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Subchronic alpha- and beta-adrenergic regulation of cardiac gap junction protein expression

A. Salameh^*, C. Frenzel, A. Boldt, B. Rassler, I. Glawe, J. Schulte, K. Mühlberg^*, H.-G. Zimmer, D. Pfeiffer^* and S. Dhein^,1

^* Medizinische Klinik I, Abteilung Kardiologie, Universitätsklinik Leipzig, Leipzig, Germany;
Klinik für Herzchirurgie, Herzzentrum Universität Leipzig, Leipzig, Germany; and
Carl-Ludwig-Institut für Physiologie, Universität Leipzig, Leipzig, Germany

¹ Correspondence: Heart Center Leipzig, Clinic for Cardiac Surgery, Struempellstr. 39, Leipzig 04289, Germany. E-mail: dhes{at}medizin.uni-leipzig.de

SPECIFIC AIMS

Connexins are proteins that form intercellular communication channels, gap junctions, which (in heart) are predominantly involved in regular conduction of the electrical impulse. Changes in these gap junctions have been considered to result in arrhythmia. Since in cardiac hypertrophy up-regulation of connexin 43 expression, but not connexin 40 has been described, and since cardiac hypertrophy often is accompanied by enhanced adrenergic stimulation, we wanted to know whether adrenergic stimulation can regulate the expression of connexins, and how the concentration-response curve is characterized. This was investigated in cultured neonatal rat cardiomyocytes exposed to - or ß-adrenoceptor agonists for 24 h as well as in adult rats exposed to 24 h treatment with these substances.

PRINCIPAL FINDINGS

1. Effects of 24 h adrenergic stimulation on cardiac connexin expression
Stimulation of cultured neonatal rat cardiomyocytes for 24 h with increasing concentrations of noradrenaline (0.1–10000 nM) led to a concentration-dependent increase in Cx43 protein content (pEC₅₀ of 9.08±0,22 M, R²=0.96, Hill slope 1) without affecting Cx40 expression as detected by Western blot analysis (Fig. 1 A, B). GAPDH expression was not altered by the treatment. Since noradrenaline can stimulate protein synthesis via both the ₁- and the ß-adrenoceptors, in subsequent experiments we investigated the precise role of ₁- and ß-stimulation. Therefore, for -stimulation, we used the selective ₁-agonist phenylephrine and for ß-stimulation, we used the ß-agonist isoproterenol (both: 0.1–10,000 nM). Both catecholamines administered for 24 h provoked a significant and concentration-dependent increase in Cx43 protein content with pEC₅₀ of 7.7 ± 0.19, R² = 0.97 (isoproterenol) and 8.17 ± 0.14, R² = 0.99 (phenylephrine) (Fig. 1C ).

View larger version (24K): [in this window] [in a new window]   Figure 1. A) Western blot results for Cx43 and Cx40 in neonatal rat cardiomyocytes exposed to noradrenaline for 24 h. All values are given as means ± SE of n = 6 experiments (for each data point) in optical density (OD) units relative to the untreated control cells (control=100%). *Significant difference vs. control, P < 0.05. B) Original Western blot for Cx43 and Cx40 for the conditions described above. C) Western blot results for Cx43 in neonatal rat cardiomyocytes exposed to the isoproterenol or phenylephrine for 24 h (n=5 for each data point).

To determine whether this rise in Cx43 protein content is due to a regulation on the transcriptional level rather than due to a reduced Cx43 protein degradation, PCR studies were performed. In these experiments we found a significant increase (P<0.05) of Cx43-mRNA relative to the housekeeping gene GAPDH under the influence of a 24 h treatment with either 10^–7 M phenylephrine or isoproterenol.

To investigate the underlying signal transduction pathway, we examined the influence of inhibitors of possibly relevant kinases. For PKC inhibition, 5 µM BIM I (bisindolylmaleimide I) was used; for PKA inhibition, 2 µM H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) was used. The inhibitors were applied concomitantly to either isoproterenol or phenylephrine. The isoproterenol-induced Cx43 expression could be significantly antagonized by H8 but not by BIM I, while the phenylephrine-induced Cx43 expression was significantly inhibited by BIM I, but not by H8.

