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Correlation of cell surface alterations with contractile response in laser microbeam irradiated myocardial cells
Printed in Sweden Copyright @ 1979 by Academic Press. IX All rights of reproduction in any form rererved @3l4-4827/79/,30341-I 1$0?.00/0
Experimental Cell Research 118 (1979) 341-351
CORRELATION CONTRACTILE
OF CELL
SURFACE
RESPONSE
IRRADIATED
ALTERATIONS
IN LASER
MYOCARDIAL
A Scanning Electron Microscope JANIS M. BURT, KENNETH
WITH
MICROBEAM CELLS Study
R. STRAHS and MICHAEL
W. BERNS
SUMMARY The contractile behavior and surface morphology of cultured neonatal rat heart cells were examined by phase contrast and scanning electron microscopy (SEM) following laser irradiation of single mitochondria. Irradiation always resulted in damage to the target mitochondrion (as determined by phase microscopy) and was associated with one of three contractile states, each of which correlated with a specific surface morphology over the irradiated mitochondrion. The results demonstrate that: (I) changes in the contractile activity of the cell correlate directly with morphological changes in the target organelle and in the membrane overlying the target organelle; (2) when the contractile activity of the cell remains unchanged, the morphology of the membrane overlying the target organelle appears normal via SEM even though the organelle is visibly damaged as judged by phase contrast microscopy: (3) the correlation between contractile behavior and surface morphology was the same regardless of which cell surface the laser beam passed through when entering the cell (i.e.. through the cell surface directly apposed to the glass or through the free cell surface directly exposed to the medium); (4) the mitochondrial lesions could be compared to lesions made in dried red blood cells irradiated from either surface. (Again the lesions appeared identical regardless of the cell surface through which the laser beam entered.) These observations suggest that laser damage is produced equally in all directions from the focal point.
Laser microbeam irradiation of cellular organelles has been used extensively in the study of cellular contractility [l-.5]. Mitochondrial irradiation with a green (532 nm) or ultraviolet (265 nm) beam focused to a spot diameter of less than 1 pm results in damage to the organelle the extent of which can be correlated with various contractile changes. A common contractile change in response to irradiation is the temporary replacement of normal synchronous beating by an arrhythmic, uncoordinated, rapid beating activity. This and other contractile responses have been shown with phase contrast and transmission electron microscopy (TEM) to correlate with characteristic 23 - 7x IX03
types of damage to the mitochondrion [ 1, 3, 61. Strahs et al. [7] demonstrated that myofibril irradiation induced contractile changes as frequently as mitochondrial irradiation suggesting that- the induced contractile responses were not necessarily related to the particular cell structure irradiated. Insight as to the mechanism by which these contractile changes occur was first obtained by Kitzes et al. [2] who demonstrated that changes in the electrical properties of the membrane-occurred with irradiation and the onset of the contractile changes. Further evidence for the involvement of the cell surface was obtained by Strahs et al. [7] who irradiated carbon par-
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Burt, Strahs and Berns
titles attached to the cell surface. In these studies, surface irradiation induced arrhythmic contractile activity in a much higher percentage of cases with much less laser energy than was required to produce similar changes following mitochondrion or myofibril irradiation, They also demonstrated that the contractile responses were dependent on extracellular calcium. The present studies were undertaken to further probe the involvement of the cell surface in the laser-induced contractile response. We report here the following: (1) It is possible to specifically irradiate and damage a mitochondrion without inducing a contractile alteration or an alteration in the morphology of the cell surface; (2) by increasing the intensity of the laser energy, more severe mitochondrial lesions can be made that are accompanied by contractile changes and changes in the surface morphology over the target organelle; (3) the appearance of the lesions produced by laser irradiation of red blood cells and mitochondria suggest that damage is produced equally in all directions from the central focus point.
O,, 55% NZ atmosphere. After 2-3 days in culture, the cells were fed daily. The cells were generally used for irradiation after 3-5 days in culture.
Microirradiation
technique
Laser microirradiation was oerformed using the 532 nm (green) wavelength of a frequency-doGbled, Qswitched. neodymium-YAG laser. Laser output was between 4-5 kW with a pulse duration of 180 nsec. Calibrated neutral density filters were used to attenuate the energy per pulse of the laser beam to between 1-2 KJ for the weakest lesions and between 2.54.5 PJ for “moderate” lesions. The diameter of the focal “point” has been estimated to be 0.5-1.0 pm. across which the energy distribution would be Gaussian. The laser microbeam configuration was similar to the one described in earlier studies and employed either a loOX Neofluar objective or a 40X phase water immersion objective [3, 91. Cells were observed prior to, during, and after irradiation on a video monitor that was interfaced with a modified Sonv time-lapse videotape system. With this system, ii was possible to monitor the contractile pattern during the entire time course of the experiment. Single cells or small clusters of cells to be used for irradiation were circled with black ink on the outside of the Rose chamber to allow for quick relocation. Irradiations were performed using either a 100x oil immersion objective and irradiating a cell through the glass or a 40x water immersion objective and irradiating a cell through the medium. Within 2 min following irradiation, the cells were fixed with 3% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2. All the cells that exhibited contractile alterations were fixed while exhibiting the altered state.
