis of myocardial dysfunction resulting from chronic ischemic heart disease and revascularization is provided. Myocardial stunning is an acute disturbance of a contractile function of ischemic myocardium at the moment of coronary circulation restoration done by various methods (coronary shunting, angioplasty, thrombolysis). Myocardial hybernation is a chronically developing foci of subnormal contractility in the region of stenotic artery. The difference between them is that stunning is a complex of structural and metabolic damages under the condition "ischemia-reperfusion" while hybernation is an adaptation of the myocardium to chronic ischemia by metabolism switching to anaerobic glycolysis. The study of pathophysiology and morphology of hybernating and stunned myocardium is necessary for developing methods of myocardium protection from ischemic damage.     

Role of the changes in the sarcoplasmic reticulum in disorders of myocardial function]

Acute focal ischemia of the myocardium, acute hemodynamic overloading of the heart, coarctation of the aorta, and fibrillation of the heart were simulated on rabbits. The animals were studied for the contractile function of the myocardium, and potential work capacity of the heart was calculated according to a special formula. The rebbits were sacrificed at the acute period of the development of a pathological process, and the contractile myocardium was investigated by electron microscope. The studies of the bioelectric activity of the heart revealed periodically appearing disorders of the cardiac rhythm. The electron-microscopy investigation showed, apart from changes in the ultrastructure of cells of the contractile myocardium reflecting their hyperfunction, marked dilatation of small canals of the sarcoplasmic reticulum, right to the formation of cisterns containing sequesters of cells. It is established that the changes referred to above in the sarcotubular system were associated with potassium dysbalance. The conclusion was drawn that the above said changes in the sarcoplasmic reticulum was a stereotype reaction of the alterative heart at the level of ultrastructures developing according to the principle of a vicious circle.     

Characteristics of the restructuring of the cardiomyocyte contractile apparatus in chronic ischemic heart disease

The contractile apparatus of cardiomyocytes was examined at the ultrastructural level in 20 heart biopsy specimens obtained during operations for aortocoronary bypass in patients with chronic ischemic heart disease (IHD). Myofilaments were found to have sustained compensatory-adaptive and destructive changes as well as changes indicative of impaired intracellular regenerations under conditions of chronic hypoxia and energy deficiency experienced by the muscle cells, with the result that myocardial contractility was substantially reduced. Together, these processes led to progressive restructuring of both the contractile apparatus of the cardiomyocytes and the cytoarchitectonics in general. This combined with signs of increasing hyperfunction of the contractile myocardium, which made the energy deficit worse and thus interfered with plastic processes in, and diminished the structural and functional capabilities of, the myocardium. On the other hand, distinct changes were noted in the cardiomyocytes that reflected their adaptation to the adverse conditions created by progressing coronary atherosclerosis. It is concluded that therapy for patients with chronic IHD should include measures aimed at promoting such adaptive changes.     

Changes in the ultrastructure of the myocardium after fibrillation and in the process of autolysis

Ultrastructure of the myocardium in the experiment was studied just after the heart ventricular fibrillation and 3 h after the animal death; the results were compared to those obtained by studying ultrastructure of human myocardium in patients dying from myocardial infarction complicated by ventricular fibrillation. The similarity of ultrastructural and autolytic changes was revealed in both series of observation. Overcontraction of myofibrils and redistribution of the mitochondria in cardiomyocytes, increase of the sarcoplasmic reticulum vesiculation, oedema of sarcoplasm, structural alterations of intercalative discs, later appearance of the symptoms of the irreversibility of lesions in the mitochondria and early penetration of colloidal lanthanum particles in rigor cells may serve ultrastructural diagnostic symptom of ventricular fibrillation. A better preservation of Purkinje's cells as compared to the contractile cardiomyocytes is also demonstrated.     

Transmural differences in the postischemic recovery of cardiac energy metabolism.

