Argon Laser Microirradiation of Mitochondria in Rat Myocardial Cells VI. Correlation of Contractility and Ultrastructure

M. W. BERNS. Argon Laser Microirradiation of Mitochondria in Rat Myocardial Cells. VI. Correlation of Contractility and Ultra- structure. A correlation is made between individual laser microbeam lesions (0.25-l pm in diameter) in single mitochondria of myocardial cells and alterations in cellular contractility. A moderate lesion is described ultrastructurally that has three concentric zones ofdamage. In addition, the outer mitochondrial membrane is broken, and the intercristae matrix outside the lesion may be affected. Two kinds of weak lesions are described. No concentric zones of damage are observed, and the cristae and mitochondrial membranes outside the lesion area are not grossly affected. All cells that exhibited an alteration in contractile patterns had moderate lesions. A correlation between laser energy density and type of lesion was observed. The nature of the lesions and explanations of the contractile responses are discussed.


Introduction
Intense laser light can be focused to an effective damage-producing spot of 0.25 pm within living cells [3, 41. When such a beam is focused into a single large mitochondrion of a myocardial cell, various changes in mitochondrial morphology and cellular contractility are observed [I, 5-81. Light microscope observations suggest at least four different kinds of mitochondrial damage. These are divided into three different general categories: "severe," "moderate" and "weak." Two types of lesions are classified as "weak" : (1) a phase dark spot within a generally "paled"* mitochondrion, (2) a completely paled mitochondrion.
The moderate lesion appears as a phase dark spot with a phase-light central region. The severe lesion appears to be destruction of the entire irradiated mitochondrion and generally results in rapid cell death.
In addition to the apparent morphological alterations to the irradiated mitochondrion, contractile changes have been observed [5, 71. These changes follow * The term "paled" is used to indicate that the mitochondrion, as viewed with the phase microscope, has undergone a considerable decrease in optical phase density. definite sequential patterns (see summary in Figure 1). In order to more fully understand and interpret these responses, it is necessary to examine single irradiated cells with the electron microscope.
In this report, we will describe the ultrastructural changes in irradiated cells and correlate these changes with the observed contractiIe responses.

Materials and Methods
An argon laser microbeam was employed in these studies [Z]. cell was relocated, scored with a diamond objective marker, cut and cemented to an Epon block. Serial sections in the silver range were cut on an LKB Ultratome III, collected on copper slotted grids coated with formvar and carbon, and examined on a Siemens Elmiskop 1 A operated at 60 KV.

Results
Light microscope morphology and contractility 241 Two types of mitochondrial lesions were produced : a dark spot within an otherwise LLpaled" mitochondrion and a dark spot with a phase light central zone. The former lesion would be classified as "weak" and the latter as LLmoderate" by our previous classification scheme [S], A correlation among the lesion type, laser energy density, and contraction change was observed (Table I). There was a predominance ofweak lesions ( 12/ 18) with the lowest energy level (6.7 pJ) and of moderate lesions ( 15/ 17) with the highest energy (12.6 pJ). Most of the contractility: changes (14/17) occurred in cells with moderate lesions, and all of the cells with weak lesions continued to contract rhythmically.
Cells irradiated in non-mitochondrial regions of the cytoplasm with all three energy densities never exhibited a contractility change. Cells undergoing sequential contraction changes exhibited six different patterns (Table 2). Of the nine cells where detailed timing data was extracted from the videotapes, eight cells exhibited the "fibrillation" § response. In addition, eight cells returned to a rhythmic rate of contraction. However, the post-irradiation rhythmic contraction rates were often considerably different than the pre-irradiation rates. The mean change in beats/minute for this group was 42. This contrasts considerably with the cells that had weak lesions and exhibited no sequential change in contractility (Table 3). In this group of cells, the mean change in beats/minute was 10.7. It is significant to note that seven (33%) of th ese cells had a post-irradiation beat rate identical to the pre-irradiation rate. This group appeared to be very similar to unirradiated control cells where the mean change in beats per minute was 9. In establishing the mean change in beat rate, an absolute value in change in frequency has been used regardless of whether the change was (+) or (-). There appeared to be no consistent pattern with respect to either an increase or decrease in beat frequency. z A contractility change is defined as an observed change in the beat frequency. This either may be an increase, decrease, or induction of an irregular beat pattern. 5 Fibrillation of a single cell is defined as an uncoordinated, rapid, irregular contraction pattern of a single cell.  A final lesion zone of moderate electron density surrounds the innermost electron dense region (see Plates 3 and 4). Much of the increased electron density of this lesion zone appears to be in the matrix between the cristae. Cristae from the unirradiated region of the mitochondrion can be seen penetrating into this lesion zone, and in some regions (see inset, Plate 3), they appear to penetrate all the way through it. In addition to the three concentric zones of the moderate lesion, the outer mitochondrial membranes appear to be broken in numerous places. Furthermore, in several areas outside the lesion zone but within the irradiated mitochondrion, there appears to be a reduced amount of electron dense material in the intercristae spaces. This may explain the paling phenomenon that is often observed in the unirradiated portions of the mitochondrion.
Weak lesion ultrastructure One weak lesion was produced in the previous cell (labeled Wr in Plates 2 and 4), and three weak lesions (Ws-W,) were produced in the cell depicted in Plates 5-9. When viewed with the phase contrast microscope, all four of the weak lesions appeared as a dark spot within an otherwise paled mitochondrion. The paIing of the rest of the mitochondrion is particularly clear in lesion Ws and Wa (Plate 6). However, when viewed with the electron microscope, lesion Wr appeared different than Ws, Ws, and W4. Lesions Ws-W4 appeared to contain alternating areas of electron dense and electron light material.
The cristae appeared broken down in these regions as no lamellar membrane pattern was evident. However in WI, cristae could be seen traversing completely through the lesion area. These cristae membranes appeared to have greater than normal electron density and considerably more spacing between.
The general integrity of the mitochondria with weak lesions was maintained. The outer mitochondrial membranes appeared to be mostly intact, and the cristae outside the lesion area were organized in a lamellar-like pattern. The lack of alteration outside of the lesion area was particularly clear in mitochondria Wi (Plate 4), Ws and W4 (Plate 7). In mitochondrion Ws (Plates 8 and 9), there appeared to be some degree of cristae disorganization. But even within this organelle, several areas with lamellar cristae patterns were evident. Serial sections through this mitochondrion revealed that the electron dense lesion material (small arrow, Plate 8) was limited in depth (Plates 8 and 9) within a single mitochondrion. A final observation on the weak lesion is that a fifth weak lesion (small arrow, Plate 7) was produced.
This lesion was produced in a mitochondrion adjacent to W4. Apparently the focal point of the laser beam extended over this mitochondrion which was not visible with the light microscope.
The general ultrastructure of this mitochondrion and lesion was similar to Ws.