Scanning electron microscopy cells. Following fixation, a 250 Frn circle was scratched into the glass around the cell with a diamond scoring objective. This procedure facilitated relocating the irradiated cell on the scanning scope since the circle was clearly visible. Cells were processed for SEM according to the procedures of Cohen et al. [lo]. The cultures were rinsed
Hart
MATERIALS
AND METHODS
Cell cultures Primary cultures of neonatal rat (l-2 days old) ventricular cells were established in Rose chambers according to a modification of the procedures used by Mark & Strasser f81. Twelve to I8 neonatal rats were sacrificed and their hearts removed and bathed in heart medium supplemented with 10% fetal calf serum (FCS) and antibiotics (penicillin and streptomycin). On a frosted slide the auricles were removed and discarded. The ventricles were cut into 1-2 mm3 pieces and subjected to six stepwise enzymatic digestions (15 min each) with 0.25% viokase. The second and third fractions were resuspended in heart medium and injected into Rose chambers at a cell density of 5-7~ lo5 cells/chamber. Non-muscle cells were allowed to attach to the top coverslip (usually plastic) during the first 2 h of culture (pre-plating) and then the chambers were flipped over, allowing the muscle cells to attach to the other coverslip (always glass). The cells were incubated at 37°C in a 5% CO,, 40%
Fig. I. Phase contrast micrographs of a heart cell (N)
prior to; (b) post laser irradiation of a single mitochondrion (urron,). This lesion type, classified as moderate, is characterized by a central phase light area surrounded by a phase dark ring. Note that the remainder of the mitochondrion is paled. Bar, 5 pm. Fig. 2. SEM of the same cell as in fig. I. (0) Low power magnification. Alignment of organelles with the organelles visible in the phase contrast micrographs is possible; (b) high magnification of the target mitochondrion. Note the outline of the mitochondrion (smnll urron’s) which corresponds to the paled portion of the mitochondrion in the phase micrographs (fig. I). Also note the raised ring and central dimple which corresponds in the phase micrographs (fig. I) to the phase dark ring and central light zone respectively. Bar, (a) 5; (h) 2.5 Frn.
Contractility and the cell surfcrce
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RESULTS Myocardial
Fig. 3. SEM of a heart cell with a moderate lesion in one mitochondrion. Note the apparent perforation of the membrane in the central dimple. Bar, 2.5 pm.
in buffer, dehydrated in a graded ethanol-water series followed by an ethanol-freon 113 series and critical point dried in a Bomar SPE-!%O/EX critical point dryer using freon 13. The coverslips were mounted on aluminum studs with silver paint and vacuum-coated with gold for 2-4 min on a Technics Hummer II. Cells were observed with an Hitachi no. HS500 SEM at accelerating voltages between 15-20 kV and tilt angles between 45 and 65 degrees. Relocation of the irradiated cell and scratched circle was further facilitated by scoring a l-2 mm semicircle into the gold coat around the cell through a dissecting microscope. The final identification of the irradiated cell while viewing with the SEM was made by comparison with photographs taken at the time of irradiation. Red blood cells. Red blood cells were air-dried on no. I thickness coverglasses. Some cells were irradiated directly through the coverglass using a 100x oil immersion objective. In these cases, the cells were immediately coated with gold and observed on the SEM. Other cells were irradiated with the 100~ oil immersion objective from the exposed cell surface by placing a drop of immersion oil directly on the cell. These cells were subjected to the same dehydration and critical point drying procedures as the myocardial cells in preparation for SEM. E.tp Cell He\ /IX 11979J
cells
Ten of 14 cells irradiated with a 100x objective through the glass were successfully located and the target mitochondrion identified on the SEM. The irradiated cells were easily identified within the scored circles (see Materials and Methods) on the basis of cell shape. The target mitochondrion was found by aligning the organelles visible in the light microscope micrographs with those visible on the SEM (compare figs 1 and 2). Because the cells are only 2-4 km thick, cell organelles, such as the nucleus, nucleoli, and individual mitochondria, are readily discernible (fig. 2). Each cell could be placed into one of three groups on the basis of lesion morphology and contractile response. As with earlier studies, there was a clear correlation between the degree of mitochondrial alteration and the contractile response [ 1, 31. The first group included four cells which began arrhythmic, uncoordinated beating activity following irradiation. The target mitochondrion of each of these cells exhibited the moderate lesion morphology [3] consisting of a phase darkened spherical area with a central light zone. The remainder of the mitochondrion exhibited a decrease in phase contrast called “paling” (fig. 1). When viewed with the SEM, the membrane overlying the target mitochondrion appears as a raised blister with a central dimple (fig. 2) which in some cases appears to be perforated (fig. 3). Fig. 4. Phase contrast micrographs of a heart cell ((1)
prior to; (6) after production of a “weak” lesion in one mitochondrion bv laser irradiation. Arrows indicate the target mitochondrion. Note the phase “paling” of the damaged mitochondrion. Bar, 5 pm. Fig. 5. SEM of (a) the same cell as in fig. 4; (h) a second cell with the same type of weak lesion. Note the apparent lack of damage- to the membrane overlying the target organelles (LWOWS).Bar. I pm.