After 25 minutes of ischemia, in the isolated rat preparation, hearts fail to reestablish adequate contractile function. To determine whether this failure was associated with a transmural variation in the metabolic response of myocardial cells to reperfusion, the authors subjected hearts to 25 minutes of global ischemia with and without 5 or 20 minutes of reperfusion. After freeze-drying the left ventricular myocardium was divided into subepicardial (EPI) and subendocardial (ENDO) regions before estimating the lactate, total adenine pool metabolites, and creatine phosphate (CP) and phosphate concentrations in each region. Other groups of hearts were perfusion-fixed with glutaraldehyde then injected with nuclear track emulsion to demonstrate that a high proportion of capillaries in both the subendocardial (89%) and subepicardial (95%) myocardium transmitted perfusate after 5 minutes of reperfusion. Reperfusion removed lactate equally from each region. Thus the differences in the capacity of reperfusion of these regions to recover CP (ENDO, 100%; EPI, 168% of preischemic values), to elevate adenosine triphosphate (ATP) (ENDO, 32%; EPI, 63%), or to retain adenosine monophosphate (AMP) (ENDO, 625%; EPI, 277%) were unlikely to be due to regional differences in microvascular function. Despite the better preservation of both structure and metabolism in the subepicardium, there was, during reperfusion, a progressive loss of purine precursors from cells in both regions of the myocardium. These results suggest that the loss of ability of the myocardium to recover significant function after relatively short periods of ischemia is due to their inability, on reperfusion, to synthesise sufficient ATP from the available precursors. This capacity for resynthesis of ATP is lost more rapidly in the subendocardial than in the subepicardial myocardium.     

Subvalvular left ventricular aneurysms.

Subvalvular left ventricular aneurysms can be subaortic or submitral with variable etiology. This is a retrospective study of 19 subvalvular aneurysms seen in 16 cases. There were 12 isolated subaortic aneurysms, 3 isolated submitral aneurysms, and in 1 case with multiple subaortic and submitral aneurysms. Subaortic aneurysms were associated with infective endocarditis, while there seems to be a strong association between submitral aneurysms and tuberculosis. Five cases of subvalvular aneurysm has associated aneurysm sinus of Valsalva-this association suggests a congenital weakness in the attachment of aortic and mitral annuli to the underlying myocardium. Cardio Pathol 2000;9:267-271     

An ultrastructural study on ischemic lesions in rabbits' hearts with pressure overload and hyperlipidemia

The effect of hypertension, hyperlipidemia, and the combination of both on acute and chronic myocardial ischemia were evaluated in a total of 30 male rabbits. After preliminary hypertension and/or hyperlipidemic load by loading of the abdominal aorta and/or cholesterol feeding, acute ischemia was produced by clipping of the left coronary artery. The banding produced elevation of carotid arterial pressure and left ventricular hypertrophy. Cholesterol feeding resulted in severe atheromatous changes in all sizes of coronary arteries. The intimal thickening was due to foam cell accumulation in all arteries examined. Animals pretreated with the combination of hypertension and hyperlipidemia displayed the most severe cardiolmegaly with advanced coronary atherosclerosis and chronic ischemic lesions of the myocardium, i.e., perivascular patchy fibrosis in the subendocardial area. Furthermore, electron microscopic detection of ultrastructural myocardial damage, involving glycogen depletion, sarcoplasmic edema, mitochondrial swelling, and contractile abnormalities, was also most frequent in this group. These changes were quantitated using the ischemic score. These results confirm the hypothesis that fatal ischemic injuries may occur clinically in human hearts with coronary insufficiency due to coexistence of hypertensive cardiomegaly and severe coronary atherosclerosis. They offer a model for further study of these combined effects.     

Ca2+ sensitivity of stunned myocardium after skinning using retrograde infusion of detergent

