Sarcolipin haploinsufficiency prevents dystrophic cardiomyopathy in mdx mice.

Sarcolipin (SLN) is an inhibitor of sarco/endoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and expressed at high levels in the ventricles of animal models for and patients with Duchenne muscular dystrophy (DMD). The goal of this study was to determine whether the germline ablation of SLN expression improves cardiac SERCA function and intracellular Ca2+ (Ca2+i) handling and prevents cardiomyopathy in the mdx mouse model of DMD. Wild-type, mdx, SLN-haploinsufficient mdx (mdx:sln+/-), and SLN-deficient mdx (mdx:sln-/-) mice were used for this study. SERCA function and Ca2+i handling were determined by Ca2+ uptake assays and by measuring single-cell Ca2+ transients, respectively. Age-dependent disease progression was determined by histopathological examinations and by echocardiography in 6-, 12-, and 20-mo-old mice. Gene expression changes in the ventricles of mdx:sln+/- mice were determined by RNA-Seq analysis. SERCA function and Ca2+i cycling were improved in the ventricles of mdx:sln+/- mice. Fibrosis and necrosis were significantly decreased, and cardiac function was enhanced in the mdx:sln+/- mice until the study endpoint. The mdx:sln-/- mice also exhibited similar beneficial effects. RNA-Seq analysis identified distinct gene expression changes including the activation of the apelin pathway in the ventricles of mdx:sln+/- mice. Our findings suggest that reducing SLN expression is sufficient to improve cardiac SERCA function and Ca2+i cycling and prevent cardiomyopathy in mdx mice.NEW & NOTEWORTHY First, reducing sarcopolin (SLN) expression improves sarco/endoplasmic reticulum Ca2+ uptake and intracellular Ca2+ handling and prevents cardiomyopathy in mdx mice. Second, reducing SLN expression prevents diastolic dysfunction and improves cardiac contractility in mdx mice Third, reducing SLN expression activates apelin-mediated cardioprotective signaling pathways in mdx heart.

Sarcolipin and uncoupling protein 1 play distinct roles in diet-induced thermogenesis and do not compensate for one another.

OBJECTIVE: It is well known that uncoupling protein 1 (UCP1) in brown adipose tissue plays an important role in diet-induced thermogenesis. In this study, whether sarcolipin (SLN), a regulator of sarco/endoplasmic reticulum Ca(2+) -ATPase pump in muscle, is also an important player of diet-induced thermogenesis was investigated, as well as whether loss of SLN could be compensated by increased UCP1 expression and vice versa. METHODS: Age- and sex-matched UCP1(-/-) , SLN(-/-) , and double knockout for both UCP1 and SLN mice maintained in C57Bl/6J background were challenged to high-fat diet for 12 weeks and then analyzed for weight gain, alterations in serum metabolites, and changes in thermogenic protein expression. RESULTS: Loss of either SLN or UCP1 alone was sufficient to cause diet-induced obesity. No compensatory upregulation of UCP1 in SLN(-/-) mice or vice versa was found. Paradoxically, loss of both mechanisms failed to exacerbate the obesity phenotype. CONCLUSIONS: Data suggest that both SLN- and UCP1-based adaptive thermogenic mechanisms were essential for achieving maximal diet-induced thermogenesis. When both mechanisms were absent, less efficient thermogenic mechanisms were activated to counter energy imbalance.

Heterozygous deletion of sarcolipin maintains normal cardiac function.

Sarcolipin (SLN) is a small proteolipid and a regulator of sarco(endo)plasmic reticulum Ca(2+)-ATPase. In heart tissue, SLN is exclusively expressed in the atrium. Previously, we inserted Cre recombinase into the endogenous SLN locus by homologous recombination and succeeded in generating SLN-Cre knockin (Sln(Cre/+)) mice. This Sln(Cre/+) mouse can be used to generate an atrium-specific gene-targeting mutant, and it is based on the Cre-loxP system. In the present study, we used adult Sln(Cre/+) mice atria and analyzed the effects of heterozygous SLN deletion by Cre knockin before use as the gene targeting mouse. Both SLN mRNA and protein levels were decreased in Sln(Cre/+) mouse atria, but there were no morphological, physiological, or molecular biological abnormalities. The properties of contractility and Ca(2+) handling were similar to wild-type (WT) mice, and expression levels of several stress markers and sarcoplasmic reticulum-related protein levels were not different between Sln(Cre/+) and WT mice. Moreover, there was no significant difference in sarco(endo)plasmic reticulum Ca(2+)-ATPase activity between the two groups. We showed that Sln(Cre/+) mice were not significantly different from WT mice in all aspects that were examined. The present study provides basic characteristics of Sln(Cre/+) mice and possibly information on the usefulness of Sln(Cre/+) mice as an atrium-specific gene-targeting model.

