Epiregulin induces leptin secretion and energy expenditure in high-fat diet-fed mice.

Adipokine leptin regulates neuroendocrine circuits that control energy expenditure, thermogenesis and weight loss. However, canonic regulators of leptin secretion, such as insulin and malonyl CoA, do not support these processes. We hypothesize that epiregulin (EREG), a growth factor that is secreted from fibroblasts under thermogenic and cachexia conditions, induces leptin secretion associated with energy dissipation. The effects of EREG on leptin secretion were studied ex vivo, in the intra-abdominal white adipose tissue (iAb WAT) explants, as well as in vivo, in WT mice with diet-induced obesity (DIO) and in ob/ob mice. These mice were pair fed a high-fat diet and treated with intraperitoneal injections of EREG. EREG increased leptin production and secretion in a dose-dependent manner in iAb fat explants via the EGFR/MAPK pathway. After 2 weeks, the plasma leptin concentration was increased by 215% in the EREG-treated group compared to the control DIO group. EREG-treated DIO mice had an increased metabolic rate and core temperature during the active dark cycle and displayed cold-induced thermogenesis. EREG treatment reduced iAb fat mass, the major site of leptin protein production and secretion, but did not reduce the mass of the other fat depots. In the iAb fat, expression of genes supporting mitochondrial oxidation and thermogenesis was increased in EREG-treated mice vs control DIO mice. All metabolic and gene regulation effects of EREG treatment were abolished in leptin-deficient ob/ob mice. Our data revealed a new role of EREG in induction of leptin secretion leading to the energy expenditure state. EREG could be a potential target protein to regulate hypo- and hyperleptinemia, underlying metabolic and immune diseases.

miR-155 Deletion in Female Mice Prevents Diet-Induced Obesity.

Obesity is a growing epidemic in developed countries. Obese individuals are susceptible to comorbidities, including cardiovascular disease and metabolic disorder. Increasing the ability of adipose tissue to expend excess energy could improve protection from obesity. One promising target is microRNA (miR)-155-5p. We demonstrate that deletion of miR-155 (-5p and -3p) in female mice prevents diet-induced obesity. Body weight gain did not differ between wild-type (WT) and miR-155 knockout (KO) mice fed control diet (CD); however, miR-155 KO mice fed high-fat diet (HFD) gained 56% less body weight and 74% less gonadal white adipose tissue (WAT) than WT mice. Enhanced WAT thermogenic potential, brown adipose tissue differentiation, and/or insulin sensitivity might underlie this obesity resistance. Indeed, miR-155 KO mice on HFD had 21% higher heat release than WT HFD mice. Compared to WT adipocytes, miR-155 KO adipocytes upregulated brown (Ucp1, Cidea, Pparg) and white (Fabp4, Pnpla2, AdipoQ, Fasn) adipogenic genes, and glucose metabolism genes (Glut4, Irs1). miR-155 deletion abrogated HFD-induced adipocyte hypertrophy and WAT inflammation. Therefore, miR-155 deletion increases adipogenic, insulin sensitivity, and energy uncoupling machinery, while limiting inflammation in WAT, which together could restrict HFD-induced fat accumulation. Our results identify miR-155 as a novel candidate target for improving obesity resistance.

Phospholamban and sarcolipin: Are they functionally redundant or distinct regulators of the Sarco(Endo)Plasmic Reticulum Calcium ATPase?

In muscle, the Sarco(Endo)plasmic Reticulum Calcium ATPase (SERCA) activity is regulated by two distinct proteins, PLB and SLN, which are highly conserved throughout vertebrate evolution. PLB is predominantly expressed in the cardiac muscle, while SLN is abundant in skeletal muscle. SLN is also found in the cardiac atria and to a lesser extent in the ventricle. PLB regulation of SERCA is central to cardiac function, both at rest and during extreme physiological demand. Compared to PLB, the physiological relevance of SLN remained a mystery until recently and some even thought it was redundant in function. Studies on SLN suggest that it is an uncoupler of the SERCA pump activity and can increase ATP hydrolysis resulting in heat production. Using genetically engineered mouse models for SLN and PLB, we showed that SLN, not PLB, is required for muscle-based thermogenesis. However, the mechanism of how SLN binding to SERCA results in uncoupling SERCA Ca(2+) transport from its ATPase activity remains unclear. In this review, we discuss recent advances in understanding how PLB and SLN differ in their interaction with SERCA. We will also explore whether structural differences in the cytosolic domain of PLB and SLN are the basis for their unique function and physiological roles in cardiac and skeletal muscle.

