Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

Phospholamban (PLN) plays a central role in Ca²⁺ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca²⁺-ATPase 2A (SERCA2A) Ca²⁺ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.Cardiovascular diseases (CVDs) leading to systolic/diastolic heart failure (HF), such as hypertensive/diabetic heart disease, stroke, and vascular atherosclerosis, are leading causes of death in the developed world (1). Many CVDs are associated with genetic predispositions. For example, in humans, the arginine to cysteine (R9C) substitution in phospholamban (PLN) has been shown to result in dilated cardiomyopathy (DCM) presenting in adolescence, leading to rapid deterioration of heart function and premature death (2). However, the etiology and molecular mechanisms of progression of DCM and other CVDs leading to HF are complex and still poorly understood, further complicating clinical assessment and management. From a biological and clinical perspective, the identification and characterization of clinically relevant, potentially druggable, pathways driving the maladaptive response in affected heart tissue are key challenges to improved diagnostic and therapeutic tools for earlier detection and preventative treatment of both inherited and chronic CVDs.Cardiac muscle contraction is controlled by Ca²⁺ flux and signaling relays, which are perturbed in HF. Internal stores of Ca²⁺ required for the proper functioning of cardiomyocytes (CMs) are normally maintained through the function of the sarco(endo)plasmic reticulum Ca²⁺-ATPase 2 (SERCA2) (3), which is responsible for the sequestration of Ca²⁺ resulting in muscle relaxation. SERCA2 activity is regulated through a reversible inhibitory interaction with PLN, which can be relieved by phosphorylation of PLN by protein kinase A (PKA) or Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) (3).Proteomic analyses have revealed changes in the abundance of other effector proteins in diverse biochemical pathways in DCM. Notably, shotgun proteomic analysis of membrane protein expression dynamics in heart microsomes isolated from mice overexpressing a superinhibitory (I40A) mutant of PLN revealed changes in G protein-coupled receptor-mediated pathways leading to activation of protein kinase C (PKC) (4). We previously reported quantitative changes in protein and cognate mRNA expression levels in cardiac ventricular tissue at different time points in the development of DCM in R9C-PLN mice representing clear clinical stages in the progression to HF (5). We showed that the latter maladaptive response was driven by elevated activity of MAPK signaling by the protein kinases p38 and JNK, in part through down-regulation of prosurvival microRNAs (6). However, the underlying upstream and downstream signaling events preceding HF were not fully explored.In the present study, we report a systematic, large-scale quantitative phosphoproteomic analysis of dysregulated protein phosphorylation-dependent signaling occurring at the early symptomatic stages of DCM progression in whole hearts from R9C mutant mice compared with wild-type littermates.