NRSF-GNAO1 Pathway Contributes to the Regulation of Cardiac Ca²⁺ Homeostasis

All supporting data are available within the article and its Supplemental Material. Please see the Major Resources Table in the Supplemental Material. For details on the experimental procedures, see Supplemental Methods.

We generated NRSF cKO mice by crossing NRSF floxed mice with transgenic mice expressing Cre recombinase under the control of the mouse αMHC (α-myosin heavy chain) promoter (αMHC-Cre-Tg; Figure S1A in the Supplemental Material)¹⁸ and subsequently confirmed the marked reductions in NRSF mRNA and protein in the NRSF cKO ventricles (Figure S1B through S1D in the Supplemental Material). Notably, by 3 weeks of age, the NRSF cKO mice started to die, and about 90% of these mice died by 10 weeks of age (Figure 1A). Gross examination of 4-week-old NRSF cKO mice revealed their hearts to be much larger than those of 4-week-old αMHC-Cre-Tg control mice (Figure 1B). The heart weight-to-body weight ratio in 4-week-old NRSF cKO mice was significantly higher than that in the control mice (Figure 1C), though their lung weight-to-body weight ratios did not differ (Figure 1D). Using a telemetric system to monitor arrhythmias in these mice, we detected frequent ventricular ectopies (premature ventricular contractions [PVCs]), runs of ventricular tachycardia, and transition from ventricular tachycardia to ventricular fibrillation in NRSF cKO mice (Figure 1E through 1G), which are similar to our findings in dnNRSF-Tg mice.⁴ Because NRSF cKO mice would be unexpectedly found dead within 24 hours of displaying apparently normal behavior and levels of activity, the manner in which they died was characterized as sudden death, which is consistent with the absences of significant increases in lung weigh-to-body weight ratios (Figure 1D) or signs of lung or liver congestion (data not shown). Echocardiography revealed a significant reduction in left ventricular ejection fraction (LVEF) in 4-week-old NRSF cKO mice (Figure 1H and 1I). Left ventricular end-diastolic dimension was larger in NRSF cKO than control αMHC-Cre-Tg mice, though wall thickness was not significantly affected (Table S1 in the Supplemental Material). NRSF cKO mice thus experience progressive LV dysfunction without LV hypertrophy.

Histological examination of 4-week-old NRSF cKO hearts showed marked heterogeneity in the size of ventricular myocytes, but no significant increase in inflammatory cell infiltration or interstitial fibrosis (Figure 1J). Electron microscopic examination of the ultrastructure of ventricular myocytes revealed that at 4 weeks, myofibrils were sparse and misarranged, Z-bands were discontinuous, and mitochondria were deformed or completely disrupted (Figure 1K), which are similar to previously reported findings in dnNRSF-Tg hearts.⁴ Using quantitative real-time reverse transcription–polymerase chain reaction, we observed that expression of Nppa (ANP [atrial natriuretic peptide]), Nppb (BNP [brain natriuretic peptide]), Hcn2 (potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2), and Cacna1h (Cav3.2), all of which are known to be NRSF target genes, was significantly higher in NRSF cKO ventricles than in control αMHC-Cre-Tg ventricles (Figure S1E through S1H in the Supplemental Material). In addition, Myh7 expression was upregulated and Atp2a2 (SERCA2 [sarco/endoplasmic reticulum, Ca²⁺-ATPase 2]) expression was downregulated in NRSF cKO ventricles as compared to control ventricles (Figure S1I and S1J in the Supplemental Material). All of these features of NRSF cKO hearts are similar to those of dnNRSF-Tg hearts and characteristic of human dilated cardiomyopathy (DCM).⁴

To identify the NRSF target genes responsible for cardiac dysfunction, we used microarray analysis to screen for genes dynamically upregulated or downregulated in the ventricular myocardium of both dnNRSF-Tg and NRSF cKO mice. Gene ontology analysis showed that several biological processes related to ion transmembrane transport were enriched (Figure S2 in the Supplemental Material). We, therefore, reviewed the screened genes seeking those that could potentially affect Ca²⁺ homeostasis and ultimately focused on Gnao1.

