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Ann Thorac Surg 1998;66:1618-1625
© 1998 The Society of Thoracic Surgeons

Triiodothyronine reverses depressed contractile performance after excessive catecholamine stimulation

Tomasz Timek, MDa, Christian-Friedrich Vahl, MDa, Andreas Bonz, MDa, Leo Schäffera, Mark Rosenberga, Siegfried Hagl, MDa

a Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany

Accepted for publication May 11, 1998.

Address reprint requests to Dr Vahl, Chirurgische Klinik, Abt Herzchirurgie, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Conflicting results have been reported regarding the acute effects of triiodothyronine (T3) on myocardial contractile performance. The present study analyzes the role of T3 in reversing the depressant effect of excessive catecholamine stimulation in isolated porcine left ventricular myocardium.

Methods. Thirty-six left ventricular trabeculae (0.4 x 6.0 mm) obtained from 6 pigs were used for measurements of isometric force development, isotonic shortening, and intracellular calcium in three experimental series (measurement conditions: 37°C; optimal length; supramaximal electrical stimulation, 1 Hz; calcium measurement, fura-2 ratio method; frequency, 225 Hz). In series 1, isometric force development was measured before and after a 60-minute incubation with 10-7 mol/L epinephrine in preparations with (n = 6) and without (n = 6) preceding fura-2 loading for calcium measurements. In series 2, the acute effects of a 30-minute administration of T3 (10-9 mol/L) on isometric force and intracellular calcium were analyzed (n = 6). In series 3, after simultaneous fura-2 loading and a 6-hour 10-7 mol/L epinephrine exposure the effects of T3 (10-9 mol/L, 30 minutes) on force development, shortening, and intracellular calcium transient were analyzed.

Results. Long-term and high-dose epinephrine exposure induced a severe contractile depression with a significant reduction of isometric force development (p < 0.05) and increased diastolic (p < 0.001) and systolic calcium (p < 0.001). In normal porcine myocardium T3 had no effect on the extent of isometric force generation but accelerated the time course of force development (p < 0.05) and increased the calcium transient (p < 0.001). After induction of myocardial depression by epinephrine exposure T3 accelerated the intracellular calcium transients and reduced diastolic calcium. Triiodothyronine increased the shortening amplitude and the force amplitude (p < 0.01).

Conclusions. Triiodothyronine reverses depressed contractile performance after preceding high-dose epinephrine exposure in isolated porcine myocardium. Increased force amplitudes and unaltered or even reduced intracellular calcium transients argue in favor of a resensitization of the contractile apparatus for calcium by T3. The study supports a potential role for T3 treatment in depressed myocardium after previous excessive catecholamine exposure (eg, brain death, catecholamine treatment, ischemia).


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Excessive catecholamine stimulation—as occurring in brain-dead organisms—can induce a severely impaired contractile performance, including irreversible damage, in mammalian myocardium [1–4]. Thus, Novitzky and coworkers [5] suggested a sympathectomy be performed to avoid myocardial injury during brain death. Allen and Kurihara [6] were the first to describe that catecholamines altered the myocardial calcium–force relationship in intact hearts. These data suggested that a desensitization of the contractile apparatus for calcium was responsible for the cardiodepressant effect of excessive catecholamine. Herzig and associates [7] confirmed this hypothesis and supported prior findings of Buss and Stull [8], who found that cyclic adenosine monophosphate (cAMP)-mediated troponin I phosphorylation induced a decrease in calcium sensitivity [9]. Theoretically such a mechanism is cardioprotective because at high concentrations of catecholamines the intracellular calcium concentration rises so dramatically that, given a normal calcium sensitivity of the contractile apparatus, the myofibrillar adenosine triphosphatase would be overstimulated, causing a rapid loss of high-energy phosphate compounds. A desensitization of the contractile apparatus for calcium is an efficient measure to reduce these effects.

