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.

Establishment and characterization of a novel breast implant-associated anaplastic large cell lymphoma cell line and PDX model (BIA-XR1) with a unique KRAS mutation.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is an uncommon T-cell lymphoma type with distinct clinical, molecular and genetic features. Establishment of BIA-ALCL cell lines and patient-derived xenograft (PDX) models are essential experimental tools to investigate the molecular pathogenesis of the disease. We characterized a novel BIA-ALCL cell line and PDX model, named BIA-XR1, derived from a patient with textured breast implant who developed lymphoma. Next-generation sequencing revealed a STAT3 mutation, commonly detected in BIA-ALCL, and a unique KRAS mutation reported for the first time in this lymphoma type. Both JAK/STAT3 and RAS/MEK/ERK oncogenic pathways were activated in BIA-XR1, which are targetable with clinically available agents.

Galectin-3 interferes with tissue repair and promotes cardiac dysfunction and comorbidities in a genetic heart failure model.

Galectin-3, a biomarker for heart failure (HF), has been associated with myocardial fibrosis. However, its causal involvement in HF pathogenesis has been questioned in certain models of cardiac injury-induced HF. To address this, we used desmin-deficient mice (des-/-), a model of progressive HF characterized by cardiomyocyte death, spontaneous inflammatory responses sustaining fibrosis, and galectin-3 overexpression. Genetic ablation or pharmacological inhibition of galectin-3 led to improvement of cardiac function and adverse remodeling features including fibrosis. Over the course of development of des-/- cardiomyopathy, monitored for a period of 12 months, galectin-3 deficiency specifically ameliorated the decline in systolic function accompanying the acute inflammatory phase (4-week-old mice), whereas a more pronounced protective effect was observed in older mice, including the preservation of diastolic function. Interestingly, the cardiac repair activities during the early inflammatory phase were restored under galectin-3 deficiency by increasing the proliferation potential and decreasing apoptosis of fibroblasts, while galectin-3 absence modulated macrophage-fibroblast coupled functions and suppressed both pro-fibrotic activation of cardiac fibroblasts and pro-fibrotic gene expression in the des-/- heart. In addition, galectin-3 also affected the emphysema-like comorbid pathology observed in the des-/- mice, as its absence partially normalized lung compliance. Collectively galectin-3 was found to be causally involved in cardiac adverse remodeling, inflammation, and failure by affecting functions of cardiac fibroblasts and macrophages. In concordance with this role, the effectiveness of pharmacological inhibition in ameliorating cardiac pathology features establishes galectin-3 as a valid intervention target for HF, with additive benefits for treatment of associated comorbidities, such as pulmonary defects. Schematic illustrating top to bottom, the detrimental role of galectin-3 (Gal3) in heart failure progression: desmin deficiency-associated spontaneous myocardial inflammation accompanying cardiac cell death (reddish dashed border) is characterized by infiltration of macrophages (round cells) and up-regulation of Lgals3 (encoding secretable galectin-3, green) and detrimental macrophage-related genes (Ccr2 and Arg1). In this galectin-3-enriched milieu, the early up-regulation of profibrotic gene expression (Tgfb1, Acta2, Col1a1), in parallel to the suppression of proliferative activities and a potential of senescence induction by cardiac fibroblasts (spindle-like cells), collectively promote des-/- cardiac fibrosis and dysfunction establishing heart failure (left panel). Additionally, galectin-3+ macrophage-enrichment accompanies the development of emphysema-like lung comorbidities. In the absence of galectin-3 (right panel), the effect of macrophage-fibroblast dipole and associated events are modulated (grey color depicts reduced expression or activities) leading to attenuated cardiac pathology in the des-/-Lgals3-/- mice. Pulmonary comorbidities are also limited.

Sting Is Commonly and Differentially Expressed in T- and Nk-Cell but Not B-Cell Non-Hodgkin Lymphomas.

