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i2- and Gαi3-Specific Regulation of Voltage-Dependent L-Type Calcium Channels in Cardiomyocytes

  • Sara Dizayee,

    Affiliation Department of Pharmacology, University of Cologne, Cologne, Germany

  • Sonja Kaestner,

    Affiliation Department of Pharmacology, University of Cologne, Cologne, Germany

  • Fabian Kuck,

    Affiliation Department of Biochemistry and Molecular Biology II, University Hospital and Clinics, University of Düsseldorf, Düsseldorf, Germany

  • Peter Hein,

    Affiliation Department of Pharmacology, University of Cologne, Cologne, Germany

  • Christoph Klein,

    Affiliation Department of Pharmacology, University of Cologne, Cologne, Germany

  • Roland P. Piekorz,

    Affiliation Department of Biochemistry and Molecular Biology II, University Hospital and Clinics, University of Düsseldorf, Düsseldorf, Germany

  • Janos Meszaros,

    Affiliation Department of Pharmacology, University of Cologne, Cologne, Germany

  • Jan Matthes,

    Affiliation Department of Pharmacology, University of Cologne, Cologne, Germany

  • Bernd Nürnberg,

    Affiliations Department of Biochemistry and Molecular Biology II, University Hospital and Clinics, University of Düsseldorf, Düsseldorf, Germany, Department of Pharmacology and Toxicology, Interfaculty Center of Pharmacogenomics and Pharmaceutical Research, University of Tübingen, Tübingen, Germany

  • Stefan Herzig

    stefan.herzig@uni-koeln.de

    Affiliation Department of Pharmacology, University of Cologne, Cologne, Germany

i2- and Gαi3-Specific Regulation of Voltage-Dependent L-Type Calcium Channels in Cardiomyocytes

  • Sara Dizayee, 
  • Sonja Kaestner, 
  • Fabian Kuck, 
  • Peter Hein, 
  • Christoph Klein, 
  • Roland P. Piekorz, 
  • Janos Meszaros, 
  • Jan Matthes, 
  • Bernd Nürnberg, 
  • Stefan Herzig
PLOS
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Correction

15 Aug 2013: Dizayee S, Kaestner S, Kuck F, Hein P, Klein C, et al. (2013) Correction: Gαi2- and Gαi3-Specific Regulation of Voltage-Dependent L-Type Calcium Channels in Cardiomyocytes. PLOS ONE 8(8): 10.1371/annotation/7be097c0-9075-41ae-91e9-d2209de952cc. https://doi.org/10.1371/annotation/7be097c0-9075-41ae-91e9-d2209de952cc View correction

Abstract

Background

Two pertussis toxin sensitive Gi proteins, Gi2 and Gi3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous Gi isoforms are functionally distinct. To test for isoform-specific functions of Gi proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC).

Methods

Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gαi2 (Gαi2−/−) or Gαi3 (Gαi3−/−). mRNA levels of Gαi/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gαi and Cavα1 protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings.

Results

In cardiac tissue from Gαi2−/− mice, Gαi3 mRNA and protein expression was upregulated to 187±21% and 567±59%, respectively. In Gαi3−/− mouse hearts, Gαi2 mRNA (127±5%) and protein (131±10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gαi2−/− mice was lowered (−7.9±0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (−10.7±0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gαi3−/− mice (−14.3±0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gαi2 (but not of Gαi3) and following treatment with pertussis toxin in Gαi3−/−. The pore forming Cavα1 protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Cavα1 and Cavβ2 subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gαi2.

Conclusion

Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gαi proteins. In particular, loss of Gαi2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.

Introduction

G protein-mediated signalling plays a central role in regulation of cardiomyocyte function. Heterotrimeric G proteins consist of three subunits, Gα, Gβ, and Gγ. Agonist-occupied receptors induce dissociation of GDP from and binding of GTP to the G protein α subunit, resulting in G protein activation. Activated Gα and Gβγ subunits couple to a plethora of effectors, including enzymes and ion channels, and hence are involved in many regulatory processes [1], [2]. The role of stimulatory Gs and inhibitory Gi proteins in cardiac signalling pathways is well studied [3], [4]. Alterations of Gαi protein expression levels are found in heart disease [5], and heart failure in humans leads to upregulation of Gαi2 and Gαi3 [6], [7], [8], [9]. Whether the upregulation of Gαi2 and Gαi3 in cardiomyocytes is causative, adaptive, or maladaptive still remains unclear.

Cardiac calcium channels are key components in complex signal transduction pathways and play an essential role in cardiac excitability and in coupling excitation to contraction [10]. One major pathway regulating calcium channels is mediated via G protein-mediated signalling. In the heart, the main sarcolemmal calcium channel is the voltage-dependent L-type calcium channel (L-VDCC). This channel is composed of three different subunits. The α1 subunit represents the pore forming subunit which contains the voltage sensor and the binding sites for calcium channel modulators [11]. It associates with two auxiliary subunits, β, and α2δ [12]. The functional properties of the pore forming subunit are differentially modified due to interaction with various β subunit isoforms [13], [14], [15], [16]. Furthermore, receptor activated Gαs protein stimulates L-VDCCs via adenylyl cyclase-mediated increases in cAMP levels and protein kinase A (PKA) activity [3]. Activation of Gi or Go modifies channel function via diverse signal cascades [17]. Thus, G protein signalling pathways are crucial in determining and balancing cardiomyocyte function in vivo.