The acute catecholamine-induced intracellular calcium overload, however, also affects other metabolic pathways and intracellular structures, inducing mitochondrial swelling and later on irreversible myocardial damage. The toxic effect of catecholamines on the myocardium has been outlined by the pioneer work of Raab [1] and Szakacs and Mehlman [2]. The observation of Bloom and Cancilla [10] that even low doses of catecholamines can induce severe functional and morphologic changes had been frequently reproduced [for review, 3] and remains extremely important as all donor hearts have a history of brain death associated with elevated plasma catecholamine levels. A reversal of myocardial performance after catecholamine exposure can only be expected as long as functional and essentially reversible alterations are present, but no (or minimal) myocardial cell death.

Novitzky and colleagues [11] suggested administration of triiodothyronine (T3) to support the contractile recovery of impaired myocardial function of donor hearts from brain-dead organisms. However, because of the numerous effects of brain death on the one hand and of T3 on the other [12, 13] the results are still important but sometimes difficult to interpret [11, 14]. Brain death-associated acute pressure- or volume-overload, which is per se associated with left ventricular distention, can consistently explain impaired myocardial performance. Even in large animal models using experimental induction of brain death it is impossible to decide whether the detrimental effects of released catecholamines are related to direct actions at the myocardial level, increased peripheral resistance and altered (more energy-consuming) ventriculoarterial coupling, a consecutive pressure- or volume-overload per se, or an increased heart rate. The present study investigates the effect of T3 on the isometric force development, isotonic shortening, and intracellular calcium transient in isolated normal left ventricular porcine myocardium after a preceding exposure to epinephrine, which was used to simulate an important aspect of brain death. We chose to perform our experiments on isolated intact fibers so as to guarantee exactly defined experimental conditions. The selected experimental model excludes important side effects of T3 (eg, on vascular resistance [afterload reduction] [15, 16]) as preload, afterload, and frequency were exactly controlled throughout the experiments.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Animal model
Thirty-six left ventricular trabeculae from 6 pigs (body weight, 22 to 29 kg; age, 4 to 6 months) were used for experiments. After premedication (phenothiazine 0.1 mL/kg, intramuscular) and tracheal intubation the pigs were anesthesized with ketamine (ketamine-HCL 2 mg/kg intravenously followed by a second bolus when median sternotomy was carried out) and pancuronium (Pancuronium Organon [Organon International B.V., Oss, the Netherlands] 0.1 mg/kg initial bolus followed by a continuous infusion at 4 µg · kg-1 · min-1) was given for muscle relaxation. After median sternotomy the pericardium was opened longitudinally and tapes were placed around the superior and inferior venae cava. To prevent distention of the left ventricle a vent was inserted through the apex of the left ventricle. A small cannula was placed in the ascending aorta allowing perfusion of the aortic root with cardioplegic solution. Then the caval tapes were snugged and after cross-clamping of the aorta a 4°C Krebs-Henseleit solution (KHS) (composition in mmol/L: NaCl, 119.0; NaHCO3, 25; KCl, 4.6; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 1.3; and glucose, 11.0; containing 30 mmol/L 2,3-butanedione monoxime [BDM]) was infused through the aortic root (2,000 mL; perfusion pressure, 40 mm Hg; duration, 10 to 14 minutes). Simultaneously the left heart was vented by placing a catheter through the apex into the cavity of the left ventricle. The right atrium was incised immediately after onset of the perfusion allowing the crystalloid solution to enter into the pericardial space. Then the heart was carefully excised in toto. After extirpation the left ventricle was incised and suitable trabeculae were immediately prepared for further measurements in one of three experimental series.

Muscle specimen
Porcine left ventricular trabeculae were prepared under binocular microscopic control. The terminal trabeculae had a diameter less than 0.5 mm and a length of about 4.0 mm. During the complete preparation process the fibers remained at 4°C in the same type of cardioprotective solution that was used during excision.

Experimental design
A similar experimental setup was used as in previous studies [17]. The trabeculae were mounted between a servo-controlled motor and a force transducer. The preparations were allowed to equilibrate in 37°C warm oxygenated KHS for at least 10 minutes. The electrically stimulated preparation (frequency, 1 Hz; amplitude, 10% above threshold; duration, 5 ms; mode, square wave) was adjusted to the length associated with maximal isometric force development (optimal length, or Lmax). When steady state conditions were obtained, measurements of calcium (see later) and mechanical parameters were carried out. Force (mN), muscle length (mm), and intracellular calcium transient (fura-2 340 nm/380 nm fluorescence ratio) were recorded simultaneously (Güth Scientific Instruments, Heidelberg, Germany). The signals of 60 subsequent twitches (sampling period, 60 seconds) were visualized on a storing oscilloscope and digitized in the computer for online data analysis and storage.

