Abstract
Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) by reducing their apparent affinity for Ca2+. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca2+ affinity of SERCA (K Ca, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (μmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.
Highlights
Sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) are major regulators of intracellular free Ca2+ in skeletal muscle, which use free energy released from the hydrolysis of ATP to transport Ca2+ ions from the cytosol into the lumen of the sarcoplasmic reticulum (SR) [1]
More recently we found that SLN protein expression in skeletal muscles coincides with SERCA2a whereby SLN and SERCA2a levels were highest in soleus and red gastrocnemius (RG), very low in extensor digitorum longus (EDL), and undetectable in white gastrocnemius (WG) [15]
Nef et al [37] reported that SERCA2a + PLN + SLN were co-expressed in human ventricular myocytes from patients with Tako-Tsubo cardiomyopathy and our study demonstrates for the first time that PLN and SLN are normally co-expressed with sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) in healthy skeletal muscle
Summary
Sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) are major regulators of intracellular free Ca2+ in skeletal muscle, which use free energy released from the hydrolysis of ATP to transport Ca2+ ions from the cytosol into the lumen of the sarcoplasmic reticulum (SR) [1]. SERCAs are 110 kDa integral membrane proteins belonging to the P-type family of cation transporters, as they are phosphorylated at a critical aspartate residue during their catalytic cycle [2]. Two SERCA isoforms predominate, namely SERCA1a and SERCA2a, which are differentially expressed in specific muscle fiber types [3,4,5,6]. The SERCA1a isoform with faster kinetics [3], is co-expressed with fast myosin heavy chain (MHC) isoforms MHCIIb, MHCIIx, and MHCIIa, while the slower SERCA2a isoform [3], is co-expressed with slow MHC isoforms MHCIβ and MHCIα [5,7]. The differences in the functional capacity of individual SERCA isoforms can be further amplified by the presence of the SERCA regulatory proteins, phospholamban (PLN) and sarcolipin (SLN) [8]
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