We have demonstrated that Rheb is inhibited in response to GD and prolonged ischemia, and inhibition of Rheb in turn inhibits mTORC1 in CMs. Forced activation of Rheb in such conditions stimulates ATP depletion and ER stress by suppression of autophagy, thereby inducing cell death. Thus, Rheb acts as a sensor of energy stress and as a critical regulator of CM survival in response to energy starvation.
We have shown previously that both GD and ischemia in CMs and in the heart, respectively, induce suppression of mTOR 19, 20
. It should be noted that mTOR is inhibited by both Rheb-dependent and independent mechanisms 19, 20
. For example, several upstream kinases, including AMPK and GSK-3β, which indirectly regulate mTOR, inhibit Rheb through phosphorylation and consequent activation of GAP activity in TSC2 2, 3, 8
. It should be noted, however, that mTOR is also inhibited through Rheb-independent mechanisms, such as Akt-dependent phosphorylation of mTOR and PRAS40, and AMPK-dependent Raptor phosphorylation 8
. Our results indicate that Rheb interacts with mTOR, that Rheb is inhibited during GD and myocardial ischemia, and that its inactivation is required for mTORC1 inhibition. Conversely, neither overexpression of Rheb nor GD affected the activity of mTORC2. We therefore propose that Rheb acts as a central and direct regulator of mTORC1 during energy starvation in CMs.
Our results suggest that forced Rheb activation exacerbates cell death and apoptosis during GD and prolonged ischemia, but it does not affect cell survival at baseline. On the other hand, downregulation of Rheb increased survival of CMs during GD, intimating the involvement of endogenous Rheb in the regulation of survival/death during GD and prolonged ischemia. Previous studies have indicated that Rheb promotes cell survival and inhibits apoptotic cell death in response to stress in several cancer cells 5, 6
. Activation of Rheb in unstressed conditions also induces hypertrophy without cell death in CMs (not shown). Thus, the function of Rheb in cells appears to be context-dependent.
Importantly, we found that downregulation of mTORC1 mediates the protective effects of Rheb inhibition during energy deprivation, since depletion of Raptor but not of Rictor, which are the adaptor proteins of complex 1 and complex 2 of mTOR, respectively, increased survival of Rheb-overexpressing CMs during GD in vitro. In addition, pharmacological and genetic inhibition of mTORC1 reduced the susceptibility to ischemic myocardial damage of Rheb-overexpressing and obese mice, and a protective effect was also observed in control animals in vivo. These results suggest that Rheb is an obvious therapeutic surrogate of mTORC1, to achieve increased CM survival during energy deprivation.
The role of mTOR in regulating the stress response is poorly understood in terminally differentiated cell types such as CMs. In particular, the role of mTOR in the regulation of CM survival has been primarily investigated through indirect means, e.g., the use of pharmacological inhibitors, which may have mTOR-independent effects 19, 20
. In addition, the role of mTOR in cardiac stress has been mostly studied in animal models of chronic ventricular remodeling in which mTORC1 is activated, while we observed mTORC1 inhibition during CM energy deprivation 9–11
. Interestingly, mTORC1 activation has been indicated as protective during cardiac mechanical overload 9, 11
. On the other hand, in our study, we demonstrated that selective and direct mTORC1 activation is detrimental during acute cardiac energy deprivation, whereas both pharmacological and genetic mTORC1 inhibition are protective. In particular, we provided the first evidence that genetic mTOR inhibition is protective during myocardial ischemia. Thus, the function of mTORC1 in CMs appears to be context-dependent. mTORC1 activation might be required for cell growth in response to mechanical overload, whereas mTORC1 inhibition is important for preservation of energy status in response to energy deprivation.
Rheb inhibition during energy deprivation is required for autophagy activation, which is protective in this condition. In fact, suppression of autophagy by knockdown of Beclin-1 completely abrogated the protective effect of Rheb knockdown in CMs during GD. Restoration of autophagy through treatment with trehalose or overexpression of Atg7, which stimulates autophagy through mTOR-independent mechanisms, significantly reduced CM death induced by forced Rheb activation. Therefore, although it is still debated whether autophagy is protective or detrimental during cardiac stress 24
, we have demonstrated that Rheb-regulated autophagy is protective during CM nutrient starvation and ischemia. In particular, we showed that Rheb-regulated autophagy is protective through the preservation of ATP content and reduction of misfolded protein accumulation, namely ER stress.