Calcium measurements
Calcium measurements were performed using the fluorescent dye fura-2/AM (F-0888, Sigma Chemicals, St. Louis, MO) and a chemical loading procedure as already described in detail [17]. Briefly, the trabeculae were incubated in darkness for 5 to 6 hours at 26°C in oxygenated KHS containing 5 µmol/L fura 2-AM. The noncytotoxic detergent cremophor EL (0.5%) (Cremophor; C-5135 Sigma) and TPEN (N,N,N,'N'-tetrakis(2-pyridylmethyl)ethylenediamine; P-4413, Sigma) was added to the incubation solution (0.43 mg TPEN/100 mL KHS) to improve the solubility of fura-2/AM and increase the transmembrane permeation. Then the muscle fiber was rinsed with oxygenated KHS for 15 minutes at 37°C, mounted between the vibrator and force transducer, and stretched in a stepwise fashion up to Lmax (stimulation frequency, 1 Hz).

After excitation at alternating wavelengths (alternation frequency, 250 Hz) of either 340 nm or 380 nm at 250 Hz the fluorescence signal was recorded at 510 nm by a photomultiplier tube. After subtracting background fluorescence the ratio (R) of both fura-2 emission fluorescence signals was monitored continuously. This ratio of the emission light is a measure of the intracellular calcium concentration. The fura-2 ratio method was selected as calcium measurements depend neither on the indicator concentration within the preparation nor on the diameter of the preparation. Muscle shortening is associated with increasing muscle diameters and therefore increasing fluorescent light intensities when only one wavelength is monitored. In contrast monitoring the fluorescence ratio of two wavelengths allows calcium measurements of preparations that change their diameter during the measurement procedure [17]. At the end of the experiments, the calibration of the calcium signal was carried out using in vivo tetanization for Rmax and rapid skinning for Rmin measurements as already described [17].

In a large number of previous methodological experiments (data not shown), calcium transients and force development always remained stable for periods greater than 60 minutes. However, to reduce potential methodological artifacts related to fura-2 photobleaching and compartmentalization endothelium-free preparations were used and the measurement period was restricted to 60 minutes.

By the term "calcium," the "intracellular free calcium concentration," which is what fura-2 measures, is meant. Fura-2 was selected as the fluorescent dye as it is capable of measuring diastolic calcium. After electrical stimulation intracellular calcium rises to a maximum and begins to decline before force development has reached its maximum. This time course of intracellular calcium is described by the term "calcium transient." A contraction is called "isometric" when the length of the trabeculum remains constant during the contraction. The term "isotonic contraction" is used when the muscle is allowed to shorten against a defined constant load. "Passive resting tension" is the passive steady-state force measured before onset of electrical stimulation.

Experimental series
Series 1: induction of desensitization
At Lmax isometric force and intracellular calcium were measured. Subsequently 10-7 mol/L epinephrine was added to the KHS and the fiber was perfused with this solution in the dark for a 60-minute incubation period while electrical stimulation (1 Hz) continued. After 60 minutes of epinephrine exposure, measurements of isometric force and intracellular calcium transient were repeated and compared with the initial values (n = 6). The same series was carried out without fura-2 loading (control, n = 6).

Series 2: T3 in normal myocardium
After fura-2 loading, measurements of isometric force development and intracellular calcium were carried out before and after a 30-minute incubation with 10-9 mol/L 3,3',5-triiodo-L-thyronine (T3; Sigma) dissolved in 33% 1 N HCl and 67% ethanol (95%). No calibration of the calcium signal was carried out in this series (n = 6). Control measurements were carried out using preparations without fura-2 loading (n = 6).

