SERCA2 mRNA Levels Research Articles

Abstract 17053: Cardiac AKAP12 Signalosome Overexpression Exacerbation of Heart Failure and Cardiac Calcium Handling

Introduction: Heart failure (HF) is responsible for 1 out of 8 deaths per year in the U.S.A. and is the major cause of death globally. Calcium cycling is crucial for proper signaling processes and efficient cardiac contraction. In failing hearts, remodeled calcium handling contributes to cardiac dysfunction. Previously we reported enhanced cardiac function in mice lacking a functional A-Kinase Anchoring Protein 12 (AKAP12). In this study, we aim to investigate the role of AKAP12 overexpression (oxAKAP12) on heart failure progression through assessing Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA2) and its regulatory protein Phospholamban (PLN). Hypothesis: Cardiac AKAP12 overexpression potentiates HF development through its action on SERCA2. Methods: HF was developed in WT and oxAKAP12-Tg mice of 8-10 weeks old males and females, through chronic Isoproterenol (ISO) administration (60 mg/kg/day for 14-days). Left ventricular homogenates were used for gene expression analysis. Furthermore, AKAP12 was transiently overexpressed in AC16 cells (human cardiomyocytes cell line), to assess protein expression levels upon treatment with 100 nM of ISO for 12-hrs. Results: Cardiac oxAKAP12 in both males and females reduced left ventricular ejection fraction (EF) by 14.5±2.5% and fractional shortening (FS) by 22.7±2.0% after 14-days of chronic ISO treatment when compared to control groups. Both RNA sequencing and RTqPCR analysis showed a significant reduction of SERCA2 and PLN mRNA levels, respectively, in left ventricular homogenates of male (53.5±2.0%, 39.0±2.5%) and female (50.0±2.5%, 45.0±2.0%) groups overexpressing AKAP12 treated with ISO (14-days) compared to control groups (p<0.005). In AC16 cells overexpressing AKAP12 and treated with ISO, SERCA2 protein expression was reduced by 37% (p=0.0413) while PLN protein expression was not significantly reduced compared to control groups. Conclusions: Cardiac oxAKAP12 negatively influences systolic function in both male and female mice, potentially through affecting calcium handling, which will be assessed in future experiments by evaluating calcium transients and sarcomere shortening in isolated primary cardiomyocytes from oxAKAP12 and control mice.

Sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) reduces the migratory capacity of CCL21-treated monocyte-derived dendritic cells.

The migration of dendritic cells (DCs) to secondary lymphoid organs depends on chemoattraction through the interaction of the chemokine receptors with chemokines. However, the mechanism of how lymphoid chemokines attract DCs to lymphoid organs remains unclear. Here, we demonstrate the mechanism of DC migration in response to the lymphoid chemokine CCL21. CCL21-mediated DC migration is controlled by the regulation of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) expression rather than through the activation of mitogen-activated protein kinases CCL21-exposed mature DCs (mDCs) exhibited decreased SERCA2 expression but not decreased phospholamban (PLB) or Hax-1 expression, which are known to be SERCA2-interacting proteins. In addition, CCL21 did not affect the mRNA levels of SERCA2 or its interacting protein Hax-1. Interestingly, SERCA2 expression was inversely related to DC migration in response to chemokine stimulation. The migratory capacity of CCL21-treated mDCs was decreased by the phospholipase C inhibitor U73122 and by the protein kinase C inhibitor BAPTA-AM. The migratory capacities of mDCs were increased in response to SERCA2 siRNA expression but were decreased by SERCA2 overexpression. In addition, DCs treated with a SERCA2-specific inhibitor (cyclopiazonic acid) had significantly increased migratory capacities as mDCs regardless of SERCA2 expression. Moreover, SERCA2 expression was dependent on DC maturation induced by cytokines or Toll-like receptor agonists. Therefore, the migratory capacities differed in differentially matured DCs. Taken together, these results suggest that SERCA2 contributes to the migration of CCL21-activated DCs as an important feature of the adaptive immune response and provide novel insights regarding the role of SERCA2 in DC functions.

