Atrial fibrillation (AF) is the most common tachyarrhythmia. AF, due to substantial remodeling processes initiated in the atria, is a typically self-sustaining and progressive disease. The development and progression of atrial fibrillation (AF) is the result of interaction among multiple signaling pathways with different kinetics. Altogether these pathophysiological pathways lead to electrical, contractile and structural impairment of the atrium and result in atrial remodeling. However, since mass-spectrometry-based proteomics has matured into a mainstream analytical tool over the past decade, it increasingly supports our efforts to gain deeper insights on the development of AF and its interacting pathomechanisms. Our group performed a stable isotope labeling with amino acids in cell culture (SILAC)-based phosphoproteome study in HL-1 mouse cardiomyocytes. HL-1 cells were subjected to 24 h continuous and 24 h interval rapid atrial pacing (RAP), respectively. The phosphoproteomes were analyzed by phosphopeptide enrichment and high-accuracy mass spectrometry (HPLC-MS/MS) in the presence of an internal SILAC-standard which consists of peptide lysates of metabolic labeled HL-1 cells with heavy amino acids. These peptides contain 13C instead of 12C which causes a known mass shift compared with the peptide that contains the light (‘normal’) version of the amino acid. Applying a cut-off of 2.0 and higher, 114 or 133 differential regulated phosphorylations were found and only 7 ...