In a previous study we addressed the role of Gαi2 in β2-adrenergic receptor-mediated signalling. Gene deletion of Gαi2 in mice reduced single L-VDCC activity in β2-adrenergic receptor-transgenic mice [18], whereas pertussis toxin (PTX) treatment reversed this effect. We speculated that this unexpected effect of PTX may have been caused by inhibiting an upregulated Gαi3. Recently, Zuberi et al. [19] showed that Gαi2 knockout leads to increased L-VDCC mRNA expression and a propensity towards ventricular arrhythmia. Muscarinic receptor-mediated inhibition of L-VDCC activity has been reported to depend on Gαi2 but not Gαi3 [20]. Though strongly suggested by these data, subtype-specific effects on cardiac L-VDCC by the highly homologous Gαi2 and Gαi3 isoforms remain unclear so far. Therefore, the present work was undertaken to elucidate whether the effects of these Gαi proteins are redundant or distinct. Using cardiomyocytes from mice lacking Gαi2 or Gαi3 and wild-type (WT) control animals, we determined structural and functional changes. Further, we examined specific signalling pathways implicated in cardiac L-VDCC modulation by Gαi protein. In this work, we provide evidence that the L-VDCC activity and kinetics are regulated in a non-redundant manner and we support this idea by demonstrating subtype-specific activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) signalling cascade.

Results

i2 deficiency decreases, while Gαi3 deficiency increases L-VDCC current density

To assess consequences of selective deletion of Gαi2 or Gαi3 genes, we measured whole-cell L-VDCC currents in cardiomyocytes from WT, Gαi2−/−, and Gαi3−/− mice. Currents recorded at different test potentials are shown as representative original recordings and current-voltage diagrams of summarized data in Fig. 1A and B, respectively. In cardiomyocytes of Gαi2−/− mice, the calcium current density at 0 mV was slightly but significantly reduced (−7.9±0.6 pA/pF, n = 11, p<0.05) compared to WT (−10.7±0.5 pA/pF, n = 22). In contrast, current density in cardiomyocytes from Gαi3−/− mice was increased (to −14.3±0.8 pA/pF, n = 14, p<0.05 vs. WT). Of note, the peak current in Gαi2−/− cardiomyocytes is shifted towards higher voltages. Comparison of time-dependent inactivation by fitting revealed no alterations of fast and slow time constants in all genotypes (e.g. at 0 mV, τfast: WT = 18.4±1.0 ms, Gαi2−/− = 26.9±4.0 ms and Gαi3−/− = 15.1±2.1 ms; τslow: WT = 95.3±4.2 ms, Gαi2−/− = 97.5±17 ms and Gαi3−/− = 94.9±9.9 ms; n = 10–13). To test whether changes in gating account for the observed differences in current density, we examined kinetic and steady state properties of activation and inactivation.

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Figure 1. Cardiac whole-cell L-type calcium currents.

Representative original current traces obtained at different test potentials (A) and IV-curves (B) reveal an increase of calcium current in ventricular myocytes from mice lacking Gαi3 and a decrease in mice lacking Gαi2 as compared to WT mice. (C) Steady-state inactivation of Gαi2−/− (n = 11) is shifted to more negative voltages as compared to Gαi3−/− (n = 13, p<0.05) and WT (n = 18, p<0.05). (D) A slowing of the recovery time constant τ is found in cardiomyocytes from Gαi2−/− mice (287±21 ms, n = 9) as compared to Gαi3−/− cells (n = 9) and WT (n = 16).

https://doi.org/10.1371/journal.pone.0024979.g001

L-VDCC kinetics are altered by Gαi2 deletion, but not by Gαi3 deletion

The steady-state inactivation properties (Fig. 1C) in Gαi2−/− cardiomyocytes were altered compared to WT cells as reflected by a significant leftward shift of V0.5 (Gαi2−/−: −23.4±1.0 mV, n = 11, WT: −19.2±0.7 mV, n = 18) and an increased slope factor (Table 1). In addition, recovery from inactivation was slowed in cardiomyocytes from mice lacking Gαi2 (τ: 287±21 ms, n = 9, p<0.05) in comparison to WT animals (τ: 215±14 ms, n = 16; Fig. 1D). In contrast, whole-cell currents in cells form Gαi3−/− mice were indistinguishable from WT regarding both steady state inactivation (V0.5: −18.2±0.9 mV, n = 13, Table 1) and recovery from inactivation (τ: 203±24 ms, n = 9). Thus, currents from Gαi2−/− mice show altered kinetic properties and this might explain the decreased current density described above (Fig. 1). Despite increased current density no alteration of current kinetics in Gαi3−/− myocytes was visible. Thus we analyzed single-channel activity to elucidate the opposing effects seen on whole-cell currents in Gαi2−/− and Gαi3−/− cardiomyocytes.