The expression patterns of stimulator of interferon genes (STING) were investigated in a cohort of 158 T- and natural killer (NK)-cell and 265 B-cell non-Hodgkin lymphomas (NHLs), as well as in control reactive lymph nodes and tonsils. STING expression was assessed by immunohistochemical methods using diagnostic biopsy specimens obtained prior to treatment. Using an arbitrary 10% cutoff, STING was differentially expressed among T/NK-cell NHLs; positive in 36 out of 38 (95%) cases of ALK+ anaplastic large cell lymphoma (ALCL), 23 out of 37 (62%) ALK-ALCLs, 1 out of 13 (7.7%) angioimmunoblastic T-cell lymphomas, 15 out of 19 (79%) peripheral T-cell lymphomas, not otherwise specified, 20 out of 36 (56%) extranodal NK/T-cell lymphomas of nasal type, 6 out of 7 (86%) T-cell lymphoblastic lymphomas, and 3 out of 4 (75%) mycosis fungoides. STING expression did not correlate with clinicopathological parameters or outcome in these patients with T/NK-cell lymphoma. By contrast, all 265 B-cell NHLs of various types were STING-negative. In addition, STING mRNA levels were very high in 6 out of 7 T-cell NHL cell lines, namely, ALK+ and ALK-ALCL cell lines, and very low or undetectable in 7 B-cell NHL cell lines, suggesting transcriptional downregulation of STING in neoplastic B-cells. At the protein level, using Western blot analysis and immunohistochemistry performed on cell blocks, STING expression was found to be restricted to T-cell NHL cell lines. Taken together, STING expression represents a novel biomarker and therapeutic target in T- and NK-cell lymphomas with direct immunotherapeutic implications since modulators of cGAS-STING activity are already available for clinical use.

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.

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.

MDMX/MDM4 is highly expressed and contributes to cell growth and survival in anaplastic large cell lymphoma.

We hypothesized that murine double minute X (MDMX), a negative p53-regulator, may be involved in dysfunctional p53-signaling in anaplastic large cell lymphoma (ALCL), anaplastic lymphoma kinase (ALK)-positive and ALK-negative, characterized frequently by non-mutated TP53 (wt-p53). By western blot analysis, MDMX was highly expressed in ALK + ALCL and expressed at variable levels in ALK- ALCL cell lines. By immunohistochemistry, high MDMX levels were observed more frequently in ALK + ALCL (36/46; 78%), compared with ALK- ALCL tumors (12/29; 41%) (p < .0018, Mann-Whitney-test). FISH analysis showed MDMX-amplification in 1 of 13 (8%) ALK- ALCL tumors, and low-level MDMX copy gains in 2 of 13 (15%) ALK- ALCL and 3 of 11 (27%) ALK + ALCL tumors. MDMX-pharmacologic inhibition or siRNA-mediated MDMX-silencing were associated with activated p53 signaling, growth inhibition and apoptotic cell death in wt-p53 ALCL cells, providing evidence that targeting MDMX may provide a new therapeutic approach for ALCL patients with wt-p53.

Untargeted Ultrahigh-Performance Liquid Chromatography-Hybrid Quadrupole-Orbitrap Mass Spectrometry (UHPLC-HRMS) Metabolomics Reveals Propolis Markers of Greek and Chinese Origin.

Chemical composition of propolis depends on the plant source and thus on the geographic and climatic characteristics of the site of collection. The aim of this study was to investigate the chemical profile of Greek and Chinese propolis extracts from different regions and suggest similarities and differences between them. Untargeted ultrahigh-performance liquid chromatography coupled to hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-HRMS) method was developed and 22 and 23 propolis samples from Greece and China, respectively, were analyzed. The experimental data led to the observation that there is considerable variability in terms of quality of the distinctive propolis samples. Partial least squares - discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) models were constructed and allowed the identification of significant features for sample discrimination, adding relevant information for the identification of class-determining metabolites. Chinese samples overexpressed compounds that are characteristic of the poplar type propolis, whereas Greek samples overexpress the latter and the diterpenes characteristic of the Mediterranean propolis type.

