Herbert K. Hagler

Intracellular calcium transients and arrhythmia in isolated heart cells.

Intracellular calcium ([Ca2+]i) elevation may mediate cardiac arrhythmias. However, direct measurement of the rapid alterations of [Ca2+]i on a beat-to-beat basis using fast temporal resolution and without signal averaging in the spontaneously beating in vivo heart is lacking. Furthermore, data from an isolated spontaneously beating myocyte preparation that develops arrhythmia similar to that in the in vivo heart are unavailable. We measured rapid changes of [Ca2+]i with fast temporal resolution in isolated spontaneously beating neonatal rat ventricular myocytes with cell-to-cell communication and characterized the interrelation between [Ca2+]i and arrhythmia. An elevated extracellular calcium ([Ca2+]o) concentration of 10.8 mM induced premature beats, a rapid beating rate (tachyarrhythmia), and chaotic or fibrillatory beating activity in a small group of myocytes. [Ca2+]i levels during systole increased from the nanomolar to micromolar concentration range before arrhythmia development. Spontaneous oscillations of [Ca2+]i during diastole could evoke a spontaneous tachyarrhythmia. In the presence of [Ca2+]i elevation, a spontaneous tachyarrhythmia could induce severe [Ca2+]i overload. Reduction of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid AM (5 microM) in the presence of 10.8 mM [Ca2+]o reversed the arrhythmia. In single ventricular myocytes superfused with 10.8 mM [Ca2+]o, oscillations of membrane potential characteristic of transient inward current occurred that were prevented by ryanodine (0.1 microM), an inhibitor of Ca2+ flux across the sarcoplasmic reticulum. This study characterizes 1) an isolated multicellular myocyte model of arrhythmia similar to that evident in in vivo hearts, 2) elevation of [Ca2+]i with systolic [Ca2+]i levels of 1-3 microM and diastolic [Ca2+]i oscillations before the initiation of arrhythmia, 3) tachyarrhythmia as a cause of severe [Ca2+]i overload, which may be important in the perpetuation and degeneration of arrhythmias, and 4) reversal of arrhythmia with reduction of [Ca2+]i. The results in the isolated myocyte model may have relevance to the generation and perpetuation of certain cardiac arrhythmias associated with calcium overload.

Free radicals alter ionic calcium levels and membrane phospholipids in cultured rat ventricular myocytes

Oxygen-derived free radicals have been implicated in damage to membrane phospholipids leading to alterations in membrane function. The purpose of this study was to investigate alterations in intracellular ionic calcium (Ca2+) levels and Ca2+ transients, cellular morphology, conjugated diene levels, arachidonate release, and lactate dehydrogenase release resulting from the exposure of cultured neonatal rat ventricular myocytes to a xanthine oxidase catalyzed free radical generating system capable of producing superoxide and hydroxyl radicals. The ability of alpha-tocopherol to prevent alterations due to free radical exposure was investigated. For measurements of Ca2+, myocytes grown on coverslips for 3-4 days were loaded with fura-2/AM and studied by microspectrofluorometry. Control myocytes superfused with a physiological buffer or buffer containing purine and iron-loaded transferrin exhibited Ca2+ transients associated with spontaneous contractions. For control, buffer perfused myocytes (n = 4), the fura-2 340/380 ratios were 0.5 +/- 0.1 (mean +/- S.E.) and 1.6 +/- 0.03 at the minimum and maximum, respectively, of the Ca2+ transient, after 1 h of perfusion. Exposure to the free radical generating solution (n = 14) altered intracellular Ca2+. The 340/380 minimum ratio was 639% of the control value after approximately 30-70 mins with cessation of normal Ca2+ transients. Bleb development was associated with increased Ca2+. Myocytes reperfused with control medium continued to exhibit an elevated minimum fura-2 ratio at 687% of control. Myocytes pretreated with 10 microM alpha-tocopherol (n = 13) for 18-24 h and exposed to free radicals did not exhibit increases in intracellular Ca2+, having a minimum 340/380 ratio of 0.5 +/- 0.1 after 60-90 mins, and although myocytes often ceased contracting, they resumed spontaneous Ca2+ transients with control medium reperfusion and also maintained normal structure. Exposure of myocyte cultures to free radical generating solutions resulted in increased levels of conjugated dienes and increased release of [3H]arachidonate and lactate dehydrogenase compared to control values after 1 h. alpha-Tocopherol treatment attenuated the increase in conjugated diene levels, and the release of [3H]arachidonate and lactate dehydrogenase. Thus, free radicals alter intracellular Ca2+, conjugated dienes and membrane structure indicating their ability to induce altered ionic homeostasis in association with myocardial membrane damage. alpha-Tocopherol decreased free radical mediated injury.