Muscle damage, metabolism, and oxidative stress in mdx mice: Impact of aerobic running.

INTRODUCTION: We tested how a treadmill exercise program influences oxygen consumption, oxidative stress, and exercise capacity in the mdx mouse, a model of Duchenne muscular dystrophy. METHODS: At age 4 weeks mdx mice were subjected to 4 weeks of twice-weekly treadmill exercise. Sedentary mdx and wild-type mice served as controls. Oxygen consumption, time to exhaustion, oxidative stress, and myofiber damage were assessed. RESULTS: At age 4 weeks, there was a significant difference in exercise capacity between mdx and wild-type mice. After exercise, mdx mice had lower basal oxygen consumption and exercise capacity, but similar maximal oxygen consumption. Skeletal muscle from these mice displayed increased oxidative stress. Collagen deposition was higher in exercised versus sedentary mice. CONCLUSIONS: Exercised mdx mice exhibit increased oxidative stress, as well as deficits in exercise capacity, baseline oxygen consumption, and increased myofiber fibrosis. Muscle Nerve 54: 110-117, 2016.

Sarcolipin is a novel regulator of muscle metabolism and obesity.

Obesity is increasing at an alarming rate, both in adults and adolescents, across the globe due to increased consumption of caloric rich diet. Obesity and its associated complications appear to be major contributing factors not only to diabetes/heart disease but also to cancer, and neurological diseases causing a huge burden on the health care system. To date, there are no effective treatments to reduce weight gain, other than caloric restriction and exercise which are often difficult to enforce. There are very few drugs available for treating obesity and those that are available only reduce obesity by ∼ 10%. Identifying mechanisms to increase energy expenditure, on top of the increase elicited by exercise, would be more beneficial to control weight gain. The purpose of this review is to highlight the role of sarcolipin (SLN), a regulator of SERCA pump, in muscle thermogenesis and metabolism. We will further discuss if enhancing SLN activity could be an effective mechanism to increase energy expenditure and control weight gain. We will also discuss the merits of adaptive thermogenesis in muscle and brown fat as potential mechanisms to increase energy expenditure during caloric overload. That said, there is still a great need for further research into the mechanism of diet induced thermogenesis and its relevance to overall metabolism and obesity.

Sarcolipin overexpression improves muscle energetics and reduces fatigue.

Sarcolipin (SLN) is a regulator of sarcoendoplasmic reticulum calcium ATPase in skeletal muscle. Recent studies using SLN-null mice have identified SLN as a key player in muscle thermogenesis and metabolism. In this study, we exploited a SLN overexpression (Sln(OE)) mouse model to determine whether increased SLN level affected muscle contractile properties, exercise capacity/fatigue, and metabolic rate in whole animals and isolated muscle. We found that Sln(OE) mice are more resistant to fatigue and can run significantly longer distances than wild-type (WT). Studies with isolated extensor digitorum longus (EDL) muscles showed that Sln(OE) EDL produced higher twitch force than WT. The force-frequency curves were not different between WT and Sln(OE) EDLs, but at lower frequencies the pyruvate-induced potentiation of force was significantly higher in Sln(OE) EDL. SLN overexpression did not alter the twitch and force-frequency curve in isolated soleus muscle. However, during a 10-min fatigue protocol, both EDL and soleus from Sln(OE) mice fatigued significantly less than WT muscles. Interestingly, Sln(OE) muscles showed higher carnitine palmitoyl transferase-1 protein expression, which could enhance fatty acid metabolism. In addition, lactate dehydrogenase expression was higher in Sln(OE) EDL, suggesting increased glycolytic capacity. We also found an increase in store-operated calcium entry (SOCE) in isolated flexor digitorum brevis fibers of Sln(OE) compared with WT mice. These data allow us to conclude that increased SLN expression improves skeletal muscle performance during prolonged muscle activity by increasing SOCE and muscle energetics.