NMR metabolic profiling of Greek propolis samples: Comparative evaluation of their phytochemical compositions and investigation of their anti-ageing and antioxidant properties.

The present study aimed to investigate the metabolic profile, as well as the antioxidant and anti-ageing activities of twenty propolis samples from different regions of Greece. Chemical profiling of methanolic extracts was investigated using HPTLC and 1H-NMR techniques. Their antioxidant activity was evaluated by free radical scavenging methods (DPPH and ABTS), whereas anti-ageing properties were assessed as anti-collagenase activity. Extracts were also investigated in vitro for their ability to inhibit tyrosinase, which is responsible for the oxidation of L-DOPA to dopachrome and the production of melanin. The HPTLC and NMR analysis revealed high variability in the phytochemical profile of the methanolic extracts, with three major groups to be observed: a) Group I, consisting of samples rich in terpenoids, which present low antioxidant but high anti-tyrosinase activity, b) Group II, consisting of samples rich in flavonoids, which form a broad cluster with major similarities at the aromatic region and showed the highest anti-oxidant and anti-collagenase activities and c) Group III, consisting of samples with lower flavonoid content than the samples of Group II, which exhibited moderate antioxidant, anti-collagenase and anti-tyrosinase activities. In conclusion, this study has shown high differentiation on the chromatographic and spectroscopic metabolic profile of Greek propolis samples of different geographical origin, which is also reflected in their biological properties. Their important effects as antioxidant, anti-tyrosinase and anti-collagenase agents make propolis an important potent ingredient in the industry of food supplements and cosmeceuticals. Moreover, a correlation of a particular chemical propolis type to a specific type of biological activity will allow to prepare standardized extracts and develop food supplements and cosmeceuticals possessing the desired pharmacological properties.

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.

Honey Extracts Exhibit Cytoprotective Properties against UVB-Induced Photodamage in Human Experimental Skin Models.

In the present study, we aimed to examine the antioxidant, antiaging and photoprotective properties of Greek honey samples of various botanical and geographical origin. Ethyl-acetate extracts were used and the and the total phenolic/flavonoid content and antioxidant capacity were evaluated. Honey extracts were then studied for their cytoprotective properties against UVB-induced photodamage using human immortalized keratinocytes (HaCaT) and/or reconstituted human skin tissue models. Specifically, the cytotoxicity, oxidative status, DNA damage and gene expression levels of specific matrix metalloproteinases (MMPs) were examined. Overall, the treatment of HaCaT cells with honey extracts resulted in lower levels of DNA strand breaks and attenuated the decrease in cell viability following UVB exposure. Additionally, honey extracts significantly decreased the total protein carbonyl content of the irradiated cells, however, they had no significant effect on their total antioxidant status. Finally, the extracts alleviated the UVB-induced up-regulation of MMPs-3, -7 and -9 in a model of reconstituted skin tissue. In conclusion, honey extracts exhibited significant photoprotective and antiaging properties under UVB exposure conditions and thus could be further exploited as promising agents for developing novel and naturally-based, antiaging cosmeceutical products.

Efficient identification of Acetylcholinesterase and Hyaluronidase inhibitors from Paeonia parnassica extracts through a HeteroCovariance Approach.