Sarcoplasmic Reticulum Phospholipid Fatty Acid Composition and Sarcolipin Content in Rat Skeletal Muscle.

In a previous study, we reported lower sarcoplasmic reticulum (SR) Ca(2+) pump ionophore ratios in rat soleus compared to red and white gastrocnemius (RG, WG) muscles which may be indicative of greater SR Ca(2+) permeability in soleus. Here we assessed the lipid composition of the SR membranes obtained from these muscles to determine if SR docosahexaenoic acid (DHA) content and fatty acid unsaturation could help to explain the previously observed differences in SR Ca(2+) permeability. Since we have shown previously that sarcolipin may also influence SR Ca(2+) permeability, we also examined the levels of sarcolipin in rat muscle. We found that SR membrane DHA content was significantly higher in soleus (5.3 ± 0.2 %) compared to RG (4.2 ± 0.2 %) and WG (3.3 ± 0.2 %). Likewise, total SR membrane unsaturation and unsaturation index (UI) were significantly higher in soleus (% unsaturation: 59.1 ± 2.4; UI: 362.9 ± 0.8) compared to RG (% unsaturation: 55.3 ± 1.0; UI: 320.9 ± 2.5) and WG (% unsaturation: 52.6 ± 1.1; UI: 310. ± 2.2). Sarcolipin protein was 17-fold more abundant in rat soleus compared to RG and was not detected in WG; however, comparisons between soleus, RG, and WG in sarcolipin-null mice revealed that, in the absence of sarcolipin, ionophore ratios are still lowest in soleus and highest in WG. Overall, our results suggest that SR membrane DHA content and unsaturation, and, in part, sarcolipin expression may contribute to SR Ca(2+) permeability and, in turn, may have implications in muscle-based metabolism and diet-induced obesity.

Sarcolipin Is a Key Determinant of the Basal Metabolic Rate, and Its Overexpression Enhances Energy Expenditure and Resistance against Diet-induced Obesity.

Sarcolipin (SLN) is a novel regulator of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) in muscle. SLN binding to SERCA uncouples Ca(2+) transport from ATP hydrolysis. By this mechanism, SLN promotes the futile cycling of SERCA, contributing to muscle heat production. We recently showed that SLN plays an important role in cold- and diet-induced thermogenesis. However, the detailed mechanism of how SLN regulates muscle metabolism remains unclear. In this study, we used both SLN knockout (Sln(-/-)) and skeletal muscle-specific SLN overexpression (Sln(OE)) mice to explore energy metabolism by pair feeding (fixed calories) and high-fat diet feeding (ad libitum). Our results show that, upon pair feeding, Sln(OE) mice lost weight compared with the WT, but Sln(-/-) mice gained weight. Interestingly, when fed with a high-fat diet, Sln(OE) mice consumed more calories but gained less weight and maintained a normal metabolic profile in comparison with WT and Sln(-/-) mice. We found that oxygen consumption and fatty acid oxidation were increased markedly in Sln(OE) mice. There was also an increase in both mitochondrial number and size in Sln(OE) muscle, together with increased expression of peroxisome proliferator-activated receptor δ (PPARδ) and PPAR γ coactivator 1 α (PGC1α), key transcriptional activators of mitochondrial biogenesis and enzymes involved in oxidative metabolism. These results, taken together, establish an important role for SLN in muscle metabolism and energy expenditure. On the basis of these data we propose that SLN is a novel target for enhancing whole-body energy expenditure.