Novel allelic mutations in murine Serca2 induce differential development of squamous cell tumors

Dominant mutations in the Serca2 gene, which encodes sarco(endo)plasmic reticulum calcium-ATPase, predispose mice to gastrointestinal epithelial carcinoma [1–4] and humans to Darier disease (DD) [14–17]. In this study, we generated mice harboring N-ethyl-N-nitrosourea (ENU)-induced allelic mutations in Serca2: three missense mutations and one nonsense mutation. Mice harboring these Serca2 mutations developed tumors that were categorized as either early onset squamous cell tumors (SCT), with development similar to null-type knockout mice [2,4] (aggressive form; M682, M814), or late onset tumors (mild form; M1049, M1162). Molecular analysis showed no aberration in Serca2 mRNA or protein expression levels in normal esophageal cells of any of the four mutant heterozygotes. There was no loss of heterozygosity at the Serca2 locus in the squamous cell carcinomas in any of the four lines. The effect of each mutation on Ca2+-ATPase activity was predicted using atomic-structure models and accumulated mutated protein studies, suggesting that putative complete loss of Serca2 enzymatic activity may lead to early tumor onset, whereas mutations in which Serca2 retains residual enzymatic activity result in late onset. We propose that impaired Serca2 gene product activity has a long-term effect on squamous cell carcinogenesis from onset to the final carcinoma stage through an as-yet unrecognized but common regulatory pathway.

Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells

Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca2+ homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca2+ concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca2+ flux was evident after the curcumin treatment (15 µM). Treatment with Ca2+ chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca2+ concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca2+ elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca2+ homeostasis and thereby promotes apoptosis in ovarian cancer cells.

No evidence for altered intracellular calcium-handling in airway smooth muscle cells from human subjects with asthma.

BackgroundAsthma is characterized by airway hyper-responsiveness and variable airflow obstruction, in part as a consequence of hyper-contractile airway smooth muscle, which persists in primary cell culture. One potential mechanism for this hyper-contractility is abnormal intracellular Ca2+ handling.MethodsWe sought to compare intracellular Ca2+ handling in airway smooth muscle cells from subjects with asthma compared to non-asthmatic controls by measuring: i) bradykinin-stimulated changes in inositol 1,4,5-trisphosphate (IP3) accumulation and intracellular Ca2+ concentration, ii) sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression, iii) mechanisms of cytoplasmic Ca2+ clearance assessed following instantaneous flash photolytic release of Ca2+ into the cytoplasm.ResultsWe found no differences in airway smooth muscle cell basal intracellular Ca2+ concentrations, bradykinin-stimulated IP3 accumulation or intracellular Ca2+ responses. Quantification of SERCA2 mRNA or protein expression levels revealed no differences in ASM cells obtained from subjects with asthma compared to non-asthmatic controls. We did not identify differences in intracellular calcium kinetics assessed by flash photolysis and calcium uncaging independent of agonist-activation with or without SERCA inhibition. However, we did observe some correlations in subjects with asthma between lung function and the different cellular measurements of intracellular Ca2+ handling, with poorer lung function related to increased rate of recovery following flash photolytic elevation of cytoplasmic Ca2+ concentration.ConclusionsTaken together, the experimental results reported in this study do not demonstrate major fundamental differences in Ca2+ handling between airway smooth muscle cells from non-asthmatic and asthmatic subjects. Therefore, increased contraction of airway smooth muscle cells derived from asthmatic subjects cannot be fully explained by altered Ca2+ homeostasis.

