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Augmentation of cardiac contractility with no change in L-type Ca²⁺ current in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8)¹

Laboratoire de Cardiologie Cellulaire et Moléculaire, INSERM U-446, Université Paris-Sud, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France; and
^* Laboratoire de Régulation des Gènes et Signalisation Cellulaire, INSERM U-99, Hôpital Henri-Mondor, F-94010 Créteil, France

³Correspondence: INSERM U-446, Université Paris-Sud, Faculté de Pharmacie, 5, Rue J.-B. Clément, F-92296 Châtenay-Malabry Cedex, France. E-mail: Fisch{at}vjf.inserm.fr

SPECIFIC AIMS

The ß-adrenergic cascade is severely impaired in virtually every form of experimental heart failure (HF) and in the human syndrome. Several studies have shown that AC5 and/or AC6 protein levels, the dominant adenylyl cyclase (AC) isoforms expressed in heart, are reduced in HF, leading to a decrease in total AC activity and a reduction in cAMP-dependent protein kinase (PKA) activity and cardiac contraction. Hence, cardiac-directed AC overexpression is a conceivable therapeutic strategy in HF. In this study, we explored the consequences at the cellular and organ level of a cardiac-directed expression of the human neuronal AC8 in the transgenic mouse line AC8TG.

PRINCIPAL FINDINGS

1. Influence of AC8 expression on the spontaneous contractility of the heart
Anatomical examination of 4- to 5-month-old AC8TG and nontransgenic (NTG) mice and their hearts showed no obvious difference between the two groups. Left ventricular systolic pressure (LVSP), heart rate (HR), and rate pressure product (RPP=LVSPxHR, used as an index of contractility) were recorded routinely in spontaneously beating isolated hearts cannulated through the aorta and perfused by the Langendorff method at constant pressure (75 mmHg). Although the coronary flow was similar in NTG and AC8TG mice, LVSP was twofold higher (Fig. 1 A), spontaneous HR was 40% faster (Fig. 1B ), and RPP was nearly threefold higher (Fig. 1C ) in AC8TG than in NTG mice, demonstrating that specific cardiac expression of AC8 strongly enhanced basal cardiac function.

View larger version (13K): [in this window] [in a new window]   Figure 1. Ex vivo basal cardiac function in NTG and AC8TG mice under spontaneous beating conditions. The hearts were perfused with oxygenated Krebs-Henseleit solution containing 1.8 mM [Ca2+]o. A) left ventricular systolic pressure (LVSP), B) Heart rate (HR), and C) rate pressure product (RPP) were measured in NTG (empty bars) and AC8TG mice (filled bars) when all parameters were stable and under spontaneous beating conditions. The bars are means ± SE NTG, n = 13; AC8TG, n = 12. ***P < 0.001.

2. Sensitivity to external calcium ([Ca²⁺]_o)
Since AC8 activity is stimulated by Ca²⁺-calmodulin, we explored the influence of a change in [Ca²⁺]_o on the contractility of both groups of animals. Increasing [Ca²⁺]_o from 0.5 to 2.5 mM resulted in a dose-dependent increase in LVSP in AC8TG and NTG hearts without alteration in HR. However, sensitivity to [Ca²⁺]_o was enhanced in AC8TG hearts, since the mean EC₅₀ for [Ca²⁺]_o was 1.05 mM in AC8TG and 1.51 mM in NTG mice (P<0.05).

3. Influence of AC8 expression on the contractility of the paced heart
Cardiac expression of AC8 results in a strong acceleration of HR, which in itself might affect the contractile properties of the hearts. Thus, additional experiments were performed at a constant stimulating frequency (680 bpm) and at 1 mM [Ca²⁺]_o. Under these conditions, AC8TG mice still developed a twofold higher LVSP than NTG mice. A detailed analysis of the kinetics of contraction and relaxation revealed that the contractile relaxation was 37% faster in AC8TG than with NTG mice (P<0.001).