Series 3: T3 in depressed myocardium
After initial control measurements of the isometric twitch amplitude (measurement conditions as described above) the muscle fibers were incubated in darkness at 26°C for 6 hours in KHS containing 10-7 mol/L epinephrine and 5 µmol/L fura 2-AM for calcium transient measurements. At the end of the incubation procedure isometric force and intracellular calcium transient were recorded. Subsequently, in one arm of the study T3 (10-9 mol/L) was added to the perfusion solution, and the fibers were perfused continuously for 30 minutes (1 Hz supramaximal electrical stimulation). After this interval the perfusion solution was again switched to KHS, and isometric force development, isotonic shortening amplitude, and intracellular calcium transient measurements were again performed (n = 6). In the control experiment, perfusion was carried out without T3 administration (n = 6).

Statistical analysis
Data are expressed as mean ± standard error of the mean. For averaging superimposed force, length, and calcium transients, special software was provided (Güth Scientific Instruments, Heidelberg, Germany). Because each fiber served as its own control, paired t test was used for statistical analysis. A value of p less than 0.05 was considered to indicate a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Series 1: high-dose epinephrine exposure in porcine myocardium
High-dose and long-term epinephrine exposure (10-7 mol/L) caused an initial dramatic rise in active tension (by a factor >2.5), resting tension, and intracellular calcium concentration, followed by a continuous decrease of developed force. After a 60-minute exposure period isometric force amplitude was significantly reduced as compared with the initial control values (4.8 ± 0.4 versus 3.7 ± 0.3 mN, n = 6, p < 0.05). Resting force of the electrically driven preparation rose from 1.8 ± 0.1 to 3.7 ± 0.3 mN (n = 6, p < 0.0001). Diastolic calcium increased considerably by a factor greater than 5 from initial control values (75 ± 10 to 404 ± 72 nmol/L, n = 6, p < 0.001). Systolic calcium increased from 860 ± 83 to 1,741 ± 131 nmol/L, n = 6, p < 0.0001). The control preparations without fura-2 loading showed an essentially similar behavior with a significant decrease of the twitch amplitude after a 60-minute epinephrine exposure (p < 0.005). Developed force was slightly higher in the control preparations (data not shown, no significant differences).

Series 2: effects of T3 in normal porcine myocardium
We observed an increase of the amplitude of the intracellular calcium transient in many of the trabeculae treated with 10-9 mol/L T3 as illustrated in Figure 1. Although there was a concomitant force increase in most preparations this increase was small. The difference in developed force failed to be significant (4.5 ± 0.5 versus 5.1 ± 0.4 mN, n = 6). However, as illustrated by the superimposed twitches in Fig 1, the velocity of force development and of the diastolic decay was significantly accelerated after T3 application. The time to peak was reduced from 210 ± 12 to 175 ± 10 ms (n = 6, p < 0.05). As compared with the initial value the amplitude of the calcium transient was 128% ± 14% of the initial control (p < 0.0001). In the control group without fura-2 loading the increase in force development after T3 administration (10-9 mol/L) was also not significant (5.2 ± 0.5 versus 6.0 ± 0.5 mN, n = 6).



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  		Fig 1. Superimposed isometric contractions of isolated trabeculae before (1) and after (2) triiodothyronine (T3) incubation in a normal porcine left ventricular muscle fiber. The slight abbreviation and the increase of the amplitude of the intracellular calcium transient and the acceleration of the time course of the isometric contraction are clearly visible.

 
Series 3: effects of T3 in epinephrine pretreated myocardium
Epinephrine incubation reduced developed force and shortening amplitude to less than 50% of the initial control. Application of T3 induced an important and statistically significant increase of the isometric force and of isotonic shortening amplitude in trabeculae that underwent a long-term and high-dose epinephrine incubation (Table 1). Similarly, as shown in Figure 2 this effect was observed in any preparation. The time courses of isometric force development and of the intracellular calcium transient were accelerated after T3 administration. In contrast to normal control porcine myocardium, systolic calcium increased after T3 application in none of the preparations pretreated with epinephrine. In some preparations systolic calcium remained nearly unaltered although there was a considerable decay of diastolic steady-state calcium (p < 0.002) (Fig 3). The decay of diastolic calcium was associated with an increased amplitude of the calcium transient and a decay in passive resting force. In two preparations the reduction of the diastolic steady-state calcium was small compared with the decay of systolic calcium, resulting in a decreased amplitude of the intracellular calcium transient. However, despite the reduced amplitude of the calcium transient the amplitude of isometric force rose after T3 administration. Thus, the generation of a given isometric force amplitude was associated with a considerably decreased intracellular calcium transient (Fig 4). An acceleration of the time course of the intracellular calcium transient and of the course of the isometric force generation was present in many of these preparations. The results are summarized in Table 1.


