Housekeeping Proteins Exhibit a High Level of Expression Variability Within Control Group and Between Ischemic Human Heart Biopsies

BACKGROUND: Human cardiac biopsies are widely used in clinical and fundamental research to decipher molecular events that characterize cardiac physiological and pathophysiological states. One of the main approaches relies on the analysis of semiquantitative immunoblots that reveals alterations in protein expression levels occurring in diseased hearts. To maintain semiquantitative results, expression level of target proteins must be standardized. The expression of HKP (housekeeping proteins) is commonly used to this purpose. METHODS AND RESULTS: We evaluated the stability of HKP expression (actin, β‐tubulin, GAPDH, vinculin, and calsequestrin) and total protein staining within control (coefficient of variation) and comparatively with ischemic human heart biopsies (P value). All HKP exhibited a high level of intragroup (ie, actin, β‐tubulin, and GAPDH) and/or intergroup variability (ie, GAPDH, vinculin, and calsequestrin). Among all, we found total protein staining to exhibit the highest degree of stability within and between groups, which makes this reference the best to study protein expression level in human biopsies from ischemic hearts and age‐matched controls. In addition, we illustrated that using an inappropriate reference protein marker misleads interpretation on SERCA2 (sarco/endoplasmic reticulum Ca(2+) ATPase) and cMyBPC (cardiac myosin binding protein‐C) expression level after myocardial infarction. CONCLUSIONS: These reemphasize the need to standardize the level of protein expression with total protein staining in comparative immunoblot studies on human samples from control and diseased hearts.