ErbB receptor tyrosine kinases orchestrate phosphorylation-based signaling in response to extracellular ligands and are key drivers in cancer biology. Although ErbB-targeted therapies have transformed cancer care, some agents cause cardiac adverse events. Yet, the acute phosphorylation programs engaged by ErbB ligands in the adult heart remain incompletely defined. Here, we applied in vivo quantitative phosphoproteomics with dual enrichment using TiO2 and anti-phosphotyrosine antibodies to map acute cardiac phosphorylation responses to epidermal growth factor (EGF; EGFR/ErbB1) and neuregulin-1(3 (NRG1(3; ErbB3/ErbB4) in adult mouse hearts. EGF triggered robust receptor tyrosine kinase signaling, convergence with insulin-associated nodes independent of insulin receptor activation, and phosphorylation of calcium-handling proteins including phospholamban, SERCA, NCX1, and CaV1.2, implicating CaMK2S. NRG1(3 elicited a coordinated ErbB-dependent response featuring activation of Akt, MAPK, and stress kinases, with engagement of sarcomere and metabolic modules. Comparative analysis identified shared core signaling alongside ligand-specific differences in kinase and transcription factor phosphorylation, with EGF displaying broader network breadth. These data provide a phosphorylation-centric framework for ligand-resolved ErbB signaling in the heart and offer mechanistic insight into how ErbB-targeted therapies may influence cardiac function.
Hypertrophic cardiomyopathy is a leading cause of sudden cardiac death, often driven by pathogenic variants in sarcomeric genes such as MYBPC3. The precise electrophysiological mechanisms underlying arrhythmogenesis in HCM, particularly in the context of MYBPC3 truncating mutations, remain incompletely defined, hindering the development of targeted therapies. Here, we leveraged human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) carrying the MYBPC3 p.R943X mutation to systematically characterize action potential remodeling and evaluate the efficacy of ion-channel–modulating drugs.High-throughput optical AP assays revealed that R943X mutant hiPSC-CMs exhibit profound baseline electrophysiological disruption, with a marked reduction in normal AP profiles and increased incidence of pro-arrhythmic morphologies, including AP prolongation, early afterdepolarizations, and delayed afterdepolarizations, compared to isogenic non-diseased controls. Pharmacological profiling demonstrated that NS-5806, a Kv4 channel modulator, exerts a biphasic, dose-dependent effect in non-diseased isogenic control cells and partially restores normal AP morphology in R943X mutants by enhancing repolarizing current and suppressing AP prolongation. In contrast, Acacetin, an ITo and late Na+ current inhibitor, preserved electrical stability in non-diseased isogenic control cells but conferred limited and dose-sensitive rescue in mutants, with higher concentrations exacerbating arrhythmic features. These findings provide direct evidence that targeted augmentation of transient outward K+ current can selectively mitigate mutation-induced arrhythmogenic remodeling in hiPSC-CMs modeling HCM. Our study highlights the therapeutic promise, and challenges, of precision ion-channel modulation in genetic cardiomyopathy and establishes a scalable platform for genotype-guided drug discovery.
Calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) is a central regulator of cardiac excitation–contraction coupling and is chronically activated in multiple cardiac pathologies. Nitrosylation has been implicated in cardiac cell signaling and function in health and disease. We tested how S-nitrosylation at two known CaMKII cysteines alter kinase activation conformation and intracellular mobility in cardiac myocytes. We directly monitored in real time, the CaMKII activation state in myocytes with GSNO treatment– before or after activation of CaMKII. Active CaMKII conformation was assessed using the fluorescent biosensor Camui and fluorescence lifetime values, and cellular mobility was assessed by fluorescence recovery after photobleach (FRAP) in CaMKII-GFP and movement away from the sarcomeric Z-line. To test the influence of each known S-nitrosylation site we used CaMKII in which each site was mutated (Cys273Ser and Cys290Ala) in Camui and CaMKII-GFP. The results reveal that in intact ventricular cardiomyocytes S-nitrosylation of CaMKII at Cys273 prior to electrical field stimulation (and β-adrenergic activation) suppresses CaMKIIδ activation and mobility, effects that are suppressed in Cys273Ser CaMKIIδ. In contrast S-nitrosylation of CaMKIIδ Cys290 was critical for GSNO-induced activation and mobility. Thus, baseline S-nitrosylation at Cys273 limits CaMKIIδ activation and mobility, while S-nitrosylation at C290 when the kinase is activated enhances activation and mobility. Thus, prevailing nitrosylation conditions in cardiac myocytes can modulate the activation and mobility of CaMKII with respect to its targets.