Rheb-induced inhibition of autophagy was accompanied by downregulation of Atg7 protein levels. Overexpression of Atg7 was sufficient to restore autophagy and to suppress Rheb-induced cell death during GD, suggesting that Rheb regulates autophagy in part through Atg7. mTORC1 was suggested to modulate autophagy through Ulk1/2 regulation 21
. The role of Ulk1/2 in mediating expression of Atg7 remains to be elucidated.
Interestingly, inadvertent activation of the Rheb/mTORC1 pathway is observed in HFD-induced obesity. Obesity is characterized by glucose intolerance and dyslipidemia, and it is associated with an increased susceptibility to myocardial ischemia 13–16
. We demonstrated that autophagy is reduced in the hearts of mice with HFD-induced obesity. These mice exhibited exacerbated myocardial injury in response to prolonged ischemia, which was normalized by rapamycin treatment or genetic mTOR inhibition, suggesting that increased mTORC1 activity may be responsible for the increased susceptibility. Remarkably, inhibition of Beclin-1 was associated with the failure of pharmacological mTORC1 inhibition to reduce ischemic injury in HFD mice, indicating that re-activation of autophagy is the crucial mechanism mediating the beneficial effects of mTORC1 inhibition in HFD-induced obesity.
Severe obesity and metabolic syndrome are associated with increased cardiovascular risk events and a poor prognosis in patients after acute MI 12, 14, 15, 25, 26
. If our results hold true in humans, it may be helpful to treat patients with obesity and metabolic syndrome using pharmacological inhibitors of Rheb or mTORC1, to stimulate autophagy during an acute episode of myocardial ischemia. Our results are also supported by an interesting previous study which showed that obesity increases vascular senescence and vascular dysfunction in response to mTOR activation27
Other previous studies showed increased basal mTORC1 activity in the liver 17, 28
, adipose tissue 29
, vasculature 27
, skeletal muscle 17, 28, 30, 31
and cardiac muscle 32–34
in both genetic and diet-induced models of obesity and dysmetabolic conditions. AMPK inhibition has been proposed as the main intracellular mechanism leading to mTORC1 activation 18, 30–34
. Our study extends this previous evidence, suggesting that Rheb is involved in the activation of mTORC1 induced by AMPK downregulation.
Several stimuli may enhance the activity of the Rheb/mTORC1 pathway in the tissues of obese and dysmetabolic animals. High caloric intake may represent one possible cause. High levels of circulating and cardiac lipids may also represent potential mechanisms. In addition, increases in circulating insulin, amino acids, cytokines and adipokines may contribute to the increased Rheb/mTORC1 activity in HFD mice 17, 18, 28, 31, 32, 35
In summary, our study demonstrates that inactivation of Rheb protects CMs during energy deprivation through activation of autophagy, reduction of energy expenditure and attenuation of ER stress (Figure XIIE
). Rheb and mTORC1 may represent therapeutic targets to reduce myocardial damage during acute myocardial ischemia, particularly in patients with obesity and metabolic syndrome.
The incidence of heart failure after acute myocardial infarction (MI) remains very high in patients. This highlights the necessity to clarify the mechanism regulating the survival and death of cardiomyocytes in response to ischemia and to find new cardioprotective therapies reducing ischemic injury. We discovered that Rheb, a small GTP-binding protein, plays a pivotal role in regulating the survival of cardiomyocytes during prolonged myocardial ischemia. Rheb activity is reduced in the ischemic heart, thereby causing the suppression of the mTORC1 pathway. Inhibition of the Rheb/mTORC1 pathway is an adaptive response during ischemia, because forced restoration of cardiac Rheb activity is detrimental under this condition. Rheb inhibition is required for the activation of autophagy, an intracellular degradation process for proteins and organelles, which is protective during energy stress through preservation of cellular energy and relief of ER stress. We discovered that obesity and metabolic syndrome (Ob/MS) are associated with cardiac activation of Rheb/mTORC1 at baseline and during ischemia. In obese mice, autophagy in the heart was suppressed and ischemic injury was exacerbated. Remarkably, inhibition of mTORC1 restores autophagy and reduces infarct size in these animals after prolonged ischemia. Thus, our results suggest that Rheb and mTORC1 may be promising therapeutic targets to reduce myocardial damage after prolonged ischemia in patients with Ob/MS who display deregulated activation of the Rheb/TORC1 pathway and consequent inhibition of autophagy.