The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported that induced pluripotent stem cellderived cardiomyocytes (iPSC-CMs) from patients with a dilated cardiomyopathy (DCM) mutation, troponin T (TnT)-R173W, display sarcomere protein misalignment and impaired contractility. Yet it is not known how TnT mutation causes dysfunction of sarcomere microdomains and how these events contribute to misalignment of sarcomeric proteins in presence of DCM TnT-R173W. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains. This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. Small molecule-based activation of AMPK can restore TnT microdomain interactions, and partially recovers sarcomere protein misalignment as well as impaired contractility in DCM TnT-R173W iPSC-CMs. Our findings suggest a novel therapeutic direction targeting sarcomere-cytoskeleton interactions to induce sarcomere re-organization and contractile recovery in DCM.Sarcomeres are the basic contractile unit of cardiac cells, whose particularly specialized function depends on a highly organized structure. The troponin-tropomyosin (Tn-Tm) complex at sarcomeric thin filaments is a critical component for excitation-contraction coupling. Stability and anchoring of the Tn-Tm complex on sarcomeres is provided by binding of the troponin T (TnT) subunit to Tm and the troponin I subunit (TnI) on actin myofilaments. Tropomyosin (TPM) together with TnI regulates actin/myosin binding and ATPase function in presence of micromolar, cytocolic Ca 2+ , which is bound by the troponin C subunit (TnC). This highly sensitive mechanisms is fine-tuned by post-translational modifications, such as PKA-mediated phosphorylation of TnI. Mutations in the Tn-Tm complex lead to severe disease, such as dilated cardiomyopathy (DCM). DCM is featured by left ventricular dilatation, contractile dysfunction, and arrhythmias 1 and represents a frequent cause of heart failure. More than 25% of DCM cases are caused by inherited mutations, particularly in sarcomeric proteins 2 . Recently, human iPSC-derived cardiomyocytes (iPSC-CMs) have been utilized for human genetic disease modeling 3-6 and drug testing 7 . Here, we analyze a sarcomeric mutation in cardiac troponin T (TnT), TnT-R173W. This mutation is located within one of the two tropomyosin binding regions of TnT, the T1 domain 8 and was the first DCM mutation reported in a human patient-specific iPSC-derived cardiomyocyte model 9 . This report www.nature.com/scientificreports www.nature.com/scientificreports/ demonstrated DCM patient-specific iPSC-CMs to display mole...

Rationale: The immature presentation of human induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) is currently a challenge for their application in disease modeling, drug screening, and regenerative medicine. Long-term culture is known to achieve partial maturation of iPSC-CMs. However, little is known about the molecular signaling circuitries that govern functional changes, metabolic output, and cellular homeostasis during long-term culture of iPSC-CMs. Objective: We aimed to identify and characterize critical signaling events that control functional and metabolic transitions of cardiac cells during developmental progression, as recapitulated by long-term culture of iPSC-CMs. Methods and Results: We combined transcriptomic sequencing with pathway network mapping in iPSC-CMs that were cultured until a late time point, day 200, in comparison to a medium time point, day 90, and an early time point, day 30. Transcriptomic landscapes of long-term cultured iPSC-CMs allowed mapping of distinct metabolic stages during development of maturing iPSC-CMs. Temporally divergent control of mitochondrial metabolism was found to be regulated by cAMP/PKA (protein kinase A)- and proteasome-dependent signaling events. The PKA/proteasome-dependent signaling cascade was mediated downstream by Hsp90 (heat shock protein 90), which in turn modulated mitochondrial respiratory chain proteins and their metabolic output. During long-term culture, this circuitry was found to initiate upregulation of iPSC-CM metabolism, resulting in increased cell contractility that reached a maximum at the day 200 time point. Conclusions: Our results reveal a PKA/proteasome- and Hsp90-dependent signaling pathway that regulates mitochondrial respiratory chain proteins and determines cardiomyocyte energy production and functional output. These findings provide deeper insight into signaling circuitries governing metabolic homeostasis in iPSC-CMs during developmental progression.

The quantitative detection methods for many microplastic (MP) polymers in the environment are inadequate. For example, effective detection methods for nylon (polyamide, PA), a widely used plastic, in different environmental samples are still lacking. In the present study, a method based on acid depolymerization−liquid chromatography−tandem mass spectrometry (LC−MS/MS) and without the separation of MPs from samples was developed to quantify nylon MPs. After removing the background monomer compounds, PA6 and PA66 were efficiently depolymerized to 6-aminocaproic acid and adipic acid, respectively, and detected by LC−MS/MS. Accordingly, the quantity of nylon MPs was accurately calculated. By using the proposed method, the recovery of spiked PA6 and PA66 MPs in the environmental samples ranged from 90.8 to 98.8%. The limits of quantification for PA6 and PA66 MPs were 0.680 and 4.62 mg/ kg, respectively. PA MPs were widely detected in indoor dust, sludge, marine sediment, freshwater sediment, fishery sediment, and fish guts and gills with concentrations of 0.725−321 mg/kg. Extremely high concentrations of PA66 MPs were detected in indoor dust and fish guts and gills, indicating the unequivocal risk of human exposure through dust ingestion and dietary exposure.