Lack of Gα_i2 leads to dilative cardiomyopathy and increased mortality in β₁-adrenoceptor overexpressing mice

Abstract

Aims

Inhibitory G (G_i) proteins have been proposed to be cardioprotective. We investigated effects of Gα_i2 knockout on cardiac function and survival in a murine heart failure model of cardiac β₁-adrenoceptor overexpression.

Methods and results

β₁-transgenic mice lacking Gα_i2 (β₁-tg/Gα_i2^−/−) were compared with wild-type mice and littermates either overexpressing cardiac β₁-adrenoceptors (β₁-tg) or lacking Gα_i2 (Gα_i2^−/−). At 300 days, mortality of mice only lacking Gα_i2 was already higher compared with wild-type or β₁-tg, but similar to β₁-tg/Gα_i2^−/−, mice. Beyond 300 days, mortality of β₁-tg/Gα_i2^−/− mice was enhanced compared with all other genotypes (mean survival time: 363 ± 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, β₁-tg, and Gα_i2^−/− mice, but significant impairment for β₁-tg/Gα_i2^−/− mice (e.g. ejection fraction 14 ± 2 vs. 40 ± 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 ± 0.06 vs. 0.48 ± 0.02% in wild-type mice), left ventricular size (length 0.82 ± 0.04 vs. 0.66 ± 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. Gα_i3 was significantly up-regulated in Gα_i2 knockout mice (protein compared with wild type: 340 ± 90% in Gα_i2^−/− and 394 ± 80% in β₁-tg/Gα_i2^−/−, respectively).

Conclusions

Gα_i2 deficiency combined with cardiac β₁-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, β₁-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gα_i2. We propose an enhanced effect of increased β₁-adrenergic drive by the lack of protection via Gα_i2. Gα_i3 up-regulation was not sufficient to compensate for Gα_i2 deficiency, suggesting an isoform-specific or a concentration-dependent mechanism.

1. Introduction

Norepinephrine concentrations and sympathetic drive are increased in human heart failure.^1–3 Although this helps to maintain contractile force, blood pressure, and blood flow to vital organs, it becomes detrimental in the long run.^4,5 β₁- and β₂-adrenoceptors are the most abundant cardiac adrenoceptors with the expression of β₁-adrenoceptors exceeding that of β₂-adrenoceptors by four-fold in the normal (human) heart.⁴ β₁-adrenoceptors exclusively couple with stimulatory G proteins (G_s), whereas β₂-adrenoceptors have been shown to directly interact with both G_s and inhibitory G (G_i) proteins.^6,7 In human heart failure, expression of sarcolemmal β₁-adrenoceptors and their coupling with G_s decreases.⁸ On the other hand, an increase of ‘promiscuous’ β₂- relative to β₁-adrenoceptors and an increased expression of G_i (particularly the isoform G_i2) are observed.^8,9 This latter finding can be interpreted as an attempt to shield the heart against catecholamines since Rau et al.¹⁰ have shown that a G_i2 increase similar to that found in human heart failure is sufficient to significantly reduce adenylyl cyclase-mediated cAMP production. This and other findings support the hypothesis that signalling via G_i proteins is cardioprotective.^11–14 Of interest, overexpression of both β₁- and β₂-adrenoceptors has been shown to induce cardiac hypertrophy and heart failure in mice, respectively.^15,16 However, levels of overexpression necessary to cause heart failure seem to be higher for β₂-adrenoceptors that in contrast to β₁-adrenoceptors couple with both G_s and G_i.^15,16 Nonetheless, cardioprotective effects mediated by G_i proteins are still a matter of debate, particularly regarding the cardiac condition, e.g. normal vs. pathological. To address this issue, we investigated whether lack of the catalytic G_i2 subunit (Gα_i2) affects cardiac function and survival in the murine heart failure model of cardiac β₁-adrenoceptor overexpression compared with wild-type mice and mice either lacking Gα_i2 or overexpressing β₁. Parts of this work have already been published as a conference abstract.¹⁷

