AKT-mTOR Signaling-Mediated Rescue of PRKAG2 R302Q Mutant-Induced Familial Hypertrophic Cardiomyopathy by Treatment with β-AR Blocker Metoprolol

PRKAG2 cardiac syndrome, as a common form of metabolic hypertrophic cardiomyopathy (HCM) caused by mutations in PRKAG2 gene, often shows myocardial hypertrophy and abnormal glycogen deposition in cardiomyocytes. However, it remains incurable due to lacking of a management guideline for treatment. Herein, a β1-AR blocker Metoprolol was applied to 5 patients with PRKAG2 cardiac syndrome identied from a PRKAG2 R302Q mutant family, resulting in signicantly postponed progression of cardiac hypertrophy. Overexpression of PRKAG2 R302Q in primary cardiomyocytes increased the activity of AMPK, induced cellular hypertrophy and glycogen storage, and promoted the phosphorylation levels of AKT-mTOR signaling. Application of either β1-AR blocker metoprolol or protein kinase A (PKA) inhibitor H89 to the cardiomyocytes rescued the HCM-like phenotypes induced by PRKAG2 R302Q, including suppression of both AKT-mTOR phosphorylation and AMPK activity. In conclusion, the current study not only determined the mechanism regulating HCM induced by PRKAG2 R302Q mutant, but also demonstrated a therapeutic strategy using β1-AR blocker to treat the patients with PRKAG2 cardiac syndrome.


Introduction
Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder with characteristics of increased ventricular wall thickness, cadiomyocyte hypertrophy and myocardial brosis [1]. PRKAG2 cardiac syndrome, as a common form of metabolic HCM caused by mutations in the PRKAG2 gene encoding the AMP-activated protein kinase (AMPK) γ2 subunit, often shows abnormal glycogen deposition in cardiomyocytes, ventricular preexcitation and progressive cardiac conduction disturbances in addition to myocardial hypertrophy [2]. AMPK is a heterotrimeric protein composed of α, β, and γ subunits, acting as the key enzyme responsible for regulating cellular energy homeostasis. AMPK activation turns on the catabolic pathway to produce ATP and turns off the anabolic pathway that requires ATP consumption, maintaining the dynamic energy balance [3]. Genetic mutants in PRKAG2 have been demonstrated to cause inappropriate activation of AMPK, leading to arrhythmias and cardiac insu ciency. For example, elevated activity of AMPK and decreased sensitivity to AMP were reported in the cardiomyotic cells with PRKAG2 K475E mutant and in the myocardium of PRKAG2 N488I mutant mice, associated with activation of mTOR/p70S6K/4EBP1 and/or Akt-mTOR-FOXO3A pathways [4,5]. In consistence, PRKAG2 mutants-induced myocardial hypertrophy can be rescued by application of the mTOR inhibitor rapamycin.
The β-adrenergic receptor (β-AR) signaling pathway is one of the pathways mediating induction of cardiac hypertrophy, which has been suggested to be associated with signaling pathways such as cAMP-PKA, MAPK, and PI3K. A phosphoproteomic analysis in vivo revealed that the kinases AMPK, AKT, and mTOR are also involved in β-AR signaling regulation of HCM [6]. In addition, β-AR showed regulation of glucose uptake and glycogen synthesis [7]. β1-AR agonists reduced insulin-induced glucose uptake and glycogen synthesis [8]. Glycogen storage in cardiomyocytes has been shown to be related with myocardial hypertrophy. Reduction of glycogen was associated with improvement of cardiac function, suggesting the therapeutic values for HCM of preventing glucose uptake by cardiomyocytes. However, Maengjo et al found that inhibition of glycogen deposition in N488I mutation model reversed ventricular preexcitation but showed limited effect on rescuing cardiac hypertrophy, suggesting the correlation between myocardial hypertrophy and glycogen storage might be in a genetic disorder-dependent manner [4].
