Radiofrequency (RF) ablation is a nonsurgical technique using catheter-directed RF energy for treating cardiac arrhythmias in children and adults. Previous reports have suggested that sequestration of calcium(Ca2+) by the sarcoplasmic reticulum may partially protect mature cardiac myocytes from the effects of RF energy. The purposes of this study were to determine whether differences exist between neonatal and adult myocyte responses to RF energy and if myocyte damage is a Ca2+-dependent process. Because immature myocardium is functionally deficient in sarcoplasmic reticulum, we hypothesized that immature myocytes would be more susceptible to damage induced by RF energy. Isolated ventricular myocytes were obtained from neonatal and adult New Zealand White rabbits by enzymatic dissociation, then placed in a perfusion chamber designed to deliver RF energy or a heated perfusate solution. Measurements of bath temperature, cell morphology, and contractile response to electrical stimuli were recorded. RF energy application associated with increased perfusate temperature resulted in cell death, but not when the temperature rise was inhibited. Thus, the acute damage to cells exposed to RF energy appears to be mediated by thermal energy. After exposure to thermal energy, neonatal cells underwent contracture at lower temperatures than did adult cells. Perfusion with solutions containing low Ca2+ concentrations, comparable to intracellular diastolic Ca2+ levels, had a protective effect for both neonatal and adult myocytes. These findings indicate that acute cell damage after exposure to RF energy is mediated by a Ca2+-dependent process. Furthermore, immature myocardium is particularly susceptible to RF-mediated cell damage, possibly secondary to reduced Ca2+ sequestration by the sarcoplasmic reticulum.
The effects of RF energy application on cardiac myocytes have not been well established. Clinical observations of transient atrioventricular nodal block and temporary or delayed injury to an accessory bypass tract after RF ablation procedures(1–4) may be explained by a zone of thermal injury between necrotic cells and unaffected viable cells. A similar zone of tissue injury has been documented in animal studies(5). The lesion size produced by RF energy has correlated well with the catheter tip temperature(6–9). Tissue heating occurs in a radial fashion around the energy source and declines in inverse proportion to increasing distance(10). Under a number of conditions, cell viability has correlated well with measures of local tissue heating(8, 9). These data have led to the hypothesis that myocardial damage during RF ablation is mediated by thermal energy.
Studies of the effects of thermal energy on adult guinea pig cardiac myocytes have implicated Ca2+ overload as a mechanism of cell contracture and subsequent cell death(11). Mature myocardium has a well developed SR, which efficiently sequesters Ca2+ from the cytoplasm. Thus, SR Ca2+ sequestration may play a role in protecting mature cardiac myocytes from the effects of RF energy. However, there are significant developmental differences between immature and mature myocardium. Notably, Ca2+ sequestration by the SR is diminished in immature myocytes(12–15). Therefore, we hypothesized that neonatal myocytes are more sensitive to thermal energy exposure than adult myocytes. We also hypothesized that perfusion with a low Ca2+ concentration solution is protective for myocytes exposed to thermal energy.
Cell preparation. Isolated ventricular myocytes were obtained from neonatal (2-5-d-old) and adult (≥2-mo-old) New Zealand White rabbits by enzymatic dissociation as described previously(12).
Perfusion chamber. Figure 1 illustrates the perfusion chamber designed to study isolated myocytes. A section of fragmented coverslip with adherent isolated myocytes was placed in a plexiglass chamber (measuring 26 mm in length × 4 mm in width × 3 mm in depth; the chamber volume was approximately 0.2 mL) mounted on the stage of an inverted microscope. On the right, at the proximal end of the chamber, the perfusion apparatus allowed for changes in perfusate solution. Heating elements surrounding the perfusion cannuli controlled the perfusate solution temperature (35-60°C). A fine tipped thermistor was mounted in close proximity to the myocytes under study. The temperature was measured with a digital thermometer (Fisher Scientific). On the left, at the distal end of the perfusion chamber, an aspiration cannula provided continuous flow of perfusate through the chamber. The chamber was equipped with two pairs of platinum electrodes, one pair for electrical stimulation of the myocytes and one for application of RF energy (2-12 W, 10-50 V, 500 kHz). The cells were stimulated with a Grass S88 stimulator (Quincy, MA) at 10-s intervals to ensure normal contractile function.
Perfusate solutions. Control Tyrode's solution (1.8 mM Ca2+) contained (in mM): NaCl, 136; KCl, 5.4; CaCl2, 1.8; NaH2PO4, 0.33; MgCl2, 1; HEPES, 10; mannitol, 4; thiamine-HCl, 0.6; glucose, 10; and pyruvic acid, 2; and was titrated to pH 7.4 with NaOH. Low Ca2+ Tyrode's solution (100 nM Ca2+), was identical to that described above with the inclusion of EGTA, 3.6 mM(pCa = 7).
