Tuberous sclerosis complex-2 (TSC2) negatively regulates mammalian target of rapamycin complex 1 (mTORC1), and its activity is reduced by protein kinase B (Akt) and extracellular response kinase ...(ERK1/2) phosphorylation to activate mTORC1. Serine 1364 (human) on TSC2 bidirectionally modifies mTORC1 activation by pathological growth factors or hemodynamic stress but has no impact on resting activity. We now show this modification biases to ERK1/2 but not Akt-dependent TSC2-mTORC1 activation. Endothelin-1-stimulated mTORC1 requires ERK1/2 activation and is bidirectionally modified by phospho-mimetic (S1364E) or phospho-silenced (S1364A) mutations. However, mTORC1 activation by Akt-dependent stimuli (insulin or PDGF) is unaltered by S1364 modification. Thrombin stimulates both pathways, yet only the ERK1/2 component is modulated by S1364. S1364 also has negligible impact on mTORC1 regulation by energy or nutrient status. In vivo, diet-induced obesity, diabetes, and fatty liver couple to Akt activation and are also unaltered by TSC2 S1364 mutations. This contrasts to prior reports showing a marked impact of both on pathological pressure-stress. Thus, S1364 provides ERK1/2-selective mTORC1 control and a genetic means to modify pathological versus physiological mTOR stimuli.
Type 2 Diabetes mellitus (T2DM) is a common comorbidity in patients after heart transplantation (HTx) and is associated with adverse long-term outcomes.
The retrospective study reported here analyzed ...the effects of vildagliptin therapy in stable patients post-HTx with T2DM and compared these with control patients for matched-pairs analysis. A total of 30 stable patients post-HTx with T2DM were included in the study. Fifteen patients (mean age 58.6 ± 6.0 years, mean time post-HTx 4.9 ± 5.3 years, twelve male and three female) were included in the vildagliptin group (VG) and 15 patients were included in the control group (CG) (mean age 61.2 ± 8.3 years, mean time post-HTx 7.2 ± 6.6 years, all male).
Mean glycated hemoglobin (HbA1c) in the VG was 7.4% ± 0.7% before versus 6.8% ± 0.8% after 8 months of vildagliptin therapy (P = 0.002 vs baseline). In the CG, HbA1c was 7.0% ± 0.7% versus 7.3% ± 1.2% at follow-up (P = 0.21). Additionally, there was a significant reduction in mean blood glucose in the VG, from 165.0 ± 18.8 mg/dL to 147.9 ± 22.7 mg/dL (P = 0.002 vs baseline), whereas mean blood glucose increased slightly in the CG from 154.7 ± 19.7 mg/dL to 162.6 ± 35.0 mg/dL (P = 0.21). No statistically significant changes in body weight (from 83.3 ± 10.8 kg to 82.0 ± 10.9 kg, P = 0.20), total cholesterol (1.5%, P = 0.68), or triglyceride levels (8.0%, P = 0.65) were seen in the VG. No significant changes in immunosuppressive drug levels or dosages were observed in either group.
Vildagliptin therapy significantly reduced HbA1c and mean blood glucose levels in post-HTx patients in this study with T2DM and did not have any negative effects on lipid profile or body weight. Thus, vildagliptin therapy presented an interesting therapeutic approach for this selected patient cohort.