Central IKKß inhibition prevents air pollution mediated peripheral inflammation and exaggeration of type II diabetes.

BACKGROUND: Prior experimental and epidemiologic data support a link between exposure to fine ambient particulate matter (<2.5 µm in aerodynamic diameter, PM2.5) and development of insulin resistance/Type II diabetes mellitus (Type II DM). We investigated the role of hypothalamic inflammation in PM2.5-mediated diabetes development. METHODS: KKay mice, a genetically susceptible model of Type II DM, were assigned to either concentrated PM2.5 or filtered air (FA) for 4-8 weeks via a versatile aerosol concentrator and exposure system, or administered intra-cerebroventricular with either IKKß inhibitor (IMD-0354) or TNFα antibody (infliximab) for 4-5 weeks simultaneously with PM2.5 exposure. Glucose tolerance, insulin sensitivity, oxygen consumption and heat production were evaluated. At euthanasia, blood, spleen, visceral adipose tissue and hypothalamus were collected to measure inflammatory cells using flow cytometry. Standard immunohistochemical methods and quantitative PCR were used to assess targets of interest. RESULTS: PM2.5 exposure led to hyperglycemia and insulin resistance, which was accompanied by increased hypothalamic IL-6, TNFα, and IKKß mRNA expression and microglial/astrocyte reactivity. Targeting the NFκB pathway with intra-cerebroventricular administration of an IKKß inhibitor [IMD-0354, n = 8 for each group)], but not TNFα blockade with infliximab [(n = 6 for each group], improved glucose tolerance, insulin sensitivity, rectified energy homeostasis (O2 consumption, CO2 production, respiratory exchange ratio and heat generation) and reduced peripheral inflammation in response to PM2.5. CONCLUSIONS: Central inhibition of IKKß prevents PM2.5 mediated peripheral inflammation and exaggeration of type II diabetes. These results provide novel insights into how air pollution may mediate susceptibility to insulin resistance and Type II DM.

High gender -specific susceptibility to curare- a neuromuscular blocking agent.

Curare, a selective skeletal muscle relaxant, has been used clinically to reduce shivering and as an anesthetic auxiliary in abdominal surgery. It is also widely used in animal experiments to block neuromuscular junction activity. Effective doses of curare diminish muscle contraction without affecting brain function, but at higher doses it is known to be lethal. However, the exact dose of curare initiating muscle relaxation vs. lethal effect has not been fully characterized in mice. In this study we carefully examined the dose-response for achieving muscle inactivity over lethality in both male and female mice (C57BL6/J). The most striking finding of this study is that female mice were highly susceptible to curare; both the ED50 and LD50 were at least 3-fold lower than male littermates. This study shows that gender-specific differences can be an important factor when administering skeletal muscle relaxants, particularly curare or other analogous agents targeted to the neuromuscular junction.

Sarcolipin protein interaction with sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is distinct from phospholamban protein, and only sarcolipin can promote uncoupling of the SERCA pump.

Sarco(endo)plasmic reticulum Ca(2+)ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Lee, A.G. (2002) Curr. Opin. Struct. Biol. 12, 547-554 and Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575-1579), but the mechanistic details are unknown. To better define how binding of SLN to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. The homo-bifunctional cross-linker (1,6-bismaleimidohexane) was employed to detect dynamic protein interaction during the SERCA cycle. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the Vmax of SERCA-mediated Ca(2+) uptake but not the pump affinity for Ca(2+); 2) SLN can bind to SERCA in the presence of high Ca(2+), but PLB can only interact to the ATP-bound Ca(2+)-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca(2+) causes uncoupling of the SERCA pump and increased heat production.

High gender -specific susceptibility to curare- a neuromuscular blocking agent

Curare, a selective skeletal muscle relaxant, has been used clinically to reduce shivering and as an anesthetic auxiliary in abdominal surgery. It is also widely used in animal experiments to block neuromuscular junction activity. Effective doses of curare diminish muscle contraction without affecting brain function, but at higher doses it is known to be lethal. However, the exact dose of curare initiating muscle relaxation vs. lethal effect has not been fully characterized in mice. In this study we carefully examined the dose-response for achieving muscle inactivity over lethality in both male and female mice (C57BL6/J). The most striking finding of this study is that female mice were highly susceptible to curare; both the EDm and LDm were at least 3-fold lower than male littermates. This study shows that gender-specific differences can be an important factor when administering skeletal muscle relaxants, particularly curare or other analogous agents targeted to the neuromuscular junction.

