Clinical and Radiographic Features of the Autosomal Recessive form of Brachyolmia Caused by PAPSS2 Mutations

Brachyolmia is a heterogeneous skeletal dysplasia characterized by generalized platyspondyly without significant long-bone abnormalities. Based on the mode of inheritance and radiographic features, at least three types of brachyolmia have been postulated. We recently identified an autosomal recessive form of brachyolmia that is caused by loss-of-function mutations of PAPSS2, the gene encoding PAPS (3-phosphoadenosine 5-phosphosulfate) synthase 2. To understand brachyolmia caused by PAPSS2 mutations (PAPSS2-brachyolmia), we extended our PAPSS2 mutation analysis to 13 patients from 10 families and identified homozygous or compound heterozygous mutations in all. Nine different mutations were found: three splice donor-site mutations, three missense mutations, and three insertion or deletion mutations within coding regions. In vitro enzyme assays showed that the missense mutations were also loss-of-function mutations. Phenotypic characteristics of PAPSS2-brachyolmia include short-trunk short stature, normal intelligence and facies, spinal deformity, and broad proximal interphalangeal joints. Radiographic features include platyspondyly with rectangular vertebral bodies and irregular end plates, broad ilia, metaphyseal changes of the proximal femur, including short femoral neck and striation, and dysplasia of the short tubular bones. PAPSS2-brachyolmia includes phenotypes of the conventional clinical concept of brachyolmia, the Hobaek and Toledo types, and is associated with abnormal androgen metabolism. (C) 2013 Wiley Periodicals, Inc

Recombinant Atrial Natriuretic Peptide Prevents Aberrant Ca²⁺ Leakage through the Ryanodine Receptor by Suppressing Mitochondrial Reactive Oxygen Species Production Induced by Isoproterenol in Failing Cardiomyocytes

<div><p>Catecholamines induce intracellular reactive oxygen species (ROS), thus enhancing diastolic Ca²⁺ leakage through the ryanodine receptor during heart failure (HF). However, little is known regarding the effect of atrial natriuretic peptide (ANP) on ROS generation and Ca²⁺ handling in failing cardiomyocytes. The aim of the present study was to clarify the mechanism by which an exogenous ANP exerts cardioprotective effects during HF. Cardiomyocytes were isolated from the left ventricles of a canine tachycardia-induced HF model and sham-operated vehicle controls. The degree of mitochondrial oxidized DNA was evaluated by double immunohistochemical (IHC) staining using an anti-VDAC antibody for the mitochondria and an anti-8-hydroxy-2′-deoxyguanosine antibody for oxidized DNA. The effect of ANP on ROS was investigated using 2,7-dichlorofluorescin diacetate, diastolic Ca²⁺ sparks assessed by confocal microscopy using Fluo 4-AM, and the survival rate of myocytes after 48 h. The double IHC study revealed that isoproterenol (ISO) markedly increased oxidized DNA in the mitochondria in HF and that the ISO-induced DNA damage was markedly inhibited by the co-presence of ANP. ROS production and Ca²⁺ spark frequency (CaSF) were increased in HF compared to normal controls, and were further increased in the presence of ISO. Notably, ANP significantly suppressed both ISO-induced ROS and CaSF without changing sarcoplasmic reticulum Ca²⁺ content in HF (p<0.01, respectively). The survival rate after 48 h in HF was significantly decreased in the presence of ISO compared with baseline (p<0.01), whereas it was significantly improved by the co-presence of ANP (p<0.01). Together, our results suggest that ANP strongly suppresses ISO-induced mitochondrial ROS generation, which might correct aberrant diastolic Ca²⁺ sparks, eventually contributing to the improvement of cardiomyocyte survival in HF.</p></div

SR Ca²⁺ content in sham and failing cardiomyocytes.

<p>A. Measurement of SR Ca²⁺ content by caffeine application. After isolation of cardiomyocytes, cardiomyocytes were loaded with 20 μM Fluo-4 AM (Molecular Probes) for 30 min at room temperature in the dark. Then, these cardiomyocytes were washed with Tyrode solution containing final concentration of 2 mM Ca²⁺. The cardiomyocytes were electrically stimulated by a field stimulator (IonOptix, MA) at a frequency of 0.5 Hz for 30 sec, and then final concentration of 20 mM caffeine were added. An arrow shows a point of addition of caffeine to the dish. B. Bar graph representation of the data in Fig 4A. Each group included 20–30 cells. At least 4 cells were evaluated for each preparation. The bars indicate the means ± SE.</p

Proposed ANP cardioprotective mechanism in heart failure.

<p>A. Leaky RyR channel in failing cardiomyocytes. B. ISO enhanced diastolic SR Ca²⁺ leak. C. ANP inhibited ISO-induced mitochondrial ROS, leading to the decrease of diastolic Ca²⁺ leak through RyR. ANP, atrial natriuretic peptide; GCA-R, membrane guanylate coupled A receptor; ISO, isoproterenol; β-AR, β adrenal receptor; PKA, protein kinase A; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; RyR, ryanodine receptor; ROS, reactive oxygen species; SR, sarcoplasmic reticulum.</p

Effects of ISO and/or ANP on ROS production in sham and failing cardiomyocytes.

<p>A. Representative images of echocardiography in a sham operated dog and a HF dog. Left ventricular ejection fraction (LVEF) in a sham operated dog was 79%, while LVEF in a HF dog was 22%. RV, right ventricle; IVS, interventricular septum; LV, left ventricle; Sham, sham operated control. B. Representative images depicting intracellular ROS production in sham and failing cardiomyocytes corresponding to Fig A. Cardiomyocytes were subjected to immunofluorescence staining with a ROS-sensitive fluorescent dye (DCFH-DA) after electrical pacing at 0.5 Hz. Upper panels: sham cardiomyocytes. Bottom panels: failing cardiomyocytes. C. Bar graph representation of the data in Fig 1B. The bars indicate the means ± SE. Each group included 20–30 cells. At least 4 cells were evaluated for each preparation. D. Representative images depicting the antioxidant effect of the free radical scavenger edaravone (100 μM), ANP (10 nM) and Mito-tempo (100 μM) after exposure to H₂O₂ (25 μM) in sham cardiomyocytes. E. Bar graph representation of the data in Fig 1D. The bars indicate the means ± SE. Changes in the fluorescence intensities of DCFH-DA were compared among cell treatment with edaravone (100 μM), ANP (10 nM) and Mito-tempo (100 μM) Each group included 20–30 cells. At least 4 cells were evaluated for each preparation.</p