Cardiac sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) plays a central role in myocardial contractility. SERCA2a actively transports Ca(2+) into the SR and regulates cytosolic Ca(2+) concentration, SR Ca(2+) load, and the rate of contraction and relaxation of the heart. In the heart, SERCA pump activity is regulated by two small molecular weight proteins: phospholamban (PLB) and sarcolipin (SLN). Decreases in the expression levels of SERCA2a have been observed in a variety of pathological conditions. In addition, altered expression of PLB and SLN has been reported in many cardiac diseases. Thus, SERCA2a is a major regulator of intracellular Ca(2+) homeostasis, and changes in the expression and activity of the SERCA pump contribute to the decreased SR Ca(2+) content and cardiac dysfunction during pathogenesis. In this review, we discuss the mechanisms controlling SERCA pump expression and activity both during normal physiology and under pathological states.

Sarcolipin (SLN) is a small molecular weight sarcoplasmic reticulum (SR) membrane protein expressed both in cardiac and skeletal muscle tissues. Recent studies using transgenic mouse models have demonstrated that SLN is an important regulator of cardiac SR Ca 2+ ATPase 2a (SERCA2a). However, there is a paucity of information regarding the SLN protein expression in small versus larger mammals and its regulation during development and cardiac pathophysiology. Therefore, the major goal of this study was to generate a SLN specific antibody and perform detailed analyses of SLN protein expression during muscle development and in the diseased myocardium. The important findings of the present study are: (i) in small mammals, SLN expression is predominant in the atria but low in the ventricle and in skeletal muscle tissues, whereas in large mammals, SLN is quite abundant in skeletal muscle tissues than the atria (ii) SLN and SERCA2a are co-expressed in all striated muscle tissues studied except ventricle and co-ordinately regulated during muscle development and (iii) SLN protein levels are ∼3 fold upregulated in the atria of heart failure dogs and ∼30% decreased in the atria of hearts prone to myocardial ischemia. In addition we found that in the phospholamban null atria, SLN protein levels are upregulated.

Sarcolipin (SLN) is an inhibitor of the sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA) and is abnormally elevated in the muscle of Duchenne muscular dystrophy (DMD) patients and animal models. Here we show that reducing SLN levels ameliorates dystrophic pathology in the severe dystrophin/utrophin double mutant (mdx:utr −/−) mouse model of DMD. Germline inactivation of one allele of the SLN gene normalizes SLN expression, restores SERCA function, mitigates skeletal muscle and cardiac pathology, improves muscle regeneration, and extends the lifespan. To translate our findings into a therapeutic strategy, we knock down SLN expression in 1-month old mdx:utr −/− mice via adeno-associated virus (AAV) 9-mediated RNA interference. The AAV treatment markedly reduces SLN expression, attenuates muscle pathology and improves diaphragm, skeletal muscle and cardiac function. Taken together, our findings suggest that SLN reduction is a promising therapeutic approach for DMD.

Sarcolipin is a novel regulator of cardiac sarcoplasmic reticulum Ca 2؉ ATPase 2a (SERCA2a) and is expressed abundantly in atria. In this study we investigated the physiological significance of sarcolipin in the heart by generating a mouse model deficient for sarcolipin. The sarcolipin-null mice do not show any developmental abnormalities or any cardiac pathology. The absence of sarcolipin does not modify the expression level of other Ca 2؉ handling proteins, in particular phospholamban, and its phosphorylation status. Calcium uptake studies revealed that, in the atria, ablation of sarcolipin resulted in an increase in the affinity of the SERCA pump for Ca 2؉ and the maximum velocity of Ca 2؉ uptake rates. An important finding is that ablation of sarcolipin resulted in an increase in atrial Ca 2؉ transient amplitudes, and this resulted in enhanced atrial contractility. Furthermore, atria from sarcolipinnull mice showed a blunted response to isoproterenol stimulation, implicating sarcolipin as a mediator of ␤-adrenergic responses in atria. Our study documented that sarcolipin is a key regulator of SERCA2a in atria. Importantly, our data demonstrate the existence of distinct modulators for the SERCA pump in the atria and ventricles.atria ͉ calcium uptake ͉ sarcoplasmic reticulum Ca 2ϩ ATPase 2 S arcolipin (SLN), a low-molecular-weight protein (31 aa), is expressed in both cardiac and skeletal muscles (1-5). It colocalizes with sarcoplasmic reticulum (SR) Ca 2ϩ ATPase (SERCA) in the cardiac SR (3) and physically interacts with the SERCA pump (6). Amino acid composition and structural analysis have suggested that SLN and phospholamban (PLB) may belong to the same family of proteins with similar functions (1,(7)(8)(9). Consistent with the notion, in vitro studies have shown that SLN can inhibit the SERCA activity by decreasing the apparent Ca 2ϩ affinity of the pump (7, 10). Protein expression analyses have demonstrated that within the heart there are chamber-specific differences in the expression pattern of SLN and PLB (4). SLN is predominantly expressed in the atrial compartment, whereas PLB is abundant in the ventricles. In addition to atria, SLN is expressed in skeletal muscle tissues (4). SLN expression is regulated during cardiac and skeletal muscle development (2-4). Furthermore, SLN expression levels are altered in atria during cardiac pathology both in animal models (2, 4, 11-13) and in humans (14), suggesting that SLN levels may play an important role in maintaining atrial Ca 2ϩ homeostasis during cardiac pathophysiology.The importance of SLN as a regulator of the cardiac SERCA pump was recently demonstrated by using adenoviral gene transfer into adult rat ventricular myocytes (3) and transgenic overexpression of SLN in the heart (15-17). These studies suggest that overexpression of SLN into ventricular myocytes resulted in decreased rates of SR Ca 2ϩ uptake, Ca 2ϩ transient amplitude, and myocyte contractility. Overexpression of SLN in the PLB-null heart revealed that SLN can inhibit SERCA pump activity in...