We confirmed that mRNA expression of Gnao1 is higher in both dnNRSF-Tg and NRSF cKO ventricles than control ventricles (Figure S3A and S3B in the Supplemental Material). By contrast, levels of Gnai2 mRNA were not significantly altered in dnNRSF-Tg or NRSF cKO ventricles as compared to control ventricles (Figure S3C and S3D in the Supplemental Material).¹³ We also confirmed that levels of Gα_o protein were significantly increased in both dnNRSF-Tg and NRSF cKO ventricles, but Gα_i2 levels were not significantly changed (Figure S3E through S3J in the Supplemental Material).

A sequence with high homology to NRSE (NRSF-binding element) was detected in the second intron of the human GNAO1 gene, on which accumulation of NRSF binding is shown in the publicly available chromatin immunoprecipitation sequencing data from the ChIP-Atlas (http://chip-atras.org/; Figure 2A). To determine whether this intronic NRSE-like sequence within GNAO1 has cis-regulatory functions mediated by NRSF, we performed reporter assays in cultured neonatal rat ventricular myocytes. The luciferase activity of the construct with the WT NRSE was significantly lower than that of the construct without NRSE or with mutated NRSE (Figure 2B). In addition, electrophoretic mobility shift assays using nuclear extract from human embryonic kidney cells 293 cells transfected with an expression vector encoding Myc-tagged NRSF and biotin-labeled NRSE as a probe showed a band shift that was blocked by addition of unlabeled NRSE but not unlabeled mutant NRSE. When the mutant NRSE was used as a probe, no band shift was observed, whereas the shifted band was super-shifted in the presence of an antibody raised against C-Myc (Figure 2C). Moreover, mRNA expression of Gnao1 increased after stimulation with a hypertrophy-inducing agent (ET-1 [endothelin-1], Ang II [angiotensin II], or PE [phenylephrine]) or administration of siRNA (small interfering RNA) targeting NRSF in neonatal rat ventricular myocytes (Figure 2D through 2F). On the other hand, Gnao1 expression was not further increased by ET-1 after preadministration of siRNA targeting NRSF in neonatal rat ventricular myocytes (Figure 2F). Taken together, these results clearly indicate that NRSF binds to GNAO1-NRSE, thereby transcriptionally repressing GNAO1 expression.

We next examined the levels of Gα_o expression in two other murine heart failure models: pressure overload induced by transverse aortic constriction (TAC) and DCM model mice carrying a knocked-in human cardiac troponin (Tnnt2) deletion mutation T(ΔK210).¹⁹ Expression of Gα_o, but not Gα_i2, was increased at both the mRNA and protein levels in ventricles of both of these mouse models (Figure S4A through S5J in the Supplemental Material), which demonstrates that increased expression of Gα_o is a widely conserved phenomenon in failing hearts.

We evaluated the effect of reduced Gα_o expression on dnNRSF-Tg hearts, by crossing Gnao1 knockout mice with dnNRSF-Tg mice. Because Gnao1 null-knockout mice reportedly exhibit abnormal behavior and die prematurely,²⁰ we analyzed Gnao1 hetero-knockout (Gnao1 hKO) mice, which are normally fertile and show no apparent abnormal phenotypes and normal survival under normal breeding conditions. As shown in Figure S5 in the Supplemental Material, expression of Gnao1 mRNA and Gα_o protein was significantly decreased by around 50% in dnNRSF-Tg mice with a Gnao1 hKO background.

Systolic blood pressures and pulse rates did not differ among the groups (Figure S6A and S6B in the Supplemental Material). Heart weight-to-body weight and lung weight-to-body weight ratios, which were significantly higher in dnNRSF-Tg than wild-type (WT) mice, were significantly lower in Gnao1 hKO; dnNRSF-Tg than dnNRSF-Tg mice (Figure S6C and S6D in the Supplemental Material). Echocardiographic analysis revealed that LVEFs were significantly larger and LV chamber dilatation was diminished in Gnao1 hKO; dnNRSF-Tg as compared to dnNRSF-Tg mice (Figure 3A and Table S2 in the Supplemental Material). Hemodynamic analysis, performed on 16-week-old mice, showed that LV end-diastolic pressure, +dP/dt and −dP/dt were significantly improved in Gnao1 hKO; dnNRSF-Tg as compared to dnNRSF-Tg mice (Figure 3B).

