Physioxia rewires mitochondrial complex composition to protect stem cell viability.

Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20 % O2) or physioxic (5 % O2) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism), higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and ß-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition.

PITX2 deficiency leads to atrial mitochondrial dysfunction.

AIM: Reduced left atrial PITX2 is associated with atrial cardiomyopathy and atrial fibrillation. PITX2 is restricted to left atrial cardiomyocytes in the adult heart. The links between PITX2 deficiency, atrial cardiomyopathy and atrial fibrillation are not fully understood. METHODS AND RESULTS: To identify mechanisms linking PITX2 deficiency to atrial fibrillation, we generated and characterized PITX2-deficient human atrial cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) and their controls. PITX2-deficient hiPSC-derived atrial cardiomyocytes showed shorter and disorganised sarcomeres and increased mononucleation. Electron microscopy found an increased number of smaller mitochondria compared to the control. Mitochondrial protein expression was altered in PITX2-deficient hiPSC-derived atrial cardiomyocytes. Single-nuclear RNA-sequencing found differences in cellular respiration pathways and differentially expressed mitochondrial and ion channel genes in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2 repression in hiPSC-derived atrial cardiomyocytes replicated dysregulation of cellular respiration. Mitochondrial respiration was shifted to increased glycolysis in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2-deficient human hiPSC-derived atrial cardiomyocytes showed higher spontaneous beating rates. Action potential duration was more variable with an overall prolongation of early repolarization, consistent with metabolic defects. Gene expression analyses confirmed changes in mitochondrial genes in left atria from 42 patients with atrial fibrillation compared to 43 patients in sinus rhythm. Dysregulation of left atrial mitochondrial (COX7C) and metabolic (FOXO1) genes was associated with PITX2 expression in human left atria. CONCLUSIONS: In summary, PITX2 deficiency causes mitochondrial dysfunction and a metabolic shift to glycolysis in human atrial cardiomyocytes. PITX2-dependent metabolic changes can contribute to the structural and functional defects found in PITX2-deficient atria.

Effect of deletion of the protein kinase PRKD1 on development of the mouse embryonic heart.

Congenital heart disease (CHD) is the most common congenital anomaly, with an overall incidence of approximately 1% in the United Kingdom. Exome sequencing in large CHD cohorts has been performed to provide insights into the genetic aetiology of CHD. This includes a study of 1891 probands by our group in collaboration with others, which identified three novel genes-CDK13, PRKD1, and CHD4, in patients with syndromic CHD. PRKD1 encodes a serine/threonine protein kinase, which is important in a variety of fundamental cellular functions. Individuals with a heterozygous mutation in PRKD1 may have facial dysmorphism, ectodermal dysplasia and may have CHDs such as pulmonary stenosis, atrioventricular septal defects, coarctation of the aorta and bicuspid aortic valve. To obtain a greater appreciation for the role that this essential protein kinase plays in cardiogenesis and CHD, we have analysed a Prkd1 transgenic mouse model (Prkd1em1) carrying deletion of exon 2, causing loss of function. High-resolution episcopic microscopy affords detailed morphological 3D analysis of the developing heart and provides evidence for an essential role of Prkd1 in both normal cardiac development and CHD. We show that homozygous deletion of Prkd1 is associated with complex forms of CHD such as atrioventricular septal defects, and bicuspid aortic and pulmonary valves, and is lethal. Even in heterozygotes, cardiac differences occur. However, given that 97% of Prkd1 heterozygous mice display normal heart development, it is likely that one normal allele is sufficient, with the defects seen most likely to represent sporadic events. Moreover, mRNA and protein expression levels were investigated by RT-qPCR and western immunoblotting, respectively. A significant reduction in Prkd1 mRNA levels was seen in homozygotes, but not heterozygotes, compared to WT littermates. While a trend towards lower PRKD1 protein expression was seen in the heterozygotes, the difference was only significant in the homozygotes. There was no compensation by the related Prkd2 and Prkd3 at transcript level, as evidenced by RT-qPCR. Overall, we demonstrate a vital role of Prkd1 in heart development and the aetiology of CHD.