4. Measurement of cell shortening
To determine whether AC8 cardiac expression also modified basal contractility at the level of the myocyte, we measured cell shortening in isolated ventricular myocytes from AC8TG and NTG mice. Since the myocytes contracted free of load, shortening amplitude was maximal and not different in both mice. However, onset of shortening developed 22% faster (P<0.01) and relaxation was 43% faster (P<0.05) in transgenic myocytes.

5. Measurement of intracellular calcium ([Ca²⁺]_i) transients
Because contraction and relaxation kinetics are largely dependent on Ca²⁺ release from and uptake into the sarcoplasmic reticulum (SR), we measured [Ca²⁺]_i transients in AC8TG and NTG myocytes, using the fluorescent Ca²⁺ indicator Fluo-3 AM. [Ca²⁺]_i transients were 30% larger in amplitude (P<0.05) and relaxed 24% faster in AC8TG than in NTG mice (P<0.05).

6. L-type Ca²⁺ current measurements
The amplitude of cardiac contraction is largely dependent on that of the L-type Ca²⁺ channel current (I_Ca,L), which initiates the calcium-induced calcium release (CICR) mechanism. Thus, we examined how cardiac expression of AC8 affects I_Ca,L in isolated ventricular myocytes using the whole-cell patch-clamp technique. Myocytes from AC8TG and NTG mice both had similar cell membrane capacitance (175 pF mean value), confirming the absence of cell hypertrophy in AC8TG mice. I_Ca,L was recorded in control 1.8 mM [Ca²⁺]_o solution during 400 ms depolarizations to 0 mV from a holding potential of -50 mV. Figure 2 A shows two representative traces of I_Ca,L recorded from NTG and AC8TG ventricular myocytes. Averaged I_Ca,L amplitudes were not statistically different (Fig. 2B ). Likewise, when normalized to cell membrane capacitance, I_Ca,L current density was similar in AC8TG and NTG ventricular myocytes (Fig. 2C ). Thus, AC8 expression did not modify basal I_Ca,L amplitude and current density at 0 mV. As shown in Fig. 2D , AC8 expression did not modify the voltage dependence of peak I_Ca,L. Since AC8 is stimulated by Ca²⁺-calmodulin, we tested the effect of an increase in [Ca²⁺]_i on I_Ca,L. However, the amplitude of I_Ca,L remained identical in AC8TG and NTG mice even when [Ca²⁺]_i was increased either by reducing the Ca²⁺ buffering capacity of the patch pipette solution or increasing the stimulation frequency (0.125–8 Hz). Thus, cardiac directed expression of AC8 leads to an enhanced cardiac function that does not involve an increase in I_Ca,L.

View larger version (23K): [in this window] [in a new window]   Figure 2. Basal L-type Ca2+ current (ICa,L) properties in NTG and AC8TG ventricular myocytes. A) Representative whole-cell ICa,L current traces recorded in NTG (left) and AC8TG myocytes (right). Currents were elicited every 8 s by a 400 ms depolarization to 0 mV from a holding potential of -50 mV. B, C) Bars indicate the mean ± SE ICa,L basal amplitude (B) and current density (C) measured in 16 NTG (empty bars) and 18 AC8TG myocytes (filled bars). D) Current-voltage (I-V) relationships of peak ICa,L in NTG () and AC8TG myocytes (). ICa,L were normalized to the cell capacitance to obtain current densities (pA/pF). Data points are means ± SE from 5 NTG and 4 AC8TG cells.

DISCUSSION

In the present study, we show that 1) cardiac specific expression of AC8 is associated with a strong enhancement of ex vivo basal cardiac function and an increased sensitivity of cardiac contraction to [Ca²⁺]_o; 2) at the level of the isolated ventricular myocyte, cell shortening developed faster and relaxed faster in AC8TG than in NTG mice; 3) [Ca²⁺]_i transient amplitude was enhanced and its decay was faster in AC8TG vs. NTG mice, which indicates a faster reuptake of Ca²⁺ by the SR; 4) despite the large increase in Ca²⁺ transients and contraction, I_Ca,L amplitude was unchanged in AC8TG even when AC8 was activated by raising [Ca²⁺]_i.