ETHNOPHARMACOLOGICAL RELEVANCE: On the basis of the relevant reference in the poem Theriaca of the ancient Greek physician Nicander and its traditional use, Paeonia parnassica was selected for the evaluation of two extracts obtained from the roots and aerial parts to inhibit hydrolytic enzymes involved in snake envenomation. The secondary metabolites which contribute to these activities were detected through a novel HeteroCovariance NMR based approach. Afterwards these ingredients were isolated, identified and evaluated for their inhibitory potency. AIM OF THE STUDY: The identification of acetylcholinesterase and hyaluronidase inhibitors from Paeonia parnassica extracts was used as a case study for the introduction of a recently developed methodology to evaluate ethnopharmacological data and exploit them for the discovery of bioactive natural compounds. This process is based on the fractionation of the selected extracts and the simultaneous phytochemical analysis and biological assessment of the resulting fractions, which permits the rapid detection of the specified secondary metabolites prior to any laborious and time-consuming purification. MATERIALS AND METHODS: The roots and aerial parts of P. parnassica were extracted using methanol: water 50:50 and the two resulted extracts were fractionated by Centrifugal Partition Chromatography. The obtained fractions were evaluated in-vitro for their ability to inhibit acetylcholinesterase and hyaluronidase enzymes and their 1H NMR spectra were recorded. The biological activity was statistically correlated with the spectral data through the HeteroCovariance Approach (HetCA). Finally the purification, identification and biological evaluation of targeted secondary metabolites were carried out. RESULTS: The general chemical structures and some explicit secondary metabolites which contribute (e.g. gallotannins, gallic acid derivatives) or not (characteristic "cage-like" monoterpenes of the genus, glycosylated flavonoids) to the anti-acetylcholinesterase and anti-hyaluronidase activities were detected through HetCA. The consequent isolation and biological evaluation of targeted compounds were performed in order to validate the effectiveness and precision of the methodology. This procedure revealed the most active ingredients of both extracts obtained from roots and aerial parts against the above mentioned biological targets, as well as other compounds possessing moderate activity. CONCLUSIONS: The results of this study contributed to the verification of the ancient text Theriaca regarding the use of Paeonia parnassica to treat the snake bite symptoms. Furthermore, the ingredients of the Paeonia parnassica extracts, which were responsible for their anti-cholinesterase and anti-hyaluronidase activities, were determined applying a HetCA methodology before their isolation. Therefore, the current work provides clear evidence that HetCA could consist an efficient tool for the exploitation of traditional medicine information in order to discover bioactive natural compounds and develop new pharmacotherapies which serve the needs of contemporary medicine.

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.

Propolis Extracts Inhibit UV-Induced Photodamage in Human Experimental In Vitro Skin Models.

The aim of this study was to assess the antioxidant, photoprotective, and antiaging effects of Greek propolis. Propolis was subjected to n-heptane or methanol extraction. Total phenolic/flavonoid content and antioxidant potential were determined in the extracts. Promising extracts were evaluated for their cytoprotective properties using human immortalized keratinocyte (HaCaT) or reconstituted human skin tissue following exposure to UVB. Assessment of cytotoxicity, DNA damage, oxidative status, and gene/protein expression levels of various matrix metalloproteinases (MMPs) were performed. The propolis methanolic fractions exhibited higher total phenolic and flavonoid contents and significant in vitro antioxidant activity. Incubation of HaCaT cells with certain methanolic extracts significantly decreased the formation of DNA strand breaks following exposure to UVB and attenuated UVB-induced decrease in cell viability. The extracts had no remarkable effect on the total antioxidant status, but significantly lowered total protein carbonyl content used as a marker for protein oxidation in HaCaT cells. MMP-1, -3, -7, and -9, monitored as endpoints of antiaging efficacy, were significantly reduced by propolis following UVB exposure in a model of reconstituted skin tissue. In conclusion, propolis protects against the oxidative and photodamaging effects of UVB and could be further explored as a promising agent for developing natural antiaging strategies.

Oxidant sensor in the cGMP-binding pocket of PKGIα regulates nitroxyl-mediated kinase activity.

Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli's salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGIα), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity was monitored in cells transfected with wildtype (WT), Cys42Ser or Cys117/195Ser PKGIα that cannot form the inter- or intradisulfide, respectively. HNO enhanced WT kinase activity, an effect significantly attenuated in inter- or intradisulfide-deficient PKGIα. To investigate whether the intradisulfide modulates cGMP binding, real-time imaging was performed in vascular smooth muscle cells expressing a FRET-biosensor comprising the cGMP-binding sites of PKGIα. HNO induced FRET changes similar to those elicited by an increase of cGMP, suggesting that intradisulfide formation is associated with activation of PKGIα. Intradisulfide formation in PKGIα correlated with enhanced HNO-mediated vasorelaxation in mesenteric arteries in vitro and arteriolar dilation in vivo in mice. HNO induces intradisulfide formation in PKGIα, inducing the same effect as cGMP binding, namely kinase activation and thus vasorelaxation.

