Leuzzi V, Carducci Ca, Carducci Cl, Pozzessere S, Burlina A, Cerone R, Concolino D, Donati MA, Fiori L, Meli C, Ponzone A, Porta F, Strisciuglio P, Antonozzi I, Blau N. Phenotypic variability, ...neurological outcome and genetics background of 6‐pyruvoyl‐tetrahydropterin synthase deficiency.
This study aimed to investigate the clinical variability and factors implied in the outcome of 6‐pyruvoyl‐tetrahydropterin synthase deficiency (PTPSd). Biochemical and clinical phenotype, treatment variables, and 6‐pyruvoyl‐tetrahydropterin synthase (PTS) genotype, were explored retrospectively in 19 Italian patients (12 males and 7 females, aged 4 months to 33 years).
According to the level of biogenic amines in cerebrospinal fluid (CSF) at the diagnosis, the patients were classified as mild (6) (normal level) or severe (13) (abnormal low level) form (MF and SF, respectively). Blood Phe ranged from 151 to 1053 µmol/l in MF (mean ± SD: 698 ± 403) and 342–2120 µmol/l in SF (mean ± SD: 1175 ± 517) (p = 0.063). Patients with MF showed a normal neurological development (a transient dystonia was detected in one), while all SF patients except one presented with severe neurological impairment and only four had a normal neurological development. The outcome of the SF was influenced by the precocity of the treatment. Serial CSF examinations revealed a decline of 5‐hydroxyindolacetic acid in MFs and an incomplete restoration of neurotransmitters in SFs: neither obviously affected the prognosis. PTS gene analysis detected 17 different mutations (seven so far unreported) (only one affected allele was identified in three subjects). A good correlation was found between genotype and clinical and biochemical phenotype.
The occurrence of brain neurotransmitter deficiency and its early correction (by the therapy) are the main prognostic factors in PTPSd.
Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in many heart pathologies. Biosensing of these parameters represents an important tool in drug ...development and disease modeling. Human embryonic stem cells and especially patient specific induced pluripotent stem cell-derived cardiomyocytes are well established as cardiac disease model.. Here, a live stem cell derived embryoid body (EB) based cardiac cell syncytium served as a biorecognition element coupled to the microcantilever probe from atomic force microscope thus providing reliable micromechanical cellular biosensor suitable for whole-day testing.
The biosensor was optimized regarding the type of cantilever, temperature and exchange of media; in combination with standardized protocol, it allowed testing of compounds and conditions affecting the biomechanical properties of EB. The studied effectors included calcium , drugs modulating the catecholaminergic fight-or-flight stress response such as the beta-adrenergic blocker metoprolol and the beta-adrenergic agonist isoproterenol. Arrhythmogenic effects were studied using caffeine. Furthermore, with EBs originating from patient's stem cells, this biosensor can help to characterize heart diseases such as dystrophies.
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•Robust cellular biosensor based on cardiomyocytes cluster and cantilever as micromechanical transducer.•Live stem cell derived embryoid body based cardiac cells syncytium applied for biorecognition.•Real-time beating force and rate as signal following effects of drugs and arrhythmic events.•Patient specific stem cells allow biomechanical characterization of heart diseases such as dystrophies.
The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo ...post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD,
APP
+
/
−
/PS1
+
/
−
). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca
2+
) leak. RyR-mediated ER Ca
2+
leak activates Ca
2+
-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca
2+
leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aβ load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.
In Gram-negative bacteria and eukaryotic organelles, β-barrel proteins of the outer membrane protein 85-two-partner secretion B (Omp85-TpsB) superfamily are essential components of protein transport ...machineries. The TpsB transporter FhaC mediates the secretion of Bordetella pertussis filamentous hemagglutinin (FHA). We report the 3.15 Å crystal structure of FhaC. The transporter comprises a 16-stranded β barrel that is occluded by an N-terminal α helix and an extracellular loop and a periplasmic module composed of two aligned polypeptide-transport-associated (POTRA) domains. Functional data reveal that FHA binds to the POTRA 1 domain via its N-terminal domain and likely translocates the adhesin-repeated motifs in an extended hairpin conformation, with folding occurring at the cell surface. General features of the mechanism obtained here are likely to apply throughout the superfamily.
Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak ...is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-S2808D+/+ mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-S2808D+/+ mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR Ca2+ store content, and increased diastolic SR Ca2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR Ca2+ leak and reduced HF progression in WT and RyR2-S2808D+/+ mice. In contrast, β-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-S2808D+/+ mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of β-blocker therapy in HF.
Abstract
Motivation
Cardiomyocytes derived from stem cells are closely followed, notably since the discovery in 2007 of human induced pluripotent stem cells (hiPSC). Cardiomyocytes (hiPSC-CM) derived ...from hiPSC are indeed more and more used to study specific cardiac diseases as well as for developing novel applications such as drug safety experiments. Robust dedicated tools to characterize hiPSC-CM are now required. The hiPSC-CM morphology constitutes an important parameter since these cells do not demonstrate the expected rod shape, characteristic of native human cardiomyocytes. Similarly, the presence, the density and the organization of contractile structures would be a valuable parameter to study. Precise measurements of such characteristics would be useful in many situations: for describing pathological conditions, for pharmacological screens or even for studies focused on the hiPSC-CM maturation process.
Results
For this purpose, we developed a MATLAB based image analysis toolbox, which gives accurate values for cellular morphology parameters as well as for the contractile cell organization.
Availability and implementation
To demonstrate the power of this automated image analysis, we used a commercial maturation medium intended to promote the maturation status of hiPSC-CM, and compare the parameters with the ones obtained with standard culture medium, and with freshly dissociated mouse cardiomyocytes.
Supplementary information
Supplementary data are available at Bioinformatics online.
Duchenne muscular dystrophy is associated with progressive deterioration in left ventricular (LV) function. The golden retriever muscular dystrophy (GRMD) dog model recapitulates the pathology and ...clinical manifestations of Duchenne muscular dystrophy. Importantly, they develop progressive LV dysfunction starting at early age.
The authors tested the cardioprotective effect of chronic administration of the ARM036, a small molecule that stabilizes the closed conformation of the cardiac sarcoplasmic reticulum ryanodine receptor/calcium release channel (RyR2) in young GRMD-dogs.
Two-month-old GRMD-dogs were treated with ARM036 or placebo for 4 months. Healthy-dogs of the same genetic background served as controls. Cardiac function was evaluated by conventional and 2-dimensional speckle-tracking echocardiography. Cardiac cellular and molecular analyses were performed at 6 months old.
Conventional echocardiography showed normal LV dimensions and ejection fraction in 6-month-old GRMD dogs. Interestingly, 2-dimensional speckle-tracking echocardiography revealed decreased global longitudinal strain and the presence of hypokinetic segments in placebo-treated GRMD dogs. Single-channel measurements revealed higher RyR2 open probability at low resting Ca2+ in GRMD cardiomyocytes than in controls. ARM036 prevented those in vivo and in vitro dysfunctions in GRMD dogs. Myofilament Ca2+-sensitivity was increased in permeabilized GRMD cardiomyocytes at short sarcomere length. ARM036 had no effect on this parameter. Cross-bridge cycling kinetics were altered in GRMD myocytes and recovered with ARM036 treatment, which coincided with the level of myosin binding protein-C-S glutathionylation.
GRMD-dogs exhibit early LV dysfunction associated with altered myofilament contractile properties. These abnormalities were prevented pharmacologically by stabilizing RyR2 with ARM036.
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