Contractility and the cell surface
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Strnhs
and Berm
The second group included five cells microscope micrograph (fig. 10). These which had no change in their contractile cells were irradiated through the glass covpatterns following irradiation. The target erslip; therefore, the SEM lesions were obmitochondrion in four of these five cells ex- served on the cell surface opposite the side hibited only “paling” (fig. 4) while the fifth of laser entry. exhibited a phase dark portion within the Lesions similar to those described above otherwise paled mitochondrion (fig. 6). were observed in red blood cells irradiated When viewed with the SEM, the four cells directly through oil (see Materials and exhibiting only paling had no surface altera- Methods). The “perforated blister” and the tions over the target mitochondrion (fig. 5). “blister” types of lesions are illustrated in The fifth cell with the phase dark area ex- fig. 11. Though the lesions were similar unhibited a small blister over the target mito- der both irradiation conditions, the surfaces of the red blood cells that were processed chondrion as seen in fig, 70. The third group included one cell which by dehydration and critical point drying underwent a transient alteration in beating were somewhat distorted and appeared to rhythmicity. In this cell, the target mito- be covered with more debris. chondrion was paled with a central phase dark area (similar to that seen in fig. 6). The cell surface over the target mitochonDISCUSSION drion exhibited a small blister (fig. 7h). In order to view the lesion from the side Lasers have been extensively used to inof laser entry, cells were irradiated from the vestigate cell contractility. In a recent re“top” side through a 40x water immersion port, Strahs et al. [7] demonstrated that objective. Three cells that exhibited pro- laser irradiation of various cell organelles longed, altered contractile activity in re- leads to alteration in the contractile activity sponse to this irradiation procedure were of the irradiated cell. They found that alobserved by SEM. The morphology of the terations occurred most frequently when lesions in these cells (fig. 8) appeared very the target was the membrane (via attached similar to the lesions found in group one carbon particles). The membrane was further implicated by Kitzes et al. [2] who above (see fig. 2). demonstrated that a depolarization of the Red blood cells cell accompanied laser irradiation and the Lesions were made in red blood cells using onset of altered contractile patterns. Simidifferent laser energy densities in order to produce damage of varying degrees of se- Fi.q. 6. Phase contrast micrographs of a heart cell (a) verity. Lesions of three types were ob- prior to; (b) after production of a weak lesion in one mitochondrion by laser irradiation. (a) Arrow indicates served with the SEM: (1) a gross perfora- target mitochondrion; (b) note the “paled” mitochontion greater than 2 pm in diameter (fig. 9~); drion with a central dark circle (arrow). This cell exhibited no contractile change. Similar lesions can be (2) lesions of approx. I km in diameter that associated with a transient contractile change. Bar, appear as raised “blisters” with small per- 5fim. 7. SEM of (u) the same cell as seen in fig. 6. forations in the center (fig. 9b); (3) lesions Fig. Note the small bump on the surface which corresponds of 1 pm and less that appear only as raised in position to the phase dark zone in the light mi(b) another cell in which the same type of “blisters” without any perforation (fig. SC). crograph; lesion was produced. This cell exhibited a transient The same lesions are depicted in a light slight change in contractile pattern. Bar, 1 pm.
Contractility and the cell surface
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Burt, Strahs and Berns
lar alterations in contractile activity occur sult in an uncommon physical effect, such as a result of membrane damage upon poor as acoustic pressure or plasma formation. impalement of heart cells (personal obserThe severity of mitochondrial lesions vation). Clearly, these studies suggest that produced by laser irradiation has been when laser irradiation of heart cells results shown to be dependent on laser energy denin contractile changes, there is a mem- sity and has been correlated with the apbrane effect; however, the exact nature of pearance of the unirradiated organelle in the this effect is unclear. phase contrast microscope [I, 3, 6, 111.The Laser irradiation of red blood cells pro- phase density of cell components is dependvides an interesting comparison for the ir- ent on the optical path length through the radiation of living cells in that they are no structure and on the refractive indices of longer alive at the time of irradiation and, the materials which compose them. Pretherefore, have no repair processes. Thus, vious results indicate [3, 61 that the conthe primary effects of irradiation on the cell centration, distribution and biological state can be examined. Furthermore, they are a (i.e., denatured or active) of materials withfairly uniform population of cells in terms of in the mitochondria are altered by irradiasize and quantity of energy absorbing pig- tion which could account for changes in ment per unit area. Thus, investigations of the phase contrast image of the organelle. the range of effects that the laser is capable TEM studies have been performed on laserof producing are possible by attenuating the induced mitochondrial lesions of varying quantity of energy actually striking the cell. severity. These studies have demonstrated The threshold lesion has a small but visible that phase dense areas correspond to eleceffect at the cell surface (figs 9c, 10~) while tron dense areas and phase light areas corthe most severe lesions result in large per- respond to electron lucent areas. Thus, alforations (figs 9a, lOa). The intermediate though the exact energy distribution across lesions demonstrate the continuum between the irradiated cell or organelle cannot be these extremes. When lesions are made measured directly, the ultrastructural morwith the same laser energy density and at a phology of the laser lesions clearly reflects focal point midway between the upper and the distribution of laser energy within the lower cell surfaces, their appearance in SEM is the same regardless of the side of