Pathological remodeling includes alterations of ion channel function and calcium homeostasis and ultimately cardiac maladaptive function during the process of disease development. Biochemical assays are important approaches for assessing protein abundance and post-translational modification of ion channels. Several housekeeping proteins are commonly used as internal controls to minimize loading variabilities in immunoblotting protein assays. Yet, emerging evidence suggests that some housekeeping proteins may be abnormally altered under certain pathological conditions. However, alterations of housekeeping proteins in aged and diseased human hearts remain unclear. In the current study, immunoblotting was applied to measure three commonly used housekeeping proteins (β-actin, calsequestrin, and GAPDH) in well-procured human right atria (RA) and left ventricles (LV) from diabetic, heart failure, and aged human organ donors. Linear regression analysis suggested that the amounts of linearly loaded total proteins and quantified intensity of total proteins from either Ponceau S (PS) blot-stained or Coomassie Blue (CB) gel-stained images were highly correlated. Thus, all immunoblotting data were normalized with quantitative CB or PS data to calibrate potential loading variabilities. In the human heart, β-actin was reduced in diabetic RA and LV, while GAPDH was altered in aged and diabetic RA but not LV. Calsequestrin, an important Ca2+ regulatory protein, was significantly changed in aged, diabetic, and ischemic failing hearts. Intriguingly, expression levels of all three proteins were unchanged in non-ischemic failing human LV. Overall, alterations of human housekeeping proteins are heart chamber specific and disease context dependent. The choice of immunoblotting loading controls should be carefully evaluated. Usage of CB or PS total protein analysis could be a viable alternative approach for some complicated pathological specimens.
Keywords: Aging; Diabetes; Heart failure; Housekeeping proteins; Human heart specimens.