2. Adrenergic stimulation increased the gap junction-mediated intercellular communication between the cultured cardiomyocytes
We next questioned whether up-regulation of Cx43 by ₁- or ß-adrenoceptor stimulation might indeed result in enhanced electrical intercellular coupling. Macroscopic gap junction currents determined in dual whole-cell voltage-clamp technique was 29 ± 4 nS in pairs of neonatal ventricular cardiomyocytes under control conditions, which was in good accordance with the values reported in the literature. After 24 h treatment of the cells with 100 nM of either phenylephrine or isoproterenol, electrical coupling was significantly enhanced to 43 ± 4 nS (n=8) and 57 ± 8 nS (n=8), respectively (Fig. 2 ). Seal resistance in all cells was between 3 and 5 G. Membrane resistance was 525 ± 34 M, and membrane capacitance was 24 ± 2 pF.

View larger version (19K): [in this window] [in a new window]   Figure 2. Upper panel) Dual whole-cell voltage clamp results for macroscopic gap junction conductance in neonatal rat cardiomyocytes exposed to either control conditions (no treatment) or 10–7 M phenylephrine or 10–7 M isoproterenol for 24 h. All values are given as means ± SE of n = 8 experiments (each data point). *Significant changes vs. control, P < 0.05. Lower panel) Original current and voltage traces for the conditions described above. Two cells were clamped in whole cell mode (holding potential=–40mV). While in cell 2 voltage was kept constant at –40 mV, in cell 1 voltage was stepped to –50 mV (200 ms pulse), thus applying a transjunctional voltage of 10 mV. The voltage and current signals given are the measured signals from both cells. Current I2 in the nonstepped cell is considered to reflect gap junction current. To assess gap junction conductance, transjunctional voltages of ±50 mV (200 ms) relative to the holding potential of –40 mV were applied.

3. 24 h adrenergic stimulation up-regulates Cx43 but not Cx40 under in vivo conditions
The next part of our investigation was the examination of the effects of 24 h ₁- or ß-adrenoceptor stimulation by 24 h in vivo infusion of phenylephrine or isoproterenol. Left ventricular weight was 635 ± 17mg for all with small, nonsignificant differences between the groups. Both treatments exhibited functional responses in the treated animals, demonstrating that the doses applied were in an effective range: left ventricular pressure was significantly enhanced by isoproterenol as was dP/dt_max, heart rate, cardiac output, and cardiac index. Phenylephrine resulted in mild positive inotropic responses, while heart rate and cardiac index were not significantly altered. However, in good agreement with the cell culture results, we found significant up-regulation of Cx43 by both isoproterenol and phenylephrine, while Cx40 again remained unchanged.

CONCLUSIONS AND SIGNIFICANCE

It has long been known that ß-adrenergic stimulation can acutely (i.e., in minutes) enhance coupling via opening of existing gap junction channels. Recently, up-regulation of Cx43 by 24 h dibutyryl-cAMP stimulation has been described, possibly indicating that chronic stimulation of ß-adrenoceptors may also lead to Cx43-up-regulation. Although it is known that -adrenergic stimulation is involved in hypertrophic responses, the effects of -adrenergic stimulation on connexin expression have not been investigated so far. Thus, the novel finding of our study is that subchronic 24 h adrenergic stimulation leads to differential up-regulation of Cx43, but not Cx40, at concentrations as found in the heart (EC₅₀ around 1 nM). This up-regulation of Cx43 can be mimicked by 24 h -adrenergic stimulation (phenylephrine) or 24 h ß-adrenergic stimulation (isoproterenol) with reasonable EC₅₀ values in the nanomolar range. On the background of a half-life time of only 90 min for Cx43, it is reasonable that changes in expression of this membrane protein take place within a window of 24 h, which may indicate that cardiomyocytes permanently adapt their communication to the current situation.