Although ventricular expression of Nppa and Myh7 mRNA was significantly higher in dnNRSF-Tg than WT hearts, that effect was largely reversed in Gnao1 hKO; dnNRSF-Tg hearts. Expression of Atp2a2 mRNA, which was reduced in dnNRSF-Tg hearts, was significantly restored in Gnao1 hKO; dnNRSF-Tg hearts (Figure S6E through S6G in the Supplemental Material). Moreover, survival rates among Gnao1 hKO; dnNRSF-Tg mice were significantly better than among dnNRSF-Tg mice (Figure 3C). When we analyzed arrhythmogenicity using a telemetric monitoring system, we found that Gnao1 hKO; dnNRSF-Tg hearts exhibited much fewer PVCs than dnNRSF-Tg hearts (Figure 3D). Histological analysis revealed substantial heterogeneity in the size of ventricular myocytes in dnNRSF-Tg hearts, which was improved in Gnao1 hKO; dnNRSF-Tg hearts (Figure 3E). There was no significant difference in ventricular fibrosis or fibrosis-related gene expression among WT, dnNRSF-Tg and Gnao1 hKO; dnNRSF-Tg mice (Figure S7A through S7D in the Supplemental Material). These data reveal that genetic reduction of Gα_o prevents the deterioration of cardiac function and increase in arrhythmogenicity otherwise seen in dnNRSF-Tg mice.

We also generated Gnao1 hKO; NRSF cKO mice by crossing Gnao1 knockout with NRSF cKO mice. As shown in Figure S8A through S8E in the Supplemental Material, expression of Gnao1 mRNA and Gα_o protein were significantly decreased with the Gnao1 hKO background. Echocardiographic parameters and survival rates were significantly better in Gnao1 hKO; NRSF cKO than NRSF cKO mice (Figure S9A through S9C in the Supplemental Material). Heart weight-to-body weight ratios were significantly smaller in Gnao1 hKO; NRSF cKO than NRSF cKO mice (Figure S9D in the Supplemental Material). Moreover, ventricular expression of Nppa mRNA was significantly lower in Gnao1 hKO; NRSF cKO than NRSF cKO hearts (Figure S9E in the Supplemental Material).

The data summarized above indicate that inhibition of Gα_o prevents the progression of cardiac dysfunction and sudden death resulting from ablation of NRSF-mediated repression in cardiac myocytes.

In normal adult ventricular myocytes, a small fraction of LTCCs are localized to caveolar microdomains.²¹ These caveolar LTCCs are thought to be more important for pathological Ca²⁺ signaling and to contribute to Ca²⁺ release from sarcoplasmic reticulum (SR) less effectively than LTCCs within another sarcolemmal microdomain, the junctional clefts, which are situated predominantly within transverse tubules (T-tubules).²² As caveolae occur more abundantly in the surface sarcolemma than T-tubules, surface sarcolemmal LTCC currents correlate with Ca²⁺ influx through LTCCs localized to the caveolar microdomains.²³ It was recently shown that chronic activation of Gα_i/o induces alterations in subcellularly localized LTCC activity that leads to cardiac dysfunction.^24,25 We, therefore, evaluated localized LTCC density in isolated ventricular myocytes by measuring LTCC currents under nondetubulated and detubulated conditions²⁶ (Figure S10 in the Supplemental Material). We found that in 20-week-old dnNRSF-Tg ventricular myocytes, surface sarcolemmal LTCC current density was significantly greater than in control WT ventricular myocytes, whereas whole-cell current density did not differ significantly between dnNRSF-Tg and WT ventricular myocytes (Figure 4A and 4B, Figure S11A and S11B and Table S3 in the Supplemental Material). Genetic depletion of Gα_o reduced surface sarcolemmal LTCC current density in ventricular myocytes from dnNRSF-Tg mice to the levels in WT ventricular myocytes, although whole-cell LTCC current density did not significantly differ between the 2 cell types (Figure 4A and 4B, Figure S11A and S11B and Table S3 in the Supplemental Material). Further investigation of the abnormal Ca²⁺ handling in dnNRSF-Tg ventricular myocytes revealed that SR Ca²⁺ content was reduced (Figure 4C). It was restored in Gnao1 hKO; dnNRSF-Tg hearts ventricular myocytes (Figure 4C). Moreover, peak Ca²⁺ transient amplitudes were decreased, and the decay times of Ca²⁺ transients were significantly prolonged as compared to those in WT cardiomyocytes (Figure 4D through 4G). These changes in dnNRSF-Tg ventricular myocytes were also ameliorated in ventricular myocytes from Gnao1 hKO; dnNRSF-Tg hearts (Figure 4D through 4G).

