Sarco/endoplasmatic reticulum calcium ATPase activity in healthy muscle and Brody disease

The sarco-/endoplasmatic reticulum calcium ATPase (SERCA) is a calcium transporter that plays a key role in the excitation-contraction-relaxation cycle by enabling muscle relaxation. Pathogenic variants in the SERCA-encoding ATP2A1 gene cause Brody disease, a rare recessive myopathy with exercise-induced muscle stiffness as the main feature. Measurement of SERCA activity is an important in vitro functional assay to aid in confirming the pathogenicity of variants in this gene. Furthermore, measurement of SERCA activity is used to study physiological and pathological excitation-contraction (de)coupling. For both, a robust assay is of the utmost importance. We aimed to fully reassess the SERCA activity assay, establish new reference values, and validate the assay by measurement of Brody disease muscle samples. All different SERCA assay parameters were extensively re-evaluated and optimized, including time linearity, the effect of sonication and pH, and the effects of homogenate, KCl/Imidazole, ATP, MgCl₂, and CaCl₂ concentrations. With the optimized assay, SERCA activity was assessed in muscle samples from healthy controls (n = 28) and patients with Brody disease (n = 4). We were able to provide new reference values and demonstrate marked decreased SERCA activity in Brody disease muscle samples (30.0 ± 4.2 mU/mg protein) compared to controls (86.7 ± 25.1 mU/mg protein). We developed a robust enzyme assay to measure SERCA activity with high discriminative power to distinguish patients with Brody disease from controls. Thus, this assay provides a reliable method of studying this important calcium pump for both clinical and scientific purposes.