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.
BACKGROUND:Patients with systemic sclerosis (SSc)–associated pulmonary arterial hypertension (PAH) have a far worse prognosis than those with idiopathic PAH (IPAH). In the intact heart, SSc-PAH ...exhibits depressed rest and reserve right ventricular (RV) contractility compared with IPAH. We tested whether this disparity involves underlying differences in myofilament function.
METHODS:Cardiac myocytes were isolated from RV septal endomyocardial biopsies from patients with SSc-PAH, IPAH, or SSc with exertional dyspnea but no resting PAH (SSc-d); control RV septal tissue was obtained from nondiseased donor hearts (6–7 per group). Isolated myocyte passive length-tension and developed tension-calcium relationships were determined and correlated with in vivo RV function and reserve. RV septal fibrosis was also examined.
RESULTS:Myocyte passive stiffness from length-tension relations was similarly increased in IPAH and SSc-PAH compared with control, although SSc-PAH biopsies had more interstitial fibrosis. More striking disparities were found between active force-calcium relations. Compared with controls, maximal calcium-activated force (Fmax) was 28% higher in IPAH but 37% lower in SSc-PAH. Fmax in SSc-d was intermediate between control and SSc-PAH. The calcium concentration required for half-maximal force (EC50) was similar between control, IPAH, and SSc-d but lower in SSc-PAH. This disparity disappeared in myocytes incubated with the active catalytic subunit of protein kinase A. Myocyte Fmax directly correlated with in vivo RV contractility assessed by end-systolic elastance (R=0.46, P=0.002) and change in end-systolic elastance with exercise (R=0.49, P=0.008) and was inversely related with exercise-induced chamber dilation (R=0.63, P<0.002), which also was a marker of depressed contractile reserve.
CONCLUSIONS: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.
The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis and autophagy
. Its hyperactivation contributes to disease in numerous organs, ...including the heart
, although broad inhibition of mTORC1 risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 that acts through modulation of RHEB (Ras homologue enriched in brain). TSC2 constitutively inhibits mTORC1; however, this activity is modified by phosphorylation from multiple signalling kinases that in turn inhibits (AMPK and GSK-3β) or stimulates (AKT, ERK and RSK-1) mTORC1 activity
. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here we show that phosphorylation or gain- or loss-of-function mutations at either of two adjacent serine residues in TSC2 (S1365 and S1366 in mice; S1364 and S1365 in humans) can bidirectionally control mTORC1 activity stimulated by growth factors or haemodynamic stress, and consequently modulate cell growth and autophagy. However, basal mTORC1 activity remains unchanged. In the heart, or in isolated cardiomyocytes or fibroblasts, protein kinase G1 (PKG1) phosphorylates these TSC2 sites. PKG1 is a primary effector of nitric oxide and natriuretic peptide signalling, and protects against heart disease
. Suppression of hypertrophy and stimulation of autophagy in cardiomyocytes by PKG1 requires TSC2 phosphorylation. Homozygous knock-in mice that express a phosphorylation-silencing mutation in TSC2 (TSC2(S1365A)) develop worse heart disease and have higher mortality after sustained pressure overload of the heart, owing to mTORC1 hyperactivity that cannot be rescued by PKG1 stimulation. However, cardiac disease is reduced and survival of heterozygote Tsc2
knock-in mice subjected to the same stress is improved by PKG1 activation or expression of a phosphorylation-mimicking mutation (TSC2(S1365E)). Resting mTORC1 activity is not altered in either knock-in model. Therefore, TSC2 phosphorylation is both required and sufficient for PKG1-mediated cardiac protection against pressure overload. The serine residues identified here provide a genetic tool for bidirectional regulation of the amplitude of stress-stimulated mTORC1 activity.
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.
RATIONALE:Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic mammalian target of rapamycin complex 1) ...activation by neurohormonal 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 GC-1 (guanylyl cyclase 1) activation.