To date, effects of all kinds of clinic treatment to PRKAG2 cardiac syndrome patients remain very limited yet mainly due to lacking of a well-accepted management guideline for the disease. Metoprolol, as a selective β1-AR blocker, interacts with β1-AR to inhibit β1-AR signaling, leading to improvement of myocardial remodeling, and rescue of cardiac hypertrophy caused by infarction or hypertensive [9,10]. Our previous study treated 5 patients in a PRKAG2 R302Q-induced HCM family with a long-term oral administration of metoprolol, resulting in signi cant prevention of myocardial hypertrophy progression [11]. However, the regulatory mechanism remains unclear.
In order to validate the function and mechanism of β-blocker in regulating myocardial hypertrophy, a uorescent-labeled adenoviral virus carrying either wild type or R302Q mutant of PRKAG2 gene was Cardiomyocyte cell culture and Infection H9C2 cells were cultured in DMEM (Gibco), supplemented with 10% fetal bovine serum (FBS, Sigma), and 1% penicillin-streptomycin (Gibco) at 5% CO2 and 37°C. The Neonatal rat cardiomyocytes (NRCMs) were isolated from the heart of newborn (1 day old) rat with PBS containing 0.01% collagenase type II. The cardiomyocytes were then seeded at a density of 1 × 10 6 cells/well in six-well culture plates coated with bronectin in plating medium, which consisted of F12 medium supplemented with 10% fetal calf serum and penicillin/streptomycin. Cells were grown to 70% con uence before infection with PRKAG2 gene γ 2 WT-(Adγ 2 WT), or γ 2 R302Q-(Adγ 2 R302Q) overexpressing adenoviruses at a multiplicity of infection of 36. Cells were harvested after forty-eight hours for further analysis.
AMPK activity analysis To assess the effect of PRKAG2 R302Q mutant on the activity of AMPK, a Cell AMPK Kinase Activity Colorimetric Quantitative Detection Kit (Haring Creature, China) was used following the manufacturer's instructions. Mean values of the absorbance from triplicates were calculated for each sample.
Cellular glycogen analysis Glycogen staining was performed using a Periodic Acid-Schiff kit (PAS, beyotime, China). The amount of glycogen storage in cells was quanti ed using a glycogen content assay kit (Solarbio, BC0340, China) according to the manufacturer's instructions.
Immuno uorescence analysis Cells were xed with 3.7% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 40 min, blocked with a 10% BSA solution for 1 hour at room temperature and then incubated with an primary antibody (1:50 dilution). Nuclei were stained with DAPI (Invitrogen, USA) for 10 minutes in the dark. Images were taken with a uorescence confocal microscope. Image-Pro Plus 6.0 software was used for quantitative analysis.
Cell proliferation assay Cell proliferation was analyzed using cell counting kit-

Results
Treatment of PRKAG2 cardiac syndrome with β1-AR blocker metoprolol. As described in our recent publication, 5 members in a family with PRKAG2 R302Q mutant were diagnosed as PRKAG2 cardiac syndrome, showing complete atrioventricular block and asymmetric ventricular septal hypertrophy [11] ( Figure 1A). Metoprolol, as a selective β1-AR blocker, was applied to these patients for a long-term treatment. As a result, progression of the cardiac hypertrophy was signi cantly postponed in all the 5 patients upon metoprolol treatment ( Figure 1B,1C), including decreased ventricular hypertrophy rate measured by the thickness of the intraventricular septum (IVSth) ( Figure 1B) and increased size of left ventricular end-diastolic dimension (LVEDD) ( Figure 1C). The patients' information and other clinical parameters have been described recently [11].