Abstract only Rationale: The Mechanistic Target of Rapamycin complex 1 (mTORC1) integrates signaling and sensory inputs to maintain cardiomyocyte homeostasis, and itself is negatively regulated by ...the signaling nexus tuberin (TSC2). We identified a novel TSC2 phosphorylation site S1365 (pS1365) targeted by protein kinase G (PKG), which suppressed hormonal growth factor (PE or ET1)-stimulated mTORC1 activity to attenuate pathological cardiomyocyte hypertrophy. This was recapitulated during growth factor stimulation with expression of a phospho-null (S1365A) or a phospho-mimetic (S1365E) TSC2 that exacerbated or blunted mTORC1 activation, respectively. The nature of TSC2 pS1365 as a potential metabolic sensor is unknown and will provide mechanistic insight into the TSC2 kinase input hierarchy that regulates the homeostatic function of mTORC1. Objective: To determine how pS1365 affects the ability of TSC2 to integrate metabolic dependent signals to regulate mTORC1. Methods/Results: TSC2 KO MEFs were infected with TSC2 WT or S1365A adenovirus, and then stimulated with ET1 (hormonal stress that also activates mTORC1), and 2-DG (AMPK stimulation). Both groups responded with similar decreases in mTORC1 activation regardless of pS1365. Phosphorylation of the AMPK site on TSC2 (S1387) was increased in all groups despite the presence of a phospho-null S1365. Both MEFs and neonatal rat cardiomyocytes (NRCMs) infected with TSC2 WT, S1365A, or S1365E adenovirus similarly increased mTORC1 activation with insulin (PI3K-Akt-TSC2 pathway) treatment. Serum withdrawal from NRCMs reduced mTORC1 activation in all groups regardless of whether a WT, S1365A, or S1365E TSC2 was expressed. In NRCMs subject to hypoxia (a combination of Erk, Akt, AMPK signaling), there was a similar observation with only nominal changes between WT, S1365A, and S1365E TSC2. Conclusions: The energy and nutrient sensing role of the TSC2-mTORC1 pathway remains intact regardless of the phospho-status of TSC2 S1365. These findings provide important mechanistic insight into the function of TSC2 pS1365 as a potent suppressor of pathological mTORC1 activation while not affecting the ability of TSC2 to respond to the metabolic dependent signals – AMPK (energy), PI3K-Akt (insulin), and serum starvation.
Abstract only Rationale: Protein kinase G 1α (PKG1α) confers anti-hypertrophic effects in hearts subjected to mechanical and neurohumoral stress. Human heart failure with a reduced ejection fraction ...(HFrEF) and mouse pressure overloaded hearts present with increased mechanistic target of rapamycin complex 1 (mTORC1) activity, protein aggregation, oxidative stress, and, as we previously described, increased PKG1α disulfide dimer formation indicative of PKG1α oxidation. Recently we demonstrated that stimulation of PKG1α phosphorylates tuberin (TSC2) at one specific serine, S1365, to inhibit mTORC1 signaling and attenuate pathological hypertrophy. Objective: To determine if the redox state of PKG1α impacts its ability to target TSC2 signaling in a chronic pressure-overload mouse model exhibiting pathologic hypertrophy. We hypothesize non-oxidized PKG1α will increase TSC2 S1365 phosphorylation to antagonize mTORC1 signaling, thereby enhancing autophagic flux to clear protein aggregates, culminating in ameliorated cardiac disease. Methods and Results: Mice expressing a non-oxidizable (redox-dead) PKG1α (cysteine 42 serine, CS) knock-in mutation and wild type (WT) littermate controls were subjected cardiac pressure overload stress via transaortic constriction (TAC) or sham surgeries. Following TAC, PKG1α CS mice exhibited reduced mTORC1 activation leading to increased autophagic flux and preventing protein aggregation, compared to WT mice. PKG1α CS TAC mice had decreased expression of the hypertrophic genes, attenuated cardiac hypertrophy (p<0.0001), and improve fractional shortening compared to WT TAC mice (28.14%±10.82 in WT vs. 47.42%±15.62 in CS; p<0.01). Treating WT TAC mice with an mTORC1 inhibitor (everolimus) abrogated mTORC1 hyperactivation, which lead to enhanced autophagic flux, attenuated hypertrophy, and improved cardiac function. Crossing PKG1α CS mice with TSC2 S1365 phospho-null mice resulted in increased cardiac hypertrophy and reduced lifespan (p<0.05). Conclusions: Preventing PKG1α oxidation attenuates mTORC1 activation to enhance autophagic flux, prevent protein aggregation, and ameliorate pathological hypertrophy in following cardiac pressure overload, dependent onTSC2 S1365 phosphorylation.