METHODS AND RESULTS:Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1α) or cysteine-42 to serine mutation redox-dead (PKG1α) were exposed to ET-1 (endothelin 1). Cells expressing PKG1α exhibited substantial mTORC1 activation (p70 S6K p70 S6 kinase, 4EBP1 elF4E binding protein-1, and Ulk1 Unc-51-like kinase 1 phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1α expression. Mice with global knock-in of PKG1α subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1α mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1α more than PKG1α myocardium following PO. TSC2 (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1α mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1α mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1α and PKG1α after PO and blocked ET-1 stimulated mTORC1 in TSC2-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.
MicroRNAs (miRs) posttranscriptionally regulate mRNA and its translation into protein, and are considered master controllers of genes modulating normal physiology and disease. There is growing ...interest in how miRs change with drug treatment, and leveraging this for precision guided therapy. Here we contrast 2 closely related therapies, inhibitors of phosphodiesterase type 5 or type 9 (PDE5-I, PDE9-I), given to mice subjected to sustained cardiac pressure overload (PO). Both inhibitors augment cyclic guanosine monophosphate (cGMP) to activate protein kinase G, with PDE5-I regulating nitric oxide (NO) and PDE9-I natriuretic peptide-dependent signaling. While both produced strong phenotypic improvement of PO pathobiology, they surprisingly showed binary differences in miR profiles; PDE5-I broadly reduces more than 120 miRs, including nearly half those increased by PO, whereas PDE9-I has minimal impact on any miR (P < 0.0001). The disparity evolves after pre-miR processing and is organ specific. Lastly, even enhancing NO-coupled cGMP by different methods leads to altered miR regulation. Thus, seemingly similar therapeutic interventions can be barcoded by profound differences in miR signatures, and reversing disease-associated miR changes is not required for therapy success.
The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis, and autophagy
1
. Its hyper-activation contributes to disease in many organs ...including the heart
1
,
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 activity
3
–
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 disease
10
–
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.
Heart failure (HF) is a leading cause of morbidity and mortality particularly in older adults and patients with multiple metabolic comorbidities. Heart failure with preserved ejection fraction ...(HFpEF) is a clinical syndrome with multisystem organ dysfunction in which patients develop symptoms of HF as a result of high left ventricular (LV) diastolic pressure in the context of normal or near normal LV ejection fraction (LVEF; ≥50%). Challenges to create and reproduce a robust rodent phenotype that recapitulates the multiple comorbidities that exist in this syndrome explain the presence of various animal models that fail to satisfy all the criteria of HFpEF. Using a continuous infusion of angiotensin II and phenylephrine (ANG II/PE), we demonstrate a strong HFpEF phenotype satisfying major clinically relevant manifestations and criteria of this pathology, including exercise intolerance, pulmonary edema, concentric myocardial hypertrophy, diastolic dysfunction, histological signs of microvascular impairment, and fibrosis. Conventional echocardiographic analysis of diastolic dysfunction identified early stages of HFpEF development and speckle tracking echocardiography analysis including the left atrium (LA) identified strain abnormalities indicative of contraction-relaxation cycle impairment. Diastolic dysfunction was validated by retrograde cardiac catheterization and analysis of LV end-diastolic pressure (LVEDP). Among mice that developed HFpEF, two major subgroups were identified with predominantly perivascular fibrosis and interstitial myocardial fibrosis. In addition to major phenotypic criteria of HFpEF that were evident at early stages of this model (3 and 10 days), accompanying RNAseq data demonstrate activation of pathways associated with myocardial metabolic changes, inflammation, activation of extracellular matrix (ECM) deposition, microvascular rarefaction, and pressure- and volume-related myocardial stress.
Heart failure with preserved ejection fraction (HFpEF) is an emerging epidemic affecting up to half of patients with heart failure. Here we used a chronic angiotensin II/phenylephrine (ANG II/PE) infusion model and instituted an updated algorithm for HFpEF assessment. Given the simplicity in generating this model, it may become a useful tool for investigating pathogenic mechanisms, identification of diagnostic markers, and for drug discovery aimed at both prevention and treatment of HFpEF.
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