Ablation of sarcolipin decreases the energy requirements for Ca2+ transport by sarco(endo)plasmic reticulum Ca2+-ATPases in resting skeletal muscle.

The purpose of this study was to examine the effects of sarcolipin (SLN) on sarco(endo) plasmic reticulum Ca(2+)-ATPase (SERCA pump) energetics in vivo and resting skeletal muscle metabolic rate. Using SLN knockout (Sln(-/-)) mice we show that SLN ablation increases the transport stoichiometry of SERCA pumps (Ca(2+) uptake/Ca(2+)-ATPase activity) and decreases the relative contribution of SERCA pumps to resting oxygen consumption (VO2) in soleus without affecting soleus or whole body VO2. These data suggest that the lower energy requirements for Ca(2+) cycling in resting skeletal muscle of Sln(-/-) mice do not impact significantly either skeletal muscle or whole body metabolic rate.

Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals.

The role of skeletal muscle in nonshivering thermogenesis (NST) is not well understood. Here we show that sarcolipin (Sln), a newly identified regulator of the sarco/endoplasmic reticulum Ca(2+)-ATPase (Serca) pump, is necessary for muscle-based thermogenesis. When challenged to acute cold (4 °C), Sln(-/-) mice were not able to maintain their core body temperature (37 °C) and developed hypothermia. Surgical ablation of brown adipose tissue and functional knockdown of Ucp1 allowed us to highlight the role of muscle in NST. Overexpression of Sln in the Sln-null background fully restored muscle-based thermogenesis, suggesting that Sln is the basis for Serca-mediated heat production. We show that ryanodine receptor 1 (Ryr1)-mediated Ca(2+) leak is an important mechanism for Serca-activated heat generation. Here we present data to suggest that Sln can continue to interact with Serca in the presence of Ca(2+), which can promote uncoupling of the Serca pump and cause futile cycling. We further show that loss of Sln predisposes mice to diet-induced obesity, which suggests that Sln-mediated NST is recruited during metabolic overload. These data collectively suggest that SLN is an important mediator of muscle thermogenesis and whole-body energy metabolism.

Ablation of sarcolipin results in atrial remodeling.

Sarcolipin (SLN) is a key regulator of sarco(endo)plasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and its expression is altered in diseased atrial myocardium. To determine the precise role of SLN in atrial Ca(2+) homeostasis, we developed a SLN knockout (sln-/-) mouse model and demonstrated that ablation of SLN enhances atrial SERCA pump activity. The present study is designed to determine the long-term effects of enhanced SERCA activity on atrial remodeling in the sln-/- mice. Calcium transient measurements show an increase in atrial SR Ca(2+) load and twitch Ca(2+) transients. Patch-clamping experiments demonstrate activation of the forward mode of sodium/calcium exchanger, increased L-type Ca(2+) channel activity, and prolongation of action potential duration at 90% repolarization in the atrial myocytes of sln-/- mice. Spontaneous Ca(2+) waves, delayed afterdepolarization, and triggered activities are frequent in the atrial myocytes of sln-/- mice. Furthermore, loss of SLN in atria is associated with increased interstitial fibrosis and altered expression of genes encoding collagen and other extracellular matrix proteins. Our results also show that the sln-/- mice are susceptible to atrial arrhythmias upon aging. Together, these findings indicate that ablation of SLN results in increased SERCA activity and SR Ca(2+) load, which, in turn, could cause abnormal intracellular Ca(2+) handling and atrial remodeling.

MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells.

The apical surface of mammalian bladder urothelium is covered by large (500-1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin-Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, resulting in the accumulation of the uroplakin-degrading multivesicular bodies. Finally, although MAL and uroplakins cofloat in detergent-resistant raft fractions, they are associated with distinct plaque and hinge membrane subdomains, respectively. These data suggest a model in which 1) MAL does not play a role in the apical sorting of uroplakins; 2) the propensity of uroplakins to polymerize forming 16-nm particles and later large 2D crystals that behave as detergent-resistant (giant) rafts may drive their apical targeting; 3) the exclusion of MAL from the expanding 2D crystals of uroplakins explains the selective association of MAL with the hinge areas in the uroplakin-delivering fusiform vesicles, as well as at the apical surface; and 4) the hinge-associated MAL may play a role in facilitating the incorporation of the exocytic uroplakin vesicles into the corresponding hinge areas of the urothelial apical surface.