Abstract only O-GlcNAcylation is a dynamic, reversible posttranslational modification (PTM) that regulates a multitude of biological processes. Fluctuations in O-GlcNAC of various calcium handling ...proteins impact their functionality in cardiomyocytes. Here, we show for the first time that TRPC6, a nonselective receptor-operated cation channel and mediator of hypertrophy and fibrosis, is constitutively O-GlcNAcylated in the ankyrin repeat domain (AR4), at Ser 14, Thr 70, and Thr 221 within the N-terminal cytoplasmic segment. Of these, only substitution of Thr 221 with alanine (T221A) results in a change of function, notably a hyperactive TRPC6 channel with 5X greater increase in consequent NFAT promoter activity, which is a marker of TRPC6 calcium signaling. Patch-clamp analysis of T221A mutant channels found a 75-80% increased conductance compared to WT. Myocardial injection of T221A in homozygous TRPC6 KO mice by AAV-9 mediated gene transfer results in systolic dysfunction, hypertrophy, and cardiac fibrosis, by loss of OGlcNAc modification at site T221. T221 is highly conserved across species and found in the AR4 domain, which forms the core structure of TRPC6 intracellular domain. Mutating the site in its closest homologs, TRPC3 and TRPC7, also activates channel activity. T221 O-GlcNAcylation also protects the nascent protein from premature proteasomal degradation. Molecular modeling from the crystal structure of human TRPC6 predicts that OGlcNACylation stabilizes electrostatic interactions with the 193-203 loop near AR4, and loop connecting AR4 to the linker helix 1 (LH1) at S199, E200, and E246. Mutating these sites to alanine also increases TRPC6-NFAT signaling similar to what was observed in the T221A mutant. In summary, this study highlights that O-GlcNAcylation of TRPC6 is an important PTM needed to stabilize channel function, and its decline results in gain-of-function related diseases.
Abstract only Brain-derived neurotrophic factor (BDNF)/ tyrosine receptor kinase B (TrkB) signaling is essential for normal cardiac contraction/relaxation. Alterations in this pathway, i.e., ...defective neuronal BDNF, account for post-ischemic cardiac injury. Less clear, however, is whether myocyte-borne BDNF has a role in this setting. We generated myocyte-selective BDNF knock-out (myoBDNF -/- ) mice, using Myh6-Cre mice crossed with BDNF floxed mice, confirming bdnf deletion via RT-PCR in isolated myocytes. Hearts from 12-wk old myoBDNF -/- mice and WT littermates underwent global ischemia (30 min) and reperfusion (2 h). At this age, the two strains had similar left ventricular (LV) sizes and fractional shortening 63±1.1 (WT) vs. 60±1.2% (myoBDNF -/- ). At reperfusion, myoBDNF -/- hearts displayed larger infarct size compared to WT (38±3 vs. 14±2%, n=9, p<.0001) and worsened LV functional recovery ( Fig. 1 ). For example, the rate-pressure product recuperated only by 14±1.5 in myoBDNF -/- mice vs. 36±3% in WT (p<.0001). The two groups had similar heart rates at 2 h reperfusion, however myoBDNF -/- mice markedly lost their contractility dP/dt max = 436±58 vs. 1407±142 mmHg/sec (WT), p< .0001, likely due to the exacerbated myocyte loss. Accordingly, post-ischemic troponin I release was significantly higher in myoBDNF -/- than in WT mice (0.9±0.04 vs. 1.3±0.04 ng/ml, p<.0002). Thus, deleting bdnf in myocytes severely limits recovery after ischemia, directly linking myocyte-borne BDNF to the heart response to injury. Therefore, preserving or enhancing autologous myocyte BDNF generation offers new avenues to counter cardiac ischemic injury and subsequent heart failure progression.