Discussion
There is a definite correlation between lesion type and change in contractility. All of the cells that exhibited a sequential change in contractility had moderate lesions placed in one mitochondrion.
No cells with weak lesions went through one of the sequential contraction patterns. However, a moderate lesion does not automatically produce a contraction change. Three cells with moderate lesions did not exhibit altered contractility.
As in earlier studies, the type of contractility responses were quite variable. In the studies reported here, six different sequences were observed. Of the nine cells carefully followed, eight exhibited the uncoordinated response termed "fibrillation", and seven of these eight returned to a rhythmic rate of contraction within several minutes of irradiation.
Though some of these cells returned to rates very close to the pre-irradiation rate, others varied considerably. This contrasts greatly with the control cells and the irradiated cells that did not exhibit contractile changes. In addition to a correlation between lesion type and contractility change, there also appears to be a correlation between energy density and lesion type. There was a preponderance ofmoderate lesions with 12.6 PJ and weak lesions with 6.7 pJ. The ultrastructural studies reveal that the moderate lesion has three concentric zones: (1) an inner light central region that contains some disrupted cristae, (2) a middle dark electron dense zone, and (3) an outer region containing both electron dense and electron light material and also some visibly altered cristae. Mitochondria with moderate lesions have varying degrees of cristae disorganization outside the lesion itself, and the outer mitochondrial membranes appear to be broken in numerous places.
Lesions classified as "weak" with the light microscope have varied ultrastructure. One kind of weak lesion (Wz-W,) appears as an electron dense region without any evidence of cristae and with scattered light areas throughout.
The other weak lesion (WI) appears to have an altered, but definite, cristae pattern. It is possible that the two types of weak lesions merely reflect a gradation of damage with the least amount of damage (non-destruction of cristae) occurring in lesion WI. This possibility is further suggested by the fact that the outer electron dense zone of the moderate lesions (Zone 3 above) is almost identical to the more severe weak lesion. In addition, careful examination of this outermost zone (see Ml, Plate 3) reveals areas that appear to have cristae patterns similar to those found in the weakest lesion (WI). The observations on the weak and moderate lesions indicate a pattern of decreasing damage emanating from the center of the moderate lesion. The observation that the outer region of the moderate lesions look similar to the weak lesions supports this idea.
The ultrastructural studies do not provide a definitive explanation for the observed contractile changes. However, the fact that the weak lesions do not result in much disruption to the irradiated mitochondrion other than in the actual lesion area itself does agree with a lack of observed contractile response. The more severe alterations produced by the moderate lesions, such as, general cristae disruption and mitochondrial membrane breakage, does allow one to more easily reconcile the contractility changes. The morphology of the moderate lesion suggests a thermal effect. If a temperature rise were occurring at the focal point, a thermal gradient