Phospholamban and sarcolipin prevent thermal inactivation of sarco(endo)plasmic reticulum Ca2+-ATPases

Abstract

Na+-K+-ATPase frommice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25-35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form protective interactions with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.

Heterozygous SOD2 deletion selectively impairs SERCA function in the soleus of female mice

“…Further investigation showed no changes in SERCA2a content or SERCA2a T‐nitration, though this was not due to a lack of total protein nitration. Like SLN, PLN has previously been demonstrated to have a protective effect on SERCA (Fu et al, 2020), and here we show that the LV has much more PLN content relative to soleus, which could be conferring cytotoxic protection. Notably, previous studies have shown that PLN is in fact expressed in the soleus (Braun, Geromella, et al, 2021; Fajardo et al, 2017), and we believe the lack of signal in the soleus speaks to the relative abundance of PLN in the LV compared to the soleus, which not only masks detection in the soleus but may also be preventing T‐nitration and oxidative damage to SERCA2a in the LV.…”

“…The reduction in Ca 2+ affinity seen in the soleus of Sod 2 +/− mice compared to WT may be partly due to the increase in SLN content, a well‐studied SERCA regulator that can reduce SERCA’s affinity for Ca 2+ (Asahi et al, 2002; Babu et al, 2007; Rathod et al, 2021), in Sod 2 +/− mice compared to WT. Despite not observing a statistically significant increase in SLN in soleus muscles from Sod2 +/− mice, it is worth noting that SLN has been shown to play a protective role for SERCA, preventing thermal inactivation of the pump (Fu et al, 2020). Furthermore, SLN is upregulated in various models of muscle wasting including spaceflight (Braun, Geromella, et al, 2021) and muscular dystrophy (Fajardo et al, 2018; Schneider et al, 2013)—both of which also demonstrate increases in nitrotyrosine (Braun, Geromella, et al, 2021; Cleverdon et al, 2021; Gehrig et al, 2012).…”

SERCA function is impaired in skeletal and cardiac muscles from young DBA/2J mdx mice

“…Lastly, we measured total protein nitrosylation and tyrosine nitration in the gastrocnemius to determine whether elevated RONS could contribute to the observed SR Ca 2+ handling dysfunction in the D2 mdx muscle. SERCA is susceptible to RONS damage and RONS are known to be high in DMD, and therefore, it is plausible that RONS modifications could be contributing to the changes in protein function we see (15,(30)(31)(32)(33)(34). Our results show that excessive and irreversible (detection under reducing conditions) RONS modifications may be impairing function of the SERCA pump.…”

“…One potential factor may be elevated oxidative/nitrosative stress. SERCA is susceptible to RONS damage and RONS are known to be high in DMD, and therefore, it is plausible that RONS modifications could be contributing to the changes in SERCA function (15,(30)(31)(32)(33)(34). Our results of elevated total protein nitrosylation and tyrosine nitration under reducing conditions are suggestive of irreversible RONS modifications that may be impairing the SERCA pump.…”

Skeletal and cardiac muscle calcium transport regulation in health and disease

“…Several studies have also noted increased oxidation and nitration of SERCA in aged skeletal muscle [ 155 , 214 , 219 ], which we have shown results in the loss of redox control of SERCA activity and expression [ 220 ]. However, our work showing that several SERCA-binding proteins, including heat shock protein 70, PLN, and SLN, can protect SERCA structure and function during cellular stress [ 152 , 159 , 221 ], suggests that these SERCA-binding proteins may play a crucial role in protecting cellular Ca 2+ homeostasis and preserving cardiac and skeletal muscle function under conditions of chronic oxidative stress and disease.…”