Loss of the serum response factor cofactor, cysteine-rich protein 1, attenuates neointima formation in the mouse.

OBJECTIVE: Cysteine-rich protein (CRP) 1 and 2 are cytoskeletal lin-11 isl-1 mec-3 (LIM)-domain proteins thought to be critical for smooth muscle differentiation. Loss of murine CRP2 does not overtly affect smooth muscle differentiation or vascular function but does exacerbate neointima formation in response to vascular injury. Because CRPs 1 and 2 are coexpressed in the vasculature, we hypothesize that CRPs 1 and 2 act redundantly in smooth muscle differentiation. METHODS AND RESULTS: We generated Csrp1 (gene name for CRP1) null mice by genetic ablation of the Csrp1 gene and found that mice lacking CRP1 are viable and fertile. Smooth muscle-containing tissues from Csrp1-null mice are morphologically indistinguishable from wild-type mice and have normal contractile properties. Mice lacking CRPs 1 and 2 are viable and fertile, ruling out functional redundancy between these 2 highly related proteins as a cause for the lack of an overt phenotype in the Csrp1-null mice. Csrp1-null mice challenged by wire-induced arterial injury display reduced neointima formation, opposite to that seen in Csrp2-null mice, whereas Csrp1/Csrp2 double-null mice produce a wild-type response. CONCLUSIONS: Smooth muscle CRPs are not essential for normal smooth muscle differentiation during development, but may act antagonistically to modulate the smooth muscle response to pathophysiological stress.

Deletion of SM-B, the high ATPase isoform of myosin, upregulates the PKC-mediated signal transduction pathway in murine urinary bladder smooth muscle.

Detrusor smooth muscle (DSM) hypertrophy induced by partial bladder outlet obstruction (PBOO) is associated with changes in the NH2-terminal myosin heavy chain isoform from predominantly SM-B to SM-A, alteration in the Ca2+ sensitization pathway, and the contractile characteristics from phasic to tonic in rabbits. We utilized the SM-B knockout (KO) mouse to determine whether a shift from SM-B to SM-A without PBOO is associated with changes in the signal transduction pathway mediated via PKC and CPI-17, which keeps the myosin phosphorylation (MLC20) level high by inhibiting the myosin phosphatase. DSM strips from SM-B KO mice generated more force in response to electrical field stimulation, KCl, carbachol, and phorbol 12,13-dibutyrate than that of age-matched wild-type mice. There was no difference in the ED50 for carbachol but the maximum response was greater for the SM-B KO mice. DSM from SM-B KO mice revealed increased mass and hypertrophy. The KO mice also showed an overexpression of PKC-alpha, increased levels of phospho-CPI-17, and an elevated level of IP3 and DAG upon stimulation with carbachol. Two-dimensional gel electrophoresis revealed an increased level of MLC20 phosphorylation in response to carbachol. Together, these changes may be responsible for the higher level of force generation and maintenance by the DSM from the SM-B KO bladders. In conclusion, our data show that ablation of SM-B is associated with alteration of PKC-mediated signal transduction and CPI-17-mediated Ca2+ sensitization pathway that regulate smooth muscle contraction. Interestingly, similar changes are also present in PBOO-induced DSM compensatory response in the rabbit model in which SM-B is downregulated.

Effect of cardiac resynchronization therapy on myocardial gene expression in patients with nonischemic dilated cardiomyopathy.