Fig. 8. SEM of a cell irradiated through the medium laser entry. TEM investigations using serial with a 40x objective. Note that the lesion appears very similar to the lesion seen in figs 2 and 3 with a sections of mitochondrial lesions produced central dimple surrounded by a raised ring. Bar, I pm. in living cells demonstrate that laser dam- Fig. 9. SEM of red blood cells with several lesions made by laser irradiation through the glass: gross perage is spherical in nature and most severe foration (a), raised blister with central dimple and percentrally. These results coupled with the foration (b), and raised blister with no dimple or perforation (c). Bar, 5 pm. Gaussian energy distribution of the laser Fig. 10. Phase contrast micrograph of the same red beam across the focal point suggest that pri- blood cells seen in fig. 9: Gross perforation (u), raised with central dimple and perforation (b), and mary absorption of laser energy occurs at blister raised blister with no perforation or dimple (L.). Bar, the focal point. Absorbed energy is most sigr”il. Scanning electron micrograph of red blood probably converted to heat and subsequent- cells with several lesions made by laser irradiation ly dissipates equally in all directions from through oil. Note the lesion type consisting of a raised without central dimple or perforation (a) and the this point. However, it should also be blister more severe lesion type consisting of a raised blister pointed out that the laser energy could re- with central dimple and perforation (b). Bar, 2 pm. El/l Cdl/t-r.\II8 (1979)
Contractility and the ceil surface
349
350
Burt,
Strahs
and Berns
target cell or organelle. They further demonstrate that the unfocused part of the laser beam (above and below the focal point) results in no visible damage to cellular structure. The present study attempts to examine the surfaces above and below the irradiated structure in “moderate” mitochondrial lesions as compared to “weak” lesions where we know that the damage zone does not exceed the limits of the target organelle. In this study, “weak” lesions which consist of a paled mitochondrion were made with low laser energy density. These lesions result in no visible surface alterations and in no alterations of cellular contractility. This corroborates the TEM evidence that the damage characteristic of this lesion type is confined to the target organelle [6]. The damage associated with “weak” lesions characterized by a paled mitochondrion with a central dark spot has also been demonstrated by TEM to be confined to the target organelle [3]. In two cells with this lesion type, a small bump was observed at the surface over the area of the dark spot within the target mitochondrion. These results can be explained by postulating that the bump is a result of localized thermally induced expansion of the organelle accompanied by a distortion of the overlying membrane. Thus, the general shape of the lesion is quite likely due to alterations of the shape of the target organelle which the membrane is lying over. When lesions of a similar size and shape to those referred to as moderate mitochondrial lesions are induced in red blood cells, there appears to be a perforation of the red cell membrane within the central depression. Lesions of this type in living heart cells always resulted in drastic contractile changes. Thus far, it has not been possible to determine whether or not a momentary
perforation of the heart cell membrane actually occurs, but such an event could account for both the temporary membrane depolarization observed by Kitzes et al. [2] and the observed contractile changes. in that study it was found that the time required for return to normal activity following irradiation varied but was between 1 and 5 min. This interval is close to the period between irradiation and fixation in the present studies so that any induced perforations might have had time to reseal and would have gone undetected in later microscopic examination. Although the results presented here can be explained by postulating a perforation, such a postulation is not necessary to explain the observations. If the damage to the organelle is thermal in nature, then the increased temperature could alter the physical characteristics of the membrane. Such physical effects are likely to affect, at least transiently, the permeability and function of the. membrane overlying the target organelle. Nathan et al. [12] demonstrated a direct effect of ultraviolet light on heart cells that was best explained by a direct alteration of membrane channel conductantes by the incident light. Clearly these possibilities are not eliminated as explanations of the observations reported here. Divergent results have been obtained by several investigators when comparing the frequency at which contractile alterations can be induced by irradiation of various subcellular organelles [5, 71. For example, Salet [5] found that nuclear irradiation gave rise to contractile alterations less frequently than did cytoplasmic irradiations. By considering the location of these two target areas with respect to their proximity to the membrane and the possible differences in pigment densities of the two targets, a logical explanation of the results can be made.
Contractility
Typical pictures of cells in monolayer culture demonstrate the differences in cell thickness in the nuclear region versus in the cytoplasmic region (present study and [13]). If the laser’s focal point were in the middle of the cell (i.e., midway between the upper and lower membranes), then more energy would have to be converted to heat for an effect on the overlying membrane to occur in a nuclear irradiation versus in a cytoplasmic irradiation. Therefore, an energy density sufficient to cause lesions and contractile changes in cytoplasmic irradiations may not be sufficient to do the same in nuclear irradiations. In conclusion, the observations reported here strongly suggest that the contractile alterations observed in heart cells following laser micron-radiation of mitochondria are not due directly to the damage of the target organelle but rather to an effect on the cell membrane. The authors would like to thank Marie Hammer-Wilson for her assistance in the laboratory and Elaine
and the cell surface
351
Kato for typing the manuscript. This research was supported by NIH grants HLl5740, GM23445, GM22754, and Air Force grant AFOSR-77-3136.