As shown earlier, AC8 is essentially expressed in the brain and is stimulated by Ca²⁺-calmodulin, unlike AC5 or AC6, which are inhibited by intracellular Ca²⁺ ([Ca²⁺]_i). During ß-adrenergic stimulation, the resulting elevation of [Ca²⁺]_i normally inhibits AC5 and AC6, and this supposedly acts as a negative feedback on L-type Ca²⁺ channel activity, [Ca²⁺]_i, and cardiac contractility. However, in AC8TG mice, an increase in [Ca²⁺]_i should stimulate AC8 activity, leading to a further increase in [Ca²⁺]_i. Intuitively, such a positive feedback might generate Ca²⁺ overload and be deleterious to the animals. However, AC8TG mice present no phenotypic alteration or any sign of cardiomyopathy despite a clear increase in cardiac AC and PKA activities. From our results, we believe this is due to the lack of cross-talk between AC8 and Ca²⁺ channels in AC8TG mice: cAMP production by AC8 does not lead to Ca²⁺ channel phosphorylation and Ca²⁺ influx via I_Ca,L does not lead to AC8 stimulation (Fig. 3 ). It is likely that adaptive changes occurred in AC8TG mice to avoid a positive feedback between AC8 and intracellular Ca²⁺, hence preserving cardiac myocytes from Ca²⁺ overload.

View larger version (62K): [in this window] [in a new window]   Figure 3. Schematic representation of the cAMP cascade in AC8TG mice. Whereas cAMP produced by endogenous AC5 and/or AC6 activates Ca2+ influx via PKA phosphorylation of the L-type Ca2+ channel, cAMP produced by AC8 does not. However, AC8 specifically fuels cAMP for PKA phosphorylation of phospholamban (PLB), alleviating the inhibitory effect of PLB on SR Ca2+-ATPase (SERCA), thereby increasing Ca2+ uptake into the SR and making more Ca2+ available for contraction.

Although cAMP produced by AC8 appears not to be accessible to Ca²⁺ channels, AC8 expression still strongly increases cardiac function, indicating that it must activate other elements of the EC coupling. From our results, we believe that AC8 activates specifically the SR function: 1) contractile relaxation was markedly accelerated in AC8TG compared with NTG mice; 2) at the level of the single ventricular myocyte, both cell shortening and [Ca²⁺]_i transient relaxed much faster in AC8TG than in NTG mice. Thus, cardiac-directed expression of AC8 might provide a unique model of cAMP compartmentation whereby cAMP produced by AC8 specifically activates Ca²⁺ uptake into the SR without increasing Ca²⁺ influx at the sarcolemma (Fig. 3) . This could be achieved by an increase in the total amount of SR Ca²⁺-ATPase, an alleviated inhibitory effect of phospholamban, its key regulator protein, due to PKA phosphorylation, or both. Another interesting aspect of this model is that AC8TG mice develop an enhanced cardiac function under basal conditions, unlike other AC transgenic mice, which need to be activated by ß-adrenergic pathway to improve cardiac function. Thus, cardiac expression of AC8 might provide a means to increase heart function even when the ß-adrenergic signaling cascade is down-regulated at the receptor level, as during HF.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0292fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.02-0292fje

² Permanent address: Laboratory of Membrane Biophysics, Institute of Cardiology, Kaunas University of Medicine, 3007 Kaunas, Lithuania. E-mail: JoJur{at}kmu.lt

This Article Full Text (PDF) All Versions of this Article: 16/12/1636 02-0292fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by GEORGET, M. Articles by FISCHMEISTER, R. Search for Related Content PubMed PubMed Citation Articles by GEORGET, M. Articles by FISCHMEISTER, R.