BACKGROUND: Cardiac resynchronization therapy (CRT) improves echocardiographic measures of ventricular structure and function in the failing heart. To determine whether or not these changes are representative of true biologic reverse ventricular remodeling or simply an artifact of an improved contraction pattern, we evaluated changes in myocardial gene expression typical of reverse remodeling before and after chronic CRT. METHODS AND RESULTS: Optimally medically treated patients with nonischemic heart failure meeting standard clinical criteria for CRT were enrolled. Before implantation of a CRT device, baseline echocardiogram and endomyocardial biopsies were obtained. These studies were repeated after 6 months of CRT. Using quantitative reverse-transcriptase polymerase chain reaction, the amount of messenger RNA for selected genes regulating contractile function (sarcoplasmic reticulum Ca2+ ATPase, alpha- and beta-myosin heavy chain [MHC] isoforms, phospholamban [PLB]), and pathologic hypertrophy (beta-MHC and atrial natriuretic peptide [ANP]) was determined from biopsy samples. Changes in gene expression (baseline to 6 months) were determined and correlated to changes in echocardiographic remodeling parameters. Ten patients were enrolled in the study, with 7 completing both baseline and follow-up biopsies and echocardiograms. On average, a significant increase was observed in alpha-MHC and PLB gene expression from baseline to 6 months (P = .016 for both). Beta-MHC levels tended to decrease with CRT (P = .078). Increased alpha-MHC levels correlated best with decreases in left ventricular end-diastolic dimension (P = .073, r = -0.71) and reductions in mitral regurgitation. No significant correlation between ejection fraction and gene expression was found. CONCLUSIONS: These changes in myocardial gene expression support the occurrence of reverse remodeling during chronic CRT. The changes are similar to those reported previously with beta-blockade, but were seen on top of standard drug therapies for heart failure.

Targeted overexpression of sarcolipin in the mouse heart decreases sarcoplasmic reticulum calcium transport and cardiac contractility.

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 beta-adrenergic agonists.

Sarcoplasmic reticulum adenosine triphosphatase overexpression in the L-type Ca2+ channel mouse results in cardiomyopathy and Ca2+ -induced arrhythmogenesis.

BACKGROUND: Overexpression of the L-type voltage-dependent calcium channel alpha(1C)-subunit (L-VDCC OE) in transgenic mice results in adaptive hypertrophy followed by a maladaptive phase associated with a decrease in sarcoplasmic reticulum adenosine triphosphatase (SERCA)2a expression at 8 to 10 months of age. Overexpressing SERCA to manipulate calcium (Ca(2+)) cycling and prevent pathologic phenotypes in some models of heart failure has been proven to be a promising genetic strategy. OBJECTIVE: In this study we investigated whether genetic manipulation that increases Ca(2+) uptake into the sarcoplasmic reticulum by overexpressing SERCA1a (skeletal muscle specific) into the L-VDCC OE background could restore or further deteriorate Ca(2+) cycling, contractile dysfunction, and electrical remodeling in the heart failure phenotype. RESULTS: We found that the survival rate of L-VDCC OE/SERCA1a OE double transgenic mice decreased by 50%. L-VDCC OE/SERCA1a OE mice displayed an accelerated phenotype of severe dilation of both ventricles associated with deteriorated left ventricular function. Voltage clamp experiments revealed enhanced increased inward Ca(2+) current density and decreased the transient outward potassium current. Action potential duration in double transgenic ventricular myocytes was prolonged, and isoproterenol induced early after depolarization. These mice demonstrated a high incidence of spontaneous left ventricular arrhythmia. Expression of the proarrhythmic signaling protein Ca(2+)/calmodulin-dependent kinase II (CaMKII) was increased while connexin43 expression was decreased, defining an important putative mechanism in the electrophysiologic disturbances and mortality. CONCLUSIONS: Despite previous reports of improved cardiac function in heart failure models after SERCA intervention, our results advocate the need to elucidate the involvement of augmented Ca(2+) cycling in arrhythmogenesis.

Transgenic mouse models for cardiac dysfunction by a specific gene manipulation.

The sarcoplasmic reticulum Ca2+ ATPase (SERCA) plays a pivotal role in calcium cycling and the beat-to-beat function of the heart. Recent studies have shown that decreased expression and activity of SERCA are associated with end-stage heart failure in humans and in experimental animal models of heart failure. There has been considerable controversy over whether a decrease in SERCA level is a cause or effect of hypertrophy. To address directly whether alterations in SERCA levels modify calcium homeostasis and heart function, we have chosen to alter the SERCA protein expression genetically using transgenic and gene-targeted knockout mouse technology. This chapter describes the methodology for generation of mouse models that overexpress different SERCA isoforms and a SERCA2 knockout mouse model with decreased SERCA levels.

Carvedilol effectively blocks oxidative stress-mediated downregulation of sarcoplasmic reticulum Ca2+-ATPase 2 gene transcription through modification of Sp1 binding.