No major changes in gating properties of single L-VDCC in Gαi3−/− cardiomyocytes

Single-channel current recordings in Gαi3−/− cardiomyocytes revealed a trend towards increased peak ensemble average currents (Fig. 2B) and higher open probability (Fig. 2C) when compared to WT cardiomyocytes (Table 2). These effects are based on a significant reduction of the mean closed time (3.8±0.5 ms vs. 6.6±0.9 ms, n = 6–7, p<0.05; Fig. 2D). Together with a significantly decreased slow time constant of the closed state and by trend a reduced first latency (Table 2) our single-channel data suggest that in Gαi3−/− exit from deeper closed states of L-VDCC is facilitated. Given that single-channel activity in Gαi2−/− mice was decreased by trend [18] our findings presented here suggest that Gαi2−/− or Gαi3−/− knockout leads to distinct changes of cardiac L-VDCC properties. Yet, these only slight functional changes alone do not elucidate the more remarkable augmentation of calcium current density in Gαi3−/−. Since the remaining Gαi isoform might have compensated for effects of Gαi3 deficiency we next checked the expression levels of Gαi2 in Gαi3 knockouts and vice versa.

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Figure 2. Single-channel properties of L-type calcium channels.

(A) Exemplary traces of barium currents show increased single-channel activity in ventricular myocytes of Gαi3−/− animals vs. WT mice. (B) The peak ensemble average current is −61±13 fA in Gαi3−/− (n = 6) and −44±9 fA in WT mice (n = 7). (C) The open probability within active sweeps is slightly enhanced in Gαi3−/− (5.4±1.0% vs. 4.0±0.9% WT) whereas (D) the mean closed time is significantly reduced (Gαi3−/− 3.8±0.5 ms vs. 6.6±0.9 ms WT). Unitary amplitude was not different with −0.83±0.02 pA (WT) and −0.79±0.03 pA (Gαi3−/−). *p<0.05 vs. WT. Box-and-whisker plots indicate minimum and maximum values as well as 25th, 50th and 75th percentiles.

https://doi.org/10.1371/journal.pone.0024979.g002

Enhanced expression levels of remaining Gαi isoform

If the generally accepted assumption holds true that Gαi2 and Gαi3 proteins are functionally redundant in the heart, we could expect a compensatory upregulation of the remaining Gαi subunit after knockout of the other. We first determined mRNA expression levels for Gαi1, Gαi2, Gαi3, and Gαo in samples from WT, Gαi2−/−, and Gαi3−/− mice using real-time PCR. In ventricular tissue from WT mice, transcripts for all Gαi protein isoforms and Gαo were found in different amounts (Fig. 3A). While Gαi2 and Gαi3 are known to be expressed in cardiomyocytes - with Gαi3 mRNA being clearly less abundant -, Gαi1 and Gαo are likely transcribed in non-cardiomyocyte ventricular cells [21]. Deletion of Gαi2 enhanced the mRNA level of Gαi3 (to 187±21%, n = 3, p<0.05 vs. WT), as expected [6], [22]. In cardiac tissue of Gαi3−/− mice Gαi2 mRNA levels were upregulated to only 127±5% (n = 4, p<0.05 vs. WT). No significant changes in Gαo or Gαi1 mRNA expression levels were detected, which is in line with the assumption, that these G proteins are not expressed in cardiomyocytes (see above and Fig. 3B).

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Figure 3. RNA expression levels of cardiac Gαi/o isoforms measured by real-time PCR.

GAPDH was used as endogenous control, and WT mice as calibrator (expression = 100%; n = 4). (A) 2−ΔCT values were calculated to analyze the relative expression of Gαi/o isoforms in WT cardiomyocytes. (B) The relative mean expression (2−ΔΔCT) reveals a significantly increased Gαi3 mRNA content in ventricular tissue of Gαi2−/− mice (n = 3) and significantly increased Gαi2 mRNA levels in Gαi3−/− mice (n = 4). (C) Representative example of Gαi2 and Gαi3 protein expression in murine ventricular tissue from WT, Gαi2−/− and Gαi3−/− mice. Cell membranes were isolated and Gαi proteins were analyzed by immunoblotting using an anti-Gαcommon antibody. Shorter exposure times were used to analyse Gαi2 protein. (D) Summarized protein expression data show an upregulation of Gαi3 in Gαi2−/− mice and an upregulation of Gαi2 in Gαi3−/− mice (n = 8). *p<0.05 vs. WT.