REFERENCES I. Berns, M W, Gross, DC L, Cheng, W K &Woodring, D, J mol cell cardiol4 (l972)-471. 2. Kitzes, M, Twinas. G & Bems. M W. J cell ohvsiol . _ 93 (1977) 99. -3. Rattner, J B, Lifsics, M, Meredith, S & Bems, M W, J mol cell cardiol 8 (1976) 239. 4. Salet, C, Exp cell res 73 (1972) 360. 5. &Jet, C, Moreno, G & Vinzens, F, Exp cell res 100 (1976) 365. 6. Adkisson, K P, Bait, D, Burgott, S L, Cheng, W K & Bems, M W, J mol cell cardiol5 (1973) 559. 7. Strahs, K R, Burt, J M & Berns, M W, Exp cell res 113(1978) 75. 8. Mark, G E & Strasser, F F, Exp cell res 44 (1966) 217. 9. Berm, M W, Exp cell res 65 (1971)470. IO. Cohen, A L, Marlow, D P & Gamer, G E, J microsc 7 (1%8) 33I. Il. Bems, M W, Gamaleja, N, Olson, R, Duffy, C & Rounds, D E, J cell nhvsiol76 (1970) 207. 12. Nathan, R D, Poole;, J P & DeHaan, R L, J gen physio167 (1976) 27. 13. Revel, J P & Wilken, K, Exp cell res 78 (1973) 1. Received June 7, 1978 Revised version received August 28, 1978 Accepted August 3 I, 1978
Exp Cell Res I I8 ( 19791
Experimental Cell Research 118 (1979) 341-351
CORRELATION CONTRACTILE
OF CELL
SURFACE
RESPONSE
IRRADIATED
ALTERATIONS
IN LASER
MYOCARDIAL
A Scanning Electron Microscope JANIS M. BURT, KENNETH
WITH
MICROBEAM CELLS Study
R. STRAHS and MICHAEL
W. BERNS
SUMMARY The contractile behavior and surface morphology of cultured neonatal rat heart cells were examined by phase contrast and scanning electron microscopy (SEM) following laser irradiation of single mitochondria. Irradiation always resulted in damage to the target mitochondrion (as determined by phase microscopy) and was associated with one of three contractile states, each of which correlated with a specific surface morphology over the irradiated mitochondrion. The results demonstrate that: (I) changes in the contractile activity of the cell correlate directly with morphological changes in the target organelle and in the membrane overlying the target organelle; (2) when the contractile activity of the cell remains unchanged, the morphology of the membrane overlying the target organelle appears normal via SEM even though the organelle is visibly damaged as judged by phase contrast microscopy: (3) the correlation between contractile behavior and surface morphology was the same regardless of which cell surface the laser beam passed through when entering the cell (i.e.. through the cell surface directly apposed to the glass or through the free cell surface directly exposed to the medium); (4) the mitochondrial lesions could be compared to lesions made in dried red blood cells irradiated from either surface. (Again the lesions appeared identical regardless of the cell surface through which the laser beam entered.) These observations suggest that laser damage is produced equally in all directions from the focal point.
Laser microbeam irradiation of cellular organelles has been used extensively in the study of cellular contractility [l-.5]. Mitochondrial irradiation with a green (532 nm) or ultraviolet (265 nm) beam focused to a spot diameter of less than 1 pm results in damage to the organelle the extent of which can be correlated with various contractile changes. A common contractile change in response to irradiation is the temporary replacement of normal synchronous beating by an arrhythmic, uncoordinated, rapid beating activity. This and other contractile responses have been shown with phase contrast and transmission electron microscopy (TEM) to correlate with characteristic 23 - 7x IX03
types of damage to the mitochondrion [ 1, 3, 61. Strahs et al. [7] demonstrated that myofibril irradiation induced contractile changes as frequently as mitochondrial irradiation suggesting that- the induced contractile responses were not necessarily related to the particular cell structure irradiated. Insight as to the mechanism by which these contractile changes occur was first obtained by Kitzes et al. [2] who demonstrated that changes in the electrical properties of the membrane-occurred with irradiation and the onset of the contractile changes. Further evidence for the involvement of the cell surface was obtained by Strahs et al. [7] who irradiated carbon par-
342
Burt, Strahs and Berns
titles attached to the cell surface. In these studies, surface irradiation induced arrhythmic contractile activity in a much higher percentage of cases with much less laser energy than was required to produce similar changes following mitochondrion or myofibril irradiation, They also demonstrated that the contractile responses were dependent on extracellular calcium. The present studies were undertaken to further probe the involvement of the cell surface in the laser-induced contractile response. We report here the following: (1) It is possible to specifically irradiate and damage a mitochondrion without inducing a contractile alteration or an alteration in the morphology of the cell surface; (2) by increasing the intensity of the laser energy, more severe mitochondrial lesions can be made that are accompanied by contractile changes and changes in the surface morphology over the target organelle; (3) the appearance of the lesions produced by laser irradiation of red blood cells and mitochondria suggest that damage is produced equally in all directions from the central focus point.
O,, 55% NZ atmosphere. After 2-3 days in culture, the cells were fed daily. The cells were generally used for irradiation after 3-5 days in culture.
Microirradiation
technique
Laser microirradiation was oerformed using the 532 nm (green) wavelength of a frequency-doGbled, Qswitched. neodymium-YAG laser. Laser output was between 4-5 kW with a pulse duration of 180 nsec. Calibrated neutral density filters were used to attenuate the energy per pulse of the laser beam to between 1-2 KJ for the weakest lesions and between 2.54.5 PJ for “moderate” lesions. The diameter of the focal “point” has been estimated to be 0.5-1.0 pm. across which the energy distribution would be Gaussian. The laser microbeam configuration was similar to the one described in earlier studies and employed either a loOX Neofluar objective or a 40X phase water immersion objective [3, 91. Cells were observed prior to, during, and after irradiation on a video monitor that was interfaced with a modified Sonv time-lapse videotape system. With this system, ii was possible to monitor the contractile pattern during the entire time course of the experiment. Single cells or small clusters of cells to be used for irradiation were circled with black ink on the outside of the Rose chamber to allow for quick relocation. Irradiations were performed using either a 100x oil immersion objective and irradiating a cell through the glass or a 40x water immersion objective and irradiating a cell through the medium. Within 2 min following irradiation, the cells were fixed with 3% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2. All the cells that exhibited contractile alterations were fixed while exhibiting the altered state.
