SUMMARYThe mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis, and autophagy1. Its hyper-activation contributes to disease in many organs including the heart1,2, 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 activity3–9. 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 disease10–13. 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.

A primary defect in human SSc-PAH resides in depressed sarcomere function, whereas this is enhanced in IPAH. These disparities correlate with in vivo RV contractility and contractile reserve and are consistent with worse clinical outcomes in SSc-PAH. The existence of sarcomere disease before the development of resting PAH in patients with SSc-d suggests that earlier identification and intervention may prove useful.

Left ventricular hypertrophy (LVH) is a major risk factor for cardiovascular morbidity and mortality. Pathological LVH engages transcriptional programs including reactivation of canonical fetal genes and those inducing fibrosis. Histone lysine demethylases (KDMs) are emerging regulators of transcriptional reprogramming in cancer, though their potential role in abnormal heart growth and fibrosis remains little understood. Here, we investigate gain and loss of function of an H3K9me2 specific demethylase, Kdm3a, and show it promotes LVH and fibrosis in response to pressure-overload. Cardiomyocyte KDM3A activates Timp1 transcription with pro-fibrotic activity. By contrast, a pan-KDM inhibitor, JIB-04, suppresses pressure overload-induced LVH and fibrosis. JIB-04 inhibits KDM3A and suppresses the transcription of fibrotic genes that overlap with genes downregulated in Kdm3a-KO mice versus WT controls. Our study provides genetic and biochemical evidence for a pro-hypertrophic function of KDM3A and proof-of principle for pharmacological targeting of KDMs as an effective strategy to counter LVH and pathological fibrosis.

Rationale: Stimulated protein kinase G-1α (PKG1α) phosphorylates tuberous sclerosis complex 2 (TSC2) at serine 1365, potently suppressing mTORC1 activation by neuro-hormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine 42 is also induced by these stressors, which blunts its cardioprotective effects. Objective: We tested the dependence of mTORC1 activation on PKG1α C42-oxidation, and its capacity to suppress such activation by soluble guanylyl cyclase-1 (GC-1) activation. Methods and Results: Cardiomyocytes expressing WT PKG1α (PKG1α WT ) or C42S redox-dead PKG1α CS/CS were exposed to endothelin-1 (ET-1). Cells expressing PKG1α WT exhibited substantial mTORC1 activation (p70 S6K, 4EBP1, and Ulk1 phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1α CS/CS expression. Mice with global knock-in of PKG1α CS/CS subjected to pressure-overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1α WT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2 S1365 phosphorylation increased in PKG1α CS/CS more than PKG1α WT myocardium following PO. TSC2 S1365A/S1365A KI mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1α CS/CS mice, protection against PO-induced mTORC1 activation, cardio-depression, and mortality in PKG1α CS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1α CS/CS after PO, and blocked ET-1 stimulated mTORC1 in TSC2 S1365A expressing myocytes. Conclusions: Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.