https://doi.org/10.1371/journal.pone.0024979.g003

Next, protein expression of Gαi isoforms was analyzed in cell membrane preparations from WT, Gαi2−/−, and Gαi3−/− ventricles by probing with Gαcommon antibodies subsequent to high resolution urea/SDS-PAGE-separation of proteins. WT mice expressed both Gαi2 and Gαi3 subunits with the protein level of Gαi2 being much higher than that of Gαi3 (Fig. 3C), in line with previous studies (e.g. [22]). As expected, in hearts from Gαi2−/− mice Gαi3 was found and Gαi2 was absent while in hearts from Gαi3−/− mice, only Gαi2 was detectable (Fig. 3C). The specificity of the detected Gαi2 and Gαi3 protein bands in Gαi2−/−, Gαi3−/− and WT cardiac tissue was confirmed by analyzing the expression of Gαi isoforms by PTX-mediated [32P]ADP ribosylation [23] (data not shown). These experiments prove that gene deletion indeed led to loss of the Gα subunit. Statistical analysis of relative expression levels obtained from immunoblots (Fig. 3D) show that Gαi2 protein is significantly upregulated in ventricles obtained from Gαi3−/− mice (to 131±10%, n = 8, p<0.05 vs. WT). However, Gαi3 protein is much more markedly upregulated upon Gαi2 deficiency (to 567±59%, n = 8, p<0.05 vs. WT). Taken together, knockout models examined in this study feature a protein upregulation of the respective other Gαi isoform. These data, together with quantitative mRNA data reported above, suggest that increased levels of Gαi2 or Gαi3 may partially compensate for the loss of the deleted other Gαi isoform. Hence, any effect seen so far could be caused by the loss of one Gαi protein and/or compensatory signalling exerted by the other. Unfortunately, double-deficient (Gαi2−/−/Gαi3−/−) mice are not viable [22] and hence cannot be used to directly address this question. However, all Gi proteins can acutely be inactivated by PTX treatment.

Acute Gi inactivation induces L-VDCC kinetic alterations in Gαi3−/− cardiomyocytes

We performed experiments with PTX for acute inactivation of Gi/o proteins in cardiomyocytes of WT and Gαi-deficient mice. Freshly isolated cardiomyocytes were incubated at 37°C with or without PTX for 3 hours and afterwards maintained and examined at room temperature. It has been shown that this protocol completely ablates Gi-mediated signalling, e.g. when triggered by adenosine receptors [24]. Of note, the 3 hours incubation phase – even in the absence of PTX – reduced current density in Gαi3-deficient cells (Fig. 4A). Notably, similar to WT cells, PTX treatment in Gαi3−/− myocytes induced a shift of V0.5 for steady state inactivation to more negative potentials (to −22.7±1.1 mV, n = 11; Fig. 4B). Likewise, the recovery from inactivation in Gαi3−/− cardiomyocytes was slowed by PTX treatment (to τ: 350±27 ms, n = 8 p<0.05 vs. WT; Fig. 4C). In contrast, the calcium current phenotype in Gαi2−/− cells was preserved with time and resistant to treatment with PTX. To summarize, PTX alters channel regulation in cells from Gαi3−/− but not from Gαi2−/− mice. In Gαi3−/−, acute PTX treatment – and thus inactivation of Gαi2−/− – mimics the kinetic changes induced by chronic gene deletion of Gαi2−/−. In contrast, the effect of Gαi3 gene deletion appears to be transient and does not interfere with channel kinetics. Because some of the calcium current changes reported here may be caused by (long-term) structural alterations rather than (acute) functional modulation of calcium channels, we next examined channel composition.

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Figure 4. Effects of acute inactivation of Gi/o proteins by PTX incubation of isolated cardiac myocytes.

(A) Effects of PTX on peak L-VDCC current density. PTX treatment by itself did not affect calcium current density. (B) Effects of PTX on steady-state inactivation, as gauged by the midpoint voltage V0.5 of a Boltzmann function. No change is seen after 3 hours of drug-free incubation compared to 0 hour. PTX leads to a significant leftward shift of V0.5 in WT (from −19.2±0.7 mV to −21.0±0.7 mV, n = 7–18) and Gαi3−/− (from −18.2±0.7 mV to −22.7±1.1 mV, n = 11–13). (C) PTX affects the recovery of the L-VDCC from inactivation. PTX inhibits the channel recovery in Gαi3−/− (τ from 189±12 ms to 350±26 ms, n = 5–11). *p<0.05 vs. WT, p<0.05 vs. Gαi3−/−, p<0.05 vs. 3 h without PTX.

https://doi.org/10.1371/journal.pone.0024979.g004

No significant structural modification of L-VDCC

To obtain insight into the effects of specific and constitutive Gαi-deficiency on L-VDCC structure and expression, we determined RNA expression levels of the pore forming Cavα1 subunit and the predominant murine cardiac Cavβ subunit, Cavβ2 [14], [16], [25], which is involved in calcium channel trafficking and gating [26], [27], [28]. The Cavβ2 subunit mRNA expression in ventricles from Gαi2−/− and Gαi3−/− animals is not altered compared to WT expression profile (Fig. 5B). In Gαi2−/− and Gαi3−/− ventricles, the pore forming Cavα1 subunit mRNA (Fig. 5B) and membrane protein (Fig. 5C and D) expression levels exhibit only slight and insignificant changes. Thus, these data do not suffice to explain the Gαi isoform-specific regulation of the current density. Therefore, we next switched to the posttranslational level and addressed signalling pathways that are involved in Gi protein-mediated action.

