Recent studies from our laboratories identified a number of small molecules that bind Gβγ and modulate Gβγ-protein interactions 19, 20
. These compounds were able to inhibit a number of Gβγ-dependent signaling events, including Gβγ-GRK2 association. GRK2 protein levels are significantly elevated in HF, and substantial evidence indicates that blocking both Gβγ and the GRK2-Gβγ interaction in diverse HF models is cardioprotective 13, 14
. In this study, we tested whether M119 and its highly related and similarly efficacious Gβγ compound inhibitor gallein could be used to target Gβγ in cardiomyocytes and thereby influence aspects of β-AR signaling and improve cardiac function in animal models of HF. Our data indicate that M119 and gallein interfere with Gβγ-GRK2 interactions in cardiomyocytes, and may enhance β-AR signaling in vitro.
M119 and gallein both halt HF progression and improve cardiac function, morphometry, histology and gene expression in animal models of either new onset or established HF.
Recruitment of GRK2 to the membrane by Gβγ is among the more well-established functions of Gβγ downstream of β-AR signaling. General inhibition of Gβγ, and of the Gβγ-GRK2 interaction, using various Gβγ inhibitory peptides (e.g. βARKct, nt-del-Phosducin, PI3Kγinact
) has proven to be a highly effective treatment for a variety of animal HF models and in isolated failing human cardiomyocytes 28
. The results of these studies have alternately been viewed as a specific effect of inhibiting GRK2 function and as a broader effect on Gβγ signaling. Consistent with the latter hypothesis, another Gβγ sequestering peptide, a truncated form of the Gβγ binding protein phosducin (nt-del-Phosducin), has also been used successfully to restore β-AR signaling in cardiomyocytes 29
. More recently, a peptide inhibitor of PI3Kγ, which is recruited to Gβγ in part through association with GRK2, has also proven to be effective in treating animal models of HF 16, 17, 27
. Collectively these results suggest that generally inhibiting pathologic Gβγ-dependent signaling in failing cardiomyocytes is salutary. Consistent with these observations, our novel small molecule based approach to targeting Gβγ signaling appears to function, at least in part, like peptide inhibitors of Gβγ signaling. Whether the observed effects are due specifically to Gβγ-GRK2 inhibition, or to other Gβγ signaling components, will be a target of our future studies. Importantly, the current study suggests selective interference with a subset of Gβγ interactions, as Gβγ-GIRK signaling was not affected (Online Figure III
Our studies of isolated adult cardiomyocytes showed that small molecule Gβγ inhibition enhanced cAMP production and cardiomyocyte contractility following acute β-AR stimulation. These data are consistent with published results showing similar effects of βARKct expression in cardiac myocytes in culture 28
and in vivo 9
. Since M119 and gallein were also found to reduce Iso-induced GRK2 membrane recruitment, we conclude that these small molecules influence β-AR signaling in cardiomyocytes similar to βARKct.
Gβγ inhibitory compounds M119 and gallein partially normalized cardiac morphology and gene expression and halted HF progression in the acute Iso pump model of HF and in CSQ mice with established HF. These data suggest promising utility of small molecule Gβγ inhibition in treating both new onset and extant HF. Importantly, β-blockers are a current standard therapy in the treatment of human HF. Combination of cardiac-restricted βARKct expression with the β-blocker metoprolol has previously demonstrated synergistic benefit for cardiac function in CSQ mice30
, suggesting a possible synergy of small molecule Gβγ inhibition with β-blocker therapy, which will be a target of our future investigation.
In addition to its role in the cardiomyocytes, recent evidence demonstrates that dysregulation of adrenergic receptor-dependent Gβγ signaling in other tissues may also contribute to cardiac dysfunction. Lymperopoulos and coworkers recently showed a significant increase of GRK2 in the chromaffin cells of the adrenal medulla, suggesting that increased Gβγ-GRK2 association contributes to dysfunctional feedback inhibition of catecholamine release via α2-AR signaling 31
. Interestingly, viral delivery of βARKct to the adrenal gland restored α2-AR feedback inhibition of catecholamine release and dramatically improved cardiac function, due at least in part to decreased chronic stimulation of cardiac β-ARs. Increased GRK2 expression is also associated with hypertension 32, 33
. β-AR signaling normally promotes vasodilation but overexpression of GRK2 within vascular smooth muscle cells leads to diminished β-AR signaling and elevated resting blood pressure, a major risk factor for HF 34
. Because heart disease can result from dysfunctional signaling in multiple organs, we and others 31
believe that systemic delivery of small molecule Gβγ inhibitors could simultaneously target multiple causes of this disease. This approach to modulating intracellular signaling is also consistent with the growing trend of therapeutics targeting intracellular components of pathological signaling in cardiovascular disease 35
In summary we have demonstrated that small molecule Gβγ inhibitors function in vivo
to improve cardiac function and halt HF progression in both new onset and extant HF in mice. Small molecule Gβγ inhibitors appear to maintain β-AR responsiveness, in part by interfering with GRK2 membrane recruitment. Future studies will determine whether Gβγ inhibitor compounds modulate other known Gβγ-dependent signaling events, including Gβγ-mediated signaling via novel ERK1/2 phosphorylation36
, and whether these effects contribute to their effects in cardiac pathophysiology. Such studies will aim to determine whether small molecule targeting of Gβγ may be an effective therapeutic paradigm for the treatment of HF.
Novelty and Significance
What is known?
- Excess signaling through cardiac G-protein βγ (Gβγ)-subunits via β-adrenergic receptors (β-AR) is a major component of the pathologic changes associated with the progression of heart failure (HF).
- Numerous reports indicate that peptide inhibitors of β-AR/Gβγ signaling can block many of the molecular changes associated with HF and can improve cardiac function.
- We recently identified two membrane permeable, structurally-related small molecules that selectively target Gβγ-protein interactions that can be used effectively in vivo.
What new information does this article contribute?
- Two structurally-related small molecules that target specific Gβγ signaling pathways are cardioprotective in mouse models of both new onset and extant HF.
- Cardiac protection conferred by these Gβγ targeting compounds is associated with partial reversal of pathologic molecular changes known to occur in HF.
Prior studies have demonstrated that interfering with Gβγ signaling downstream of β-AR activation is cardioprotective in HF models. Previous approaches generally used large peptides that required viral vector delivery to target cells/organs. Here we demonstrate that systemic delivery of small molecule inhibitors of Gβγ signaling can disrupt pathologic molecular changes underlying HF. Further, we demonstrate that these small molecules reduce the progression of HF in two distinct mouse models. Our study provides rationale for further development of Gβγ-targeting compounds as a therapeutic approach for HF. This could lead to new cardiac therapies that, in combination with existing drugs, may improve patient health.