Further analysis of Ca²⁺ transients revealed that what are likely secondary SR Ca²⁺ release events and Ca²⁺ sparks during the declining phase of the Ca²⁺ transient (occurring after the mouse action potential would be expected to have fully repolarized) were observed more frequently in dnNRSF-Tg than WT cells (Figure 4H through 4J). These ryanodine receptor-mediated events may also cause arrhythmogenic delayed (or early) afterdepolarizations. The resting cytosolic Ca²⁺ concentration in unstimulated myocytes was also increased in dnNRSF-Tg cardiomyocytes, as previously reported (Figure 4K).²⁷ These pathophysiological changes in SR function would be expected to lead to increased arrhythmogenicity and cardiac dysfunction. Notably, all of the dysfunctional changes observed in dnNRSF-Tg ventricular myocytes were ameliorated in ventricular myocytes from Gnao1 hKO; dnNRSF-Tg mice (Figure 4I through 4K).

As described above, increases in Ca²⁺ influx through surface sarcolemmal LTCCs is known to activate pathological Ca²⁺ signaling, including CaMKII activation, which leads to impairment of SR activity through hyperphosphorylation of RyR2 (ryanodine receptor-2).²² We found that levels of CaMKII phosphorylation at Thr286, which represents CaMKII activation, and RyR2 phosphorylation at Ser2814, which is a site phosphorylated by CaMKII, were significantly increased in the dnNRSF-Tg ventricular myocardium (Figure 5A through 5C). These alterations were reversed in the Gnao1 hKO; dnNRSF-Tg ventricular myocardium (Figure 5A through 5C). However, there were no significant differences in ventricular PKA (protein kinase A) activities among the dnNRSF-Tg, Gnao1 hKO; dnNRSF-Tg, and WT mice (Figure 5D). These results demonstrate that Gα_o-induced alterations in subcellular localization of LTCC current density lead to activation of pathological Ca²⁺ signaling, including CaMKII activation, and impaired Ca²⁺ handling in dnNRSF-Tg ventricular myocytes.

To further evaluate the functional role of increased cardiac Gα_o expression, we generated transgenic mice in which human Gα_o is overexpressed in a cardiac-specific manner under the control of the cardiac-specific αMHC promoter (GNAO1-Tg; Figure S12A in the Supplemental Material). Although the amino acid sequences of human and murine Gα_o are the same, the nucleotide sequences of the human and murine genes differ slightly, enabling endogenous murine Gnao1 to be distinguished from overexpressed human GNAO1 through mRNA analysis. Four independent GNAO1-Tg lines, all with similar phenotypes, were obtained (data not shown). Among them, we analyzed in detail line 21, which expressed intermediate levels of the transgene (Figure S12B in the Supplemental Material). GNAO1-Tg mice showed progressive cardiac dysfunction with LV dilatation and mild hypertrophy in the absence of load (Figure 6A), whereas systolic blood pressures and pulse rates were comparable between GNAO1-Tg and WT (Figure S12C and S12D in the Supplemental Material).

To investigate the primary effect of increased cardiac GNAO1, we analyzed ventricular myocardium and isolated ventricular myocytes from 8-week-old GNAO1-Tg mice, in which cardiac function was still comparable to that in their WT littermates. At that time, heart weight-to-body weight ratios were already significantly higher in GNAO1-Tg than WT mice, thus preceding the decline in cardiac function (Figure S12E in the Supplemental Material). Expression of two cardiac stress marker genes, Nppa and Myh7, was significantly elevated in GNAO1-Tg ventricles (Figure S12F and S12G in the Supplemental Material). Endogenous mouse Gnao1 mRNA levels were also significantly increased in GNAO1-Tg ventricles, though Gnai2 mRNA levels were not (Figure S12H and S12I in the Supplemental Material). Ventricular Rcan1.4 mRNA expression, which correlates with activity in the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway, was significantly higher in GNAO1-Tg than WT hearts (Figure S12J in the Supplemental Material). Western blot analysis showed that protein levels of Gα_o were significantly increased in GNAO1-Tg hearts and that ventricular myocardial CaMKII phosphorylation was significantly increased in GNAO1-Tg hearts (Figure 6B), as was sarcolemmal LTCC current density, though whole-cell LTCC current density was not altered (Figure 6C and 6D, Figure S11C and S11D and Table S4 in the Supplemental Material). Ventricular myocardial PKA activity also did not significantly differ between GNAO1-Tg and WT hearts (Figure 6E). These data clearly demonstrate that cardiac overexpression of Gα_o is sufficient to alter the subcellular distribution of localized LTCC current densities; activate Ca²⁺-dependent pathological signaling pathways, including CaMKII; and impair cardiac function.