Blinded Contractility Analysis in hiPSC-Cardiomyocytes in Engineered Heart Tissue Format: Comparison With Human Atrial Trabeculae.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) may serve as a new assay for drug testing in a human context, but their validity particularly for the evaluation of inotropic drug effects remains unclear. In this blinded analysis, we compared the effects of 10 indicator compounds with known inotropic effects in electrically stimulated (1.5 Hz) hiPSC-CM-derived 3-dimensional engineered heart tissue (EHT) and human atrial trabeculae (hAT). Human EHTs were prepared from iCell hiPSC-CM, hAT obtained at routine heart surgery. Mean intra-batch variation coefficient in baseline force measurement was 17% for EHT and 49% for hAT. The PDE-inhibitor milrinone did not affect EHT contraction force, but increased force in hAT. Citalopram (selective serotonin reuptake inhibitor), nifedipine (LTCC-blocker) and lidocaine (Na+ channel-blocker) had negative inotropic effects on EHT and hAT. Formoterol (beta-2 agonist) had positive lusitropic but no inotropic effect in EHT, and positive clinotropic, lusitropic, and inotropic effects in hAT. Tacrolimus (calcineurin-inhibitor) had a negative inotropic effect in EHTs, but no effect in hAT. Digoxin (Na+-K+-ATPase-inhibitor) showed a positive inotropic effect only in EHTs, but no effect in hAT probably due to short incubation time. Ryanodine (ryanodine receptor-inhibitor) reduced contraction force in both models. Rolipram and acetylsalicylic acid showed noninterpretable results in hAT. Contraction amplitude and kinetics were more stable over time and less variable in hiPSC-EHTs than hAT. HiPSC-EHT faithfully detected cAMP-dependent and -independent positive and negative inotropic effects, but limited beta-2 adrenergic or PDE3 effects, compatible with an immature CM phenotype.

S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure.

Cardiac myosin-binding protein C (cMyBP-C) regulates actin-myosin interaction and thereby cardiac myocyte contraction and relaxation. This physiologic function is regulated by cMyBP-C phosphorylation. In our study, reduced site-specific cMyBP-C phosphorylation coincided with increased S-glutathiolation in ventricular tissue from patients with dilated or ischemic cardiomyopathy compared to nonfailing donors. We used redox proteomics, to identify constitutive and disease-specific S-glutathiolation sites in cMyBP-C in donor and patient samples, respectively. Among those, a cysteine cluster in the vicinity of the regulatory phosphorylation sites within the myosin S2 interaction domain C1-M-C2 was identified and showed enhanced S-glutathiolation in patients. In vitro S-glutathiolation of recombinant cMyBP-C C1-M-C2 occurred predominantly at Cys(249), which attenuated phosphorylation by protein kinases. Exposure to glutathione disulfide induced cMyBP-C S-glutathiolation, which functionally decelerated the kinetics of Ca(2+)-activated force development in ventricular myocytes from wild-type, but not those from Mybpc3-targeted knockout mice. These oxidation events abrogate protein kinase-mediated phosphorylation of cMyBP-C and therefore potentially contribute to the reduction of its phosphorylation and the contractile dysfunction observed in human heart failure.-Stathopoulou, K., Wittig, I., Heidler, J., Piasecki, A., Richter, F., Diering, S., van der Velden, J., Buck, F., Donzelli, S., Schröder, E., Wijnker, P. J. M., Voigt, N., Dobrev, D., Sadayappan, S., Eschenhagen, T., Carrier, L., Eaton, P., Cuello, F. S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure.

Ranolazine antagonizes catecholamine-induced dysfunction in isolated cardiomyocytes, but lacks long-term therapeutic effects in vivo in a mouse model of hypertrophic cardiomyopathy.