Scanning electron microscopy cells. Following fixation, a 250 Frn circle was scratched into the glass around the cell with a diamond scoring objective. This procedure facilitated relocating the irradiated cell on the scanning scope since the circle was clearly visible. Cells were processed for SEM according to the procedures of Cohen et al. [lo]. The cultures were rinsed
Hart
MATERIALS
AND METHODS
Cell cultures Primary cultures of neonatal rat (l-2 days old) ventricular cells were established in Rose chambers according to a modification of the procedures used by Mark & Strasser f81. Twelve to I8 neonatal rats were sacrificed and their hearts removed and bathed in heart medium supplemented with 10% fetal calf serum (FCS) and antibiotics (penicillin and streptomycin). On a frosted slide the auricles were removed and discarded. The ventricles were cut into 1-2 mm3 pieces and subjected to six stepwise enzymatic digestions (15 min each) with 0.25% viokase. The second and third fractions were resuspended in heart medium and injected into Rose chambers at a cell density of 5-7~ lo5 cells/chamber. Non-muscle cells were allowed to attach to the top coverslip (usually plastic) during the first 2 h of culture (pre-plating) and then the chambers were flipped over, allowing the muscle cells to attach to the other coverslip (always glass). The cells were incubated at 37°C in a 5% CO,, 40%
Fig. I. Phase contrast micrographs of a heart cell (N)
prior to; (b) post laser irradiation of a single mitochondrion (urron,). This lesion type, classified as moderate, is characterized by a central phase light area surrounded by a phase dark ring. Note that the remainder of the mitochondrion is paled. Bar, 5 pm. Fig. 2. SEM of the same cell as in fig. I. (0) Low power magnification. Alignment of organelles with the organelles visible in the phase contrast micrographs is possible; (b) high magnification of the target mitochondrion. Note the outline of the mitochondrion (smnll urron’s) which corresponds to the paled portion of the mitochondrion in the phase micrographs (fig. I). Also note the raised ring and central dimple which corresponds in the phase micrographs (fig. I) to the phase dark ring and central light zone respectively. Bar, (a) 5; (h) 2.5 Frn.
Contractility and the cell surfcrce
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RESULTS Myocardial
Fig. 3. SEM of a heart cell with a moderate lesion in one mitochondrion. Note the apparent perforation of the membrane in the central dimple. Bar, 2.5 pm.
in buffer, dehydrated in a graded ethanol-water series followed by an ethanol-freon 113 series and critical point dried in a Bomar SPE-!%O/EX critical point dryer using freon 13. The coverslips were mounted on aluminum studs with silver paint and vacuum-coated with gold for 2-4 min on a Technics Hummer II. Cells were observed with an Hitachi no. HS500 SEM at accelerating voltages between 15-20 kV and tilt angles between 45 and 65 degrees. Relocation of the irradiated cell and scratched circle was further facilitated by scoring a l-2 mm semicircle into the gold coat around the cell through a dissecting microscope. The final identification of the irradiated cell while viewing with the SEM was made by comparison with photographs taken at the time of irradiation. Red blood cells. Red blood cells were air-dried on no. I thickness coverglasses. Some cells were irradiated directly through the coverglass using a 100x oil immersion objective. In these cases, the cells were immediately coated with gold and observed on the SEM. Other cells were irradiated with the 100~ oil immersion objective from the exposed cell surface by placing a drop of immersion oil directly on the cell. These cells were subjected to the same dehydration and critical point drying procedures as the myocardial cells in preparation for SEM. E.tp Cell He\ /IX 11979J
cells
Ten of 14 cells irradiated with a 100x objective through the glass were successfully located and the target mitochondrion identified on the SEM. The irradiated cells were easily identified within the scored circles (see Materials and Methods) on the basis of cell shape. The target mitochondrion was found by aligning the organelles visible in the light microscope micrographs with those visible on the SEM (compare figs 1 and 2). Because the cells are only 2-4 km thick, cell organelles, such as the nucleus, nucleoli, and individual mitochondria, are readily discernible (fig. 2). Each cell could be placed into one of three groups on the basis of lesion morphology and contractile response. As with earlier studies, there was a clear correlation between the degree of mitochondrial alteration and the contractile response [ 1, 31. The first group included four cells which began arrhythmic, uncoordinated beating activity following irradiation. The target mitochondrion of each of these cells exhibited the moderate lesion morphology [3] consisting of a phase darkened spherical area with a central light zone. The remainder of the mitochondrion exhibited a decrease in phase contrast called “paling” (fig. 1). When viewed with the SEM, the membrane overlying the target mitochondrion appears as a raised blister with a central dimple (fig. 2) which in some cases appears to be perforated (fig. 3). Fig. 4. Phase contrast micrographs of a heart cell ((1)
prior to; (6) after production of a “weak” lesion in one mitochondrion bv laser irradiation. Arrows indicate the target mitochondrion. Note the phase “paling” of the damaged mitochondrion. Bar, 5 pm. Fig. 5. SEM of (a) the same cell as in fig. 4; (h) a second cell with the same type of weak lesion. Note the apparent lack of damage- to the membrane overlying the target organelles (LWOWS).Bar. I pm.
