Culture and characterization of fetal human atrial and ventricular cardiac muscle cells

Summary Atrial and ventricular cardiac muscle cells isolated from 14- to 18-wk old fetal human hearts were grown in culture and characterized. Once established in culture the flattened cells contracted spontaneously and possessed differentiated ultrastructural characteristics including organized sarcomeres, intercalated discs, and transverse tubules with couplings. Atrial granules were present in the cultured atrial cells. Some cultured ventricular myocytes also contained electron-dense granules associated with Golgi cisternae, which were similar in size and appearance to atrial granules. The cultured ventricular myocytes divided and expressed the genes for thymidine kinase, histone H4, myosin heavy chain, muscle-specific creatine kinase, atrial natriuretic factor, and insulin-like growth factor II. These results establish that differentiated fetal human heart muscle cells can be cultured in sufficient quantities for biochemical, molecular, and morphological analyses. This work was supported by a postdoctoral fellowship from the American Heart Association, Louisiana Affiliate (JBD) and the National Institutes of Health, Bethesda, MD (HL-35632) (WCC).     

Effects of endotoxin on neutrophil-mediated I/R injury in isolated perfused rat hearts

With the use of a syngeneic model, we demonstrate that rat polymorphonuclear neutrophils (PMNs) exacerbate ischemia-reperfusion injury in the isolated rat heart. However, PMNs (19 x 10(6) cells) from lipopolysaccharide (LPS)-treated rats (LPS-PMNs; 100 mg/kg administered 7 h before exsanguination) induce less reperfusion injury in the isolated heart. Average recovery of left ventricular developed pressure after 20 min of ischemia and 60 min of reperfusion was 51 +/- 4% in hearts receiving PMNs from saline-treated control rats (saline-PMNs) versus 78 +/- 2% in hearts receiving LPS-PMNs. Ischemic hearts reperfused with LPS-PMNs recovered to the same extent as did hearts reperfused with Krebs buffer only. LPS-PMNs and saline-PMNs showed no difference in basal or phorbol ester-induced superoxide production. Whereas twice the number of LPS-PMNs was positive for nitroblue tetrazolium, the percent positive for L-selectin, a receptor integral in PMN-adhesion to endothelium, was 50% less in LPS-PMNs than in controls. After reperfusion, three-fourths of the saline-PMNs remained within the hearts, whereas only one-fourth of LPS-PMNs were trapped. These data suggest that PMNs from LPS-treated rats do not exacerbate ischemia-reperfusion injury as do control PMNs, possibly, due to impaired PMN adhesion to endothelium as a result of decreased L-selectin receptors.     

Double membrane-bounded intestinal microvilli in Oncopeltus fasciatus

A double plasma membrane (DPM) surrounding intestinal microvilli of the migratory milkweed bug, Oncopeltus fasciatus, is described. Mutant and wild types of the phytophagous insect have been studied by conventional SEM and TEM procedures with the use of membrane-enhancing staining methods. Longitudinal and transverse sections revealed a DPM surrounding microvilli and continuing over the apical portions of the intestinal cell. The outer membrane of the DPM contributes to an intestinal lining or peritrophic membrane (PTM), which apparently accumulates in layers. SEM studies reveal a rugose intestinal surface and complete PTM in both starved and fed insects. Only rarely are exposed microvilli seen by SEM. SEM examinations also enable the observation of numerous blebs on the luminal side of the PTM apparently held in position by a neck-like attachment and apparently derived from the outer membrane of the DPM. Preliminary TEM studies of microvilli revealed unique microvesicle-like structures, lying just inside the inner membrane of the DPM, which may be of membrane origin based on their typical trilaminar appearance after en bloc staining with uranyl acetate. Highly ordered microfilaments were observed to occupy the most central aspect of the microvilli.     

Sarcoplasmic reticulum and intermediate filament organization in cultured neonatal cardiac muscle cells

Cardiac muscle cells from 3-day-old rat neonates were cultured for periods of 2 to 56 days. In order to facilitate ultrastructural studies on the organization of the sarcoplasmic reticulum, the cells were prepared for transmission electron microscopy according to a regimen including postfixation in reduced osmium ferrocyanide. The nonjunctional sarcoplasmic reticulum (NJSR) was organized as a loose, fenestrated sleeve around the exterior of bundles of myofilaments and was particularly prominent at the level of the Z line. The only recognizable junctional elements of the sarcoplasmic reticulum were in a peripheral location. Reduced osmium ferrocyanide was also useful in distinguishing intermediate (10 nm) filaments, since it understained Z substance, which often obscured these structures. Intermediate filaments were arranged both at the Z line and the intercalated disc, in parallel strands, approximately at right angles to the myofilaments.     

Vascular alterations in response to air desiccation injury in the carotid artery of the C3H/HeJ mouse

The purpose of this study was to develop a model of vascular injury in 8-week-old C3H/HeJ mice (weight, 25 to 30 g) by using air desiccation. The carotid arteries were excised 1 to 8 weeks postinjury and evaluated by Verhoeff's stain and immunocytochemistry. In the first group of mice studied (n = 107), neointimal formation occurred and peaked at Day 14. In addition, medial cell division (measured by bromodeoxyuridine labeling) peaked at Day 3, whereas intimal cell proliferation increased gradually throughout the experimental period of 21 days. In addition, extensive thrombus formation occurred within 3 days after injury. The next experiment involved 124 mice and evaluated the effect of anticoagulants on the neointimal and thrombotic response. Mice received aspirin, heparin, or vehicle-only time-release pellets. Both anticoagulants significantly decreased the neointimal and thrombotic responses. The results of this study validate our animal model as being consistent with the Response to Injury Hypothesis of atherogenesis.     