Myofilament Ca2+ desensitization contributes to the contractile dysfunction of ischemic/reperfused ("stunned") myocardium. We examined the presence of reduced Ca2+ sensitivity of isometric force in chemically skinned fibers obtained from stunned myocardium using different modes of applying the detergent Triton X-100. Langendorff-perfused rat hearts underwent 20 min ischemia/20 min reperfusion, which caused a 35 +/- 3% decrease in left ventricular developed pressure, compared to continuously perfused control hearts. Stunned and control hearts were randomly allocated to two different permeabilization protocols: In group A, trabeculae were dissected and immersed in skinning solution containing 1% Triton X-100 for 20 min. Group B hearts remained fixed to the aortic cannula and skinning solution was infused retrogradely for 6 min prior to dissection of trabeculae. Extraction of cytosolic marker proteins was more complete in group-B than in group-A preparations. Group-A preparations from stunned hearts exhibited significant Ca2+ desensitization (pCa50 = 5.07 and 5.15 in stunned and control myocardium, respectively). In group B no such difference was observed, all preparations showing higher Ca2+ sensitivity and maximum force than group-A preparations (pCa50 = 5.32 in stunned versus 5.33 in control hearts). Prolonging group-A skinning to 150 min also abolished the difference in Ca2+ sensitivity between stunned and control myocardium. In conclusion, compared to a conventional protocol, skinning by perfusion results in more complete permeabilization and better preservation of myocardial contractile function. Ischemia/reperfusion at this moderate degree of contractile dysfunction induces Ca2+ desensitization at least partially by factors that can be extracted by thorough skinning.     

Abnormal morphological patterns of the left ventricular myocardium in the critical stenosis of the aortic valve and the intact ventricular septum.

The evaluation of prognostic factors in infants with critical stenosis of the aortic valve and intact ventricular septum is often misleading due to a complex interaction among lesions in the mitral and aortic valves, and the left ventricular myocardium. The clinical parameters on the left ventricular function, such as ejection fraction and left ventricular end-diastolic volume, are of particular interest as their effects on survival are very controversial. We performed a clinicopathologic analysis of two autopsied cases of this disease. Besides the morphological hallmarks of the aortic and mitral valves, these two cases showed two extreme types of pathology in the left ventricular myocardium, which might have significant impacts on the clinical evaluation of the left ventricular function. Case 1 showed endocardial fibroelastosis associated with abnormal intertrabecular spaces (so-called spongy myocardium), obscuring accurate estimation of the left ventricular end-diastolic volume. Case 2 showed ischemic necrosis of the apical part of the left ventricular myocardium. This infarct was associated with acute and chronic subendocardial ischemia and mild endocardial fibroelastosis. Aggravation of the left ventricular failure could be caused by the recent ischemic insult. The evaluation of the left ventricular function, therefore, should include the evaluation of the morphologic status of the myocardium as regards to whether there is ischemia, endocardial fibroelastosis or hypertrophied trabeculae in addition to stenotic lesions in the aortic and mitral valves.     

Actin-containing cells in human pulmonary fibrosis.

Lung tissue from five patients with varying degrees of diffuse pulmonary fibrosis was examined for the presence of nonmuscle cells containing the contractile protein actin. Histochemical studies using light- and electron-microscopic techniques suggested that the fibrotic tissue, in areas lacking smooth muscle, had within it numerous cells with cytoplasmic microfilaments composed of actin. Similar cells rarely were observed in control lung tissue. The orientation of these cells within the parenchyma, as well as the arrangement and density of the filaments in the cell, suggests a contractile function. Cells with contractile properties might play a role in the altered dynamics of the lung in diffuse fibrotic pulmonary disease.     

Structural correlates of regional myocardial dysfunction in patients with critical coronary artery stenosis: Chronic hibernation?

The morphological changes in the myocardium of hypocontractile segments, especially the possible structural counterparts underlying chronic ischemia, are not well documented. Light and electron microscopy was performed on myocardium derived from the anterior wall of the left ventricle of 98 patients during coronary artery bypass grafting. Wall motion data were collected from the region corresponding to the biopsied zone. The changes seen in a substantial part of the cardiomyocytes corresponded to “dedifferentiation” rathern than degeneration characteristics. The affected cardiomyocytes showed a partial to complete loss of sarcomers, sarcoplasmic reticulum, and T-tubules and presented abundant plaques of glycogen, strands of rough endoplasmic reticulum, lots of minimitochondria, and a tortous nucleus. The volume of the cells was similar to that of normal cells. The number of the affected cells was consistently higher in endocardial parts than in epicardial ones. The cell changes occurred in the myocardium of patients both with andn without a previous Q-wave infarction. There was a significant relation between anterior wall motion abnormalities and the incidence of affected cells in the endocardium, but not in the epicardium. A significant relatiosnhip was found in noninfarcted patients between the presence of affected cells and the amount of connective tissue. Furthermore, in these patietns the number of affected cells depended on the degree of stenosis. It is proposed that segments in which these structural changes prevail will not recover immediately after revascularization but that they might how a delayed recovery of function, because structural remodeling requires time in order to regain sufficient contractile material.     