Metabolic Communication by SGLT2 Inhibition.

BACKGROUND: SGLT2 (sodium-glucose cotransporter 2) inhibitors (SGLT2i) can protect the kidneys and heart, but the underlying mechanism remains poorly understood. METHODS: To gain insights on primary effects of SGLT2i that are not confounded by pathophysiologic processes or are secondary to improvement by SGLT2i, we performed an in-depth proteomics, phosphoproteomics, and metabolomics analysis by integrating signatures from multiple metabolic organs and body fluids after 1 week of SGLT2i treatment of nondiabetic as well as diabetic mice with early and uncomplicated hyperglycemia. RESULTS: Kidneys of nondiabetic mice reacted most strongly to SGLT2i in terms of proteomic reconfiguration, including evidence for less early proximal tubule glucotoxicity and a broad downregulation of the apical uptake transport machinery (including sodium, glucose, urate, purine bases, and amino acids), supported by mouse and human SGLT2 interactome studies. SGLT2i affected heart and liver signaling, but more reactive organs included the white adipose tissue, showing more lipolysis, and, particularly, the gut microbiome, with a lower relative abundance of bacteria taxa capable of fermenting phenylalanine and tryptophan to cardiovascular uremic toxins, resulting in lower plasma levels of these compounds (including p-cresol sulfate). SGLT2i was detectable in murine stool samples and its addition to human stool microbiota fermentation recapitulated some murine microbiome findings, suggesting direct inhibition of fermentation of aromatic amino acids and tryptophan. In mice lacking SGLT2 and in patients with decompensated heart failure or diabetes, the SGLT2i likewise reduced circulating p-cresol sulfate, and p-cresol impaired contractility and rhythm in human induced pluripotent stem cell-derived engineered heart tissue. CONCLUSIONS: SGLT2i reduced microbiome formation of uremic toxins such as p-cresol sulfate and thereby their body exposure and need for renal detoxification, which, combined with direct kidney effects of SGLT2i, including less proximal tubule glucotoxicity and a broad downregulation of apical transporters (including sodium, amino acid, and urate uptake), provides a metabolic foundation for kidney and cardiovascular protection.

Human model of primary carnitine deficiency cardiomyopathy reveals ferroptosis as a novel mechanism.

Primary carnitine deficiency (PCD) is an autosomal recessive monogenic disorder caused by mutations in SLC22A5. This gene encodes for OCTN2, which transports the essential metabolite carnitine into the cell. PCD patients suffer from muscular weakness and dilated cardiomyopathy. Two OCTN2-defective human induced pluripotent stem cell lines were generated, carrying a full OCTN2 knockout and a homozygous OCTN2 (N32S) loss-of-function mutation. OCTN2-defective genotypes showed lower force development and resting length in engineered heart tissue format compared with isogenic control. Force was sensitive to fatty acid-based media and associated with lipid accumulation, mitochondrial alteration, higher glucose uptake, and metabolic remodeling, replicating findings in animal models. The concordant results of OCTN2 (N32S) and -knockout emphasizes the relevance of OCTN2 for these findings. Importantly, genome-wide analysis and pharmacological inhibitor experiments identified ferroptosis, an iron- and lipid-dependent cell death pathway associated with fibroblast activation as a novel PCD cardiomyopathy disease mechanism.

Extracellular Matrix Profiling and Disease Modelling in Engineered Vascular Smooth Muscle Cell Tissues.