In light of the recent finding that Gα_o is involved in vesicle trafficking independently of canonical G protein signaling,²⁸ we hypothesized that Gα_o would primarily alter the subcellular localization of LTCCs in ventricular myocytes. However, the structures of T-tubules in isolated ventricular myocytes from 20-week-old dnNRSF-Tg mice, which we used to measure LTCC current densities in Figure 4, are disorganized (Figure S13A in the Supplemental Material). For that reason, we immunostained LTCC a1c subunits (Ca_V1.2) in ventricular myocytes isolated from 8-week-old dnNRSF-Tg and GNAO1-Tg mice, as both exhibit cardiac function comparable to that in their WT littermates, and the structure of their T-tubules is preserved (Figure 6A and Figure S13B and S13C in the Supplemental Material).⁴ Colocalization analysis of Ca_V1.2 and CAV3 (caveolin-3) and measurement of relative fluorescence intensities comparing the sarcolemmal and cytosolic (T-tubule) regions revealed that subcellular localization of LTCCs in 8-week-old dnNRSF-Tg or GNAO1-Tg mice did not significantly differ from that in their WT littermates (Figure S14A through S14H in the Supplemental Material). Moreover, Western blotting showed that the total abundance of Ca_V1.2 protein in the ventricular myocardium did not differ between 8-week-old dnNRSF-Tg or GNAO1-Tg mice and their WT littermates (Figure S15A through S15D in the Supplemental Material). Because both 8-week-old dnNRSF-Tg and GNAO1-Tg mice already showed elevated surface sarcolemmal LTCC current density (Figure S16A through S16D in the Supplemental Material, Figure 6C and 6D and Tables S4 and S5 in the Supplemental Material), these results suggest that Gα_o initially increases the activity of surface sarcolemmal LTCCs, which precedes alteration in the structure of the T-tubules and local distribution of LTCC proteins during the progression in pathological cardiac remodeling.

To determine whether Gα_o also plays important roles in other heart failure models, we evaluated the phenotypes in Gnao1 hKO mice subjected to pressure overload–induced cardiac hypertrophy and heart failure induced by TAC, as we revealed that Gnao1 mRNA levels and Gα_o protein levels were both increased in the ventricular myocardium after TAC (Figure S4A through S4E in the Supplemental Material). There were no deaths among WT or Gnao1 hKO mice during the 12-week follow-up after TAC. In Gnao1 hKO ventricles, Gnao1 mRNA levels and Gα_o protein levels were decreased to about 50% of that in WT ventricles (Figure S17A through S17E in the Supplemental Material). Echocardiographic analysis showed that after 4 weeks after TAC, LV wall thickness was significantly greater in WT than Gnao1 hKO mice, although LVEF was preserved and did not differ between the 2 genotypes (Figure S18A and Table S3 in the Supplemental Material). Eight or 12 weeks after TAC, however, LVEF had deteriorated, and left ventricular end-diastolic dimension was enlarged in WT mice (Figure S18A through S18C and Table S6 in the Supplemental Material). By contrast, both LVEF and left ventricular end-diastolic dimension were maintained in Gnao1 hKO mice, even 12 weeks after TAC, at which time heart weight-to-body weight and lung weight-to-body weight ratios were significantly lower in Gnao1 hKO than WT mice (Figure S18D and S18E and Table S6 in the Supplemental Material). Histological analysis 12 weeks after TAC showed that TAC-induced cardiac fibrosis was significantly attenuated in Gnao1 hKO as compared to WT hearts (Figure S18F and S18G in the Supplemental Material). Moreover, 12 weeks after TAC, the altered ventricular myocardial expression of cardiac remodeling-related genes (Nppa, Myh7, and Atp2a2) and fibrosis-related genes (Col1a1 and Tgfb3) seen in WT mice were significantly reversed in Gnao1 hKO (Figure S18H through S18L in the Supplemental Material). These data showing that genetic reduction of Gα_o prevented the progression of pathological cardiac remodeling and heart failure during pressure overload clearly imply a significant role of Gα_o in this pathological process.

