Toward countering muscle and bone loss with spaceflight: GSK3 as a potential target.

We examined the effects of ∼30 days of spaceflight on glycogen synthase kinase 3 (GSK3) content and inhibitory serine phosphorylation in murine muscle and bone samples from four separate missions (BION-M1, rodent research [RR]1, RR9, and RR18). Spaceflight reduced GSK3ß content across all missions, whereas its serine phosphorylation was elevated with RR18 and BION-M1. The reduction in GSK3ß was linked to the reduction in type IIA fibers commonly observed with spaceflight as these fibers are particularly enriched with GSK3. We then tested the effects of inhibiting GSK3 before this fiber type shift, and we demonstrate that muscle-specific Gsk3 knockdown increased muscle mass, preserved muscle strength, and promoted the oxidative fiber type with Earth-based hindlimb unloading. In bone, GSK3 activation was enhanced after spaceflight; and strikingly, muscle-specific Gsk3 deletion increased bone mineral density in response to hindlimb unloading. Thus, future studies should test the effects of GSK3 inhibition during spaceflight.

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

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) restores intracellular Ca2+ ([Ca2+ ]i ) to resting levels after muscle contraction, ultimately eliciting relaxation. SERCA pumps are highly susceptible to tyrosine (T)-nitration, impairing their ability to take up Ca2+ resulting in reduced muscle function and increased [Ca2+ ]i and cellular damage. The mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2), converts superoxide radicals into less reactive H2 O2 . Heterozygous deletion of SOD2 (Sod2+/- ) in mice increases mitochondrial oxidative stress; however, the consequences of reduced SOD2 expression in skeletal and cardiac muscle, specifically the effect on SERCA pumps, has yet to be investigated. We obtained soleus, extensor digitorum longus (EDL), and left ventricle (LV) muscles from 6 to 7 month-old wild-type (WT) and Sod2+/- female C57BL/6J mice. Ca2+ -dependent SERCA activity assays were performed to assess SERCA function. Western blotting was conducted to examine the protein content of SERCA, phospholamban, and sarcolipin; and immunoprecipitation experiments were done to assess SERCA2a- and SERCA1a-specific T-nitration. Heterozygous SOD2 deletion did not alter SERCA1a or SERCA2a expression in the soleus or LV but reduced SERCA2a in the EDL compared with WT, though this was not statistically significant. Soleus muscles from Sod2+/- mice showed a significant reduction in SERCA's apparent affinity for Ca2+ when compared to WT, corresponding with significantly elevated SERCA2a T-nitration in the soleus. No effect was seen in the EDL or the LV. This is the first study to investigate the effects of SOD2 deficiency on muscle SERCA function and shows that it selectively impairs SERCA function in the soleus.

Characterizing SERCA Function in Murine Skeletal Muscles after 35-37 Days of Spaceflight.

It is well established that microgravity exposure causes significant muscle weakness and atrophy via muscle unloading. On Earth, muscle unloading leads to a disproportionate loss in muscle force and size with the loss in muscle force occurring at a faster rate. Although the exact mechanisms are unknown, a role for Ca2+ dysregulation has been suggested. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump actively brings cytosolic Ca2+ into the SR, eliciting muscle relaxation and maintaining low intracellular Ca2+ ([Ca2+]i). SERCA dysfunction contributes to elevations in [Ca2+]i, leading to cellular damage, and may contribute to the muscle weakness and atrophy observed with spaceflight. Here, we investigated SERCA function, SERCA regulatory protein content, and reactive oxygen/nitrogen species (RONS) protein adduction in murine skeletal muscle after 35-37 days of spaceflight. In male and female soleus muscles, spaceflight led to drastic impairments in Ca2+ uptake despite significant increases in SERCA1a protein content. We attribute this impairment to an increase in RONS production and elevated total protein tyrosine (T) nitration and cysteine (S) nitrosylation. Contrarily, in the tibialis anterior (TA), we observed an enhancement in Ca2+ uptake, which we attribute to a shift towards a faster muscle fiber type (i.e., increased myosin heavy chain IIb and SERCA1a) without elevated total protein T-nitration and S-nitrosylation. Thus, spaceflight affects SERCA function differently between the soleus and TA.

The role of phospholamban and GSK3 in regulating rodent cardiac SERCA function.