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The second group included five cells microscope micrograph (fig. 10). These which had no change in their contractile cells were irradiated through the glass covpatterns following irradiation. The target erslip; therefore, the SEM lesions were obmitochondrion in four of these five cells ex- served on the cell surface opposite the side hibited only “paling” (fig. 4) while the fifth of laser entry. exhibited a phase dark portion within the Lesions similar to those described above otherwise paled mitochondrion (fig. 6). were observed in red blood cells irradiated When viewed with the SEM, the four cells directly through oil (see Materials and exhibiting only paling had no surface altera- Methods). The “perforated blister” and the tions over the target mitochondrion (fig. 5). “blister” types of lesions are illustrated in The fifth cell with the phase dark area ex- fig. 11. Though the lesions were similar unhibited a small blister over the target mito- der both irradiation conditions, the surfaces of the red blood cells that were processed chondrion as seen in fig, 70. The third group included one cell which by dehydration and critical point drying underwent a transient alteration in beating were somewhat distorted and appeared to rhythmicity. In this cell, the target mito- be covered with more debris. chondrion was paled with a central phase dark area (similar to that seen in fig. 6). The cell surface over the target mitochonDISCUSSION drion exhibited a small blister (fig. 7h). In order to view the lesion from the side Lasers have been extensively used to inof laser entry, cells were irradiated from the vestigate cell contractility. In a recent re“top” side through a 40x water immersion port, Strahs et al. [7] demonstrated that objective. Three cells that exhibited pro- laser irradiation of various cell organelles longed, altered contractile activity in re- leads to alteration in the contractile activity sponse to this irradiation procedure were of the irradiated cell. They found that alobserved by SEM. The morphology of the terations occurred most frequently when lesions in these cells (fig. 8) appeared very the target was the membrane (via attached similar to the lesions found in group one carbon particles). The membrane was further implicated by Kitzes et al. [2] who above (see fig. 2). demonstrated that a depolarization of the Red blood cells cell accompanied laser irradiation and the Lesions were made in red blood cells using onset of altered contractile patterns. Simidifferent laser energy densities in order to produce damage of varying degrees of se- Fi.q. 6. Phase contrast micrographs of a heart cell (a) verity. Lesions of three types were ob- prior to; (b) after production of a weak lesion in one mitochondrion by laser irradiation. (a) Arrow indicates served with the SEM: (1) a gross perfora- target mitochondrion; (b) note the “paled” mitochontion greater than 2 pm in diameter (fig. 9~); drion with a central dark circle (arrow). This cell exhibited no contractile change. Similar lesions can be (2) lesions of approx. I km in diameter that associated with a transient contractile change. Bar, appear as raised “blisters” with small per- 5fim. 7. SEM of (u) the same cell as seen in fig. 6. forations in the center (fig. 9b); (3) lesions Fig. Note the small bump on the surface which corresponds of 1 pm and less that appear only as raised in position to the phase dark zone in the light mi(b) another cell in which the same type of “blisters” without any perforation (fig. SC). crograph; lesion was produced. This cell exhibited a transient The same lesions are depicted in a light slight change in contractile pattern. Bar, 1 pm.
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lar alterations in contractile activity occur sult in an uncommon physical effect, such as a result of membrane damage upon poor as acoustic pressure or plasma formation. impalement of heart cells (personal obserThe severity of mitochondrial lesions vation). Clearly, these studies suggest that produced by laser irradiation has been when laser irradiation of heart cells results shown to be dependent on laser energy denin contractile changes, there is a mem- sity and has been correlated with the apbrane effect; however, the exact nature of pearance of the unirradiated organelle in the this effect is unclear. phase contrast microscope [I, 3, 6, 111.The Laser irradiation of red blood cells pro- phase density of cell components is dependvides an interesting comparison for the ir- ent on the optical path length through the radiation of living cells in that they are no structure and on the refractive indices of longer alive at the time of irradiation and, the materials which compose them. Pretherefore, have no repair processes. Thus, vious results indicate [3, 61 that the conthe primary effects of irradiation on the cell centration, distribution and biological state can be examined. Furthermore, they are a (i.e., denatured or active) of materials withfairly uniform population of cells in terms of in the mitochondria are altered by irradiasize and quantity of energy absorbing pig- tion which could account for changes in ment per unit area. Thus, investigations of the phase contrast image of the organelle. the range of effects that the laser is capable TEM studies have been performed on laserof producing are possible by attenuating the induced mitochondrial lesions of varying quantity of energy actually striking the cell. severity. These studies have demonstrated The threshold lesion has a small but visible that phase dense areas correspond to eleceffect at the cell surface (figs 9c, 10~) while tron dense areas and phase light areas corthe most severe lesions result in large per- respond to electron lucent areas. Thus, alforations (figs 9a, lOa). The intermediate though the exact energy distribution across lesions demonstrate the continuum between the irradiated cell or organelle cannot be these extremes. When lesions are made measured directly, the ultrastructural morwith the same laser energy density and at a phology of the laser lesions clearly reflects focal point midway between the upper and the distribution of laser energy within the lower cell surfaces, their appearance in SEM is the same regardless of the side of Fig. 8. SEM of a cell irradiated through the medium laser entry. TEM investigations using serial with a 40x objective. Note that the lesion appears very similar to the lesion seen in figs 2 and 3 with a sections of mitochondrial lesions produced central dimple surrounded by a raised ring. Bar, I pm. in living cells demonstrate that laser dam- Fig. 9. SEM of red blood cells with several lesions made by laser irradiation through the glass: gross perage is spherical in nature and most severe foration (a), raised blister with central dimple and percentrally. These results coupled with the foration (b), and raised blister with no dimple or perforation (c). Bar, 5 pm. Gaussian energy distribution of the laser Fig. 10. Phase contrast micrograph of the same red beam across the focal point suggest that pri- blood cells seen in fig. 9: Gross perforation (u), raised with central dimple and perforation (b), and mary absorption of laser energy occurs at blister raised blister with no perforation or dimple (L.). Bar, the focal point. Absorbed energy is most sigr”il. Scanning electron micrograph of red blood probably converted to heat and subsequent- cells with several lesions made by laser irradiation ly dissipates equally in all directions from through oil. Note the lesion type consisting of a raised without central dimple or perforation (a) and the this point. However, it should also be blister more severe lesion type consisting of a raised blister pointed out that the laser energy could re- with central dimple and perforation (b). Bar, 2 pm. El/l Cdl/t-r.\II8 (1979)