Aortic smooth muscle cells (SMCs) have an intrinsic role in regulating vessel homeostasis and pathological remodelling. In two-dimensional (2D) cell culture formats, however, SMCs are not embedded in their physiological extracellular matrix (ECM) environment. To overcome the limitations of conventional 2D SMC cultures, we established a 3D in vitro model of engineered vascular smooth muscle cell tissues (EVTs). EVTs were casted from primary murine aortic SMCs by suspending a SMC-fibrin master mix between two flexible silicon-posts at day 0 before prolonged culture up to 14 days. Immunohistochemical analysis of EVT longitudinal sections demonstrated that SMCs were aligned, viable and secretory. Mass spectrometry-based proteomics analysis of murine EVT lysates was performed and identified 135 matrisome proteins. Proteoglycans, including the large aggregating proteoglycan versican, accumulated within EVTs by day 7 of culture. This was followed by the deposition of collagens, elastin-binding proteins and matrix regulators up to day 14 of culture. In contrast to 2D SMC controls, accumulation of versican occurred in parallel to an increase in versikine, a cleavage product mediated by proteases of the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family. Next, we tested the response of EVTs to stimulation with transforming growth factor beta-1 (TGFß-1). EVTs contracted in response to TGFß-1 stimulation with altered ECM composition. In contrast, treatment with the pharmacological activin-like kinase inhibitor (ALKi) SB 431542 suppressed ECM secretion. As a disease stimulus, we performed calcification assays. The ECM acts as a nidus for calcium phosphate deposition in the arterial wall. We compared the onset and extent of calcification in EVTs and 2D SMCs cultured under high calcium and phosphate conditions for 7 days. Calcified EVTs displayed increased tissue stiffness by up to 30 % compared to non-calcified controls. Unlike the rapid calcification of SMCs in 2D cultures, EVTs sustained expression of the calcification inhibitor matrix Gla protein and allowed for better discrimination of the calcification propensity between independent biological replicates. In summary, EVTs are an intuitive and versatile model to investigate ECM synthesis and turnover by SMCs in a 3D environment. Unlike conventional 2D cultures, EVTs provide a more relevant pathophysiological model for retention of the nascent ECM produced by SMCs.

Nitroxyl Donor CXL-1020 Lowers Blood Pressure by Targeting C195 in Cyclic Guanosine-3',5'-Monophosphate-Dependent Protein Kinase I.

BACKGROUND: We previously demonstrated that nitroxyl causes vasodilation, at least in part, by inducing the formation of an intradisulfide bond between C117 and C195 in the high affinity cyclic guanosine monophosphate-binding site of PKGI (cyclic guanosine monophosphate-dependent protein kinase I). The aim of this study was to determine whether nitroxyl donors lower blood pressure via this novel PKGI activation mechanism in vivo. METHODS: To determine this, a C195S PKGI knock-in mouse model was generated that ubiquitously and constitutively expresses a mutant kinase resistant to nitroxyl-induced intradisulfide activation. RESULTS: Knock-in and wild-type littermates did not differ in appearance, body weight, in PKGI protein expression or blood gas content. Organ weight was similar between genotypes apart from the cecum that was significantly enlarged in knock-in animals. Mean arterial pressure and heart rate monitored in vivo over 24 hours by radio-telemetry revealed neither a significant difference between genotypes at baseline nor during angiotensin II-induced hypertension or sepsis. CXL-1020, a clinically relevant nitroxyl donor, did not lower blood pressure in normotensive animals. In contrast, administering CXL-1020 to hypertensive wild-type mice reduced their blood pressure by 10±4 mm Hg (P=0.0184), whereas the knock-in littermates were unaffected. CONCLUSIONS: Oxidation of C195 in PKGI contributes to the antihypertensive effects observed in response to nitroxyl donors, emphasising the potential importance of nitroxyl donors in pathological scenarios when cyclic guanosine monophosphate levels are reduced and insufficient to activate PKGI.

CMYA5 is a novel interaction partner of FHL2 in cardiac myocytes.