2. Methods

2.1. Mouse models used

Animals were kept in individually ventilated cages with a 12/12 h dark/light cycle and food and water ad libitum. Mice ubiquitary lacking Gα_i2 (Gα_i2^−/−) have originally been generated on a 129Sv background, but subsequently been backcrossed to C57BL/6J.^13,18,19 Mice with a cardiac-specific overexpression of the human β₁-adrenoceptor (β₁-tg) have originally been generated on a FVB/N background.¹⁵ For the current study, we backcrossed this line on a C57BL/6J background (>10 generations) to allow for mating with Gα_i2 knockout mice to generate β₁-transgenic animals deficient of Gα_i2^−/− (β₁-tg/Gα_i2^−/−). As control animals we used age-matched wild-type littermates. Animals of both sexes were used for our study. For genotyping, tail-clips from 3-week-old mice were used. Genomic DNA was prepared and genotyping PCR for Gα_i2 and the β₁-receptor was performed as described previously.^13,20 Animals were killed by cervical dislocation. Animal breeding, maintenance, and experiments were approved by the responsible federal state authority (Landesamt fuer Natur-, Umwelt- und Verbraucherschutz Nordrhein-Westfalen; reference: 87-51.04.2010.A078). All animal experiments comply with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

2.2. Survival analysis

All genotypes were monitored for a minimum period of 2 years. Kaplan–Meier survival curves were used to determine mean survival time of each mouse line. Spontaneous death was defined as the event of interest, while killing an animal was a censored event. Mice used for breeding were excluded from our analysis. Numbers of mice included in our survival analysis were 200, 34, 37, and 192 for C57BL/6 (wild type), Gα_i2^−/−, β₁-tg/Gα_i2^−/−, and β₁-tg mice, respectively. Numbers of spontaneous deaths that occurred during an observation period were 9 (C57BL/6), 10 (Gα_i2^−/−), 21 (β₁-tg/Gα_i2^−/−), and 22 (β₁-tg).

2.3. Echocardiography

At an age of 302 ± 19 days, mice (n = 5–7 of every genotype) were examined by echocardiography under light inhalation anaesthesia with oxygen and 1.5% isoflurane through a nose cap. Chests were epilated and the animals were placed on a heating table to prevent hypothermia and cardiodepressive effects. For the experiments, a commercial echocardiography system (Philips iE33 ultrasonic system, ‘Qlab Cardiac Analysis’ Software; Philips Healthcare, Hamburg, Germany) equipped with a 15 MHz linear array transducer (L15-io7) allowing frame rates of 270 Hz was used. The transducer was moved along the parasternal long and short axis of the left ventricle, and loops of 3 s duration were recorded in one-dimensional (M-mode) and two-dimensional planes. To monitor the heart rate of the animals and thus anaesthesia during measurements, an ECG was derived. For reconstructive three-dimensional echocardiography, multiple short-axis slices were recorded every 500 µm using a millimetre screw-tripod.^21,22

2.4. Ventricle-to-body weight ratio

Before killing a mouse, its body weight was measured. For determining ventricular weight, hearts were excised immediately after killing by cervical dislocation, atria were cut, and intraventricular blood removed. We analysed 11, 8, 7, and 14 hearts of C57BL/6 (wild-type), Gα_i2^−/−, β₁-tg/Gα_i2^−/−, and β₁-tg mice, respectively, including those from mice examined by echocardiography.

2.5. Quantitative real-time PCR

For quantitative real-time PCR (qPCR), we used ventricles that were stored at −80°C immediately after excision. qPCR analysis was performed to determine relative ventricular mRNA expression levels of the cardiomyopathy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), the G_i proteins Gα_i2 and Gα_i3, and the cardiac protein kinase A (PKA) targets ryanodine receptor 2 (RYR2), troponin I (TnI, TNNI3), and phospholamban (PLB). All steps of analysis were performed following the manufacturer's protocol by QIAGEN (Hilden, Germany). mRNA isolation was performed with the RNeasy^® Fibrous Tissue Kit (QIAGEN). Quality and quantity of the purified mRNA were controlled using a NanoDrop 8000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). For reverse transcription, the QuantiTect^® Reverse Transcription Kit was used (QIAGEN). qPCR was run in triple repeats with the QuantiTect SYBR^® Green PCR Kit (QIAGEN). Specific primer pairs for Gα_i2, BNP, RYR2, TNNI3, and PLB were designed using Roche Assay Design Center: Gα_i2: 5′-AAG ACC TGT CCG GTG TCA T-3′ for sense and 5′-GGG ATG TAG TCA CTC TGT GC-3′ for antisense. BNP: 5′-GTC AGT CGT TTG GGC TGT AAC-3′ for sense and 5′-AGA CCC AGG CAG AGT CAG AA-3′ for antisense. RYR2: 5′-TTC ACA CCT GTT CCT GTG GA-3′ for sense and 5′-TTT CTC TTA TCC TTT CCA GGT GA-3′ for antisense. TNNI3: 5′-GAG CCA CAC GCC AAG AAA-3′ for sense and 5′-GCC CCT TCT CTC CAC GTC-3′ for antisense. PLB: 5′-CTG TGA CGA TCA CCG AAG C-3′ for sense and 5′-TGG TCA AGA GAA AGA TAA AAA GTT GA-3′ for antisense. Primer pairs for Gα_i3 and ANP were reported previously.^23–25 S29 served as a housekeeping gene. The qPCR was started with an initial step of incubation at 95°C for 15 min. Next, 45 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 25 s, and elongation at 72°C for 10 s were run with a transition rate of 20°C per second. Finally, a melting curve analysis was applied to check for product purity at 64°C for 1 min with a transition rate of 0.1°C per second.

