Project description:Pathogenic mutations in alpha kinase 3 (ALPK3) cause cardiomyopathy and a range of musculoskeletal defects. How ALPK3 mutations result in disease remains unclear and little is known about this atypical kinase. Using a suite of engineered human pluripotent stem cells (hPSCs) we show that ALPK3 localizes to the sarcomere, specifically at the M-Band. Both sarcomeric organization and calcium kinetics were disrupted in ALPK3 deficient hPSC derived cardiomyocytes. Further, cardiac organoids derived from ALPK3 knockout hPSCs displayed reduced force generation. Phosphoproteomic profiling of wildtype and ALPK3 null hPSC derived cardiomyocytes revealed ALPK3-dependant phospho-peptides were enriched for proteins involved in sarcomere function and protein quality control. We demonstrate that ALPK3 binds to the selective autophagy receptor SQSTM1 (Sequestome 1) and is required for the sarcomeric localization of SQSTM1. We propose that ALPK3 is a myogenic kinase with an integral role in the intracellular signaling networks underlying sarcomere maintenance required for continued cardiac contractility.
Project description:Pathogenic mutations in alpha kinase 3 (ALPK3) cause cardiomyopathy and a range of musculoskeletal defects. How ALPK3 mutations result in disease remains unclear and little is known about this atypical kinase. Using a suite of engineered human pluripotent stem cells (hPSCs) we show that ALPK3 localizes to the sarcomere, specifically at the M-Band. Both sarcomeric organization and calcium kinetics were disrupted in ALPK3 deficient hPSC derived cardiomyocytes. Further, cardiac organoids derived from ALPK3 knockout hPSCs displayed reduced force generation. Phosphoproteomic profiling of wildtype and ALPK3 null hPSC derived cardiomyocytes revealed ALPK3-dependant phospho-peptides were enriched for proteins involved in sarcomere function and protein quality control. We demonstrate that ALPK3 binds to the selective autophagy receptor SQSTM1 (Sequestome 1) and is required for the sarcomeric localization of SQSTM1. We propose that ALPK3 is a myogenic kinase with an integral role in the intracellular signaling networks underlying sarcomere maintenance required for continued cardiac contractility.
Project description:Overexpressed either GFP, wild-type (WT) NFIX or a phospho-dead mutant of NFIX in which eight serine residues surrounding S286 were mutated to alanine (S265/267/268/271/272/273/274/275A) was performed C2C12 cells prior to the induction of myogenesis. Cells were harvest 2 days post induction of myogensis with 2% horse serum.
Project description:To characterize phosphorylation-based signaling events across different exercise modalities we subjected eight healthy young men (age, 26.3±1.3 years; BMI, 23.5±0.7 kg/m2; maximal oxygen uptake (VO2 max), 42.6±1.5 ml/kg/min) that did not perform regular physical activity apart from local bicycling to an acute bout of endurance (90 min ~60% of VO2 max), sprint (3 x 30-s all-out cycling), or resistance exercise (6 sets of 10 RM knee extensions) in the fasting state. All participants completed the three types of exercise in a randomized crossover design with 14 days washout between each exercise bout.
Project description:We recently identified C18ORF25 as a new exercise-regulated phophoprotein. To investigate potential in vivo functions of C18ORF25, we used CRISPR/Cas9 to generate a whole-body knock-out (KO) mouse model on a C57BL/6J background. Proteomic analysis was performed to identify potental changes in the proteome. To gain further insights into the possible signalling pathways regulated by C18ORF25, Soleus muscles from WT and KO mice (n=4) were isolated, and the muscle from one leg was maintained at resting tension with no stimulation as a control while the muscle from the contralateral leg was subject to electrical stimulation ex vivo. Muscles were quickly snap frozen and subject to single-shot label-free phosphoproteomic analysis to compare the signalling responses between the genotypes.
Project description:To gain insight into possible regulators of liver extracellular vesicle (EV)-mediated glucose effectiveness, we evaluated the phosphoproteomic profile of soleus muscle incubated in low and high glucose, without or with liver EVs
Project description:Recent studies in non-human model systems have shown therapeutic potential of modified mRNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the α-galactosidase (GLA) gene in a human cardiac model generated from induced-pluripotent stem cell-derived from two patients with Fabry disease. In line with the clinical phenotype, cardiomyocytes from Fabry patient’s induced pluripotent stem cells show accumulation of the glycosphinolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Further, the patient-specific cardiomyocytes have significant upregulation of lysosomal associated proteins. Upon modRNA treatment, a subset of lysosomal proteins were partially restored to wildtype levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA treated cardiomyocytes demonstrating that α-galactosidase enzymatic activity was restored. Together, our results validate the utility of patient IPSC-derived cardiomyocytes as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.
Project description:Myogenesis is governed by signalling networks whose regulations are tightly controlled in a time-dependent manner. While different protein kinases have been identified to regulate various aspects of myogenesis, knowledge on the global signalling networks and their downstream substrates during myogenesis remains incomplete. Here, we map the myogenic differentiation of C2C12 cells using mass spectrometry (MS)-based phosphoproteomics and proteomics. From these data, we infer global kinase activity and predict substrates of key kinases that are involved in myogenesis. We found that multiple mitogen-activated protein kinases (MAPKs) mark the initial wave of signalling cascades. Further phosphoproteomic and proteomic profiling with MAPK1/3 and MAPK8/9 specific inhibitions unveil their shared and distinctive roles on myogenesis.
Project description:Our understanding of heart failure (HF) has been provided by indirect surrogates, such as post-mortem histology, cardiovascular imaging, and molecular characterisation in vivo and in vitro, rather than directly in pre-mortem human cardiac tissue. Using our heart bank of pre-mortem hearts procured according to the most stringent protocols, we examined ischemic (ICM) and dilated cardiomyopathy (DCM) -- the most common causes of HF and leading causes of cardiac transplantation1. We performed unbiased, comprehensive, paired proteomic and metabolomic analysis of 51 left ventricular (LV) samples from 44 cryopreserved pre-mortem human ICM and DCM hearts, including age-matched, healthy, histopathologically-normal donor controls of both genders for comparison. Data integration via pathway and correlation network analysis revealed overlapping and divergent disease pathways in ICM and DCM, and, strikingly, precise sex-specific differences within each disease that unveil the interaction of gender with HF. Identified core functional nodes in each disease may serve as novel therapeutic targets, and we provide all proteomic and metabolomic results via an interactive online repository (https://mengboli.shinyapps.io/heartomics/) as a publicly available resource.