Gαi2 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 Gαi2. We propose an enhanced effect of increased β1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or a concentration-dependent mechanism.

BackgroundTwo 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).MethodsVentricular 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.ResultsIn 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.ConclusionOur 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.

Membranous adenylyl cyclases play a major role in G-protein-coupled receptor signalling and regulate various cellular responses, such as cardiac contraction. Cardiac apoptosis and development of cardiac dysfunction is prevented in mice lacking AC 5, a predominant isoform in the heart. In the search for a potent and selective AC 5 inhibitor, we recently identified 2'(3')-methylanthraniloyl-inosine-5'-triphosphate(MANT-ITP) as the most potent AC 5 inhibitor with a K ( i ) of 13 nM. Therefore, AC inhibition of MANT-ITP was assessed in ventricular cardiomyocytes and compared to three other MANT-nucleotides to evaluate its effect on cardiac signalling. Basal and isoproterenol-induced L-type calcium currents (I (Ca,L)) in murine ventricular cardiomyocytes were recorded by whole-cell patch-clamp technique, using four different MANT-nucleotides. The effects of the MANT-nucleotides on I (Ca,L) were unexpectedly complex. All MANT-nucleotides exhibited an inhibitory effect on basal I (Ca,L). Additionally, several MANT-nucleotides, i.e., MANT-ITPγS, MANT-ATP, and MANT-ITP, caused a strong initial increase in basal I (Ca,L) within the first 2.5 min that appeared to be unrelated to AC 5 inhibition. However, we detected a significant reduction on isoproterenol-induced I (Ca,L) with MANT-ITP, supporting the notion that AC 5 plays an important role in agonist-stimulated activation of I (Ca,L). Collectively, MANT-nucleotides are useful tools for the characterization of recombinant ACs, for fluorescence studies and crystallography, but in intact cardiomyocytes, caution must be exerted since MANT-nucleotides apparently possess additional effects than AC 5 inhibition, limiting their usefulness as tools for intact cell studies.

Background: Two pertussis toxin sensitive G i proteins, G i2 and G i3 , are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G i isoforms are functionally distinct. To test for isoformspecific functions of G i 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 Ga i2 (Ga i2 2/2 ) or Ga i3 (Ga i3 2/2 ). mRNA levels of Ga i/o isoforms and L-VDCC subunits were quantified by real-time PCR. Ga i and Ca v a 1 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 Ga i22/2 mice, Ga i3 mRNA and protein expression was upregulated to 187621% and 567659%, respectively. In Ga i32/2 mouse hearts, Ga i2 mRNA (12765%) and protein (131610%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Ga i2 2/2 mice was lowered (27.960.6 pA/pF, n = 11, p,0.05) compared to wild-type cells (210.760.5 pA/pF, n = 22), whereas it was increased in myocytes from Ga i3 2/2 mice (214.360.8 pA/pF, n = 14, p,0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Ga i2 (but not of Ga i3 ) and following treatment with pertussis toxin in Ga i3 2/2 . The pore forming Ca v a 1 protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca v a 1 and Ca v b 2 subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Ga i2 . Conclusion:Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Ga i proteins. In particular, loss of Ga i2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.

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