2.6. Western blot analysis

Ventricles from three different animals per genotype were isolated, separately homogenized, and individually analysed. Tissue was homogenized in protein lysis buffer containing 21 mM Tris–HCl, pH 8.3, 0.67% SDS, 238 mM β-mercapoethanol, and 0.2 mM PMSF. Gα protein isoform separation was performed in gels containing 6 M urea. In total, 20 µg per lysate was loaded. To verify Gα_i3 antibody specificity, ventricle lysates isolated from Gα_i3-deficient mice were loaded.²⁶ The proteins were visualized by immunodetection using the following primary antibodies described elsewhere:^27–30 rabbit anti-Gα_i2 (1 : 8000) and rabbit anti-Gα_i3 (1 : 5000). Equal loading was verified with antibodies against rabbit anti-β-actin (1 : 10 000). The protein levels of Gα_i2 and Gα_i3 were quantified using the densitometric analysis software (Image Lab; Bio-Rad, Gräfelfing, Germany) and were normalized to the β-actin levels of the same samples. For each ventricle, immune blots were run in triplicates or quadruplicates. Homogenates of the four different genotypes were always loaded on the same gel and analysed in parallel.

2.7. Radioligand-binding experiments

Hearts from 4- to 6-month-old mice of both sexes were isolated in ice-cold phosphate buffer saline, frozen in liquid nitrogen, and stored at −80°C until membrane preparation. Ventricular tissue was homogenized in a 0.32 M sucrose solution and centrifuged for 11 min at 300 × g. Supernatants were centrifuged for 41 min at 80 000 × g and pellets resuspended in aqua destillata. All preparation steps were carried out at 4°C. Finally, aliquots of 0.5 mL were frozen in liquid nitrogen and stored at −80°C. Protein content was determined according to Lowry and ranged from 5.4 to 7.7 mg/mL. For saturation-binding experiments, membranes were incubated in Tris–HCl buffer (Tris 50 mM, pH 7.4; EDTA 5 mM) of a final volume of 1.5 mL at 23°C containing 50–300 μg protein. Eight concentrations (0.025–1.5 nM) of [³H]CGP 12177 (specific activity: 37.7 Ci/mmol; PerkinElmer, Rodgau, Germany) were used to quantify β-adrenoceptor expression. Non-specific binding was measured in the presence of 10 µM propranolol or atropine (Sigma-Aldrich, Steinheim, Germany). Incubation was stopped after 90 min by rapid filtration through polyethylenimine (0.1%)-pretreated glass microfiber sheets using a Brandel cell harvester. Filter-bound radioactivity was detected by liquid scintillation counting. Fraction of β₂-adrenoceptor binding of [³H]CGP 12177 was estimated in the presence of 70 nM ICI 118,551 (Sigma-Aldrich). Quality of the used homogenates was similar, as confirmed by muscarinic receptor levels determined with six concentrations (0.5–1.5 nM) of [³H]N-methylscopolamine (specific activity: 84.1 Ci/mmol; PerkinElmer). Binding curves were analysed by non-linear curve fitting using the software GraphPad Prism^®. Data points were fitted by a one-site-specific binding model.

2.8. Statistical analysis

Data are given as mean ± S.E.M. Survival times were calculated by Kaplan–Meier estimation. Differences were determined by the log-rank test. Cardiac functional parameters obtained from echocardiography and ventricle-to-body weight ratios were compared by ANOVA followed by post hoc tests (Bonferroni). mRNA expression ratios using C_t values obtained by qPCR were compared using the REST-2009^® software.³¹ Distribution between sexes was compared by Fisher's exact test. P-values <0.05 were defined to indicate statistically significant differences.

