Longevity of adult ventricular rat heart muscle cells in serum-free primary culture

The study had two aims: first, to improve the longevity of isolated adult cardiomyocytes in serum-free culture, and, second, to investigate whether catecholamines which promote hypertrophy in vivo can prolong survival of isolated adult rat cardiomyocytes in serum-free culture. The basic cell culture medium consists of serum-free medium 199 with 10(-7) M insulin. In this medium 50% of the initially plated cardiomyocytes survive in elongated form for 2 days. Omission of glutamine and supplementation of the basic medium with 5 mM creatine, 2 mM carnitine and 5 mM taurine extends survival of elongated cells to 14 days. In supplemented medium, normal cell ATP content is maintained (27 nmol/mg protein after 15 days), but cells gradually atrophy and reduce their protein mass. The trophic effects of catecholamines (epinephrine, norepinephrine, phenylephrine; 10 microM, added on day 3 of culture) were investigated. After addition of catecholamines the cells spread. Spreading can be prevented by prazosin (10 microM) and phentolamine (10 microM) but not by propranolol (10 microM), indicating that spreading is stimulated via the alpha 1-adrenoreceptor. Cells also spread in the presence of the phorbol ester phorbol myristate acetate (10 microM). Catecholamines reduce the progressive cell atrophy and protein loss. With 10 microM phenylephrine, cellular ATP content remained constant at 27 nmol/mg protein until day 15. The results indicate that agents which stimulate protein kinase C (alpha 1-agonists, phorbol esters) stimulate cell spreading, protein synthesis and long-term survival of cardiomyocytes in vitro.     

心肌梗死后残存心室肌细胞L型钙通道的改变

目的　探讨L型钙通道在急性心肌梗死 (AMI)后室性心律失常发生中的作用及其机制。方法　开胸冠脉结扎制备兔AMI模型 ,于 1周和 2个月处死动物分离心室肌细胞 ,以膜片钳技术记录梗死及周边区心外膜细胞L -型钙通道电流 (ICa -L)的变化。结果　AMI兔梗死周边区心外膜细胞L型钙电流受到抑制 ,电流密度 -电压关系 (I -V)曲线上移 ,其峰值电流密度在正常对照组、AMI后 1周和 2个月分别为 - ( 5 5 8± 1 5 3) pA /pF(n =10 )、- ( 3 5 2± 0 93) pA/ pF (n =6 ,与对照组比较P <0 0 5 )和 - ( 4 84± 1 4 8)pA/ pF(n =11,与对照组比较P <0 0 5 ) ,但I -V曲线的形态轨迹不变。其失活曲线左移 ,失活速度加快 ,半数最大失活电位 3组分别为 -( 13 1± 4 2 )mV、- ( 2 5 9± 7 0 )mV和 - ( 2 1 3± 5 6 )mV ,P <0 0 5。结论　AMI后梗死周边带心外膜细胞L型钙通道受抑制 ,可能为AMI后室性心律失常发生的机制之一 ;AMI后 2个月钙通道的异常程度减轻 ,有恢复正常的趋势     

Signaling pathways in failing human heart muscle cells

Experimental studies have delineated important signaling pathways in cardiomyocytes and their alterations in heart failure; however, there is now evidence that these observations are not necessarily applicable to human cardiac muscle cells. For example, angiotensin II (A II) does not exert positive inotropic effects in human ventricular muscle cells, in contrast to observation in rats. Thus, it is important to elucidate cardiac signaling pathways in humans in order to appreciate the functional role of neurohumoral or mechanical stimulation in human myocardium in health and disease. In the present article, we review signal pathways in the failing human heart based on studies in human cardiac tissues and in vivo physiological studies related to A II, nitric oxide, and β-adrenergic stimulation. (Trends Cardiovasc Med 1997; 7:151-160). ? 1997, Elsevier Science Inc.     