We further addressed the functional significance of Gα_o by crossing Gnao1 knockout with DCM model mice carrying a knocked-in troponin T deletion mutation (ΔK210), which causes human familial DCM. Gnao1 mRNA levels and Gα_o protein levels were both increased in ventricular myocardium of ΔK210 mice (Figure S4F through S4J in the Supplemental Material). In Gnao1 hKO; ΔK210 ventricles, both Gnao1 mRNA and Gα_o protein levels were significantly decreased to about 50% of that in ΔK210 ventricles, whereas Gnai2 mRNA and Gα_i2 levels did not significantly differ between the 2 genotypes (Figure S19A through S19E in the Supplemental Material). ΔK210 mice reportedly show cardiac dysfunction similar to human DCM and sudden death due to lethal arrhythmias.¹⁹ Survival rates were significantly better among Gnao1 hKO; ΔK210 than ΔK210 mice, and echocardiographic analysis revealed LVEF to be significantly higher in Gnao1 hKO; ΔK210 than ΔK210 mice (Figure S20A through S20C and Table S7 in the Supplemental Material). Thinning of the LV wall, which was observed in ΔK210 mice, was significantly ameliorated in Gnao1 hKO; ΔK210 mice (Figure S20B and S20D and Table S7 in the Supplemental Material).

Increases in heart weight-to-body weight and lung weight-to-body weight ratios seen in ΔK210 mice were both ameliorated in Gnao1 hKO; ΔK210 mice (Figure S20E and S20F in the Supplemental Material). In addition, the altered ventricular myocardial expression of cardiac remodeling-related genes (Nppa, Myh7 and Atp2a2) seen in ΔK210 mice was significantly reversed in Gnao1 hKO; ΔK210 mice (Figure S20G through S20I in the Supplemental Material). These data demonstrating the significant role played by Gα_o in the progression of heart failure and sudden death caused by cardiac troponin T mutation support our hypothesis that the increase in Gα_o contributes to the progression of cardiac dysfunction and pathological cardiac remodeling in these cases.

It remains unclear whether Gα_i and Gα_o operate through identical mechanisms, but Gα_i and Gα_o are thought to have similar functions in the heart; that is, inhibition of Gα_s-mediated signaling pathways related to increases in cardiac contractility.²⁹ Although activation of Gα_s increases cardiac contractility and heart rates, persistent activation leads to heart failure. Thus, although increased Gα_i/o activity may depress cardiac function by inhibiting Gα_s-mediated signaling in the short term, it may exert cardioprotective effects in the longer term by reducing myocardial energy expenditure, myocyte apoptosis, and arrhythmias in the same way that β blockers do.²⁹ Indeed, Gα_i2 was reported to have cardioprotective effects in mouse models of ischemic-reperfusion, tachycardia-induced cardiomyopathy, and cardiac β1-adrenoceptor overexpression.^13,15,16 However, enhancing Gα_i2 signaling can have detrimental effects during the development of heart failure.¹⁷ Thus, the role of Gα_i2 in the pathological process underlying heart disease is still controversial. Several reports have shown that Gα_o activates different signaling pathways than Gα_i2. In contrast to Gα_i, the Gα_o subunit appears not to inhibit adenylyl cyclase in vitro.^30,31 In addition, transgenic mice overexpressing a constitutively active Gα_o mutant showed hypercontractility related to increased whole-cell LTCC activity in cardiac myocytes with no effect on PKA.³² This makes elucidation of the function of cardiac Gα_o of great interest, but up to now little has been known about the pathophysiological role of Gα_o in the heart. In the present study, we clearly demonstrated that ventricular Gα_o expression is increased in heart failure in mice, with no statistical difference in Gα_i2 expression and that the increase in Gα_o is sufficient to induce alterations in subcellularly localized LTCC activity. This in turn leads to activation of pathological Ca²⁺-dependent signaling pathways, including CaMKII activation, which impairs RyR2 and SR function, thereby promoting pathological cardiac remodeling and heart failure.²² Moreover, increasing Gα_o in the heart did not affect PKA activity, which is inactivated by Gα_i2. These data suggest the function of Gα_o within ventricular myocytes is distinct from that of Gα_i2.

The distribution of localized LTCC current activity is regulated by spatial alteration of both the number and open probability of LTCCs in the sarcolemma.³³ One recent study reported that in failing cardiomyocytes exhibiting a loss of T-tubule structure, there is a net redistribution of Ca_V1.2 from the T-tubules to the surface sarcolemma and a selective elevation in the open probability of the surface channels.³⁴ Our study suggests that increased expression of Gα_o increases surface sarcolemmal LTCC activity and that these increases in Gα_o expression and LTCC activity precede the structural and functional alterations during the progression of heart failure. Further study to identify the detailed molecular mechanisms by which increased Gα_o selectively activates surface sarcolemmal LTCCs will be necessary.