3. Results

3.1. Gross phenotype

Genotypes were distributed as expectable according to the Mendelian law (not shown). Overall distribution between sexes was nearly equal (51% male and 49% female) and did not differ between genotypes (not shown). All mice behaved normally and no overt phenotype was observed. Deaths occurred suddenly and were only in single cases preceded by unspecific symptoms like reduced movement or seclusive behaviour. Gastrointestinal symptoms as described previously for mice lacking Gα_i2 were not seen in our study, presumably due to keeping the mice in individually ventilated cages.³²

3.2. Survival analysis

Kaplan–Meyer estimation revealed a significantly enhanced mortality of β₁-transgenic mice deficient of Gα_i2^−/− (β₁-tg/Gα_i2^−/−) compared with all other genotypes (Figure 1). Mean survival time was reduced to 363 ± 21 days (22 deaths out of 37 mice in total) compared with 669 ± 31 for wild-type mice (9/200), 489 ± 37 for Gα_i2^−/− (10/34), and 561 ± 20 for β₁-tg mice (21/192). Though survival time of mice only lacking Gα_i2 was significantly reduced, too, this effect was significantly more dramatic in β₁-tg/Gα_i2^−/− mice (P = 0.02 in a log-rank test compared with Gα_i2^−/−). Of note, survival curves of β₁-tg/Gα_i2^−/− and mice only lacking Gα_i2 were indistinguishable up to an age of ∼300 days. Similar to recent findings by another group, survival curves of wild-type and β₁-tg mice started to differ around an age of 13 months,³³ though in our study the difference in mean survival times derived from Kaplan–Meier analysis did not reach statistical significance. Fifty per cent of deaths in the β₁-tg/Gα_i2^−/− group already occurred up to an age of 300 days. Since survival curves thus indicated a rather early onset of detrimental effects, we defined 300 days as the target age for further experiments. Moreover, this allowed for a sufficient number of animals available for in vivo examination. Of note, the choice of an age of 300 days excluded a considerable number of β₁-tg/Gα_i2^−/− (and to a lesser extent Gα_i2^−/−) animals from further analysis due to early death. This might have biased the obtained data, e.g. if animals were still living at Day 300 because they have been less affected than those that had already died.

Figure 1.

Kaplan–Meier survival curves of wild-type, knockout, and transgenic mice. Vertical bars indicate a censoring event (usually representing killing of an animal for experimental purposes). Mean survival times were significantly decreased for Gα_i2^−/− and β₁-tg/Gα_i2^−/− mice, each compared with all other genotypes. However, compared with Gα_i2^−/− mice, survival time was significantly lower in β₁-tg/Gα_i2^−/− mice (log-rank test). The number of animals underlying survival analysis was 200, 34, 37, and 192 for wild-type (C57BL/6), Gα_i2^−/−, β₁-tg/Gα_i2^−/−, and β₁-tg mice, respectively. Vertical dashed lines label age range of mice used for further investigations (Figures 2, 3, 5–7).

3.3. Echocardiographic analysis of cardiac morphology and function

At a mean age of 300 days (range: 273–326), the ventricle-to-body weight ratio of β₁-tg/Gα_i2^−/− mice was significantly increased (0.71 ± 0.06%, n = 5) compared with wild-type mice (0.48 ± 0.02%, n = 11), Gα_i2^−/− (0.50 ± 0.01%, n = 8), and β₁-tg mice (0.44 ± 0.01%, n = 14), thus indicating cardiac hypertrophy in β₁-transgenic mice lacking Gα_i2 (Figure 2). This was confirmed in echocardiographic examinations of anaesthetized mice of the same age. Ventricles of β₁-tg/Gα_i2^−/− mice appeared to be clearly enlarged (see Supplementary material online, Figure S1). End-systolic and end-diastolic ventricular volumes were similar when comparing wild-type, Gα_i2^−/−, and β₁-tg mice, but significantly enhanced in β₁-tg/Gα_i2^−/− mice compared with all other genotypes (Figure 3A and B). Furthermore, ejection fraction was significantly reduced in β₁-tg/Gα_i2^−/− mice (Figure 3C). Significantly increased ventricular length and reduced myocardial thickness in β₁-tg/Gα_i2^−/− mice indicated a dilative cardiomyopathy (data not shown; compare Supplementary material online, Figure S1).

Figure 2.

Ventricle-to-body weight ratios of knockout and transgenic mice indicate a significant cardiac hypertrophy of β₁-tg/Gα_i2^−/− mice. The number of animals used for analysis was 11, 8, 5, and 14 for wild-type (C57BL/6), Gα_i2^−/−, β₁-tg/Gα_i2^−/−, and β₁-tg mice, respectively. Age range of examined mice was 301 ± 3 days. Asterisks indicate a significant difference compared with all other genotypes in Bonferroni post-tests following ANOVA (P < 0.01).