Differences in regulatory mechanisms of atrial and ventricular muscle contraction in bovine heart

The purpose of this study was to characterize the regulatory mechanisms of atrial muscle contraction. Natural actomyosin (NAM) and tropomyosin-troponin (TM-TN) complex were prepared from atrial and ventricular muscle of the same bovine heart. The results were as follows: (1) Atrial NAM was more sensitive to Ca2+ than was ventricular NAM: the pCa required for 50% ATPase activation was 5.96 +/- 0.10 vs. 5.63 +/- 0.07, (mean +/- SE; n = 6; p less than 0.01); (2) reconstitution of desensitized actomyosin of rabbit skeletal muscle plus atrial or ventricular TM-TN complex produced higher Ca2+ sensitivity in atrial muscle than in ventricular muscle: the pCa required for 50% ATPase activation was 6.48 +/- 0.10 vs. 6.23 +/- 0.15 (n = 3; p less than 0.05); (3) the amount of inorganic phosphate covalently bound to atrial NAM was equivalent to that bound to ventricular NAM; (4) SDS-polyacrylamide gel electrophoresis of the two NAMs revealed several protein bands of different mobility from 16,000 to 30,000 daltons; and (5) the superprecipitation response of atrial NAM was characterized by a stepwise change in turbidity after the addition of MgATP, in contrast to the biphasic pattern of ventricular NAM. These data suggest that the free Ca ion concentration required for atrial muscle contraction is lower than that required for ventricular muscle contraction and that the difference is attributable to differences in atrial and ventricular regulatory proteins.     

Nuclear pores in ventricular muscle cells from adult hypertrophied hearts

Nuclear pores were observed in myocardial cells in both spontaneous and experimentally induced hypertrophy. The ultrastructure of these pores was studied in cross sections and tangential sections of hypertrophied rabbit, hamster and guinea pig heart cells. The morphology was similar to that previously seen in normal cardiac cells and in other cell types. Clusters of nuclear pores were frequently seen, and the closest center-to-center spacing between pores was 1400 Å. Annular granules with associated fibrils were observed on both the cytoplasmic and nucleoplasmic sides of the nuclear envelope. Many of the pores contained granules ～ 300 Å in diameter which sometimes appeared hollow. The presence of large numbers of nuclear pores in hypertrophied cardiac cells supports the hypothesis that transport of material necessary for RNA-mediated protein synthesis may occur through the pores. Furthermore the mature cardiocyte provides an opportunity to study nuclear pore function in nuclei that do not synthesize DNA or divide.     

Embryonic and embryonic-like stem cells in heart muscle engineering

Cardiac muscle engineering is evolving rapidly and may ultimately be exploited to (1) model cardiac development, physiology, and pathology; (2) identify and validate drug targets; (3) assess drug safety and efficacy; and (4) provide therapeutic substitute myocardium. The ultimate success in any of these envisioned applications depends on the utility of human cells and their assembly into myocardial equivalents with structural and functional properties of mature heart muscle. Embryonic stem cells appear as a promising cell source in this respect, because they can be cultured reliably and differentiated robustly into cardiomyocytes. Despite their unambiguous cardiogenicity, data on advanced maturation and seamless myocardial integration of embryonic stem cell-derived cardiomyocytes in vivo are sparse. Additional concerns relate to the limited control over cardiomyogenic specification and cardiomyocyte maturation in vitro as well as the risk of teratocarcinoma formation and immune rejection of stem cell implants in vivo. Through the invent of embryonic-like stem cells - such as parthenogenetic stem cells, male germline stem cells, and induced pluripotent stem cells - some but certainly not all of these issues may be addressed, albeit at the expense of additional concerns. This review will discuss the applicability of embryonic and embryonic-like stem cells in myocardial tissue engineering and address issues that require particular attention before the potential of stem cell-based heart muscle engineering may be fully exploited. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".     