Myocyte inclusion criteria and definitions. Myocytes were perfused with a 1.8 mM Ca2+ concentration solution at physiologic temperature (35-37°C) and electrically stimulated at twice threshold voltage to ensure normal contractile function. Inclusion criteria for myocytes in the study were a rod-shaped appearance, a smooth cell membrane, a visible nucleus, and the ability to contract consistently in response to electrical stimuli. Myocyte response to radiofrequency energy or thermal energy was recorded as normal (no change in cell morphology or response to electrical stimulus); nonviable (cell death with full contracture, ball-shaped appearance, and no visible mechanical response to electrical stimulus); or partially affected (cell does not meet the criteria for nonviable, but displayed morphologic or physiologic characteristics, which are not completely normal, i.e. a change in cell morphology or a diminished response to electrical stimulus). For statistical analysis, the response of the neonatal and adult myocytes was also grouped as either unaffected (normal) or affected(including myocytes labeled partially affected and those labeled nonviable).
RF energy protocol. Initial studies sought to determine whether injury to cardiac myocytes after RF energy application was due to thermal energy. Neonatal myocytes were exposed to RF energy for 60 s, and cell death was recorded as a function of bath temperature and RF power. The isolated cardiac myocytes were divided into two groups. The cells in one group were not perfused during application of RF energy. Myocytes in the other group were perfused with a physiologic solution to maintain a physiologic temperature(29-38°C) and to prevent a rise in bath temperature during application of RF energy.
Thermal energy protocol. To approximate the thermal energy distribution of in vivo RF energy application, another protocol(example shown in Fig. 2) was designed to deliver a pulse of heated perfusate solution to the myocytes. This was accomplished by initially perfusing the myocytes with a physiologic Ca2+ concentration solution (1.8 mM) at physiologic temperatures (35-37°C) for at least 30 s. Only myocytes which met the inclusion criteria (listed above) were used. The myocytes were pulsed with a heated perfusate solution at a constant temperature (range, 38-55°C) for 30 s. To examine the role of Ca2+ overload using this protocol, the perfusate for some cells was changed to a low Ca2+ concentration solution (100 nM Ca2+) for 60 s before the pulse of thermal perfusate. Myocytes were continuously stimulated during the low Ca2+ concentration infusion, and all myocytes perfused with low Ca2+ solution were noted to cease visible contractions before the application of thermal energy. The low Ca2+ concentration infusion was continued for 30 s after the application of thermal energy was discontinued. The response of myocytes to the pulse of heated perfusate was recorded 120 s after the return to physiologic temperature and physiologic Ca2+ concentration. To study the developmental differences, both neonatal and adult myocytes were studied using this protocol.
Data analyses. Values are presented as mean ± SEM unless indicated otherwise in the text. Statistical significance was determined usingχ2 analysis, t test, or ANOVA. The response of isolated myocytes as a function of developmental stage, extracellular Ca2+ concentration, and maximum perfusion temperature could not be analyzed using three-way ANOVA because each group contained different numbers of myocytes. Accordingly, statistical analysis of the differences in temperature between groups of affected cells was performed by two-way ANOVA, using age and Ca2+ as grouping factors. In addition, a comparable two-way ANOVA was performed on the unaffected cells to determine whether the cells in any of the groups were exposed to higher temperatures in a systematic fashion.
In the preceding analysis, temperature was a dependent variable. Thus, the previous data interpretation may have been influenced by differences in the distribution of temperatures to which each group was exposed. Moreover, these raw data were not amenable to analysis by regression of the response data because these results were in the form of categorical data with only three outcomes.
To more precisely compare the interactions between age, perfusate Ca2+ concentration, and temperature as they affect cell viability, the cumulative response of cells within each group was compared as a function of increasing temperature. For each temperature, the number of cells exhibiting a response at that temperature or any lower temperature was plotted as a function of that particular temperature. Partially affected cells were analyzed separately from nonviable cells. To compensate for differences in the number of cells in the groups, each curve was normalized to the maximal value for that group. Thus, at the highest temperatures, the normalized response was 100%. For statistical analysis, a linear regression of the data was used to calculate the temperature at which 50% of the cells had become partially affected or nonviable. Low temperatures below which no cells were affected or high temperatures above which all cells were affected were not used in the regression data. Values for the temperature at which 50% of the cells responded were then compared using a paired t test for data grouped by age, Ca2+ concentration, or manner of response (partially affected versus nonviable).