Carvedilol is a beta-adrenoceptor blocker and a potent antioxidant that improves cardiac function in patients with heart failure. The restoration of sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene expression may be an underlying mechanism of its beneficial effects on cardiac function. In primary cultured neonatal rat cardiac myocytes, treatment with either carvedilol or its beta-receptor inactive metabolite, BM910228, attenuated the hydrogen peroxide-mediated decrease in SERCA2 mRNA and protein levels, while metoprolol, a pure beta-blocker, had no effect. Moreover, carvedilol itself significantly enhanced SERCA2 gene transcription, suggesting that carvedilol specifically restores SERCA2 gene transcription. Site-directed mutagenesis revealed that two Sp1 sites in the SERCA2 gene promoter region mediated the response to carvedilol under oxidative stress. Further, electrophoretic mobility shift assays revealed that Sp1 and Sp3 transcription factors correlated with carvedilol-mediated changes in the promoter assays. These studies may provide a mechanistic explanation for the beneficial effects of carvedilol in heart failure.

The effects of intracellular Ca2+ on cardiac K+ channel expression and activity: novel insights from genetically altered mice.

We tested the hypothesis that chronic changes in intracellular Ca(2+) (Ca(2+)(i)) can result in changes in ion channel expression; this represents a novel mechanism of crosstalk between changes in Ca(2+) cycling proteins and the cardiac action potential (AP) profile. We used a transgenic mouse with cardiac-specific overexpression of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) isoform 1a (SERCA1a OE) with a significant alteration of SERCA protein levels without cardiac hypertrophy or failure. Here, we report significant changes in the expression of a transient outward K(+) current (I(to,f)), a slowly inactivating K(+) current (I(K,slow)) and the steady state current (I(SS)) in the transgenic mice with resultant prolongation in cardiac action potential duration (APD) compared with the wild-type littermates. In addition, there was a significant prolongation of the QT interval on surface electrocardiograms in SERCA1a OE mice. The electrophysiological changes, which correlated with changes in Ca(2+)(i), were further corroborated by measuring the levels of ion channel protein expression. To recapitulate the in vivo experiments, the effects of changes in Ca(2+)(i) on ion channel expression were further tested in cultured adult and neonatal mouse cardiac myocytes. We conclude that a primary defect in Ca(2+) handling proteins without cardiac hypertrophy or failure may produce profound changes in K(+) channel expression and activity as well as cardiac AP.

Overexpression of sarcolipin decreases myocyte contractility and calcium transient.

OBJECTIVE: Sarcolipin (SLN) is a novel 31-amino-acid protein associated with the sarcoplasmic reticulum (SR) whose function in cardiac muscle is poorly defined. In this study, we tested the hypothesis that SLN is a regulator of SR Ca(2+) transport function by overexpressing SLN in adult rat ventricular myocytes which express low levels of SLN. METHODS: Expression of SLN mRNA in rat tissues was analyzed by Northern blot as well by RT-PCR analysis. To define the role of SLN in cardiac muscle contractility, we overexpressed SLN in adult rat ventricular myocytes using adenoviral gene transfer techniques. Localization of SLN in the adult rat ventricular myocytes was determined using confocal microscopy. Myocyte contractility and calcium transients were measured using edge detection and Fura 2AM. RESULTS: Our results demonstrate that overexpression of SLN decreased the cell shortening significantly when compared to control myocytes, whereas the time to peak contraction was not altered. In addition, SLN overexpression prolonged the time of 50% relaxation. Calcium transient analysis shows that time to 50% decay of [Ca(2+) ]i was markedly prolonged in SLN-overexpressing myocytes (control -245.0+/-3.78 vs. SLN -199.0+/-3.25 ms, p<0.001). However, there were no significant differences in peak amplitudes of [Ca(2+)](i) between SLN-overexpressing and control myocytes. We further demonstrate that SLN is localized within the SR membrane similar to PLB and SR Ca(2+) ATPase. Co-immunoprecipitation studies indicate that SLN can physically interact with phospholamban. CONCLUSIONS: We conclude that SLN may play an important role in regulating the SR calcium ATPase pump, possibly by interacting with phospholamban.