Characteristics of junctional regions between Purkinje and ventricular muscle cells of canine ventricular subendocardium

The normal cardiac activation sequence requires propagation of the action potential from the subendocardial Purkinje network into the underlying ventricular muscle cells. This process occurs at specific junctional sites distributed over the endocardial surface of both ventricles. At these junctional sites, action potentials can be recorded from cells that appear to be interposed between the Purkinje cells and the ventricular muscle cells. The action potential upstrokes recorded from these "transitional" cells have characteristic double phases produced by electrotonic interactions with the Purkinje cells and the ventricular muscle cells. We have shown that these junctional regions in the canine subendocardium appear to be fixed anatomic sites with locations independent of the activation sequence of the Purkinje network. In addition, the activation delay between the Purkinje cells and the ventricular muscle cells at a junctional site and the patterns of the action potential upstrokes of transitional cells at a junctional site are independent of the activation sequence of the Purkinje network. We have also demonstrated that at some locations there are multiple Purkinje activation signals recorded with a surface electrode and that these multiple activation signals represent discrete groups of Purkinje cells, some of which contribute to the junctional process while others appear to be substantially uncoupled from neighboring Purkinje cell groups and the underlying transitional cells.     

An effect of the cardiotoxic protein volvatoxin a on the function and structure of heart muscle cells

Volvatoxin-A, the heat labile cardiotoxin present in the mushroom Volvariella volvacea, causes a competitive, dose and time-dependent inhibition of the Ca²⁺-accumulating activity of a sarcoplasmic-reticulum rich microsomal fraction isolated from guinea pig ventricular muscle. The inhibition is accompanied by an activation of the Ca²⁺-dependent ATPase enzyme. Concentrations of toxin which inhibit the Ca²⁺-transporting activity of the microsomes render them leaky to Ca²⁺, but do not effect the rate of incorporation of P³². Ten μg/ml toxin failed to alter the activity of the Na⁺ + K⁺-activated, ouabain sensitive ATPase enzyme. It damaged the fine morphology of the mitochondria, inhibited the ability of isolated mitochondria to accumulate Ca²⁺, and had little effect on the ability of isolated plasma membranes to bind Ca²⁺. These findings may explain why volvatoxin A increases the diastolic resting tension in heart muscle.     

Fine structural analysis of rabbit synovial cells. II. Fine structure and rosette-forming cells of explant and monolayer cultures

The fine structure and plasma membrane receptors of explant and monolayer cells of normal rabbit synovium were studied. In explants about 10% of the cells were round and formed rosettes with IgG and C₃ markers, whereas the remaining cells were stellate, resembled young fibroblasts, and had no receptors for IgG and complement. Monolayer cells looked like fibroblasts, produced fine extracellular fibrils and hyaluronate, and formed no rosettes. Thus early cultures contain both macrophages and fibroblasts but only the latter persist in monolayer.     

Fine structure of cells and their histologic organization within internodal pathways of the heart: clinical and electrocardiographic implications.

The fine structure of the normal internodal pathways was studied in 1 human and 2 canine hearts and correlated with histologic observations on more than 100 human and 10 canine hearts. From the electron microscopic studies six different kinds of myocardial cells were classified from two locations: the Eustachian ridge (posterior internodal pathway) and the Bachmann bundle (anterior internodal pathway). Five of the six kinds of cells (working myocardial cells, Purkinje-like cells, either broad or slender transitional cells and P cells, all previously described) were present in both locations. A sixth cell, pleomorphic and dark in appearance, with a special intertwined relation to P cells, is newly designated as an ameboid cell. It was found solely in the Eustachian ridge. In the same area a rare direct contact between a nerve and a myocardial cell was observed. The importance of these different kinds of cells, their respective cell connections, and their topographic locations inside the internodal pathways are discussed relative to certain functions such as rapid conduction and subsidiary pacemaking. The possible influence of these factors on clinical electrocardiographic changes is considered.     

Echocardiographic evaluation of ventricular septal aneurysms.

The spontaneous closure of ventricular septal defects is frequently associated with septal aneurysm formation. In this paper we discuss the M-mode and two-dimensional echocardiographic findings in nine children with aneurysms of the ventricular septum in association with ventricular septal defects. In all patients the diagnosis was confirmed by angiography. The ventricular septal aneurysms were detected by both M-mode and two-dimensional echocardiography. With M-mode echocardiography, septal aneurysms could be recognized by a pattern of multiple systolic echoes within the right ventricle. With two-dimensional echocardiography, the protrusion of the septal aneurysm into the right ventricle could be seen from several views and the location and the relative size of the aneurysm assessed. Echocardiographic techniques useful in the detection of ventricular septal aneurysms are discussed and examples presented.