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Figure 5. Calcium channel subunit expression.

(A) 2−ΔCT values were calculated against GAPDH expression to analyze the relation of L-type calcium channel subunits in WT cardiomyocytes (n = 4). (B) No significant changes in the relative mean expression (2−ΔΔCT) were observed in ventricular tissues from Gαi2−/− (n = 3) and Gαi3−/− mice (n = 4). (C) Representative example of Cavα1 protein expression in murine ventricular tissue from WT, Gαi2−/− and Gαi3−/− mice. (D) Quantification of Cavα1 protein levels show no change in ventricles from Gαi3−/−, Gαi2−/− and WT mice (n = 3).

https://doi.org/10.1371/journal.pone.0024979.g005

Akt activation is unaltered by deletion of Gαi2 or Gαi3

Cardiac Gαi is known to activate the PI3-kinase Akt/PKB pathway [29], and Akt-mediated β-subunit phosphorylation prevents Cavα1 degradation [30]. A Gi-isoform-specific regulation of Akt could explain the calcium current increase in the case of Gαi3−/− and decrease in case of Gαi2−/−. To examine the functional significance of Gαi-dependent activation of Akt in vivo, animals were treated with either saline or the muscarinic receptor agonist carbachol (CCh, 0.5 mg/kg body weight) by i.p. injection; 15 min later animals were killed, and signalling activity was assessed in heart preparations. Phosphorylation of Akt and its downstream effector glycogen synthase kinase-3α/β (GSK3α/β), were increased in cardiac tissue from all mouse strains after treatment with CCh (Fig. 6A). Although the basal phosphorylation of Akt in Gαi2−/− and Gαi3−/− cardiac tissue was slightly increased compared to WT (to 138±44% and 143±58% of WT, respectively), we could not observe statistically significant differences between CCh stimulated WT (233±14% to basal), Gαi2−/− (201±27%) and Gαi3−/− (195±25%) mice (each n = 3). Importantly, the total amount of Akt was not changed in all mice models. Thus, Gi-subtype specific channel regulation seems to be independent of Akt phosphorylation in the investigated knockout models.

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Figure 6. Signalling events downstream of Gαi stimulation.

Representative western blot of cardiac tissues of animals treated either with saline or CCh (0.5 mg/kg body weight) for 15 min. (A) Western Blot and densitometry analyses of ventricular homogenates show that Akt phosphorylation was significantly and to a similar extent increased after CCh stimulation in all genotypes (n = 3). Total amount of Akt protein was unaltered and was used as loading control. (B) Western blot showing ERK1/2 phosphorylation and total ERK1/2 expression and bar graphs of combined results expressed as increase in pERK1/2 normalized to total ERK1/2 and compared to saline treated WT mice. The increase of ERK1/2 activation in CCh treated cells was markedly inhibited by Gαi2 gene deletion compared to WT and Gαi3−/− mice (n = 3). *p<0.05 vs. WT NaCl.

https://doi.org/10.1371/journal.pone.0024979.g006

Lack of Gαi2 protein abolishes ERK1/2 activation

Recently, a marked increase of L-type calcium channel density that involved PKC-dependent activation of the ERK1/2 pathway was reported [31]. To determine its involvement, we measured the activation of ERK1/2 protein in total cardiac tissue. Gαi2−/− mice demonstrated a significantly blunted increase of ERK1/2 phosphorylation in CCh-stimulated animals (128±5% to basal, n = 3) compared to WT (268±6%, n = 3) and Gαi3−/− (192±20%, n = 3) cardiomyocytes. Of note, in Gαi2−/− and Gαi3−/− cardiac tissues, the basal ERK1/2 phosphorylation levels were increased compared to WT basal phosphorylation (Fig. 6B), while the total amount of ERK1/2 in the heart was the same in all mouse models tested. These results indicate a Gαi2-dependent ERK1/2 phosphorylation and strongly suggest that ERK1/2 plays an important role in isoform-specific Gαi protein signalling.

Discussion

The two inhibitory G protein isoforms Gi2 and Gi3 are both upregulated in heart failure [6], [7], [8]. One functionally important target of Gi protein signalling is the L-VDCC, the crucial trigger of cardiac excitation-contraction coupling. Gi-protein-mediated inhibition of L-VDCC has been demonstrated for β2-adrenergic [4] and muscarinic [20] receptor signalling. In this context, we previously provided single-channel evidence that Gαi2 does not confer the L-VDCC inhibition observed in mice with chronic overexpression of the β2-adrenergic receptor [18]. On the other hand, cardiac Gαi2 (but not Gαi3) seems necessary and sufficient to mediate the muscarinic receptor-mediated L-VDCC inhibition [20], presumably through the classical adenylyl cyclase pathway. So far, no isoform-specific function could be assigned to cardiac Gαi3; however, Gi3 has been shown to be an exclusive and specific regulator of autophagy in the liver [22], [32].