Effects of endotoxin on neutrophil-mediated ischemia/reperfusion injury in the rat heart in vivo

We have previously shown that a nonlethal dose of lipopolysaccharide (LPS) decreases L-selectin expression of neutrophils (PMNs), thereby preventing PMN-mediated reperfusion injury in the isolated heart. In the present study we determined whether or not that dose of LPS would protect hearts during in vivo ischemia and reperfusion by preventing PMN-induced reperfusion injury. Rats receiving saline vehicle showed marked myocardial injury (necrotic area/area at risk = 82%+/-2%) and significant depression in left ventricular function as assessed in the isolated isovolumic heart preparation at constant flow rates of 5, 10, 15, and 20 ml/min. The administration of LPS (100 microg/kg body wt) 7 hr prior to ischemia resulted in a reduction in myocardial damage (necrotic area/area at risk = 42%+/-3%) and preservation of function. Myocardial function was similar to that of sham ischemic saline- and LPS-treated rats. Moreover, PMN infiltration as determined by histology was quantitatively more severe in hearts of saline-treated rats than in hearts of LPS-treated rats. Isolated hearts from vehicle- and LPS-treated animals undergoing sham ischemia in vivo recovered to the same extent after in vitro ischemia/reperfusion, suggesting that LPS did not induce protection by altering intrinsic properties of the heart. Our results indicate that LPS-induced protection of the heart from in vivo PMN-mediated ischemia/reperfusion injury may be due to decreased L-selectin expression of PMNs in LPS-treated animals.     

CFTR modulates lung secretory cell proliferation and differentiation

We have permanently reversed the lethal phenotype in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-deficient (knockout) mouse after in utero gene therapy with an adenovirus containing the cftr gene. The gene transfer targeted somatic stem cells in the developing lung and intestine, and these epithelial surfaces demonstrated permanent developmental changes after treatment. The survival statistics from the progeny of heterozygote-heterozygote matings after in utero cftr gene treatment demonstrated an increased mortality in the homozygous normal pups, indicating that overexpression during development was detrimental. The lungs of these pups revealed accelerated secretory cell proliferation and differentiation. The extent of proliferation and differentiation in the secretory cells of the lung parenchyma after in utero transfer of the cftr gene was evaluated with morphometric and biochemical analyses. These studies provide further support of the regulatory role of the cftr gene in the development of the secretory epithelium.     

ELECTROPHORETIC ANALYSIS OF ENZYMES FROM THREE SPECIES OF CHLAMYDOMONAS

Isoenzyme patterns of acetone-extracted proteins revealed a close similarity between Chlamydomonas eugametos and C. moewusii but a distant relationship between the two and C. reinhardtii. Chlamydomonas eugametos and C. moewusii had identical banding patterns of malate dehydrogenase (MDH) and leucine aminopeptidase (LAP) in starch gels. These two species exhibited the same MDH distribution spectrum in analytical disc polyacrylamide gels but neither species showed definitive LAP or glutamate dehydrogenase (GDH) activity. There were differences in the starch gel alpha esterase (α-EST) patterns of C. eugametos and C. moewusii due to an additional weak band at Rf 0.75 in the latter species and a slight variation in the position of another band at Rf 0.80–0.82. Some variations between the two species also occurred in the α-EST banding in disc gels at Rf 0.70–0.85 and at Rf 0.06–0.14 with C. moewusii exhibiting the greatest number of bands. Chlamydomonas reinhardtii displayed patterns of all four enzymes but the band characteristics were distinctively different from those of C. eugametos and C. moewusii. There appeared to be no obvious isoenzyme difference between mating types of either species. It is concluded that C. eugametos and C. moewusii are not identical species but are closely related in regard to the enzymes assayed. Isoenzyme analysis is considered to be a useful approach to algal systematics.     

Ultrastructural morphometric analysis of cultured neonatal and adult rat ventricular cardiac muscle cells

Neonatal and adult rat ventricular cardiac muscle cells cultured on laminin differed from similar myocytes grown on plastic in the amount and distribution of their mitochondria and transverse tubules. Point-count morphometry was used at the electron microscopic level to quantify these differences. Adult myocytes grown on laminin contained more mitochondria per unit volume than adult myocytes grown on plastic. No significant differences were observed in the volume percent of myofibrils in either adult or neonatal ventricular myocytes when grown on laminin and compared to those grown on plastic. The transverse tubule system in neonatal and adult myocytes was reduced significantly when both groups were cultured on laminin. Furthermore, neonatal and adult myocytes cultured on laminin were flatter than those cultured on plastic. This may indicate a relationship between the surface/volume ratio and transverse tubule development in cultured myocytes. These studies establish that point-count morphometry can be used to quantify changes in the organelle volume densities of cultured cardiac muscle cells.