Ablation of phospholamban and sarcolipin results in cardiac hypertrophy and decreased cardiac contractility.

AIMS: Improving the sarco(endo)plasmic reticulum (SR) Ca(2+)-ATPase (SERCA) function has clinical implications in treating heart failure. The present study aimed to determine the effect of constitutive activation of the SERCA pump on cardiac contractility in normal mice and during pressure-overload-induced cardiac hypertrophy. METHODS AND RESULTS: The SERCA pump was constitutively activated in both atrial and ventricular chambers of the mouse heart by ablating its key regulators, phospholamban (PLN) and sarcolipin (SLN). The double-knockout (dKO) mice for PLN and SLN showed increased SERCA pump activity, Ca(2+) transients and SR Ca(2+) load, and developed cardiac hypertrophy. Echocardiographic measurements showed that the basal cardiac function was not affected in the young dKO mice. However, the cardiac function worsened upon ageing and when subjected to pressure overload. CONCLUSION: Our studies suggest that the constitutive activation of the SERCA pump is detrimental to cardiac function. Our findings also emphasize the need for dynamic regulation of the SERCA pump by PLN and/or SLN to maintain cardiac contractility in normal conditions and during pathophysiological states.

Disproportional expression of proteolipid protein and DM-20 in the X-linked, dysmyelinating shaking pup mutant.

The shaking pup is an X-linked canine mutant with a severe hypomyelination of the CNS. Proteolipid protein (PLP) and the related DM-20 protein were examined in this mutant by densitometric scanning of Western blots stained with PLP antiserum. In the spinal cord of 4-week-old mutants, PLP was reduced to less than 1% of the control level, which is a greater deficiency than was found for other myelin proteins. On Western blots of control spinal cord, PLP stained much more intensely than DM-20. However, on Western blots of the mutant spinal cord, a component with the electrophoretic mobility of DM-20 stained slightly more intensely with PLP antiserum than PLP itself. This component was shown to be DM-20 by its lack of reactivity with an antiserum raised to a synthetic peptide corresponding to part of the PLP sequence that is missing in DM-20. Thus PLP and DM-20 are expressed in approximately equal and greatly reduced amounts in the mutant spinal cord. Although PLP or DM-20 could not be detected in brain from the 4-week-old mutant, similar disproportional expression of these two proteins was demonstrated in both spinal cord and brain from a 10-week-old mutant pup. Immunostaining of tissue sections showed that the small amounts of PLP and/or DM-20 synthesized in the mutant are present in the thin myelin sheaths. The results suggest that the shaking pup could have a primary defect in the PLP gene leading to a severe deficiency of PLP and DM-20 as well as disproportional expression of these two proteins.

Abnormal compact myelin in the myelin-deficient rat: absence of proteolipid protein correlates with a defect in the intraperiod line.

The cervical spinal cords of 23-day-old myelin-deficient (md) rats, an X chromosome-linked myelin mutant, and their normal littermates were studied by light and electron microscopy, immunocytochemistry, and in situ hybridization. Light microscopy showed that there were scattered myelinated fibers in the md rat, particularly in the lateral and ventral columns. Ultrastructural examination of these fibers showed that the myelin often had many lamellae that were tightly compacted, but in which the intraperiod line was abnormally fused at most places, resulting in a minor alteration of the myelin periodicity. Immunocytochemical staining of adjacent sections following a variety of fixation methods showed that the myelinated fibers were positive for myelin basic protein but negative for proteolipid protein (PLP). In situ hybridization using cDNA probes to these proteins showed a severe diminution of the mRNAs for both proteins. These findings provide further support for an abnormality in genetic regulation of PLP as has been described in another X chromosome-linked mutant, the jimpy mouse. Despite the lack of PLP, however, a few myelinated fibers are formed in the md rat, but the myelin formed in general lacks a normal intraperiod line, a site at which this protein is thought to be located.