We used an exon-specific gene-targeting strategy to generate a mouse model deficient only in the SM-B myosin isoform. Here we show that deletion of exon-5B (specific for SM-B) in the gene for the heavy chain of smooth muscle myosin results in a complete loss of SM-B myosin and switching of splicing to the SM-A isoform, without affecting SM1 and SM2 myosin content. Loss of SM-B myosin does not affect survival or cause any overt smooth muscle pathology. Physiological analysis reveals that absence of SM-B myosin results in a significant decrease in maximal force generation and velocity of shortening in smooth muscle tissues. This is the first in vivo study to demonstrate a functional role for the SM-B myosin isoform. We conclude that the extra seven-residue insert in the surface loop 1 of SM-B myosin is a critical determinant of crossbridge cycling and velocity of shortening.

A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca 2؉ -ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by ϳ35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca 2؉ homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca 2؉ stores and Ca 2؉ release in myocytes of SERCA2 heterozygous mice were decreased by ϳ40 -60% and ϳ30 -40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by ϳ40%. However, the rate of Ca 2؉ transient decline () was not altered significantly, suggesting that compensation was occurring in the removal of Ca 2؉ from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by ϳ40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased ϳ2-and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na ؉ -Ca 2؉ exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca 2؉ homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na ؉ -Ca 2؉ exchanger.In heart, muscle relaxation is largely dependent on the action of the sarcoplasmic reticulum (SR) 1 Ca 2ϩ ATPase (SERCA)to resequester cytosolic calcium released during contraction. Increased activity of SERCA, either by transgenic overexpression of SERCA isoforms in the heart (1-3) or by ablation of its regulatory protein, phospholamban (PLB) (4), has been shown to enhance cardiac rates of contraction and relaxation (1-4). To examine the effects of decreased SERCA2 activity on cardiac function, we have recently developed a transgenic mouse model with a null allele of the SERCA2 gene (5). Although complete loss of SERCA function in homozygous animals is embryonic lethal, disruption of one copy of the SERCA2 gene results in decreased cardiac SERCA2 mRNA (ϳ45%), protein (ϳ35%), and SR Ca 2ϩ uptake (ϳ35%) (5). These changes are associated in vivo with impaired cardiac performance (5). Because SERCA2 activity controls both the rate of calcium removal and the amount of calcium stores available within the SR, we hypothesize that the level of SERCA2 activity is a critical determinant of cardiac contractility. Therefore, one goal of this study is to determine if reduced SERCA2 levels compromise cardiac contractility by directly altering calcium handling and contractile functions of individual myocytes during excitationcontraction coupling.During excitation-contraction coupling, Ca 2ϩ entry through the L-type Ca 2ϩ channel activates Ca 2ϩ release from SR Ca 2ϩ stores, via the ryanodine receptor (RyR). This rise in cytosolic Ca 2ϩ i...

Abnormal intracellular Ca(2+) handling is an important factor in the progressive functional decline of dystrophic muscle. In the present study, we investigated the function of sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA) in various dystrophic muscles of mouse models of Duchenne muscular dystrophy. Our studies show that the protein expression of sarcolipin, a key regulator of the SERCA pump is abnormally high and correlates with decreased maximum velocity of SR Ca(2+) uptake in the soleus, diaphragm and quadriceps of mild (mdx) and severe (mdx:utr-/-) dystrophic mice. These changes are more pronounced in the muscles of mdx:utr-/- mice. We also found increased expression of SERCA2a and calsequestrin specifically in the dystrophic quadriceps. Immunostaining analysis further showed that SERCA2a expression is associated both with fibers expressing slow-type myosin and regenerating fibers expressing embryonic myosin. Together, our data suggest that sarcolipin upregulation is a common secondary alteration in all dystrophic muscles and contributes to the abnormal elevation of intracellular Ca(2+) concentration via SERCA inhibition.

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca 2؉ ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by ␤-adrenergic agonists.The sarco(endo)plasmic reticulum (SR) 2 Ca 2ϩ ATPase (SERCA) plays a dominant role in transporting Ca 2ϩ into the SR during the contraction-relaxation cycle of the heart. The rate and amount of Ca 2ϩ transported into the SR determines both the rate of muscle relaxation and the SR Ca 2ϩ load available for the next cycle of contraction (1-4). It is well established that SERCA function is regulated by phospholamban (PLB), whose inhibitory effect is reversed by phosphorylation by protein kinase A and the calcium/calmodulin-dependent protein kinase (CAMKII) during adrenergic activation (5-7). Recent studies have shown that in addition to PLB, sarcolipin (SLN) could also play an important role in the regulation of SERCA pump activity (8 -12).SLN is a 31-amino acid protein expressed in both cardiac and skeletal muscle (11,(13)(14)(15). We have recently demonstrated that SLN is localized in the cardiac SR membrane, and its distribution pattern is similar to SERCA2a and PLB (11). SLN mRNA is differentially expressed in small as opposed to larger mammals. In rodents, SLN mRNA is abundant in the atria with very low levels in the ventricle and skeletal muscles (11,14,15). In contrast, in larger mammals including humans, SLN mRNA is abundant in fast-twitch skeletal muscle compared with atria and ventricle (13). SLN expression is developmentally regulated (11), and its expression levels are modified under certain pathological conditions of the muscle (16,17). Decreased expression of SLN mRNA has been shown in the atria of patients with atrial fibrillation (16). A recent study also showed that SLN mRNA was up-regulated ϳ50-fold in the hypertrophied ventricles of Nkx2-5-null mice (17). Structural similarities between SLN and PLB indicate that they are homolog...