AIMS: Hypertrophic cardiomyopathy (HCM) is often accompanied by increased myofilament Ca(2+) sensitivity and diastolic dysfunction. Recent findings indicate increased late Na(+) current density in human HCM cardiomyocytes. Since ranolazine has the potential to decrease myofilament Ca(2+) sensitivity and late Na(+) current, we investigated its effects in an Mybpc3-targeted knock-in (KI) mouse model of HCM. METHODS AND RESULTS: Unloaded sarcomere shortening and Ca(2+) transients were measured in KI and wild-type (WT) cardiomyocytes. Measurements were performed at baseline (1 Hz) and under increased workload (30 nM isoprenaline (ISO), 5 Hz) in the absence or presence of 10 µM ranolazine. KI myocytes showed shorter diastolic sarcomere length at baseline, stronger inotropic response to ISO, and drastic drop of diastolic sarcomere length under increased workload. Ranolazine attenuated ISO responses in WT and KI cells and prevented workload-induced diastolic failure in KI. Late Na(+) current density was diminished and insensitive to ranolazine in KI cardiomyocytes. Ca(2+) sensitivity of skinned KI trabeculae was slightly decreased by ranolazine. Phosphorylation analysis of cAMP-dependent protein kinase A-target proteins and ISO concentration-response measurements on muscle strips indicated antagonism at ß-adrenoceptors with 10 µM ranolazine shifting the ISO response by 0.6 log units. Six-month treatment with ranolazine (plasma level >20 µM) demonstrated a ß-blocking effect, but did not reverse cardiac hypertrophy or dysfunction in KI mice. CONCLUSION: Ranolazine improved tolerance to high workload in mouse HCM cardiomyocytes, not by blocking late Na(+) current, but by antagonizing ß-adrenergic stimulation and slightly desensitizing myofilaments to Ca(2+). This effect did not translate in therapeutic efficacy in vivo.

Cardiac myosin-binding protein C (MYBPC3) in cardiac pathophysiology.

More than 350 individual MYPBC3 mutations have been identified in patients with inherited hypertrophic cardiomyopathy (HCM), thus representing 40­50% of all HCM mutations, making it the most frequently mutated gene in HCM. HCM is considered a disease of the sarcomere and is characterized by left ventricular hypertrophy, myocyte disarray and diastolic dysfunction. MYBPC3 encodes for the thick filament associated protein cardiac myosin-binding protein C (cMyBP-C), a signaling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of contraction and relaxation. This review aims to provide a succinct overview of how mutations in MYBPC3 are considered to affect the physiological function of cMyBP-C, thus causing the deleterious consequences observed inHCM patients. Importantly, recent advances to causally treat HCM by repairing MYBPC3 mutations by gene therapy are discussed here, providing a promising alternative to heart transplantation for patients with a fatal form of neonatal cardiomyopathy due to bi-allelic truncating MYBPC3 mutations.

Heart failure-specific changes in protein kinase signalling.

Among the myriad of molecular alterations occurring in heart failure development, aggravation of the disease is often attributed to global or local changes in protein kinase activity, thus making protein kinases attractive targets for therapeutic intervention. Since protein kinases do not only have maladaptive roles, but also contribute to the physiological integrity of cells, it is a challenging task to circumvent undesired inhibition of protein kinase activity. Identification of posttranslational modifications and/or protein-protein interactions that are exclusively apparent under pathophysiological conditions provides exciting information for alternative non-kinase inhibitory treatment strategies that eliminate maladaptive functions of a protein kinase, but preserve the beneficial ones. Here, we focus on the disease-specific regulation of a number of protein kinases, namely, Ca(2+)/calmodulin-dependent protein kinase II isoform δ (CaMKIIδ), G protein-coupled receptor kinase 2 (GRK2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein kinase D (PKD) and protein kinase C isoform ß2 (PKCß2), which are embedded in complex signal transduction pathways implicated in heart failure development, and discuss potential avenues for novel treatment strategies to combat heart disease.