Several reports have shown a tight link between CaMKII activation and increased surface sarcolemmal LTCC activity in failing ventricular myocytes.^34,35 Our study demonstrates that surface sarcolemmal LTCC current density and levels of CaMKII phosphorylation are increased in ventricular myocytes from GNAO1-Tg hearts before the decline in cardiac function. Moreover, dnNRSF-Tg hearts exhibited increased surface sarcolemmal LTCC activity and abnormal Ca²⁺ handling, and these abnormalities were reversed by genetic knockdown of Gα_o with suppression of CaMKII activation and hyperphosphorylation of RyR2, which is known to decrease SR Ca²⁺ content and increase the resting cytosolic Ca²⁺ concentration due to Ca²⁺ leakage from the SR. These lines of evidence all support our notion that Gα_o-induced increases in local Ca²⁺ influx via surface sarcolemmal LTCCs activate CaMKII and hyper-phosphorylates RyR2, which in turn leads to Ca²⁺ handling abnormalities, increased arrhythmogenicity, and cardiac dysfunction (Figure S21 in the Supplemental Material).

CaMKII also plays a vital role in transcriptional pathways during the development of pathological cardiac remodeling.³⁶ We previously reported that CaMKII signaling induced by hypertrophic stimuli disrupts class II HDAC (histone deacetylase)-NRSF complexes and attenuates NRSF-mediated repression, which leads to reactivation of the fetal gene program.³ Our data showing that inhibition of NRSF derepresses Gα_o expression, leading to CaMKII activation, suggest Gα_o acts as an amplifier within the CaMKII-NRSF circuit that drives pathological cardiac remodeling (Figure S21 in the Supplemental Material). Our finding that increased expression of endogenous mouse Gnao1 in GNAO1-Tg ventricles, in which human GNAO1 is overexpressed, precedes the decline in cardiac function strongly supports this notion. Our study using multiple heart failure models suggests that this pathological NRSF-GNAO1-CaMKII circuit underlies heart failure derived from various causes.

In this study, we showed that Gα_o is a key component of a Ca²⁺-dependent transcriptional circuit consisting of NRSF and CaMKII, which drives pathological cardiac remodeling. Publicly available single-cell RNA sequence data showed greater expression of Gα_o and other NRSF target genes, such as Nppa, Acta1, and Cacna1h, in ventricular cardiomyocytes from human DCM patients than from healthy controls (Figure S22 in the Supplemental Material, data for Nppa, Acta1, and Cacna1h are not shown here).³⁷ These data raise the possibility that the NRSF-GNAO1 pathway broadly underlies the development of human heart failure and that selective inhibition of Gα_o could be a novel and effective therapeutic approach to treating heart failure. Given that Gα_o reportedly plays a pivotal role in the central nervous system, it will be necessary to develop drugs that target Gα_o but do not cross the blood-brain barrier.²⁰

H. Inazumi, K. Kuwahara, Y. Nakagawa, Y. Kuwabara, M. Yamada, and N. Kurebayashi designed experiments. H. Inazumi and Y. Kuwabara performed most of the experiments. T. Numaga-Tomita, T Kashihara, N. Kurebayashi, and M. Nonaka performed electrophysiological experiments. T Nakada and M. Oya performed immunostaining experiments. N. Kurebayashi and M. Sugihara performed echocardiography of the Homozygous knock-in mice with the deletion mutation Lys-210 in the endogenous cardiac troponin T gene (ΔK210). H. Inazumi, K. Kuwahara, Y. Nakagawa, Y. Kuwabara, T Kashihara, T. Numaga-Tomita, T Nakada, M. Oya, M. Yamada, and N. Kurebayashi analyzed the data. S. Morimoto provided ΔK210. H. Inazumi, K. Kuwahara, and Y. Nakagawa wrote the article. All authors reviewed and approved the final draft. We would like to thank Richard Neubig for the useful discussions. We thank Ayako Ohta, Yukari Kubo, and Mebae Kobayashi for their excellent secretarial works and Mizuho Takemura for their excellent technical support.

Supplemental Methods

Table S1–S7

Figure S1–S22

References 4,19,25,38–44