The different behaviour of L-VDCC currents obtained with isolated myocytes from Gαi2−/− and Gαi3−/− mice shown here and previously [18] demonstrates contrasting functional roles of these two Gαi isoforms. Although the issue has also been addressed by others [19], [20], [33], our study is the first to demonstrate small but significant changes of basal whole-cell current density: a reduction in myocytes from Gαi2−/− mice and an increase in myocytes from Gαi3−/− mice. The altered steady-state inactivation and recovery observed with Gαi2−/− under basal conditions (Fig. 1C and D), and with Gαi3−/− myocytes after PTX treatment (Fig. 4B and C), point to a modulation of gating properties specific to Gαi2. In contrast to data presented here, Nagata et al. [20] did not detect a significant difference in L-VDCC activity between myocytes from WT, Gαi2−/− and Gαi3−/− mice. With respect to Gαi2−/−, this finding can be explained by the different prepulse potentials: the prepulse voltages (−50 mV used by Nagata and co-workers vs. −40 mV in our case) - intended to inactivate primarily sodium currents - lie within the descending part of steady-state inactivation (Fig. 1C). This likely translates into the more reduced peak in the current voltage plot in our study. Interestingly, Zuberi et al. [19] compared Gαi2−/− mice with Gαi1−/−/Gαi3−/− double knockout mice and found distinct effects on surface ECGs: in Gαi2−/− animals (but not in the double knockout mice), the effective refractory period was reduced and ventricular arrhythmias were induced more easily. In summary, Gαi2 protein deletion showed dramatic consequences on channel regulation in vivo and ex vivo.

There are no changes of cardiac L-VDCC composition regarding the main cardiac L-VDCC subunits, Cavα1 and Cavβ2 (Fig. 5B and D) that would explain the obtained effects on current density. Furthermore, because of the compensatory upregulation of the remaining Gαi isoform in case of either Gαi2 or Gαi3 deficiency (Fig. 3B and D), it is difficult to attribute the observed changes in L-VDCC regulation/function to the higher expression of one Gαi isoform or to the loss of the other or to both. Therefore, given the novel functional effects reported here for cardiac Gαi2 and Gαi3, we have to consider a number of molecular pathways. For instance, activation of Stim1 [34] may lead to altered L-VDCC function and subcellular distribution. Enhanced endocytosis and degradation of calcium channels can also be mediated by activation of PIKfyve [35] and subsequent Cavα1 targeting to lysosomes. Further, the RGK proteins Rad and Rem expressed in the heart [36] are appealing candidates, because they negatively regulate both membrane expression and gating of L-VDCC [37], [38] while little is known about how these small GTP-binding proteins are regulated themselves [39]. In our present study, we focused on two other important molecular pathways since they are significantly regulated by Gi-signalling. First, PI3-kinase Akt/PKB signalling is known to be activated by cardiac Gαi [29], and Catalucci et al. [30] revealed a mechanism through which the PI3-kinase Akt/PKB pathway modulates Ca2+ entry in cardiac cells via L-VDCC. Our data showed a CCh-induced Akt phosphorylation independent of the deletion of either Gαi isoform (Fig. 6A). Second, it was demonstrated that deactivation of Gi leads to a significant reduction in ERK1/2 phosphorylation and that this effect was Gs- and Gq- independent [29], [40]. In the present study, we have shown that Gαi2 deletion prevents phosphorylation of ERK1/2. Recently, Smani et al. [31] found a leftward shift and a marked increase in L-VDCC density induced by urocortin, which involved PKC-dependent activation of the MAPK-ERK1/2 pathway. Based on these findings we propose that loss of activation of L-VDCC by ERK1/2 might be a mechanism involved in functional regulation of calcium current in Gαi2 deficient mice.

A change of Gαs mediated signalling might account for altered calcium currents when a Gαi is lacking. With this caveat in mind, we demonstrated in our previous study that Gαs protein expression remained unaffected in hearts from mice deficient in the major isoform Gαi2 [18]. Yet, possible changes in associated proteins like Gβγ subunits might indirectly affect Gα mediated signalling [41]. Indeed, we observed that expression of Gβ1/2 was slightly reduced in Gαi2−/−, but not Gαi3−/− (data not shown). In any case, our single-channel analysis (in particular, the decrease in open time) does not support the idea of enhanced Gαs mediated, cAMP-mediated signalling in Gαi3-deficient hearts (Table 2).

The data presented here could not elucidate all effects seen in the knockout animals. Thus, eventually, Gi3's role in L-VDCC regulation remains unclear, mainly in light of the absence of acute PTX effects in Gαi2−/− mice. However, due to the effects seen by incubation without PTX, our findings suggest that the increment in channel activity observed in the absence of Gαi3 might be driven by an in vivo mechanism, which is not preserved ex vivo (Fig. 4A). It has also to be pointed out that the immunoblot data reveal total cardiac membrane channel protein levels, which does not necessarily match up the fraction of functional channels located in the sacrolemma. Given our currently available methods to analyze the subcellular localization of calcium channels and their regulation, all of these ideas require further work, which for technical reasons has to be done in recombinant systems.

