Summary Exercise is essential in regulating energy metabolism and whole body insulin sensitivity. To explore the exercise signaling network we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates including AKAP1 using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPK-dependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry.

Insulin resistance (IR) is a complex pathophysiological state that arises from both environmental and genetic perturbations and leads to a variety of diseases, including type-2 diabetes (T2D). Obesity is associated with enhanced adipose tissue inflammation, which may play a role in disease progression. Inflammation modulates protein glycosylation in a variety of cell types, and this has been associated with biological dysregulation. Here, we have examined the effects of an inflammatory insult on protein glycosylation in adipocytes. We performed quantitative N-glycome profiling of membrane proteins derived from mouse 3T3-L1 adipocytes that had been incubated with or without the proinflammatory cytokine TNF-alpha to induce IR. We identified the regulation of specific terminal N-glycan epitopes, including an increase in terminal di-galactose-and a decrease in biantennary alpha-2,3-sialoglycans

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea–induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage–dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.

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Background - Transcriptome sequencing can improve genetic diagnosis of Mendelian diseases but requires access to tissue expressing disease-relevant transcripts. We explored genetic testing of hypertrophic cardiomyopathy (HCM) using transcriptome sequencing of patient-specific human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). We also explored whether antisense oligonucleotides (AOs) could inhibit aberrant mRNA splicing in hiPSC-CMs. Methods - We derived hiPSC-CMs from patients with HCM due to MYBPC3 splice-gain variants, or an unresolved genetic cause. We used transcriptome sequencing of hiPSC-CM RNA to identify pathogenic splicing and used AOs to inhibit this splicing. Results - Transcriptome sequencing of hiPSC-CMs confirmed aberrant splicing in two people with previously identified MYBPC3 splice-gain variants (NM_000256.3:c.1090+453C>T and NM_000256.3:c.1224-52G>A). In a patient with an unresolved genetic cause of HCM following genome sequencing, transcriptome sequencing of hiPSC-CMs revealed diverse cryptic exon splicing due to an MYBPC3 NM_000256.3:c.1928-569G>T variant, and this was confirmed in cardiac tissue from an affected sibling. AO treatment demonstrated almost complete inhibition of cryptic exon splicing in one patient-specific hiPSC-CM line. Conclusions - Transcriptome sequencing of patient specific hiPSC-CMs solved a previously undiagnosed genetic cause of HCM and may be a useful adjunct approach to genetic testing. AO inhibition of cryptic exon splicing is a potential future personalised therapeutic option.