Chronic right ventricular pressure overload results in a hyperplastic rather than a hypertrophic myocardial response

Myocardial hyperplasia is generally considered to occur only during fetal development. However, recent evidence suggests that this type of response may also be triggered by cardiac overload after birth. In congenital heart disease, loading conditions are frequently abnormal, thereby affecting ventricular function. We hypothesized that chronic right ventricular pressure overload imposed on neonatal hearts initiates a hyperplastic response in the right ventricular myocardium. To test this, young lambs (aged 2-3 weeks) underwent adjustable pulmonary artery banding to obtain peak right ventricular pressures equal to left ventricular pressures for 8 weeks. Transmural cardiac tissue samples from the right and left ventricles of five banded and five age-matched control animals were studied. We found that chronic right ventricular pressure overload resulted in a twofold increase in right-to-left ventricle wall thickness ratio. Morphometric right ventricular myocardial tissue analysis revealed no changes in tissue composition between the two groups; nor were right ventricular myocyte dimensions, relative number of binucleated myocytes, or myocardial DNA concentration significantly different from control values. In chronic pressure overloaded right ventricular myocardium, significantly (P < 0.01) more myocyte nuclei were positive for the proliferation marker proliferating cellular nuclear antigen than in control right ventricular myocardium. Chronic right ventricular pressure overload applied in neonatal sheep hearts results in a significant increase in right ventricular free wall thickness which is primarily the result of a hyperplastic myocardial response.     

慢性压力负荷性心衰大鼠心肌细胞凋亡的研究

目的探讨慢性心衰时是否有心肌细胞凋亡。方法利用末端转移酶介导的切口末端标记法（ＴＵＮＥＬ）及ＴＥＭ法,检测慢性压力负荷性心衰大鼠左、右心室心肌的凋亡细胞。结果在心衰大鼠的左、右心室心肌组织中存在有凋亡的心肌细胞,而在对照组大鼠心肌组织中未见到心肌细胞调亡。结论慢性心衰与心肌凋亡之间有紧密联系。     

Three-dimensional myofiber architecture of the embryonic left ventricle during normal development and altered mechanical loads

Mechanical load influences embryonic ventricular growth, morphogenesis, and function. To date, little is known regarding how the embryonic left ventricular (LV) myocardium acquires a three-dimensional (3D) fiber architecture distribution or how altered mechanical load influences local myofiber architecture. We tested the hypothesis that altered mechanical load changes the maturation process of local 3D fiber architecture of the developing embryonic LV compact myocardium. We measured transmural myofiber angle distribution in the LV compact myocardium in Hamburger-Hamilton stages 21, 27, 31, and 36 chick embryos during normal development or following either left atrial ligation (LAL; LV hypoplasia model) or conotruncal banding (CTB; LV hyperplasia model). The embryonic LV was stained with f-actin and then z-serial optical sectioning was performed using a laser confocal scanning microscope. We reconstructed local 3D myofiber images and computed local transmural myofiber angle distribution. Transmural myofiber angles in compact myocardium (in LV sagittal sections) were oriented in a circumferential direction until stage 27 (-10 to 10 degrees). Myofibers in the outer side of compact myocardium shifted to a more longitudinal direction by stage 36 (10 to 40 degrees), producing a transmural gradient in myofiber orientation. Developmental changes in transmural myofiber angle distribution were significantly delayed following LAL, while the changes in angle distribution were accelerated following CTB. Results suggest that mechanical load modulates the maturation process of myofiber architecture distribution in the developing LV compact myocardium.     