Regarding the signal transduction, our data indicate that the -adrenergic effect is mediated via PKC, since it could be inhibited by the PKC inhibitor BIM I, while the ß-adrenergic effect is transduced by PKA, indicated by its sensitivity to the PKA inhibitor H8. The fact that the treatment also leads to enhanced expression of Cx43mRNA is in favor of the assumption that either a de novo synthesis is induced and (at least partially) involved (Fig. 3 ) or alternatively that Cx43mRNA is somehow stabilized. These data correspond well to data from the literature showing that hypertrophic responses to -adrenergic stimulation are transduced via PKC, and that cAMP, which is involved in the ß-adrenoceptor-adenylylcyclcase-PKA pathway, can induce expression of Cx43. In the case of the 1-adrenoceptor, this agrees well with previous data from our group showing up-regulation of Cx43 by endothelin and angiotensin, via ET_A or AT₁ receptor stimulation, since both are G_q/11-coupled. Thus, the 1-adrenoceptor would be the third G_q/11-coupled receptor linked to PKC and leading to up-regulation of Cx43. Therefore, it might be a general principle that G_q/11-coupled receptor stimulation leads to Cx43 up-regulation.

View larger version (21K): [in this window] [in a new window]   Figure 3. Scheme of the hypothetic role of adrenergic stimulation in regulation of Cx43 gap junction protein expression. PKC, protein oxide).

The functional data obtained from the dual whole cell voltage clamp experiments indicate that enhanced Cx43 protein leads to enhanced electrical coupling and thus is of functional relevance. We conclude that at least a portion of the increased Cx43 seems to form functional channels. This also means that the degree of gap junctional intercellular communication can be regulated by enhanced expression of connexins, in this special case Cx43 in response to either - or ß-adrenergic stimulation, which might resemble an interesting new principle of regulating the biophysical properties and opens new therapeutic approaches, since enhanced connexin synthesis as described by others in cardiac hypertrophy and as suggested to be arrhythmogenic might be counteracted by antiadrenergic treatment.

Another point to consider is the in vivo relevance. Although neonatal rat cardiomyocytes are a well established model for investigation of gap junction expression, one might argue that in adult cells this regulation could be different. However, it is impossible to study this effect in adult cardiomyocytes since these are normally postmitotic cells, which after isolation, decrease their Cx43 expression and several days after isolation in culture change their morphology to a more embryonic type and then increase Cx43 expression again, so that in adult cells there are no stable conditions regarding connexin expression. To circumvent this point, we decided to infuse catecholamines in vivo in adult rats and investigate connexin expression after 24 h constant infusion. These results confirmed our in vitro findings and also revealed clear Cx43 up-regulation following - or ß-adrenoceptor stimulation without affecting Cx40 expression. The similarity of the in vitro and in vivo findings further supports of the use of neonatal rat cardiomyocytes as a model for cardiac connexin expression regulation.

Cx43 but not Cx40 seems the main target for connexin expression regulation by adrenergic stimulation in the heart. Thus, enhanced adrenergic stimulation in cardiac disease may contribute to the differential regulation of cardiac connexins with enhanced Cx43 and unaltered Cx40 as described in cardiac hypertrophy. Our findings may also relate to patients suffering from early stages of heart failure in which ß₁-adrenoceptors are still functional but confronted with enhanced local catecholamine levels. These patients are at high risk for ventricular fibrillation, and treatment with ß-adrenoceptor antagonists such as metoprolol has been shown to reduce mortality. To understand this, it must be kept in mind that not only the expression level of connexins, but also the localization of the functional gap junction channels with regard to the cell axis is of importance for the regular propagation of the cardiac electrical impulse. In contrast, in late stages with manifest heart failure (ejection fraction <30%), ß-adrenoceptors are down-regulated with decreased responsiveness of the ß-adrenoceptor adenylylcyclase system, which would implicate a reduction in Cx43 expression, as described by several groups. This may also lead to conduction failure and arrhythmia, especially if Cx43 is decreased locally by >50% and inhomogeneity is increased, which also was observed but might not be found in all patients.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4871fje;

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