Protein phosphatase 2A affects myofilament contractility in non-failing but not in failing human myocardium

Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation–contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (ΔpCa50 = 0.05 ± 0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ΔpCa50 = 0.10 ± 0.03) and dilated (DCM, ΔpCa50 = 0.06 ± 0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56α was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts

The homozygous K280N troponin T mutation alters cross-bridge kinetics and energetics in human HCM

Hypertrophic cardiomyopathy (HCM) is a genetic form of left ventricular hypertrophy, primarily caused by mutations in sarcomere proteins. The cardiac remodeling that occurs as the disease develops can mask the pathogenic impact of the mutation. Here, to discriminate between mutation-induced and disease-related changes in myofilament function, we investigate the pathogenic mechanisms underlying HCM in a patient carrying a homozygous mutation (K280N) in the cardiac troponin T gene (TNNT2), which results in 100% mutant cardiac troponin T. We examine sarcomere mechanics and energetics in K280N-isolated myofibrils and demembranated muscle strips, before and after replacement of the endogenous troponin. We also compare these data to those of control preparations from donor hearts, aortic stenosis patients (LVHao), and HCM patients negative for sarcomeric protein mutations (HCMsmn). The rate constant of tension generation following maximal Ca2+ activation (k ACT) and the rate constant of isometric relaxation (slow k REL) are markedly faster in K280N myofibrils than in all control groups. Simultaneous measurements of maximal isometric ATPase activity and Ca2+-activated tension in demembranated muscle strips also demonstrate that the energy cost of tension generation is higher in the K280N than in all controls. Replacement of mutant protein by exchange with wild-type troponin in the K280N preparations reduces k ACT, slow k REL, and tension cost close to control values. In donor myofibrils and HCMsmn demembranated strips, replacement of endogenous troponin with troponin containing the K280N mutant increases k ACT, slow k REL, and tension cost. The K280N TNNT2 mutation directly alters the apparent cross-bridge kinetics and impairs sarcomere energetics. This result supports the hypothesis that inefficient ATP utilization by myofilaments plays a central role in the pathogenesis of the disease

Triggering of the dsRNA Sensors TLR3, MDA5, and RIG-I Induces CD55 Expression in Synovial Fibroblasts

Background: CD55 (decay-accelerating factor) is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS. Methods: FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR) ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C)) and 5'-triphosphate RNA were used to activate the cytoplasmic double-stranded (ds)RNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry. Results: CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1 beta and especially by the TLR3 ligand poly(I:C). Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55. Conclusions: Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocyte

NOX2, p22phox and p47phox are targeted to the nuclear pore complex in ischemic cardiomyocytes colocalizing with local reactive oxygen species.

BACKGROUND: NADPH oxidases play an essential role in reactive oxygen species (ROS)-based signaling in the heart. Previously, we have demonstrated that (peri)nuclear expression of the catalytic NADPH oxidase subunit NOX2 in stressed cardiomyocytes, e.g. under ischemia or high concentrations of homocysteine, is an important step in the induction of apoptosis in these cells. Here this ischemia-induced nuclear targeting and activation of NOX2 was specified in cardiomyocytes. METHODS: The effect of ischemia, mimicked by metabolic inhibition, on nuclear localization of NOX2 and the NADPH oxidase subunits p22(phox) and p47(phox), was analyzed in rat neonatal cardiomyoblasts (H9c2 cells) using Western blot, immuno-electron microscopy and digital-imaging microscopy. RESULTS: NOX2 expression significantly increased in nuclear fractions of ischemic H9c2 cells. In addition, in these cells NOX2 was found to colocalize in the nuclear envelope with nuclear pore complexes, p22(phox), p47(phox) and nitrotyrosine residues, a marker for the generation of ROS. Inhibition of NADPH oxidase activity, with apocynin and DPI, significantly reduced (peri)nuclear expression of nitrotyrosine. CONCLUSION: We for the first time show that NOX2, p22(phox) and p47(phox) are targeted to and produce ROS at the nuclear pore complex in ischemic cardiomyocytes

Hypertrophic Cardiomyopathy: A Vicious Cycle Triggered by Sarcomere Mutations and Secondary Disease Hits