Activation of AMPK in cardiomyocytes by overexpression of PRKAG2 R302Q. In order to determine the regulation of AMPK activity by PRKAG2 R302Q mutant in cardiomyocytes, wild type (WT) or R302Q mutant (MUT) PRKAG2 gene were cloned into an adenovirus vector, and then transfeted into primary NRCMs for further analysis ( Figure 2A). As shown in Figure 2B and 2C, overexpression of PRKAG2 was con rmed at mRNA and protein levels in both WT and MUT vector-transfected cells. Moreover, AMPK activity showed upregulation by both WT and MUT PRKAG2 ( Figure 2D). In consistence, phosphorylation of AMPK was promoted by both WT and MUT PRKAG2 ( Figure 2E). Notably, MUT PRKAG2 promoted phosphorylation of AMPK and activity of AMPK with a higher level than WT PRKAG2 ( Figure 2D, 2E).
PRKAG2 R302Q induced myocardial hypertrophy and glycogen storage in cardiomyocytes. In order to determine the mechanism regulating PRKAG2 R302Q mutant-induced myocardial hypertrophy, NRCMs and H9C2 cells overexpressing WT or MUT PRKAG2 were analyzed with myocardial hypertrophy, cell proliferation and glycogen storage. Cell hypertrophy showed induction by MUT PRKAG2, which was measured through α-SMA staining ( Figure 3A, 3B). Glycogen storage in cardiomyocytes was increased by WT or MUT PRKAG2 ( Figure 3C, 3D). In consistence, increased expression levels of myocardial hypertrophy markers, such as ANP, BNP and β-MHC, were observed in those cells ( Figure 3E). To determine the regulation of cell proliferation by PRKAG2, both EDU staining and CCK8 assay were applied to the cardiomyocytes. As shown in Figure 3F and 3G, PRKAG2 overexpression signi cantly promoted the proliferation of cardiomyocytes.
Rescue of PRKAG2 R302Q-induced myocardial hypertrophy by β1-AR blocker and PKA inhibitor. In order to validate the therapeutic effects and explore the mechanisms of β1-AR blockers to cure the patients with PRKAG2 cardiac syndrome, either β1-AR blocker metoprolol or PKA inhibitor H89 was applied to the PRKAG2 R302Q MUT-expressing cardiomyocytes, followed by analysis of cellular hypertrophy, glycogen storage and cell proliferation. As shown in Figure 4, both metoprolol or H89 treatment can partly or completely rescue the PRKAG2 R302Q MUT-induced phenotypes including AMPK activity ( Figure 4A), cellular size ( Figure 4B, 4C), glycogen storage ( Figure 4D, 4E), expression levels of ANP, BNP and β-MHC ( Figure 4F), and cell proliferation ( Figure 4G-4I).
PRKAG2 R302Q activated AKT-mTOR signaling in cardiomyocytes. In order to explore the molecular mechanisms through which PRKAG2 R302Q induces myocardial hypertrophy, we rstly analyzed AKT-mTOR signaling in cardiomyocytes overexpressing WT or MUT PRKAG2. As shown in Figures 5A-5D, MUT PRKAG2 signi cantly activated AKT-mTOR signaling by promoting the phosphorylation levels of AKT, mTOR and downstream target genes p70S6K and 4EBP1. In consistence with the cellular phenotypes in Figure 4, both β1-AR blocker metoprolol and PKA inhibitor H89 treatment reversed the MUT PRKAG2induced phosphorylation of AKT, mTOR, p70S6K and 4EBP1 ( Figure 5E-5H).