Figure 3.

Morphological and functional parameters obtained by echocardiography. End-systolic (A) and -diastolic (B) left ventricular volumes and the ejection fraction (C) were significantly impaired in β₁-tg/Gα_i2^−/− mice compared with all other genotypes indicating overt heart failure. Heart rate (D) was similar for all investigated genotypes. The number of animals used for analysis was 5, 6, 6, and 7 for wild-type (C57BL/6), Gα_i2^−/−, β₁-tg/Gα_i2^−/−, and β₁-tg mice, respectively. Age range of examined mice was 307 ± 3 days. Asterisks indicate significant differences compared with all other genotypes in Bonferroni post-tests following ANOVA (*P < 0.05, **P < 0.01).

3.4. Expression of β-adrenoceptors and comparison to β-transgenic mice on an FVB/N background

Using saturation radioligand-binding experiments (Figure 4), we confirmed significant overexpression of β-adrenoceptors in 4- to 6-month-old β₁-tg mice (B_max 609 ± 18 vs. 8.4 ± 1.6 fmol/mg in C57BL/6 wild-type mice). In wild-type ventricles, ∼80% of radioligand binding was due to β₁-adrenoceptors, while in transgenic mice virtually all detected receptors were β₁-adrenoceptors (data not shown). Of note, on the original FVB/N background, we found the overexpression level to be more than two-fold higher (B_max: 1425 ± 68 fmol/mg) than on a C57BL/6 background. Furthermore, in FVB/N-based β₁-transgenic mice, mean survival time was significantly reduced (402 ± 15 days). Similar to (C57BL/6-based) β₁-tg/Gα_i2^−/− mice, ventricle-to-body weight ratio was increased (0.62 ± 0.03 vs. 0.52 ± 0.02% in FVB/N wild-type mice; P < 0.05; n = 10 and 7, respectively) and cardiac function impaired (e.g. ejection fraction: 21 ± 1 vs. 41 ± 2% in FVB/N wild-type mice; P < 0.01; n = 5 and 6, respectively) at a mean age of 307 ± 6 days (range 276–330).

Figure 4.

Ventricular β-adrenoceptor expression obtained by radioligand binding in (A) wild-type and (B) β₁ overexpressing mice (β₁-tg) at an age of 4–6 months. In both C57BL/6- and FVB/N-based β₁-tg mice, β-adrenoceptor expression was strikingly increased compared with the corresponding wild-type. Of note, the (over-) expression level was higher in tissue from transgenic mice with an FVB/N background. Each homogenate (10–12 ventricles per genotype) was used in three independent experiments in duplicates.

3.5. Expression of G_i proteins

Expression of Gα_i2 mRNA was not detectable in Gα_i2 knockout mice (Figure 5A). We found that the lack of Gα_i2 was accompanied by an increased expression of Gα_i3 mRNA, both on a wild-type and on a β₁-transgenic background (Figure 5B). Expression of neither Gα_i2 nor Gα_i3 mRNA was significantly altered in hearts of mice being only transgenic for the β₁-adrenoceptor. Immunoblot analysis of ventricle homogenates revealed Gα_i2 protein expression in ventricles of C57BL/6 and β₁-tg mice, whereas the Gα_i2 protein was absent in Gα_i2^−/− and β₁-tg/Gα_i2^−/− ventricles (Figure 5C and E). Gα_i3 protein levels were significantly up-regulated in Gα_i2^−/− (340 ± 90%) and β₁-tg/Gα_i2^−/− (394 ± 80%) ventricles compared with C57BL/6 and β₁-tg animals and absent in Gα_i3-deficient ventricles (Figure 5D and F).

Figure 5.

Ventricular expression of cardiac G_i. (A) Gα_i2 mRNA was not detectable in knockout mice on either wild-type or β₁-transgenic background and expressed at wild-type level in mice only transgenic for the β₁-adrenoceptor. (B) Gα_i3 mRNA expression was significantly increased compared with wild-type mice in animals lacking Gα_i2 on both a wild-type and β₁-transgenic background, respectively. The number of animals used for qPCR analysis was 3–4 for each genotype. Age range of examined mice was 302 ± 4 days. Asterisks indicate significant differences compared with mRNA expression levels of age-matched wild-type mice in an REST^® analysis (*P < 0.05, **P < 0.01). (C) Representative immunoblots for the Gα_i2 protein in ventricle homogenates. The Gα_i2 protein was absent in Gα_i2^−/− and β₁-tg/Gα_i2^−/− mice. (E) Statistical analysis of Gα_i2 expression levels. (D) Representative immunoblot for the Gα_i3 protein in ventricle homogenates. To verify Gα_i3 antibody specificity, Gα_i3-deficient ventricle homogenates were loaded in addition. (F) Statistical analysis of Gα_i3 expression levels. A significant up-regulation of Gα_i3 protein levels was detectable in Gα_i2^−/− and β₁-tg/Gα_i2^−/− mice. For western blots, individual homogenates from three animals per genotype were analysed in 3–4 independent experiments. β-Actin was used to demonstrate equal loading of the gels. Asterisks indicate significant differences in Bonferroni post-tests following ANOVA (*P < 0.05; **P < 0.01).