Myocyte cellular hypertrophy and hyperplasia contribute to ventricular wall remodeling in anemia-induced cardiac hypertrophy in rats.

To determine the effects of chronic anemia on the functional and structural characteristics of the heart, 1-month-old male rats were fed a diet deficient in iron and copper, which led to a hemoglobin concentration of 4.63 g/dl, for 8 weeks. At sacrifice, under fentanyl citrate and droperidol anesthesia, systolic, diastolic, and mean arterial blood pressures were decreased, whereas differential pressure was increased. Left ventricular systolic pressure and the ventricular rate of pressure rise (mmHg/s) were reduced by 9% and 14%, respectively. Moreover, developed peak systolic ventricular pressure and maximal dP/dt diminished 14% and 12%. After perfusion fixation of the coronary vasculature and the myocardium, at a left ventricular intracavitary pressure equal to the in vivo measured end diastolic pressure, a 10% thickening of the left ventricular wall was measured in association with a 13% increase in the equatorial cavitary diameter and a 44% augmentation in ventricular mass. The 52% hypertrophy of the right ventricle was characterized by an 11% thicker wall and a 37% larger ventricular area. The 33% expansion in the aggregate myocyte volume of the left ventricle was found to be due to a 14% myocyte cellular hypertrophy and a 17% myocyte cellular hyperplasia. These cellular parameters were calculated from the estimation of the number of myocyte nuclei per unit volume of myocardium in situ and the evaluation of the distribution of nuclei per cell in enzymatically dissociated myocytes. Myocyte cellular hyperplasia provoked a 9% increase in the absolute number of cells across the left ventricular wall. In contrast, myocyte cellular hypertrophy (42%) was responsible for the increase in myocyte volume of the right ventricle. The proliferative response of left ventricular myocytes was not capable of restoring diastolic cell stress, which was enhanced by the changes in ventricular anatomy with anemia. In conclusion, chronic anemia induced an unbalanced load on the left ventricle, which evoked a hyperplastic reaction of preexisting myocytes, in an attempt to normalize diastolic wall and myocyte stress.     

Apoptosis and epicardial contributions act as complementary factors in remodeling of the atrioventricular canal myocardium and atrioventricular conduction patterns in the embryonic chick heart

Background : During heart development, it has been hypothesized that apoptosis of atrioventricular canal myocardium and replacement by fibrous tissue derived from the epicardium are imperative to develop a mature atrioventricular conduction. To test this, apoptosis was blocked using an established caspase inhibitor and epicardial growth was delayed using the experimental epicardial inhibition model, both in chick embryonic hearts. Results : Chicken embryonic hearts were either treated with the peptide caspase inhibitor zVAD‐fmk by intrapericardial injection in ovo (ED4) or underwent epicardial inhibition (ED2.5). Spontaneously beating embryonic hearts isolated (ED7–ED8) were then stained with voltage‐sensitive dye Di‐4‐ANEPPS and imaged at 0.5–1 kHz. Apoptotic cells were quantified (ED5–ED7) by whole‐mount LysoTracker Red and anti‐active caspase 3 staining. zVAD‐treated hearts showed a significantly increased proportion of immature (base to apex) activation patterns at ED8, including ventricular activation originating from the right atrioventricular junction, a pattern never observed in control hearts. zVAD‐treated hearts showed decreased numbers of apoptotic cells in the atrioventricular canal myocardium at ED7. Hearts with delayed epicardial outgrowth showed also increased immature activation patterns at ED7.5 and ED8.5. However, the ventricular activation always originated from the left atrioventricular junction. Histological examination showed no changes in apoptosis rates, but a diminished presence of atrioventricular sulcus tissue compared with controls. Conclusions : Apoptosis in the atrioventricular canal myocardium and controlled replacement of this myocardium by epicardially derived HCN4‐/Trop1‐ sulcus tissue are essential determinants of mature ventricular activation pattern. Disruption can lead to persistence of accessory atrioventricular connections, forming a morphological substrate for ventricular pre‐excitation. Developmental Dynamics 247:1033‐1042, 2018. © 2018 Wiley Periodicals, Inc.