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target cell or organelle. They further demonstrate that the unfocused part of the laser beam (above and below the focal point) results in no visible damage to cellular structure. The present study attempts to examine the surfaces above and below the irradiated structure in “moderate” mitochondrial lesions as compared to “weak” lesions where we know that the damage zone does not exceed the limits of the target organelle. In this study, “weak” lesions which consist of a paled mitochondrion were made with low laser energy density. These lesions result in no visible surface alterations and in no alterations of cellular contractility. This corroborates the TEM evidence that the damage characteristic of this lesion type is confined to the target organelle [6]. The damage associated with “weak” lesions characterized by a paled mitochondrion with a central dark spot has also been demonstrated by TEM to be confined to the target organelle [3]. In two cells with this lesion type, a small bump was observed at the surface over the area of the dark spot within the target mitochondrion. These results can be explained by postulating that the bump is a result of localized thermally induced expansion of the organelle accompanied by a distortion of the overlying membrane. Thus, the general shape of the lesion is quite likely due to alterations of the shape of the target organelle which the membrane is lying over. When lesions of a similar size and shape to those referred to as moderate mitochondrial lesions are induced in red blood cells, there appears to be a perforation of the red cell membrane within the central depression. Lesions of this type in living heart cells always resulted in drastic contractile changes. Thus far, it has not been possible to determine whether or not a momentary
perforation of the heart cell membrane actually occurs, but such an event could account for both the temporary membrane depolarization observed by Kitzes et al. [2] and the observed contractile changes. in that study it was found that the time required for return to normal activity following irradiation varied but was between 1 and 5 min. This interval is close to the period between irradiation and fixation in the present studies so that any induced perforations might have had time to reseal and would have gone undetected in later microscopic examination. Although the results presented here can be explained by postulating a perforation, such a postulation is not necessary to explain the observations. If the damage to the organelle is thermal in nature, then the increased temperature could alter the physical characteristics of the membrane. Such physical effects are likely to affect, at least transiently, the permeability and function of the. membrane overlying the target organelle. Nathan et al. [12] demonstrated a direct effect of ultraviolet light on heart cells that was best explained by a direct alteration of membrane channel conductantes by the incident light. Clearly these possibilities are not eliminated as explanations of the observations reported here. Divergent results have been obtained by several investigators when comparing the frequency at which contractile alterations can be induced by irradiation of various subcellular organelles [5, 71. For example, Salet [5] found that nuclear irradiation gave rise to contractile alterations less frequently than did cytoplasmic irradiations. By considering the location of these two target areas with respect to their proximity to the membrane and the possible differences in pigment densities of the two targets, a logical explanation of the results can be made.
Contractility
Typical pictures of cells in monolayer culture demonstrate the differences in cell thickness in the nuclear region versus in the cytoplasmic region (present study and [13]). If the laser’s focal point were in the middle of the cell (i.e., midway between the upper and lower membranes), then more energy would have to be converted to heat for an effect on the overlying membrane to occur in a nuclear irradiation versus in a cytoplasmic irradiation. Therefore, an energy density sufficient to cause lesions and contractile changes in cytoplasmic irradiations may not be sufficient to do the same in nuclear irradiations. In conclusion, the observations reported here strongly suggest that the contractile alterations observed in heart cells following laser micron-radiation of mitochondria are not due directly to the damage of the target organelle but rather to an effect on the cell membrane. The authors would like to thank Marie Hammer-Wilson for her assistance in the laboratory and Elaine
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Kato for typing the manuscript. This research was supported by NIH grants HLl5740, GM23445, GM22754, and Air Force grant AFOSR-77-3136.
REFERENCES I. Berns, M W, Gross, DC L, Cheng, W K &Woodring, D, J mol cell cardiol4 (l972)-471. 2. Kitzes, M, Twinas. G & Bems. M W. J cell ohvsiol . _ 93 (1977) 99. -3. Rattner, J B, Lifsics, M, Meredith, S & Bems, M W, J mol cell cardiol 8 (1976) 239. 4. Salet, C, Exp cell res 73 (1972) 360. 5. &Jet, C, Moreno, G & Vinzens, F, Exp cell res 100 (1976) 365. 6. Adkisson, K P, Bait, D, Burgott, S L, Cheng, W K & Bems, M W, J mol cell cardiol5 (1973) 559. 7. Strahs, K R, Burt, J M & Berns, M W, Exp cell res 113(1978) 75. 8. Mark, G E & Strasser, F F, Exp cell res 44 (1966) 217. 9. Berm, M W, Exp cell res 65 (1971)470. IO. Cohen, A L, Marlow, D P & Gamer, G E, J microsc 7 (1%8) 33I. Il. Bems, M W, Gamaleja, N, Olson, R, Duffy, C & Rounds, D E, J cell nhvsiol76 (1970) 207. 12. Nathan, R D, Poole;, J P & DeHaan, R L, J gen physio167 (1976) 27. 13. Revel, J P & Wilken, K, Exp cell res 78 (1973) 1. Received June 7, 1978 Revised version received August 28, 1978 Accepted August 3 I, 1978
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