Cardiac contractile function is largely mediated by the regulation of Ca2+ cycling throughout the lifespan. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is paramount to cardiac Ca2+ regulation, and it is well established that SERCA dysfunction pathologically contributes to cardiomyopathy and heart failure. Phospholamban (PLN) is a well-known inhibitor of the SERCA pump and its regulation of SERCA2a-the predominant cardiac SERCA isoform-contributes significantly to proper cardiac function. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase involved in several metabolic pathways, and we and others have shown that it regulates SERCA function. In this mini-review, we highlight the underlying mechanisms behind GSK3's regulation of SERCA function specifically discussing changes in SERCA2a and PLN expression and its potential protection against oxidative stress. Ultimately, these recent findings that we discuss could have clinical implications in the treatment and prevention of cardiomyopathies and heart failure.

Low-dose lithium feeding increases the SERCA2a-to-phospholamban ratio, improving SERCA function in murine left ventricles.

NEW FINDINGS: What is the central question of this study? Inhibition of glycogen synthase kinase-3 (GSK3) has been shown to improve cardiac SERCA2a function. Lithium can inhibit GSK3, but therapeutic doses used in treating bipolar disorder can have toxic effects. It has not been determined whether subtherapeutic doses of lithium can improve cardiac SERCA function. What is the main finding and its importance? Using left ventricles from wild-type mice, we found that subtherapeutic lithium feeding for 6 weeks decreased GSK3 activity and increased cardiac SERCA function compared with control-fed mice. These findings warrant the investigation of low-dose lithium feeding in preclinical models of cardiomyopathy and heart failure to determine the therapeutic benefit of GSK3 inhibition. ABSTRACT: The sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) pump is responsible for regulating calcium (Ca2+ ) within myocytes, with SERCA2a being the dominant isoform in cardiomyocytes. Its inhibitor, phospholamban (PLN), acts by decreasing the affinity of SERCA for Ca2+ . Changes in the SERCA2a:PLN ratio can cause Ca2+ dysregulation often seen in patients with dilated cardiomyopathy and heart failure. The enzyme glycogen synthase kinase-3 (GSK3) is known to downregulate SERCA function by decreasing the SERCA2a:PLN ratio. In this study, we sought to determine whether feeding mice low-dose lithium, a natural GSK3 inhibitor, would improve left ventricular SERCA function by altering the SERCA2a:PLN ratio. To this end, male wild-type C57BL/6J mice were fed low-dose lithium via drinking water (10 mg kg-1  day-1 LiCl for 6 weeks) and left ventricles were harvested. GSK3 activity was significantly reduced in LiCl-fed versus control-fed mice. The apparent affinity of SERCA for Ca2+ was also increased (pCa50 ; control, 6.09 ± 0.03 versus LiCl, 6.26 ± 0.04, P < 0.0001) along with a 2.0-fold increase in SERCA2a:PLN ratio in LiCl-fed versus control-fed mice. These findings suggest that low-dose lithium supplementation can improve SERCA function by increasing the SERCA2a:PLN ratio. Future studies in murine preclinical models will determine whether GSK3 inhibition via low-dose lithium could be a potential therapeutic strategy for dilated cardiomyopathy and heart failure.

SERCA2a tyrosine nitration coincides with impairments in maximal SERCA activity in left ventricles from tafazzin-deficient mice.

The sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) is imperative for normal cardiac function regulating both muscle relaxation and contractility. SERCA2a is the predominant isoform in cardiac muscles and is inhibited by phospholamban (PLN). Under conditions of oxidative stress, SERCA2a may also be impaired by tyrosine nitration. Tafazzin (Taz) is a mitochondrial-specific transacylase that regulates mature cardiolipin (CL) formation, and its absence leads to mitochondrial dysfunction and excessive production of reactive oxygen/nitrogen species (ROS/RNS). In the present study, we examined SERCA function, SERCA2a tyrosine nitration, and PLN expression/phosphorylation in left ventricles (LV) obtained from young (3-5 months) and old (10-12 months) wild-type (WT) and Taz knockdown (TazKD ) male mice. These mice are a mouse model for Barth syndrome, which is characterized by mitochondrial dysfunction, excessive ROS/RNS production, and dilated cardiomyopathy (DCM). Here, we show that maximal SERCA activity was impaired in both young and old TazKD LV, a result that correlated with elevated SERCA2a tyrosine nitration. In addition PLN protein was decreased, and its phosphorylation was increased in TazKD LV compared with control, which suggests that PLN may not contribute to the impairments in SERCA function. These changes in expression and phosphorylation of PLN may be an adaptive response aimed to improve SERCA function in TazKD mice. Nonetheless, we demonstrate for the first time that SERCA function is impaired in LVs obtained from young and old TazKD mice likely due to elevated ROS/RNS production. Future studies should determine whether improving SERCA function can improve cardiac contractility and pathology in TazKD mice.