RF energy protocol. Application of RF energy (41.9 ± 0.4 V) to neonatal myocytes resulted in a rise in temperature of the bath solution to 58.8 ± 3.4°C and subsequent death of all cells (n = 16). In contrast, the application of even greater amounts of RF energy (51.4± 0.4 V) to neonatal myocytes that were continuously perfused did not produce a rise in temperature above physiologic levels (35.2 ± 1.6°C), and all cells remained normal (n = 18). The difference in response to RF energy between the two groups was statistically significant(p < 0.0001) by χ2 analysis. Thus, despite the application of a higher level of RF energy, cell death was prevented by suppressing an associated rise in bath temperature. These results suggest that the acute effects of RF energy are mediated by thermal energy.
Thermal energy protocol. The responses of neonatal and adult myocytes to elevated temperatures were determined as a function of 1) the maximum temperature that the perfusate solution achieved during the thermal energy pulse and 2) the Ca2+ concentration of the perfusate solution. The cells are grouped by response, age, and perfusate Ca2+ concentration. The mean temperatures for cells in each group using this approach are shown in Figure 3.
This analysis demonstrates that the mean temperature of the affected neonatal myocytes was lower than the mean temperature of the affected adult myocytes (Fig. 3A). This difference was statistically significant for both control Ca2+ concentration solution (neonatal 45.6± 0.2°C, n = 15; adult 47.9 ± 0.6°C,n = 22) (p < 0.01) and low Ca2+ concentration solution (neonatal 48.1 ± 0.7°C, n = 23; adult 50.3± 0.4°C, n = 10) (p < 0.01). This result suggests that immature myocytes are more susceptible to thermal energy damage than are mature myocytes.
In addition, when myocytes were perfused with low Ca2+ concentration solutions, a protective effect was noted. Greater temperatures were necessary to produce a deleterious effect when myocytes were perfused with low Ca2+ concentration solutions compared with myocytes perfused with control Ca2+ concentration solutions. This was statistically significant for neonatal (p < 0.01) and adult (p < 0.001) myocytes. This difference reflects the protective effect of perfusion with low Ca2+ concentration solutions. That is, neonatal or adult myocytes perfused with low Ca2+ concentration solutions were able to tolerate a higher mean temperature compared with neonatal and adult myocytes perfused with control Ca2+ concentration solutions.
In contrast, there was no statistically significant difference in mean temperatures between the unaffected neonatal and unaffected adult myocytes (Fig. 3B). This was true for both control and low Ca2+ concentration solutions (control Ca2+ neonatal 43.2± 0.8°C, n = 10; adult 42.9 ± 1.0°C,n = 7; and low Ca2+ neonatal 45.1 ± 0.7°C,n = 16; adult 45.0 ± 0.6°C, n = 10). The unaffected myocytes perfused with low Ca2+ concentration solutions had a higher mean temperature compared with those perfused with control Ca2+ concentration solutions. This difference was statistically significant for both neonatal (p < 0.05) and adult (p< 0.05) myocytes.
To more precisely compare the interactions between age, perfusate Ca2+ concentration, and temperature on cell viability, the cumulative response of cells within each group was compared as a function of increasing temperature. Figure 4 compares unaffected (normal) with nonviable myocytes. Figure 5 compares unaffected (normal) myocytes with those that are partially affected. Both graphs demonstrate that neonatal myocytes were affected at lower temperatures compared with adult myocytes. In addition, myocytes perfused with physiologic Ca2+ concentration solutions (1.8 mM) were affected at lower temperatures compared with myocytes perfused with low Ca2+ concentration solutions (100 nM). These trends can be examined quantitatively by comparing the temperatures at which half the cells in each group showed a response (R50) as calculated by linear regression. In agreement with the data presented in Figure 3, the R50 temperature was lower for neonatal compared with adult myocytes (46.4 ± 0.7°C versus 48.7 ± 1.2°C, p < 0.05, n = 4, by paired t test). Furthermore, control myocytes had a lower R50 than myocytes perfused with low Ca2+ concentration solutions (46.4 ± 0.6°C versus 48.7 ± 1.3°C,p < 0.05, n = 4, by paired t test). Thus, both increased age of the myocytes and perfusion with low Ca2+ concentration solutions were positive factors in protecting myocytes from the effects of hyperthermia.
The major findings of this study are that: 1) acute cellular injury from RF energy application is thermally mediated, 2) neonatal myocytes are more sensitive to thermal energy application than are adult myocytes, and 3) perfusion of myocytes with a low Ca2+ concentration solution reduced the toxic effects of thermal energy on neonatal and adult myocytes.