Four-and-a-half LIM domains protein 2 (FHL2) is an anti-hypertrophic adaptor protein that regulates cardiac myocyte signalling and function. Herein, we identified cardiomyopathy-associated 5 (CMYA5) as a novel FHL2 interaction partner in cardiac myocytes. In vitro pull-down assays demonstrated interaction between FHL2 and the N- and C-terminal regions of CMYA5. The interaction was verified in adult cardiac myocytes by proximity ligation assays. Immunofluorescence and confocal microscopy demonstrated co-localisation in the same subcellular compartment. The binding interface between FHL2 and CMYA5 was mapped by peptide arrays. Exposure of neonatal rat ventricular myocytes to a CMYA5 peptide covering one of the FHL2 interaction sites led to an increase in cell area at baseline, but a blunted response to chronic phenylephrine treatment. In contrast to wild-type hearts, loss or reduced FHL2 expression in Fhl2-targeted knockout mouse hearts or in a humanised mouse model of hypertrophic cardiomyopathy led to redistribution of CMYA5 into the perinuclear and intercalated disc region. Taken together, our results indicate a direct interaction of the two adaptor proteins FHL2 and CMYA5 in cardiac myocytes, which might impact subcellular compartmentation of CMYA5.

Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling.

BACKGROUND: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. METHODS: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. RESULTS: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin ß1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of ß-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that ß1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with ß-blockers had a distinct cardiac ECM profile. CONCLUSIONS: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that ß-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation.

The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+ -load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow-up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non-failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca2+ -binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+ -scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue.

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.

Regulation of Cardiac PKA Signaling by cAMP and Oxidants.

Pathologies, such as cancer, inflammatory and cardiac diseases are commonly associated with long-term increased production and release of reactive oxygen species referred to as oxidative stress. Thereby, protein oxidation conveys protein dysfunction and contributes to disease progression. Importantly, trials to scavenge oxidants by systemic antioxidant therapy failed. This observation supports the notion that oxidants are indispensable physiological signaling molecules that induce oxidative post-translational modifications in target proteins. In cardiac myocytes, the main driver of cardiac contractility is the activation of the ß-adrenoceptor-signaling cascade leading to increased cellular cAMP production and activation of its main effector, the cAMP-dependent protein kinase (PKA). PKA-mediated phosphorylation of substrate proteins that are involved in excitation-contraction coupling are responsible for the observed positive inotropic and lusitropic effects. PKA-actions are counteracted by cellular protein phosphatases (PP) that dephosphorylate substrate proteins and thus allow the termination of PKA-signaling. Both, kinase and phosphatase are redox-sensitive and susceptible to oxidation on critical cysteine residues. Thereby, oxidation of the regulatory PKA and PP subunits is considered to regulate subcellular kinase and phosphatase localization, while intradisulfide formation of the catalytic subunits negatively impacts on catalytic activity with direct consequences on substrate (de)phosphorylation and cardiac contractile function. This review article attempts to incorporate the current perception of the functionally relevant regulation of cardiac contractility by classical cAMP-dependent signaling with the contribution of oxidant modification.

A Proteomics-Based Assessment of Inflammation Signatures in Endotoxemia.

We have previously shown that multimers of plasma pentraxin-3 (PTX3) were predictive of survival in patients with sepsis. To characterize the release kinetics and cellular source of plasma protein changes in sepsis, serial samples were obtained from healthy volunteers (n = 10; three time points) injected with low-dose endotoxin (lipopolysaccharide [LPS]) and analyzed using data-independent acquisition MS. The human plasma proteome response was compared with an LPS-induced endotoxemia model in mice. Proteomic analysis of human plasma revealed a rapid neutrophil degranulation signature, followed by a rise in acute phase proteins. Changes in circulating PTX3 correlated with increases in neutrophil-derived proteins following LPS injection. Time course analysis of the plasma proteome in mice showed a time-dependent increase in multimeric PTX3, alongside increases in neutrophil-derived myeloperoxidase (MPO) upon LPS treatment. The mechanisms of oxidation-induced multimerization of PTX3 were explored in two genetic mouse models: MPO global knock-out (KO) mice and LysM Cre Nox2 KO mice, in which NADPH oxidase 2 (Nox2) is only deficient in myeloid cells. Nox2 is the enzyme responsible for the oxidative burst in neutrophils. Increases in plasma multimeric PTX3 were not significantly different between wildtype and MPO or LysM Cre Nox2 KO mice. Thus, PTX3 may already be stored and released in a multimeric form. Through in vivo neutrophil depletion and multiplexed vascular proteomics, PTX3 multimer deposition within the aorta was confirmed to be neutrophil dependent. Proteomic analysis of aortas from LPS-injected mice returned PTX3 as the most upregulated protein, where multimeric PTX3 was deposited as early as 2 h post-LPS along with other neutrophil-derived proteins. In conclusion, the rise in multimeric PTX3 upon LPS injection correlates with neutrophil-related protein changes in plasma and aortas. MPO and myeloid Nox2 are not required for the multimerization of PTX3; instead, neutrophil extravasation is responsible for the LPS-induced deposition of multimeric PTX3 in the aorta.

Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents.

The contraction and relaxation of the heart is controlled by stimulation of the ß1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of ß1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

Interference with ERK-dimerization at the nucleocytosolic interface targets pathological ERK1/2 signaling without cardiotoxic side-effects.

Dysregulation of extracellular signal-regulated kinases (ERK1/2) is linked to several diseases including heart failure, genetic syndromes and cancer. Inhibition of ERK1/2, however, can cause severe cardiac side-effects, precluding its wide therapeutic application. ERKT188-autophosphorylation was identified to cause pathological cardiac hypertrophy. Here we report that interference with ERK-dimerization, a prerequisite for ERKT188-phosphorylation, minimizes cardiac hypertrophy without inducing cardiac adverse effects: an ERK-dimerization inhibitory peptide (EDI) prevents ERKT188-phosphorylation, nuclear ERK1/2-signaling and cardiomyocyte hypertrophy, protecting from pressure-overload-induced heart failure in mice whilst preserving ERK1/2-activity and cytosolic survival signaling. We also examine this alternative ERK1/2-targeting strategy in cancer: indeed, ERKT188-phosphorylation is strongly upregulated in cancer and EDI efficiently suppresses cancer cell proliferation without causing cardiotoxicity. This powerful cardio-safe strategy of interfering with ERK-dimerization thus combats pathological ERK1/2-signaling in heart and cancer, and may potentially expand therapeutic options for ERK1/2-related diseases, such as heart failure and genetic syndromes.

Cardiac cGMP Signaling in Health and Disease: Location, Location, Location.

3',5'-Cyclic guanosine monophosphate (cGMP) is a ubiquitous second messenger, which critically regulates cardiac pump function and protects from the development of cardiac hypertrophy by acting in various subcellular microdomains. Although clinical studies testing the potential of cGMP elevating drugs in patients suffering from cardiac disease showed promising results, deeper insight into the local actions of these drugs at the subcellular level are indispensable to inspire novel therapeutic strategies. Detailed information on the spatio-temporal dynamics of cGMP production and degradation can be provided by the use of fluorescent biosensors that are capable of monitoring this second messenger at different locations inside the cell with high temporal and spatial resolution. In this review, we will summarize how these emerging new tools have improved our understanding of cardiac cGMP signaling in health and disease, and attempt to anticipate future challenges in the field.

Divergent off-target effects of RSK N-terminal and C-terminal kinase inhibitors in cardiac myocytes.