Discussion
As a highly genetically heterogeneous disease, 50%~60% of HCM are caused by mutations in myosin genes [12]. Recent evidence indicated that metabolism-related mutants can also result in myocardial hypertrophy, called "metabolic HCM". PRKAG2 cardiac syndrome is a typical form of metabolic HCM caused by mutations in the PRKAG2 gene, which encodes the γ2 subunit of AMPK [13]. PRKAG2 cardiac syndrome has a high incidence of sudden cardiac death [14][15]. However, there is still no o cial guidelines available for management and treatment of PRKAG2 cardiac syndrome [16]. In the present study, we are the rst to apply β1-AR blocker metoprolol to ve patients with PRKAG2 cardiac syndrome, resulting in great therapeutic effects. In vitro studies using NRCMs and H9C2 cell line demonstrated rescue of the PRKAG2 R302Q-induced cardiomyocyte hypertrophy and glycogen storage by treatment with β-AR inhibitors. Furthermore, AKT-mTOR signaling pathway was demonstrated to involve in the regulation of PRKAG2 cardiac syndrome. AKT is a serine/threonine kinase involved in the regulation of multiple cellular functions, including metabolism, glucose uptake, proliferation and protein synthesis [17,18]. In the heart, mTOR is a large-molecule serine-threonine protein kinase that is activated by phosphorylation of TSC2 by AKT [19]. Activation of mTOR leads to increased proliferation and elevated size of cardiomyocytes, which are associated with cardiac hypertrophy [20]. p70S6K and 4EBP1, as two of the main target genes downstream of mTOR, are important regulators of protein synthesis in the heart [21,22]. In the current study, overexpression of PRKAG2 R302Q in cardiomyocytes activated AKT-mTOR-p70S6K/4EBP1 signaling, leading to increased cell size and promoted cell proliferation ( Figure 6).
The cardio-protective effects of β-blockers have been well de ned in treatment of patients with coronary heart disease, heart failure, and hypertension, showing clinical outcomes including anti-myocardial ischemia, anti-hypertension, anti-arrhythmias, and increased left ventricular ejection fraction [23,24]. In the model of pressure overload-induced myocardial hypertrophy, β1-AR was highly activated and enriched in the heart, accounting for about 70% of the total cardiac β-ARs [25]. Continuous activation of β1-AR led to myocardial remodeling, myocardial hypertrophy and even heart failure [26]. In consistence, metoprolol showed therapeutic effects here in our study to PRKAG2 R302Q-induced HCM, adding a node to the regulatory network between β-blockers and heart diseases.
Abnormal activation of AMPK has been considered as a main reason causing myocardial hypertrophy, cardiac conduction disturbances, arrhythmias and even sudden death [27,28]. Contradictory results about the effect of PRKAG2 mutants on the AMPK activity were reported. Introduction of PRKAG2 mutants into CCL13 cell line decreased the AMPK activity [29]. However, PRKAG2 cardiac syndrome was shown in the PRKAG2 mutants-transgenic mice, associated with increased AMPK activity in the heart of young mice and decreased sensitivity to AMP [30]. In consistence, here we demonstrated activation of AMPK by PRKAG2 R302Q in cardiomyocytes, In conclusion, the current study demonstrated that application of β1-AR blocker metoprolol is able to ameliorate or even reverse myocardial hypertrophy and glycogen storage induced by PAKAG2 R302Q mutant via activation of the AKT-mTOR signaling pathway, suggesting the potential of metoprolol to be developed as a clinical rst-line drug in treatment of PRKAG2 cardiac syndrome. Nevertheless, there is no doubt that further study through clinical trials will be still required to con rm the therapeutic effects and administrative strategy.
Declarations Figure 1 β1-AR blocker metoprolol suppressed the cardiac hypertrophy in patients with PRKAG2 cardiac syndrome.
A: Schematic representation of the work ow to treat the patients with metoprolol. B: Annual thickness change of the intraventricular septum (IVSth) in the 5 patients before and after treatment with metoprolol. C: Change of left ventricular end-diastolic dimension (LVEDD) in the 5 patients before and after treatment with metoprolol. Data are presented as the mean ± SD. *p<0.05 (n=5).   Application of metoprolol or H89 rescued the PRKAG2 R302Q-induced cell proliferation in cardiomyocytes. Both EDU staining and CCK8 assays were performed. Data are presented as the mean ± SD (n=3). *p<0.05, **p<0.01. ns means non-signi cant. All comparisons were made versus WT group. In the current study, overexpression of PRKAG2 R302Q in cardiomyocytes activated AKT-mTOR-p70S6K/4EBP1 signaling, leading to increased cell size and promoted cell proliferation ( Figure 6).