3.6. Expression of hypertrophy markers and PKA targets

According to our morphological and functional findings, qPCR analysis revealed a significant up-regulation of the cardiomyopathy markers ANP (Figure 6A) and BNP (Figure 6B) in ventricular tissue of β₁-tg/Gα_i2^−/− compared with wild-type mice. However, mRNA expression levels of ANP were also significantly increased in Gα_i2^−/− mice and showed a tendency to be higher in β₁-tg animals (Figure 6A). BNP levels were unchanged in Gα_i2^−/−, but significantly increased in β₁-tg mice (Figure 6B). We tested for mRNA expression of the calcium release channel (ryanodine receptor) RYR2, the cardiac TnI TNNI3, and PLB as known targets of PKA-mediated phosphorylation (Figure 7). Compared with C57BL/6 wild-type mice, no differences were seen for RYR2 mRNA, but there was a significantly reduced expression of TnI in ventricles of β₁-tg/Gα_i2^−/− (63 ± 7% of wild type), and of PLB in ventricles of both β₁-tg and β₁-tg/Gα_i2^−/− (43 ± 6 and 47 ± 12% of wild type, respectively).

Figure 6.

Expression of cardiac hypertrophy markers in murine ventricles. Increased mRNA expression of ANP (A) and BNP (B) compared with wild-type levels confirmed morphological parameters indicating cardiac hypertrophy in β₁-tg/Gα_i2^−/− mice. The number of animals used for analysis was 3–4 for each genotype. Age range of examined mice was 302 ± 4 days. Asterisks indicate significant differences compared with mRNA expression levels of age-matched wild-type mice in an REST^® analysis (*P < 0.05, **P < 0.01).

Figure 7.

Ventricular mRNA expression of the PKA targets RYR2, TnI, and PLB. (A) RYR2 expression was similar in all genotypes. (B) TnI expression was significantly reduced in β₁-tg/Gα_i2^−/− ventricles compared with wild-type (C57BL/6) tissue. (C) In both β₁-adrenoceptor overexpressing mouse lines (β₁-tg and β₁-tg/Gα_i2^−/−), ventricular PLB expression was significantly reduced compared with wild-type (C57BL/6) tissue. The number of animals used for analysis was 3–4 for each genotype. Age range of examined mice was 288 ± 9 days. Asterisks indicate significant differences compared with mRNA expression levels of age-matched wild-type mice in an REST^® analysis (*P < 0.05).

4. Discussion

In line with the idea of cardioprotective signalling mediated by G_i proteins, we show here that the lack of Gα_i2 in mice overexpressing cardiac β₁-adrenoceptors was detrimental: cardiac contractility was significantly depressed and survival time dramatically reduced. Mice deficient for either Gα_i2 or Gα_i3 have been reported to have no overt cardiac phenotype in vivo (echocardiography) and ex vivo (isolated whole hearts and myocytes).³⁴ This is in perfect agreement with our current data obtained from Gα_i2-deficient mice in the same range of age. Survival time of Gα_i2-deficient mice was reduced, but reduction was significantly more pronounced in β₁-tg/Gα_i2^−/− mice. Of interest, survival curves of β₁-tg/Gα_i2^−/− and mice only lacking Gα_i2 were indistinguishable up to an age of ∼300 days. Since survival curves of β₁-tg mice start to drop rather late (this study and Lee et al.³³), it is tempting to speculate that the early effect on mortality (up to Day 300) is due to Gα_i2 deficiency while the detrimental effect observed beyond 300 days of age is related to the cardiac overexpression of β₁-adrenoceptors. Given that cardiac function of mice only lacking Gα_i2 was unaffected at an age of 300 days, a cause of death other than reduced pump function has to be supposed. In a previous study, mice with both a Gα_i2 knockout and a cardiac-specific overexpression of β₂-adrenoceptors have been shown to be not viable.¹³ This fatal outcome might be explained by direct coupling of β₂-adrenoceptors to G_i proteins. The drastic effect of Gα_i2 deficiency on top of cardiac β₁-adrenoceptor overexpression seen in our current study is rather surprising given that β₁-adrenoceptors are thought to exclusively couple with G_s proteins. However, recent work suggests a role for G_i in modulation of β₁-mediated effects. Melsom et al.³⁵ showed that inhibition of G_i proteins by pertussis toxin (PTX) not only enhanced cAMP accumulation following selective stimulation of either β₁- or β₂-adrenoceptors, but also increased inotropic potency of β₁- and β₂-adrenergic agonists, respectively. Another work by the same group indicates that G_i exerts intrinsic receptor-independent inhibitory activity on adenylyl cyclase.³⁶