Significance: Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by left ventricular hypertrophy, diastolic dysfunction, and myocardial disarray. Disease onset occurs between 20 and 50 years of age, thus affecting patients in the prime of their life. HCM is caused by mutations in sarcomere proteins, the contractile building blocks of the heart. Despite increased knowledge of causal mutations, the exact path from genetic defect leading to cardiomyopathy is complex and involves additional disease hits. Recent Advances: Laboratory-based studies indicate that HCM development not only depends on the primary sarcomere impairment caused by the mutation but also on secondary disease-related alterations in the heart. Here we propose a vicious mutation-induced disease cycle, in which a mutation-induced energy depletion alters cellular metabolism with increased mitochondrial work, which triggers secondary disease modifiers that will worsen disease and ultimately lead to end-stage HCM. Critical Issues: Evidence shows excessive cellular reactive oxygen species (ROS) in HCM patients and HCM animal models. Oxidative stress markers are increased in the heart (oxidized proteins, DNA, and lipids) and serum of HCM patients. In addition, increased mitochondrial ROS production and changes in endogenous antioxidants are reported in HCM. Mutant sarcomeric protein may drive excessive levels of cardiac ROS via changes in cardiac efficiency and metabolism, mitochondrial activation and/or dysfunction, impaired protein quality control, and microvascular dysfunction. Future Directions: Interventions restoring metabolism, mitochondrial function, and improved ROS balance may be promising therapeutic approaches. We discuss the effects of current HCM pharmacological therapies and potential future therapies to prevent and reverse HCM. Antioxid. Redox Signal. 31, 318-358

FLS express functional cytoplasmic dsRNA sensors.

<p>RA-derived synovial fibroblasts were stimulated for 16 h with the indicated concentrations (µg/ml) of poly(I:C), poly(I:C) with fugene, or 3pRNA with fugene to trigger, respectively, TLR3, MDA5, and RIG-I. Transcription levels of (<b>A</b>) TLR3, MDA5, and RIG-I, and (<b>B</b>) the anti-viral/pro-inflammatory response genes IFN β, IP-10, and TNFα was measured by quantitative and semiquantitative PCR, respectively. Depicted is the fold change gene expression compared to medium control (mean ± SD, n = 4) (<b>A</b>) or representative photographs (<b>B</b>). *, p<0.05, **, p<0.005.</p

Expression of complement regulatory proteins on cultured FLS of patients with different forms of arthritis.

<p>CD55, CD46, and CD59 expression was measured by flow cytometry on cultured FLS from patients with RA, OA, PsA, and SpA. Indicated is the fold difference in mean fluorescence intensity (MFI) over respective isotype control Ig (cMFI) (mean, n = 4−5).</p

Poly(I:C)-induced upregulation of CD55 on synovial fibroblasts increases the binding capacity for CD97.

<p>Synovial fibroblasts were stimulated for 2 days with 100 µg/ml poly(I:C). Affinity for CD97 was measured with multivalent fluorescent probes loaded with recombinant CD97-3EGF or EMR2-2EGF (control). To confirm specificity, cells were preincubated with mAb CLB-CD97L/1, directed against the first SCR of CD55. On top, representative histogram plots are shown. The bars represent the fold difference in mean fluorescence intensity (MFI) for CD97-3EGF over EMR2-2EGF (mean ± SD, n = 3). *, p<0.05.</p

CD55 is upregulated by poly(I:C) and IL-1β on synovial fibroblasts.

<p>RA-derived synovial fibroblasts (<b>A, C-F</b>) and dermal fibroblasts (<b>B</b>) were starved overnight and subsequently stimulated for 2 days with 100 ng/ml TNFα, 100 ng/ml IFNγ, 100 ng/ml IL-1β, 1 ng/ml IL-6, 100 U/ml IFNα, 100 µg/ml LTA (TLR2 ligand), 100 µg/ml poly(I:C) (TLR3 ligand), 10 µg/ml LPS (TLR4 ligand), 100 µg/ml imiquimod (TLR7 ligand), or 10 µg/ml CpG oligonucleotides (TLR9 ligand). Expression of CD55 (<b>A</b> and <b>B</b>), CD46 (<b>C</b>) and CD59 (<b>D</b>) was studied by flow cytometry. <b>E,</b> Upregulation of CD55 in response to increasing concentrations of poly(I:C). <b>F,</b> Inhibition of CD55 upregulation by chloroquine (HCQ), an inhibitor of endosomal acidification, added prior to poly(I:C) stimulation. Indicated is the relative protein expression as percentage of the medium control (mean ± SD, n = 6 (<b>A</b>) and 3–5 (<b>B-F</b>)). *, p<0.05; **, p<0.005.</p

Stimulation of cytoplasmic dsRNA receptors in FLS upregulates CD55 expression and, through MDA5, induces cell death.

<p>RA-derived synovial fibroblasts were stimulated for 2 days with the indicated concentrations (µg/ml) of poly(I:C), poly(I:C) with fugene, or 3pRNA with fugene to trigger, respectively, TLR3, MDA5, and RIG-I. <b>A,</b> Expression of CD55 analyzed by flow cytometry (mean ± SD, n = 6). <b>B,</b> Representative flow cytometry plots of annexin V and propidium iodide staining. <b>C,</b> Percentages of annexin V and/or propidium iodide-positive cells analyzed by flow cytometry (mean ± SD, n = 6). <b>D, E,</b> Effect of the pan-caspase inhibitor QVD on cell death and CD55 expression induced by intracellular delivery of poly(I:C) (mean ± SD, n = 3). *, p<0.05; **, p<0.005; ***, p<0.001.</p