Taken together, our data reported here and in a previous paper [18] point to the (patho-) physiological importance of subtype-specific Gi protein signalling in the heart. In particular, in terminal heart failure, Gαi2 upregulation now appears as an attractive mechanism linked to remodelling of L-VDCC [13], [14], [15], [16]. Therefore, the present study provides new insights into potential mechanisms linking modulation of L-VDCC to the inhibitory G protein isoform Gi2 in cardiomyocytes, and highlights Gi2-specific signalling via ERK1/2. Further research needs to focus on detailed signalling pathways involving ERK1/2.

Methods

Ethic statement

Animal breeding, maintenance and experiments were approved by the responsible federal state authority (Landesamt für Natur-, Umwelt- und Verbraucherschutz Nordrhein-Westfalen; reference No. K 27, 24/04 and 8.87-51.05.20.09.232) and the responsible local authority (Umwelt- und Verbraucherschutzamt der Stadt Köln; reference No. 576/1.36.6.3.-47/08 Be). All animal experiments conform with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Animals

Generation, breeding and characterization of Gαi2- or Gαi3- deficient mice have been described previously [18], [22], [42], [43], [44], [45]. All Gαi-deficient mouse strains used were backcrossed onto a C57Bl6 background for >10 generations. Knockout and WT control mice were maintained at the animal facilities of the Heinrich-Heine-University, Düsseldorf, and of the Department of Pharmacology at the University of Cologne. Mice analyzed in this study were of both sexes, 3–9 months of age and weighted 20–35 g.

Genotyping

Tail-clip analysis was performed on 3–4 weeks old mice. Genomic DNA was prepared and genotyping PCR for Gαi2 and Gαi3 was performed as described previously [18], [43].

Real time PCR

Primer for Gαi1, Gαi2, Gαi3 isoforms and Cavα1 subunit were described previously [25], [46]. Specific primers for the Gαo and Cavβ2 subunit were designed using Primer Express Software v3.0 (Applied Biosystems, Foster City, USA). For Gαo: 5′-TGGCATCGTAGAAACCCACTT-3′ (sense) and 5′-CGACGTCAAACAGCCTGAAG-3′ (antisense) and for Cavβ2: 5′-GGGAGGCAGTACGTAGAGAAGCT-3′ (sense) and 5′-TGCAAATGCAACAGGTTTT GTC-3′ (antisense). Total cellular RNA was extracted from murine heart (ventricle) according to the manufacturer's protocol (Qiagen QIAshredder and RNeasy Mini Kit, Qiagen, Hilden, Germany). For qualitative analysis of RNA integrity, 2 µg of total RNA was separated on a 1% formaldehyde agarose gel. Total RNA was subsequently converted into cDNA by ImProm-II Reverse Transcription Kit (Promega, Mannheim, Germany). Real-time PCR was carried out using the 7500 Real-Time PCR system (Applied Biosystems) under standard conditions with 200 pM PCR primers. Each sample was analyzed in triplets using SYBR green (Applied Biosystems) as fluorescent detector and GAPDH as endogenous control.

Cell membrane preparation

Murine cardiac ventricles were disrupted and homogenised in a lysis buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.5 mM DTT, and an EDTA-free protease inhibitor cocktail (Roche, Penzberg, Germany) using an ultra-turrax blender. Cellular membranes were isolated by two steps of centrifugation at 450 g and 30.000 g. Membrane pellets were subsequently dissolved in a buffer consisting of a freezing supplement (70 mM Tris (pH 7.4), 12 mM MgCl2, 60% Glycerol, 240 µg/µl DNAse) and the lysis buffer in a ratio of 1∶6 in order to stabilize membrane-associated proteins.

Phosphorylation assay

Animals were injected i.p. with 0.5 mg/kg carbachol (CCh) diluted in 0.9% normal saline. Sham injections were performed with 400 µl 0.9% saline per 30 g weight. After 15 min ventricular tissue was harvested and lysed in buffer (50 mM HEPES, 1% Triton, 50 mM NaCl, 10 mM Na3VO4, 0.1% SDS, 0.1 M NaF, 10 mM EDTA, complete mini protease inhibitor, pH 7.4) and left for 30 min at 4°C. Total cell lysates were extracted from the supernatant by centrifugation at 13.500 g for 20 min.

Immunoblotting

i and Cavα1 proteins isolated from cell membranes were separated on 6 M urea/9% SDS-PAGE gels (protein content per lane was 90 µg) and on 8% SDS-PAGE gels (protein content per lane was 100 µg), respectively [22]. Protein kinase B/Akt and ERK1/2 in total cell lysates were separated on 10% SDS-PAGE gels (100 µg protein content per lane). Separated proteins were blotted onto nitrocellulose membranes (Hybond C extra; Amersham Bioscience). Gαi proteins were detected with an anti-Gαcommon antibody [45] (1∶1000) and Cavα1 with an anti-Cavα1 antibody (1∶200; Sigma Aldrich). For detection of phosphorylation, membranes were incubated with phospho-Akt (Ser473), phospho-GSK3α/β (Ser21/9) and phospho-ERK1/2 (Thr202/Tyr204) antibodies. Membranes were reprobed with Akt and ERK1/2 antibodies (each 1∶1000; Cell Signalling) after stripping. Emitted light of stained membranes were captured on films and developed in different expositions. Protein densities were calculated using Aida Image Analyzer (Raytest, Straubenhardt, Germany) software. The Ras-GTP-activating protein (RasGAP; [47]) was used as a loading control for Cavα1. Equal loading on blotting membrane for Gαi proteins was controlled by a non-specific protein staining using Ponceau S. Only blots with equal loading were analyzed. To confirm Gαi band specificity, we performed ADP ribosylation of PTX-sensitive G proteins as described [23].