4.1. β₁-Adrenoceptor overexpression

Like in mice overexpressing the cardiac β₂-adrenoceptor, the phenotype of β₁-transgenic mice seems to depend on the extent of overexpression. A higher expression level of β₁-adrenoceptors led to an earlier and more pronounced hypertrophy of cardiomyocytes and a higher sensitivity of contractile response to dobutamine.¹⁵ We found that after back-crossing on a C57BL/6 background, cardiac β-adrenoceptor overexpression was lower compared with FVB/N-based β₁-transgenic mice. This might explain why survival curves of β₁-transgenic mice on a C57BL/6 background start to drop later (in accordance to findings of another group that independently backcrossed the β₁-transgeni mice mouse of Engelhardt et al. on a C57BL/6 background).^15,33 In good agreement, cardiac function was already impaired at an age of 300 days in FVB/N-, but not in C57BL/6-based β₁-transgenic mice.

4.2. cAMP-dependent adrenergic signalling

That there was overt cardiomyopathy in (C57BL/6-based) β₁-transgenic mice additionally lacking Gα_i2 thus might be explained by a dual mechanism: increased β₁-adrenergic drive and lack of protection by Gα_i2. Engelhardt et al.³⁷ found an increased expression of the cardiac Na⁺-/H⁺-exchanger NHE1 in β₁-adrenoceptor transgenic mice and showed that NHE1 inhibition prevented heart failure in this mouse model. Since NHE1 activity is modulated by cAMP, one might speculate that, in the β₁-overexpression model, lack of Gα_i2 further aggravates NHE1-dependent effects leading to accelerated development of cardiac dysfunction and eventually mortality in our study.³⁸ Though only an indirect hint, we looked at the expression of other potential phosphorylation targets at the mRNA level. Altered phosphorylation of the calcium release channel RYR2 has been attributed to heart failure and cardiac arrhythmia, though there is an ongoing controversial discussion.³⁹ In our mouse models, RYR2 mRNA expression was unchanged, but we cannot exclude that enhanced phosphorylation due to a maintained increase of β-adrenergic tone might be involved in the early mortality we observed in β₁-transgenic mice lacking Gα_i2. TnI seems to mediate both lusitropic and inotropic responses following β-adrenergic stimulation in murine hearts,⁴⁰ though the role of TnI and its phosphorylation in healthy and failing hearts is matter of debate.⁴¹ We found a significant reduction of TnI expression at the mRNA level in β₁-transgenic mice lacking Gα_i2. This might be related to their detrimental outcome since it has been shown that TnI knockout leads to heart failure in adult mice.⁴² Both β₁-transgenic mouse lines investigated in our study (β₁-tg and β₁-tg/Gα_i2^−/−) showed a significant reduction of PLB mRNA expression. Engelhardt et al.⁴³ found no alteration of PLB expression in β₁-transgenic mice at an age of 2 months, but an increased PLB phosphorylation indicating enhanced β-adrenergic signalling. The decrease of PLB expression observed at an age of ∼9–10 months in our study might be compensatory by restoring the function of the SR calcium pump SERCA2A.⁴⁴ Indeed, knockout of PLB in β-transgenic mouse hearts has been shown to rescue its heart failure phenotype.⁴⁵ However, there seems to be a critical difference between mice and men because human PLB null has been shown to result in lethal cardiomyopathy.⁴⁶ Of note, PKA-dependent phosphorylation of β₂-adrenoceptors has been shown to drive a switch from G_s to G_i signalling,⁴⁷ though this mechanism is matter of debate. β₁-Adrenoceptor overexpression thus might enhance β₂-adrenoceptor-mediated G_i signalling. This would explain the pronounced effect of Gα_i2 deficiency in β₁-transgenic mice compared with mice with an otherwise wild-type background seen in our study.