Porous Hydroxyapatite as a Bone Graft Substitute in Cranial Reconstruction: A Histometric Study

To assess the potential of a porous hydroxyapatite matrix to serve as a bone graft substitute, bilateral 15 ± 20 mm craniectomy defects were reconstructed in 17 dogs with blocks of implant and split-rib autografts. Specimens were retrieved at 3, 6, 12, 24, and 48 months, and undecalcified sections were prepared for microscopy and histometry. The implant and graft cross-sectional areas did not change with time, documenting their equivalent ability to maintain cranial contour. Bone ingrowth extended across the implant from one cranial shelf to the other in 15 specimens. Little apparent bone ingrowth was seen in most graft specimens. Two implants and three grafts were nonunited, possibly due to lack of fixation or the orientation of the histology sections. The implant specimens were composed of 39.3 percent hydroxyapatite matrix, 17.2 percent bone ingrowth, and 43.5 percent soft-tissue ingrowth. The graft specimens were composed of 43.7 percent bone and 56.3 percent soft tissue. This study supported the thesis that a porous hydroxyapatite matrix may-function in part as a bene graft substitute. The brittle hydroxyapatite matrix undoubtedly became stronger with bone ingrowth, but the degree of cranial protection achieved was not measured in this study. The size of the cranial defect used in this study did not permit estimation of the distance over which bone ingrowth may be reliably expected. There remains a need for greater understanding of the causes of nonunion, the extent of predictable ingrowth depth, and the strength of the resultant implant-bone composite.

Porous hydroxylapatite as a bone graft substitute in mandibular contour augmentation: A histometric study

The buccal contour of the mandible was augmented in 17 dogs with 5 X 7.5 X 20 mm blocks of porous hydroxylapatite (HA) on one side and two-layered split rib autografts on the other. Both specimens were retrieved at three, six, 12, 24, and 48 months. Undecalcified sections were prepared for microradiography, light and UV microscopy, and histometry. A transmitted light video image digitizing system was used to trace implant and graft perimeters and calculate cross sectional areas. This system was also used to measure graft density and calculate bone and soft tissue compositions. The HA matrix, bone and soft tissue compositions of implant specimens were measured with a backscattered scanning electron microscope imaging digitizing system. All grafts became increasingly resorbed with time whereas all implants remained intact. Mature osteotonic bone ingrowth was present in all implants except one which failed to unite with the mandibular cortex. The mean graft areas decreased from 30.8 mm2 at three months to 0.7 mm2 at 48 months, while the implant areas averaged 35.5 mm2 and remained stable. The graft specimens were composed of 46.6% bone and 53.4% soft tissue or fluid space. The implant specimens were composed of 34.5% HA matrix, 28.6% bone, and 33.9% soft tissue. The HA matrix had a surface area of 9.8 mm2/mm3 that was 61.9% covered with bone ingrowth and 38.1% covered with soft tissue or fluid space. In contrast to the rapid resorption of graft onlays, the porous HA matrix demonstrated a long-term permanence with maintenance of contour and osseous incorporation over the four-year duration of this study.

Sinus histiocytosis of pelvic lymph nodes after hip replacement. A histiocytic proliferation induced by cobalt-chromium and titanium.

Six men who had undergone hip replacements for degenerative joint disease or trauma subsequently had radical prostatectomies or cystoprostatectomies with bilateral pelvic lymph node dissections for adenocarcinoma of the prostate or transitional cell carcinoma of the urinary bladder. The hip prostheses implanted in three patients were known to contain cobalt-chromium alloy and titanium. The pelvic lymph nodes ipsilateral to the hip prosthesis in five patients and the bilateral pelvic nodes in the only patient with bilateral hip prosthesis had dark brown or black cut surfaces. These lymph nodes did not contain carcinoma but showed florid sinus histiocytosis characterized by large polygonal histiocytes filling and expanding sinuses and interfollicular regions. The foamy histiocytes contained cobalt-chromium and titanium micropar-ticles by light microscopy, ultrastructure, and energy-dispersive x-ray microanalysis. The lymph nodes uninvolved by the histiocytic reaction lacked the heavy metal microparticles. Four cases were found to have a small number of polyethylene particles, which might have contributed to the histiocytic response. By immunohis-tochemistry, the foamy cells displayed immunoreactivity for lysozyme, α-1-antitrypsin, α-1-antichymotrypsin, and cathepsin D, providing additional support for their histiocytic derivation. To our knowledge, this is the first time that microparticles of cobalt-chromium and titanium that migrate from hip prostheses to pelvic lymph nodes have been shown to elicit a distinctive type of florid sinus histiocytosis. Pathologists should be aware of this characteristic foreign-body tissue response to avoid confusion with other types of sinus histiocytosis or with metastatic carcinoma.

Subcellular electrolyte alterations during progressive hypoxia and following reoxygenation in isolated neonatal rat ventricular myocytes.