P90 ribosomal S6 kinases (RSK) are ubiquitously expressed and regulate responses to neurohumoral stimulation. To study the role of RSK signalling on cardiac myocyte function and protein phosphorylation, pharmacological RSK inhibitors were tested. Here, the ATP competitive N-terminal kinase domain-targeting compounds D1870 and SL0101 and the allosteric C-terminal kinase domain-targeting FMK were evaluated regarding their ability to modulate cardiac myocyte protein phosphorylation. Exposure to D1870 and SL0101 significantly enhanced phospholamban (PLN) Ser16 and cardiac troponin I (cTnI) Ser22/23 phosphorylation in response to D1870 and SL0101 upon exposure to phenylephrine (PE) that activates RSK. In contrast, FMK pretreatment significantly reduced phosphorylation of both proteins in response to PE. D1870-mediated enhancement of PLN Ser16 phosphorylation was also observed after exposure to isoprenaline or noradrenaline (NA) stimuli that do not activate RSK. Inhibition of ß-adrenoceptors by atenolol or cAMP-dependent protein kinase (PKA) by H89 prevented the D1870-mediated increase in PLN phosphorylation, suggesting that PKA is the kinase responsible for the observed phosphorylation. Assessment of changes in cAMP formation by FRET measurements revealed increased cAMP formation in vicinity to PLN after exposure to D1870 and SL0101. D1870 inhibited phosphodiesterase activity similarly as established PDE inhibitors rolipram or 3-isobutyl-1-methylxanthine. Assessment of catecholamine-mediated force development in rat ventricular muscle strips revealed significantly reduced EC50 for NA after D1870 pretreatment (DMSO/NA: 2.33 µmol/L vs. D1870/NA: 1.30 µmol/L). The data reveal enhanced cardiac protein phosphorylation by D1870 and SL0101 that was not detectable in response to FMK. This disparate effect might be attributed to off-target inhibition of PDEs with impact on muscle function as demonstrated for D1870.

Analysis of fibrosis in control or pressure overloaded rat hearts after mechanical unloading by heterotopic heart transplantation.

Mechanical unloading (MU) by implantation of left ventricular assist devices (LVAD) has become clinical routine. This procedure has been shown to reverse cardiac pathological remodeling, with the underlying molecular mechanisms incompletely understood. Most studies thus far were performed in non-standardized human specimens or MU of healthy animal hearts. Our study investigates cardiac remodeling processes in sham-operated healthy rat hearts and in hearts subjected to standardized pathological pressure overload by transverse aortic constriction (TAC) prior to MU by heterotopic heart transplantation (hHTx/MU). Rats underwent sham or TAC surgery. Disease progression was monitored by echocardiography prior to MU by hHTx/MU. Hearts after TAC or TAC combined with hHTx/MU were removed and analyzed by histology, western immunoblot and gene expression analysis. TAC surgery resulted in cardiac hypertrophy and impaired cardiac function. TAC hearts revealed significantly increased cardiac myocyte diameter and mild fibrosis. Expression of hypertrophy associated genes after TAC was higher compared to hearts after hHTx/MU. While cardiac myocyte cell diameter regressed to the level of sham-operated controls in all hearts subjected to hHTx/MU, fibrotic remodeling was significantly exacerbated. Transcription of pro-fibrotic and apoptosis-related genes was markedly augmented in all hearts after hHTx/MU. Sarcomeric proteins involved in excitation-contraction coupling displayed significantly lower phosphorylation levels after TAC and significantly reduced total protein levels after hHTx/MU. Development of myocardial fibrosis, cardiac myocyte atrophy and loss of sarcomeric proteins was observed in all hearts that underwent hHTX/MU regardless of the disease state. These results may help to explain the clinical experience with low rates of LVAD removal due to lack of myocardial recovery.

Cysteine-Based Redox Sensing and Its Role in Signaling by Cyclic Nucleotide-Dependent Kinases in the Cardiovascular System.

Oxidant molecules are produced in biological systems and historically have been considered causal mediators of damage and disease. While oxidants may contribute to the pathogenesis of disease, evidence continues to emerge that shows these species also play important regulatory roles in health. A major mechanism of oxidant sensing and signaling involves their reaction with reactive cysteine thiols within proteins, inducing oxidative posttranslational modifications that can couple to altered function to enable homeostatic regulation. Protein kinase A and protein kinase G are regulated by oxidants in this way, and this review focuses on our molecular-level understanding of these events and their role in regulating cardiovascular physiology during health and disease.

Correction: Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.

[This corrects the article DOI: 10.1371/journal.pone.0145937.].