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  		Table 1. Synopsis of the Results of Series 3

 


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  		Fig 2. Effect of triiodothyronine (T3) application in an isolated porcine left ventricular trabeculum that was incubated for 6 hours with epinephrine. Note that after T3 application the isometric force amplitude (upper trace) and the amplitude of shortening (lower trace) increased considerably. (Epi = epinephrine.)

 


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  		Fig 3. Illustration of the effect of triiodothyronine (T3) application after a 6-hour period of epinephrine incubation on intracellular calcium transient and force as illustrated by an original recording. Note that systolic calcium (peak value of the calcium transient) remains nearly unaltered in this muscle, while diastolic calcium declines. This is accompanied by a dramatic decline of resting tension and a considerable increase of the isometric force amplitude. (Epi = epinephrine.)

 


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  		Fig 4. Digitized and averaged recordings of 60 sequential isometric contractions before and after triiodothyronine (T3) application (upper trace, intracellular calcium transient; lower trace, isometric force). Trace (1) represents averaged recordings after long-term, high-dose epinephrine exposure; curve (2) indicates recordings measured after T3 application. Note that after T3 treatment the amplitude of the intracellular calcium transient declines considerably. The diastolic decay of the calcium transient is accelerated. However, despite the considerable reduction of the amplitude of the intracellular calcium transient the force amplitude did not decline. There was even a slight increase. The time course of both contraction and relaxation was accelerated. An unchanged force amplitude measured at a reduced intracellular calcium transient provides direct evidence that the contractile apparatus becomes more sensitive to calcium after T3 application (sensitization of the contractile apparatus to calcium). (Epi = epinephrine.)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
Studies analyzing the cardiovascular effects of brain death, catecholamine exposure, and thyroid hormone administration in donor hearts have been extensive during the past 20 years. The still controversial data may be explained by different experimental and clinical conditions. It is an advantage of the present study that exactly defined measurement conditions could be used throughout the course of the experiment. An important finding of the present investigation is that in normal porcine myocardium the acute effects of T3 on calcium transients and force generation are small. In contrast, T3 was highly efficient in reversing contractile performance after a preceding catecholamine exposure.

The present study reproduces observations of previous investigators that long-term and high-dose catecholamine exposure can induce an acute severe depression of myocardial contractile function [3–5, 9]. The reduced force amplitudes despite elevated intracellular calcium concentrations after catecholamine exposure strongly suggest that the contractile apparatus became less sensitive to calcium, explaining the contractile depression (series 1). The recovery of contractile performance occurring after T3-administration despite the unchanged or even reduced intracellular calcium levels suggests that sensitivity of the contractile apparatus to calcium had increased (resensitization). If so, these experimental observations have important implications for donor heart management and for the improvement of myocardial performance in those situations that are associated with increased plasma catecholamine levels.

Catecholamine-induced desensitization of the contractile apparatus to calcium
Cyclic AMP-mediated actions of catecholamines include numerous processes, which even have opposite effects and have been compared by Katz with "a man who blows hot and cold with one breath" [9].

Acute effects of catecholamine stimulation include improved contractile performance with raised values for muscle shortening, which have been uniformly ascribed to raised intracellular calcium levels. However, if catecholamine stimulation persists additional factors have to be considered: the cAMP-dependent phosphorylation of the inhibitory subunit of troponin (troponin I) [18] correlates negatively with the calcium sensitivity and the maximum velocity of actomyosin ATPase activity, which determines the velocity of contraction; on the other hand, a cAMP-dependent phosphorylation of myosin light chains (MLC) is accompanied by enhancement of the ATPase activity and correlates positively with contractile performance [19–21].