Cardiomyocyte isolation

Single ventricular myocytes were isolated from hearts of 3–9 months old mice by enzymatic dissociation using a method described previously [48]. Only rod shaped cardiomyocytes were used for the experiments. Cells were maintained at room temperature and subjected to patch-clamp analysis. If indicated, a fraction of isolated ventricular myocytes was incubated without or with 1.5 µg/ml of PTX (Sigma Aldrich, St. Louis, USA) for 3 hours at 37°C [24].

Single-channel measurements

Single-channel patch clamp recordings were done in the cell-attached configuration as reported [18]. The composition of bath solution was (mM): K-glutamate 120, KCl 25, MgCl2 2, HEPES 10, EGTA 2, CaCl2 1, Na2-ATP 1, glucose 10 (pH 7.4 with KOH). Patch pipettes (7–9 MΩ) contained (mM): BaCl2 70, HEPES 10, sucrose 110 (pH 7.4 with TEA-OH). Barium currents were recorded at room temperature using a holding potential of −100 mV and depolarizing test pulses to +20 mV (duration 150 ms, frequency 1.66 Hz, 180 sweeps per experiment minimum). Data were sampled at 10 kHz and filtered at 2 kHz using an Axopatch 200A amplifier (Axon Instruments, Sunnyvale, CA, U.S.A.). Only experiments with one single active channel in the patch were analyzed (identified by the lack of stacked openings).

Whole-cell current measurements

Conventional whole-cell patch clamp recordings were performed with cells maintained at room temperature in bath solution containing (mM): NaCl 137, CsCl 5.4, CaCl2 2, MgCl2 1, glucose 10, HEPES 10 (pH 7.4 with NaOH). Pipette (2–3 MΩ) solution was composed of (mM): CsCl 120, MgCl2 1, Mg-ATP 4, EGTA 10, HEPES 5 (pH 7.2 with CsOH). Giga-Ohm seals (resistance 2–5 GΩ) were formed by gentle suction. At the beginning of each experiment, membrane capacitance was measured by means of fast depolarizing ramp pulses from −80 to −85 mV over 25 ms. Cells were depolarized from a holding potential of −80 mV to a 50 ms prepulse to −40 mV in order to inactivate sodium channels. This was followed by test pulse voltages ranging from −40 to +50 mV in 10 mV steps (pulse duration 150 ms). For time-dependent inactivation, the declining raw currents at −10, 0, +10, +20 and +30 mV were fitted by a double-exponential function, yielding fast and slow time constant. For investigation of gating kinetics, standard two-pulse protocols were used: the voltage-dependent inactivation was measured after a prepulse of variable amplitude and 250 ms duration, followed by a test pulse of fixed amplitude for 50 ms. The midpoint voltage V0.5 was determined by fitting a Boltzmann function to the data. Recovery from inactivation was determined by two 200 ms depolarizing voltage pulses to 0 mV. The interpulse interval at a holding potential of −45 mV was increased from 50 to 375 ms in 25 ms steps. Recovery was fitted by a mono-exponential function, yielding the recovery time constant τ.

Statistical analysis

Data are presented as means ± SEM. Patch-clamp data were analyzed using pClamp software (CLAMPEX 6 and FETCHAN, Axon Instruments). Analysis of L-VDCC kinetics was performed as described previously [49] using GraphPad Prism 4. A one-sample t-test was used to analyze normalized data. Differences between genotypes were analyzed by one-way ANOVA followed by Dunnett's or Tukey's post test, and between treatments within one given genotype by two-tailed Student's t tests. For electrophysiological statistics, number of cardiomyocytes from a minimum of 3 animals, and for molecular biology statistics, number of animals was evaluated. P values<0.05 were considered statistically significant.

Acknowledgments

The authors wish to thank Olga Felda and Sigrid Kirchmann-Hecht for excellent technical assistance, Jens Reifenrath for help with animal breeding, and Christian Fabisch, Andreas Markl, Katja Pexa, Markus Schubert and Oliver Stöhr for discussion and support.

Author Contributions

Conceived and designed the experiments: SD SK RPP BN SH. Performed the experiments: SD SK FK CK. Analyzed the data: SD SK FK PH CK RPP J. Matthes J. Meszaros SH. Wrote the paper: SD PH J. Matthes RPP BN SH. Critical revisions of the manuscript: SD PH J. Matthes RPP BN SH.

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