This study characterizes the sequential alterations of, and relations between, multiple electrolytes in cytoplasm, mitochondria, and whole cells during hypoxia and on reoxygenation in isolated neonatal rat ventricular myocytes. Subcellular electrolyte content and distribution were measured by electron probe x-ray microanalysis, membrane phospholipid degradation by tritiated arachidonic acid release, and cell morphology by electron microscopy. At 1-2 hours of hypoxia, the myocyte population showed a loss of cytoplasmic potassium, magnesium, and chlorine without alteration of cytoplasmic sodium or calcium. Mitochondria showed increased potassium with unchanged magnesium content. There was no morphological evidence of cell injury or tritiated arachidonic acid release. At 3-5 hours of hypoxia, the myocyte population showed a further loss of cytoplasmic potassium and magnesium and an increase in cytoplasmic sodium, chlorine, and calcium. At a single-cell level, the increase in cytoplasmic sodium preceded the increase in cytoplasmic calcium. Mitochondria showed increased sodium and chlorine and decreased magnesium before increased calcium content; potassium loss was manifest only at 5 hours of hypoxia. At 3-5 hours of hypoxia, there was also tritiated arachidonic acid release and morphological evidence of cell injury. Reoxygenation for 1 hour after 5 hours of hypoxia partially reversed the mean alterations of all electrolytes, except calcium, in the cytoplasm of the myocyte population, whereas analysis was required at a single-cell level to show a partial reversal in calcium levels in cytoplasm of reoxygenated cells. Reoxygenation for 1 hour after 5 hours of hypoxia partially reversed the mean alterations of all electrolytes, including calcium, in the mitochondria of the myocyte population. Recovery of potassium in the cytoplasm correlated with reduction of mitochondrial calcium content on reoxygenation and best predicted recovery of cellular homeostasis of sodium, chlorine, magnesium, and calcium. This study demonstrates that in this experimental model of hypoxia 1) initial losses of cytoplasmic potassium and magnesium occur in the absence of cell injury; 2) increases of sodium, chlorine, and calcium occur in association with cell injury, with sodium increasing before calcium; 3) membrane phospholipid degradation and electrolyte derangement, including increased calcium, may contribute to reversible and irreversible phases of cell injury; 4) analysis of calcium at a subcompartmental level and at a single-cell level is required to correlate reduction of calcium on reoxygenation with recovery of cell homeostasis; 5) reduction of calcium content in mitochondria may predict recovery of cell homeostasis; and 6) recovery of potassium on reoxygenation best predicts recovery of cell membrane function and cell homeostasis.

Ultramicrotomy for biological electron microscopy.

This chapter describes the practical details involved in the successful sectioning of plastic-embedded specimens for electron microscopy. The focus will be on those parts of the process that are really key to the successful sectioning of any biological material, i.e., the proper shape and size of the specimen, sharp knives and how to make them, and the orientation of specimen and production of ultrathin sections onto water-filled boats attached to either glass or diamond knives. This chapter is designed to assist the beginner in microtomy in understanding more fully the terms and basic principles involved in producing ultrathin sections of most plastic-embedded biological specimens.

X-Ray Microanalysis and Free Calcium Measurements in Cultured Neonatal Rat Ventricular Myocytes

The response of cardiac myocytes to changes in calcium homeostasis is thought to be an important factor in cardiac myocyte injury under various conditions of ischemia, hypoxia and reperfusion injury (Allen and Orchard, 1983; Barry et al., 1987; Buja et al., 1985; Farber, 1982; Jennings and Reiner, 1981; Nayler, 1981). During the past several years advances in cryofixation, data collection and interpretation of electron beam excited x-ray microanalysis have been developed in our laboratory (Hagler et al., 1979; Hagler et al., 1983; Hagler and Buja, 1986). The use of x-ray microanalysis techniques have been important in understanding changes in total elemental content on a compartment by compartment and a cell by cell basis (Wendt-Gallitelli and Jacob, 1982; Wendt-Gallitelli and Wolburg, 1984; Wheeler-Clark and Tormey, 1987; Somlyo et al., 1985). A model of hypoxic and ischemic injury in our laboratory has been a primary culture of neonatal rat ventricular myocytes (Buja et al., 1985). Observations will be presented in this paper from this cultured cell model to make general points about biological x-ray microanalysis. The data analysis schemes which we have devised to look at a changing population of cells will be illustrated from this work. We will also present some data on intracellular free calcium measurements and their correlation with x-ray microanalysis measurements in this model.

Subcellular calcium shifts in ischemia and reperfusion

Subcellular calcium shifts and pathological calcification are observed under conditions of myocardial ischemia and/or reperfusion cell injury and represent the more severe manifestations of abnormal calcium (Ca2+) homeostasis. The use of specialized techniques for measurement of intracellular electrolytes such as electron probe x-ray microanalysis and the use of the fluorescent Ca2+ indicator, fura 2, are providing new insights into regulation of intracellular Ca2+ and the role of altered Ca2+ homeostasis in the pathogenesis of myocardial cell injury. During the progression of myocardial cell injury, investigations indicate that increased intracellular Ca2+ develops in association with other manifestations of membrane injury, including other electrolyte alterations, altered cell volume regulation, and altered membrane phospholipids.