The negative inotropic effect of troponin I phosphorylation [18] seems to override the positive effects of MLC phosphorylation when both phosphorylation processes occur simultaneously. This was explained by the activity of the protein phosphatase 2B, which is also calcium-dependent and acts on phosphorylated MLCs in a way that reverses the positive inotropic effect at high intracellular calcium loads [19]. Thus, there is a kind of automatic check on hyperphosphorylation of MLCs when calcium accumulates in the myoplasm of the heart cells [22, 23]. Figure 5 summarizes these divergent processes.



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  		Fig 5. Illustration of some effects mediated by high-dose catecholamine stimulation. Improved contractile performance is caused by increased intracellular calcium levels after catecholamine stimulation. When catecholamine exposure persists a cyclic adenosine monophosphate (cAMP)-dependent phopsphorylation of troponin I is induced, which in turn is associated with negative inotropic effects (reduced calcium sensitivity of the contractile apparatus and retarded actomyosin adenosine triphosphatase activity). A parallel cAMP-dependent phosphorylation of myosin light chains (LC) induces an enhancement of the myofibrillar adenosine triphosphatase activity correlating positively with the contractile performance. The catalytic activity of the protein phosphatase 2B increases with rising intracellular calcium concentrations and acts on phosphorylated myosin-light chains when intracellular calcium remains elevated. As a result the negative inotropic effect of troponin I phosphorylation overrides the positive effects of myosin light chain phosphorylation when both phosphorylation processes occur in the presence of high intracellular calcium loads.

 
These regulatory mechanisms brought about by phosphorylation of troponin I and dephosphorylation of MLCs may have a role in protecting the myocardium against excess levels of myoplasmic calcium (eg, the catecholamine storm during brain death). Unfortunately the phosphorylation of troponin I outlasts the reversal of positive inotropism brought about by removal of ß-adrenergic stimulation [23]. Thus, even at normalized intracellular calcium concentrations the reduced responsiveness of the contractile apparatus persists, explaining—at least in part—the severely depressed contractile performance in these situations [4]. This consistently explains the negative inotropic effect as demonstrated in the present study after long-term and high-dose epinephrine stimulation, which may well correspond to the situation occurring in the myocardium during brain death.

Acute effects of thyroid hormone in normal porcine myocardium
Our results confirm previous data from Snow and coworkers [24] obtained in rabbit papillary muscle. The effects of T3 in normal porcine myocardium were small and included—although with the small number of experiments this was not significant—a slightly increased isometric force reached in shorter time associated with an increased amplitude of the intracellular calcium transient. The accelerated time course of the intracellular calcium transient and of isometric force development (Fig 1) is consistent with reports that T3 can increase cardiac performance acutely, independent of induction of protein neosynthesis [11, 13, 24–27]. An increased activity of the sarcoplasmic reticular calcium ATPase after T3 administration may be involved [28]. However, no significant increase of myosin ATPase activity was observed when thyroxine was administered to young rats, which under normal conditions exhibit a high level of cardiac myosin adenosine triphosphatase activity [29, 30]. A T3-mediated stimulation of mitochondrial adenine nucleotide translocase (supplying more adenosine triphosphate to the cytosol), a significantly increased 2-deoxyglucose uptake, or other acute metabolic effects of T3 also have to be considered [24, 25, 27, 31, 32]. However, it is an interesting finding that the effect on isometric force generation was so small that it failed to be significant under the given experimental conditions in normal control porcine myocardium. This seems to indicate that T3 is not able to increase the sensitivity of the contractile apparatus to calcium in normal porcine myocardium. The small parallel rise of the intracellular calcium transient and isometric force development may be consistently explained by the acute T3 mediated effect on the sodium–calcium exchanger [33].

