IntroductionSarcoplasmic reticulum Ca2+ATPase 2a (SERCA2a) plays a critical role in regulating cardiac contraction. Changes in the levels of SERCA2a and its main regulator, phospholamban (PLN), have been implicated in heart failure (HF). Previous studies in small and heterogenous cohorts of HF patients have reported decreased gene expression levels of SERCA2a and PLN but findings at the protein level have been less consistent. Thus, we aimed to investigate SERCA2a and PLN protein levels in a large cohort of patients with HF.MethodsWe examined the transcript and protein levels of SERCA2a and PLN in cardiac explants obtained from 114 HF patients with dilated cardiomyopathy (DCM), 65 HF patients with ischaemic heart disease (IHD) and 57 non-failing controls. Transcript levels were assessed via quantitative polymerase chain reaction. Protein levels were measured by untargeted multiplexed quantitative proteomics using tandem mass tags (TMT). For validation, we developed a targeted proteomics assay using parallel reaction monitoring (PRM) on LV tissues from 10 patients carrying the single arginine deletion in PLN (R14del) mutation and compared to 13 non-failing controls.ResultsTranscript levels of SERCA2a were reduced in DCM patients compared to controls (FC=0.81, P=0.005), as were PLN levels (FC=0.78, P<0.001). No significant changes were observed in IHD patients. However, TMT proteomics revealed no change in SERCA2a protein levels in both groups of human HF. Similarly, PLN levels were unaltered between groups. Even in carriers of the R14del PLN mutation, SERCA2a protein levels were unaltered. The targeted PRM approach was highly specific and able to discern PLN peptides that are affected by the R14del PLN mutation. Total PLN levels were decreased in R14del patients compared to controls (FC=0.72, P=0.002). When the targeted PRM method was applied to our larger cohort of patients with end-stage HF, it confirmed the absence of significant differences in SERCA2a and PLN protein levels between patients with DCM, IHD and controls.ConclusionIn this largest proteomics analysis of cardiac tissues from patients with end-stage HF to date, we observed no changes in SERCA2a and PLN protein abundance compared to control hearts. Our findings challenge the existing paradigm that a reduction of SERCA2a is a hallmark of human HF. While similar protein abundance does not rule out altered SERCA2a activity, our findings provide a possible explanation for the failure of SERCA2a gene therapy in clinical trials.Conflict of InterestNone