4.3. G_i-mediated adrenergic signalling

Irrespective of the (disputed) role of β₂-adrenoceptor regulation by β₁-adrenoceptors, the accepted role of G_i proteins for β₂-adrenoceptor signalling should be considered. Communal et al.⁴⁸ have described opposing β-adrenoceptor-mediated effects on apoptosis in rat cardiomyocytes in terms of enhancement via β₁- but attenuation via β₂-adrenoceptors. Indeed, the combination of β₁-adrenoceptor blockers and β₂-adrenoceptor agonists has been shown to be superior to only β₁-adrenoceptor antagonists in treating cardiomyopathy in rodents.^49,50 Though the above-mentioned anti-apoptotic effect by β₂-adrenergic signalling was attributed to G_i-dependent activation of p38, the finding that overexpression of a dominant negative p38 isoform rescued cardiomyopathy of β₂- but not β₁-adrenoceptor transgenic mice argues against a role of this mechanism in our study.^51,52 Kohler et al.¹⁴ have recently shown that following an ischaemia/reperfusion (I/R) protocol, cardiac infarct size was significantly increased in mice deficient for Gα_i2. Also in a murine I/R model, similar findings have been obtained with an induced cardiac expression of a G_i inhibitor peptide.¹¹ Taken together with our current study on β₁-adrenoceptor overexpressing mice, these data suggest that the cardioprotective role of G_i proteins might become evident under certain circumstances only, i.e. cardiac stress and/or disease. Not all studies support the idea of G_i proteins being protective in cardiomyopathy and heart failure. The beneficial effect of combined treatment with a β₁-adrenoceptor blocker and a β₂-adrenoceptor agonist following myocardial infarction in rats was also seen when using fenoterol that has been shown to mediate β₂-adrenergic effects independent of G_i proteins.^50,53 Of note, the reported G protein selectivity of stereoisomers of fenoterol has recently been challenged.⁵⁴ In a mouse model of dilative cardiomyopathy due to cre-recombinase overexpression, an increase of G_i signalling by knock-in of a mutated Gα_i2 did not improve but even worsen ventricular hypertrophy and mortality.⁵⁵ In a pathophysiologically more relevant model, Hussain et al.⁵⁶ found no change in G_i activity in rat heart failure following myocardial infarction: despite a significant increase of Gα_i2 protein by 50% G_i inhibition by PTX treatment, here did neither change baseline contractility nor inotropic response following β-adrenergic stimulation in ventricular strips from failing hearts.

4.4. Role of G_i2 vs. G_i3

The putative differential roles of the most abundant cardiac G_i isoforms Gα_i2 and Gα_i3 are still a matter of debate. As mentioned above, mice with cardiac-specific overexpression of β₂-adrenoceptors were not viable, if they in addition were deficient of Gα_i2.¹³ In the same study, heterozygous knockout of Gα_i2 in β₂-transgenic mice caused a reduced activity of ventricular L-type Ca²⁺ channels that was attributed to an increased expression of Gα_i3. These data led us to the hypothesis that Gα_i2 is cardioprotective, whereas Gα_i3 is rather of regulatory relevance. In a subsequent study, this hypothesis was supported by our finding that, in Gα_i2 knockout mice, cardiac Gα_i3 expression was up-regulated and accordingly ventricular Ca²⁺-current density was decreased while, in mice lacking Gα_i3, Ca²⁺-current density was enhanced despite an increased Gα_i2 expression.²⁴ Kohler et al.¹⁴ confirmed an increased cardiac Gα_i2 expression in mice lacking Gα_i3 and found ventricular infarct size following ischaemia to be reduced here compared with wild-type mice or mice lacking Gα_i2.

5. Conclusion

In conclusion, our data support the idea of the G_i protein Gα_i2 to be cardioprotective. Of interest, this was observed in β₁-adrenoceptor overexpressing mice, i.e. a mouse model of cardiac stress not directly involving G_i signalling. The lack of Gα_i2 seemed to aggravate rather than cause cardiac dysfunction. The observed up-regulation of Gα_i3 was not sufficient to compensate for Gα_i2 deficiency, suggesting an isoform-specific or a concentration-dependent mechanism to be elucidated in further studies.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Funding

This study was supported in part by the Intramural Research Program of the NIH (Project Z01-ES-101643) to L.B. and by Deutsche Forschungsgemeinschaft (DFG; He 1578/13-1 and 2) to S.H.