Reversed contractile performance after T3 administration
The positive effects on the contractile performance after T3 administration were considerable and significant when T3 was used after long-term epinephrine stimulation (desensitized porcine myocardium). The alteration of the shape of the intracellular calcium transient may be explained by a combination of direct effects on calcium channels [32–34] and acute metabolic effects [13, 24, 25, 27, 31]. Triiodothyronine was able to reverse the contractile depression, resulting in significantly more force generation for a given calcium transient. The exact pathway for these acute positive inotropic effects of T3 is not clear. Several potential targets of a T3 action have to be considered: (1) The calcium reuptake rate into the sarcoplasmic reticulum [28] and the sodium–calcium exchange [33] are accelerated after T3 administration explaining the significant decline of diastolic calcium (Table 1). Even when systolic calcium remains constant a reduction of diastolic calcium results in an increase of the amplitude of the calcium transient that is associated with increased isometric force amplitudes. (2) A direct interaction with myosin as observed in a previous study after application of small peptides [35] is theoretically possible. However, we argue against such a mechanism in this study, as the peptides also had positive inotropic effects in normal isolated atrial myocardium. However, in the present study a clear positive inotropic effect was observed only after a preceding desensitization. (3) A T3 action on the contractile apparatus level by influencing myosin heavy chain expression [36] has been described. In a chronic rat model (hyperthyroid rats) Butkow and coworkers [37] have already demonstrated an increased calcium sensitivity in isolated working hearts. However, in the present study, a T3-mediated synthesis of new proteins does not seem likely as the incubation period of 30 minutes seems too short for such an effect. A direct effect through a calmodulin-dependent membrane calcium adenosine triphosphatase activity as described by Rudinger and associates [38] in rabbit myocardium has to considered. As this stimulatory action was mediated in a calmodulin-dependent way, calmodulin-dependent effects such as phosphorylation of MLCs could consistently explain the results [39]. As discussed previously even the incomplete recovery of force may be consistently explained by the observation that MLC phosphorylation cannot override the negative inotropic effects of catecholamine-induced troponin I phosphorylation. However, the previous experimental data of Litten and colleagues [40], who found an unchanged extent of light chain phosphorylation in rabbit hearts in chronic thyrotoxicosis, argue against such a possibility. However, their data may not be comparable to our study conditions, as we used an acute experimental model.

Methodological aspects
It is one of the advantages of the present study that an experimental model with exactly defined and controlled conditions was selected for the analysis. In large animal models it is difficult to maintain even the basic determinants of cardiac contractility (preload, afterload, frequency) constant. In addition any experimental protocol for brain death induction induces the activation of numerous physiologic feedback loops on neurohumoral, metabolic, and electrophysiologic levels with concomitant liberation of several mediator substances. Thus even clear experimental findings in large animal models are often difficult to interpret.

The methodological limitations of measurements of intracellular calcium concentrations using fura-2 have been addressed in detail in prior studies [17]. Compartmentalization and inhomogeneous distribution of the fluorescent dye still present a severe methodological limitation. For that reason, we show the absolute values calculated for intracellular calcium concentration, which should be interpreted as an orientation regarding the range of the calcium values.

Summarizing, cardiac muscle seems to use (1) the modulation of intracellular calcium for beat-to-beat regulation [6, 17, 37], (2) phosphorylation and dephosphorylation of MLCs and troponin for midterm adaptation [18–23], and (3) alterations of the composition and properties of intracellular proteins (including the contractile apparatus) for long-term adaptation [20, 25, 29, 37]. In accordance with the literature the present study supports the view that T3 can interfere with all three levels of controlling contractility.

As suggested by the data presented here T3 can reverse depressed myocardial performance after a preceding high-dose and long-term epinephrine stimulation. We hypothesize that (1) reversal of the decreased sensitivity of the contractile proteins to calcium and (2) a reduction of diastolic steady-state calcium by increased calcium reuptake into the sarcoplasmic reticulum and activation of the sodium–calcium exchanger [33] are important for the beneficial effects T3 and may—at least in part—explain the contractile recovery observed in human hearts after T3 application [13, 31, 41, 42]. Further studies are required to evaluate whether T3 is also efficient in other clinical situations associated with catecholaminergic stress including ischemia, catecholamine treatment, extracorporeal circulation, and surgical treatment.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
Tomasz Timek (Stanford University, CA) was supported by a fellowship from the Humbold Foundation. This work was supported by the German Research Foundation (SFB 414, research projects Q2 and Q4). We thank Karin Sonnenberg, Ulrike Gaffga, and Nicole Stumpf for their expert experimental assistance.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

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