The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis, and autophagy
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. Its hyper-activation contributes to disease in many organs ...including the heart
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, though broad mTORC1 inhibition risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 by modulating Rheb (Ras homolog enriched in brain). TSC2 constitutively inhibits mTORC1, but this activity is modified by phosphorylation from multiple signaling kinases to in turn inhibit (AMPK, GSK3β) or stimulate (Akt, ERK, RSK-1) mTORC1 activity
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. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here, we reveal phosphorylation or gain-or-loss of function mutations at either of two adjacent serines in TSC2 (S1365/1366 mouse; 1364/1365 human), with no prior known function, is sufficient to bi-directionally potently control growth-factor and hemodynamic-stress stimulated mTORC1 activity and consequent cell growth and autophagy. Basal mTORC1 activity, however, is unchanged. In heart, myocytes, and fibroblasts, phosphorylation occurs by protein kinase G (PKG), a primary effector of nitric oxide and natriuretic peptide signaling whose activation is protective against heart disease
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. PKG suppression of hypertrophy and stimulation of autophagy in myocytes requires TSC2 phosphorylation. Homozygous knock-in (KI) mice expressing a phospho-silenced TSC2 (S1365A) mutation develop far worse heart disease and mortality from sustained pressure-overload (PO) due to hyperactive mTORC1 that cannot be rescued by PKG stimulation. Heterozygote SA-KI are rescued, and KI-mice expressing a phospho-mimetic (S1365E) mutation are protected. Neither KI model alters resting mTORC1 activity. Thus, TSC2 phosphorylation is both required and sufficient for PKG-mediated cardiac protection against pressure-overload. These newly identified serines provide a genetic tool to bi-directionally regulate the amplitude of stress-stimulated mTORC1 activity.
IntroductionTumor cells survive hypoxic, nutrient challenged environments by shifting metabolism to aerobic glycolysis while reducing oxidative phosphorylation; known as the Warburg effect. A major ...controller of this shift is mechanistic target of rapamycin complex-1 (mTORC1). We recently discovered a single serine on tuberous sclerosis complex 2 (TSC2) at position 1365 that acts as a rheostat on growth-stimulated mTORC1 activation, and its phosphorylation potently protects hearts exposed to pressure overload. Its impact on cardiac metabolic adaptation to ischemic injury is unknown.HypothesisWe tested that TSC2 S1365 modifies metabolic substrate utilization favoring glycolysis when mutated so it cannot be phosphorylated, enhancing mTORC1 activation and inducing a Warburg effect to improve post-ischemic outcome.Methods & ResultsHearts from knock-in (KI) mice expressing a phospho-silencing mutation (S1365A, TSC2) or a phospho-mimetic mutation (S1365E, TSC2) were exposed to 30 or 45 min myocardial ischemia (IS) +/- 2 or 6 hours reperfusion (IR) ex vivo or in vivo, respectively. Ex vivo TSC2 hearts had greater function after IR, similar to that from classical pre-conditioning. In vivo, TSC2 mice had higher ejection fraction during IS vs TSC2, and this increased further upon reperfusion (EFTSC235.1%±7.1, n=8; TSC250.1%±5.7, n=7; p<0.001). TSC2 hearts had higher gene expression of lactate dehydrogenase B, and higher lactate and pyruvate levels indicating more glycolysis. Oncometabolites rose more in TSC2 mice, and hypoxia-inducible factor 1 alpha (HIF-1α) levels rose. During IS, 14-3-3- binding to TSC2 was reduced compared to TSC2, revealing a novel regulation of TSC2 by S1365 and capacity to modulate TSC2 activity and thus mTORC1 activation by this mechanism.ConclusionsTSC2 S1365 modulates a metabolic switch during IS and particularly IR. Its suppression induces a cardiac Warburg effect, favoring glycolysis and enhancing function in IR hearts. The pathway provides a new means to enhance cell function/survival in metabolically challenging environments.
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