Previous reports(2, 7, 9) have suggested that damage from RF energy is mediated by thermal injury. This hypothesis is supported by the correlation between the amplitude of temperature measured in tissue exposed to RF energy and the extent of the zone of the myocardial lesion surrounding the ablation catheter tip(8, 16–18). In 1989, Haines and Watson(9) created RF lesions in canine right ventricle free-wall preparations and estimated the temperature at which myocardium became nonviable tissue to be 47.9°C with a range of 46.6 to 48.9°C. Other reports have demonstrated cell injury when intact tissue is superfused with a heated solution(11, 19, 20). In 1993, Nath et al.(11) used thermal energy to simulate RF energy and found that adult guinea pig papillary muscle went into contracture at perfusate temperatures >50°C (tissue temperature was not recorded). In addition, thermistor ablation catheters allow for continuous temperature monitoring during RF energy application, and some clinical trials note a similar critical temperature associated with successful RF ablation of an arrhythmogenic foci(1, 2). However, other investigators have suggested that the toxic effects of application of electrical fields may not be thermally mediated but may be secondary to transient or permanent depolarization-induced changes in cell membrane permeability or electroporation(21, 22).
The current study suggests that temperature plays a primary role in RF-mediated cell damage. RF-mediated toxicity occurred only when sufficient RF power was applied to raise the bath temperature. Furthermore, RF-mediated myocardial cell contracture was prevented when a rise in bath temperature was inhibited by perfusion with a cool bath solution. When isolated myocytes were exposed to a pulse of heated solution, the mean temperature for affected cells was 45.6 ± 0.7°C in neonates and 47.8 ± 0.8°C in adults. The results of the current study using isolated neonatal and adult myocytes correlate well with those from intact muscle preparations, thus validating the use of isolated myocytes in studies of RF and thermally mediated myocardial injury. However, other factors such as the accumulation of extracellular toxins and cell to cell interactions may contribute to the damage caused during in vivo exposure to RF energy. Although the current model of isolated rabbit myocytes cannot be used exclusively, it does provide an alternative method for studying the effects of RF energy on the cellular level.
Studies using thermal energy application in adult guinea pig papillary muscle have implicated Ca2+ overload as a mechanism of cell contracture and subsequent cell death(11). In that preparation, the SR was hypothesized to play a protective role in sequestering intracellular Ca2+ in cells exposed to thermal energy. In the current study, neonatal myocytes underwent cell contracture and death at lower temperatures than did myocytes isolated from mature myocardium. Neonatal myocytes, which are deficient in functional SR and therefore have less ability to sequester Ca2+, may be more sensitive to Ca2+ overload. Thus, the finding that myocytes perfused with a low Ca2+ concentration solution were able to tolerate a higher mean temperature than were myocytes perfused with control Ca2+ solution supports the hypothesis that thermal injury is mediated by a Ca2+ overload.
Although the internal Ca2+ concentration was not measured directly, the role of Ca2+ overload in thermally mediated myocyte toxicity was examined for both immature and mature myocardium by lowering extracellular Ca2+ concentrations to intracellular levels during the exposure to elevated temperatures. Analysis of either the difference in temperature between affected and unaffected cells or the cumulative cellular response after exposure to elevated temperatures demonstrated the protective effect of perfusion with low Ca2+ solution. These findings indicate the importance of Ca2+ in thermally mediated acute myocyte toxicity. In addition, these results may have significant clinical implications in terms of the relative effectiveness or toxicity of RF ablation techniques in immature myocardium. It is possible that effective RF ablation of immature myocardium may be achieved at lower temperatures as compared with temperatures required for successful ablation in adult myocardium. Other researchers have suggested that enhanced deleterious effects of RF ablation lesions in immature myocardium results from hypertrophic scar proliferation(23). In the current study, changes in the perfusate Ca2+ concentration influenced the acute viability of isolated myocytes during exposure to thermal energy. This isolated myocyte model may prove useful for investigating other factors involved in cell viability during exposure to RF energy.
In summary, the current data show that hyperthermia is the primary mechanism for cell injury during application of RF energy, and this thermal injury is mediated by Ca2+ overload. In addition, neonatal cardiac myocytes are more sensitive to thermal energy than are adult myocytes.
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The authors thank Tania Helgren for assistance in the preparation of this manuscript.
Supported in part by the American Heart Association, Greater Los Angeles Affiliate Grants 1075-CS1, 981-CS1, and 788 IG, the National Institutes of Health Grant HL-02723, the Laubisch Fund, the Variety Club, and the J. H. Nicholson Endowment.
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Maginot, K., Klitzner, T., Friedman, W. et al. The Relationship between Temperature and Calcium in Acute Cell Damage after Exposure to Radiofrequency or Thermal Energy in Isolated Neonatal and Adult Rabbit Cardiac Myocytes. Pediatr Res 43, 527–531 (1998). https://doi.org/10.1203/00006450-199804000-00015