Acute - Nongenomic Effect of Thyroid Hormones
Against Calcium Overload in
Newborn Rat Cultured Myocytes

Tova Zinman^a, Asher Shainberg^a, Narcis Tribulova^b, Mordechai Manoach^c

^a Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
^bInstitute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovak Republic
^cDepartment of Physiology, Sackler School of Medicine, Tel Aviv, Israel

Correspondence: M Manoach, Dept. of Physiology, Tel Aviv Medical School, Tel Aviv, P.O. Box 69978, Tel Aviv, Israel.
E-mail: manoach@post.tau.ac.il, phone +972 3 640 9108

1.  Introduction

Ventricular fibrillation (VF) is one of the major causes of sudden death in humans. Although it was believed that VF could not terminate spontaneously, evidence suggests that there are two types of VF: a sustained VF (SVF) that requires electrical defibrillation and a transient one (TVF) that spontaneously reverts into a sinus rhythm [Wigger, 1929]. TVF exhibits quite synchronized electrical fibrillating activity, with a large part of the ventricular mass acting in synchrony [Manoach and Wyatt, 1985]. "Synchronized" fibrillation occurs in hearts with functional cell to cell coupling, ensuring continuous propagation of electrical signals through the myocardium in a manner that brings the cardiomyocytes to act in synchrony. Recent experiments have shown that antiarrhythmic/defibrillating compounds that can transform SVF into a TVF, decrease intracellular free Ca²⁺ concentration [Ca²⁺]_i and prevent Ca²⁺ overload by increasing the SR Ca²⁺ re-uptake [Manoach et al., 1997]. Following this assumption, we postulated that any defibrillating drug should enhance or re-establish intercellular coupling, and prevent intercellular electrical uncoupling, most probably by increasing intracellular cAMP levels, decreasing elevated [Ca²⁺]_i and/or preventing Ca²⁺ overload [Manoach et al., 1999].

In a preliminary study we showed that thyroid hormones (T₃ and T₄) prevented Ca²⁺ overload caused by elevated extracellular Ca²⁺ concentration. Since protection against Ca²⁺ overload is an important feature for antiarrhythmic-defibrillating compounds, the aim of this study was to examine the acute effect of T₃ and T₄ in various cases of Ca²⁺ overload and to elucidate the mechanisms involved.

2.  Material and Methods

The experiments were carried out in cultured silent and contracting myocytes using indo-1 as an indicator of [Ca²⁺]_i [Shneyvays et al 2001]. The [Ca²⁺]_i was elevated either by increase of extracellular Ca²⁺ ([Ca²⁺]_o) to 3.8 mM, by decrease of extracellular Na⁺ ([Na⁺])_o to 10 mM, or by administration of 10 mM caffeine. Either T₃ or T₄ (10 or 100 nM) was added to the culture medium. The experiments were performed with or without the presence of 1 μM thapsigargine (inhibitor of SR Ca uptake), 20 μM H-89 (PKA inhibitor) or 10 μM thioridazine (CaM kinase inhibitor).

3.  Results

The results showed that administration of 10 or 100 nM T₃ or T₄ at elevated [Ca²⁺]_i (either due to a direct increase of [Ca²⁺]_o, decrease of [Na⁺]_o or caffeine administration) immediately decreased the [Ca²⁺]_i toward its diastolic control level. Increase of [Ca²⁺]_o following administration of T₃ or T₄ leads to a slow and smaller elevation of [Ca²⁺]_i.

The protective effect of T₃ and T₄ was abolished in the presence of all inhibitors used (thapsigargine, H-89 and thioridazine). Since the action of the PKA inhibitor was stronger and faster than the CaM kinase inhibitor, we suggest that the main effect of T₃ and T₄ is most likely through phospholamban phosphorylation by PKA.

4.  Discussion

In general, decrease of [Ca²⁺]_i can be achieved either by uptake of Ca²⁺ into the sarcoplasmic reticulum via SR Ca²⁺ATPase (SERCA2a) or by removal via sarcolemmal Na⁺-Ca²⁺ exchanger (NCX) [Bers 1991]. Following the result that the elevated [Ca²⁺]_i was decreased back to its basic diastolic level even in the case of low [Na⁺]_o (when NCX works in backward mode [Reuter, 2002]) and was abolished in the presence of thapsigargine we hypothesized that the T₃, T₄ protection against Ca overload is achieved due to an increase of SERCA2a activity. SERCA2a activity can be increased either via phosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and/or Ca²⁺/calmodulin-dependent kinase (CaM kinase) as well as via direct CaM kinase phosphorylation of the Ca²⁺-ATPase [Hawkins et al., 1995]. To examine these possibilities, further experiments were performed in the presence of PKA and CaM kinase inhibitors. The results that all of these inhibitors prevented the protective effect of T₃ and T₄ support our hypothesis. Since PKA inhibitor exhibited a stronger and faster effect than CaM kinase inhibitor, we suggest that the main effect of T₃ and T₄ is most likely through phospholamban phosphorylation.

5.  Conclusions

In conclusion, the present study indicates that thyroid hormones decrease elevated [Ca²⁺]_i and abolish Ca²⁺ overload in newborn rat cardiomyocytes most likely by direct, acute and nongenomic increase of SR Ca²⁺ uptake. The possibility that acute administration of T₃ and T₄ can have an antiarrhythmic/defibrillating effect is now under investigation.

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