PALMITATE MEDIATED REPRESSION OF PPAR ACTIVITY AND SERCA2A EXPRESSION IS ASSOCIATED WITH CYTOKINE ACTIVATION IN PRIMARY CARDIOMYOCYTES

Patients with diabetes and the metabolic syndrome frequently exhibit hyperlipidemia, which is believed to contribute to cardiac inflammation. Palmitate, one of the most abundant fatty acids in human diets has been shown to induce the expression of inflammatory cytokines in skeletal muscle cells, which may cause skeletal muscle insulin resistance. In addition, it has been proposed that palmitate mediated cytotoxicity is due in part to its inhibitory effects on β-oxidation. Here we were interested in determining if palmitate induces inflammation in primary cardiomyocytes and whether this has any effect on PPAR activity and hence β-oxidation. Rat neonatal cardiomyocytes were treated with palmitate for various time points and at various concentrations. We found that palmitate induced cardiomyocyte inflammation in a dose and time dependent manner as measured by TNF-α and IL-6 mRNA expression by qRT-PCR. PPAR activity was determined by assessing the expression of PPAR target genes (i.e. Acsl, Acadl, Cpt1b, Ucp2). We found that palmitate caused an initial induction of PPAR activity (at 4-8 hours) as evidenced by a significant increase in PPAR target gene expression. However, this was followed by a later repression of PPAR activity with a significant decrease in PPAR target gene expression after 24 hour palmitate exposure (Figure). In contrast, oleate, a non-toxic monounsaturated fatty acid inhibited the expression of pro-inflammatory cytokines. Furthermore oleate also induced PPAR target gene expression at early time points, but in contrast to palmitate, oleate’s effect on PPAR induction persisted after 24 hours and hence showed no signs of later repression. Because previous studies have shown a link between inflammation, PPAR activity, and Serca2a expression and because of the importance of Serca2a in cardiomyocyte physiology and health we evaluated Serca2a mRNA and protein expression. Palmitate induced a significant decrease in both mRNA and protein levels of Serca2a, while oleate had no effect on Serca2 mRNA levels. To evaluate if this translated into changes in Serca2 activity we assessed calcium transients in Fluo4 loaded cardiomyocytes. We found that calcium uptake, an indicator of Serca2a activity, was significantly prolonged in palmitate treated cardiomyocytes. This is the first study to demonstrate palmitate induced inflammation in primary mammalian cardiomyocytes. The resulting inflammation was associated with a late stage repression of PPAR activity and progressive repression of Serca2a expression which can lead to attenuated β-oxidation and calcium uptake, respectively.

Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal.

Differences in locomotor performance between individuals: importance of parvalbumin, calcium handling and metabolism

Locomotor performance is linked to fitness and health of animals and is expected to be under strong selection. However, interindividual variation in locomotor performance is pronounced in many species. It was our aim to investigate the relative importance of energy metabolism and calcium handling in determining sprint and sustained locomotion in the zebrafish (Danio rerio). Sprint and sustained performance (U(crit)) varied independently from each other. Using in vivo electroporation, we found that increased parvalbumin protein concentration improved both sprint and sustained locomotion. This is the first demonstration that parvalbumin plays a role in determining whole-animal performance. High sprint performance fish had greater mRNA concentrations of the metabolic regulators PPARδ and PGC1β compared with fish with poor sprint performance. High sustained performance fish, in contrast, had greater concentrations of PGC-1α and PGC-1β. The increased expression of these metabolic regulators indicates an enhancement of the metabolic machinery in high performance animals. Sprint performance is also enhanced by creatine kinase activity, which may be associated with increased PPARδ mRNA concentration. Ryanodine receptor (RyR) and sarcoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) mRNA concentrations were significantly increased in high sustained performance fish, while parvalbumin 2, dihydropyridine (DHPR) receptor and SERCA2 mRNA levels were increased in fish with high sprint velocities. Sustained performance was more sensitive to experimentally induced decreases in RyR and DHPR activity than sprint performance. We provide mechanistic explanations of why locomotor performance differs between individuals, which is important for understanding ecological and sporting success, disease and the evolutionary processes underlying selection.

Cre-loxP DNA recombination is possible with only minimal unspecific transcriptional changes and without cardiomyopathy in Tg(αMHC-MerCreMer) mice

Cre-loxP technology for conditional gene inactivation is a powerful tool in cardiovascular research. Induction of gene inactivation can be carried out by per oral or intraperitoneal tamoxifen administration. Unintended transient cardiomyopathy following tamoxifen administration for gene inactivation has recently been reported. We aimed to develop a protocol for tamoxifen-induced gene inactivation with minimal effects on gene transcription and in vivo cardiac function, allowing studies of acute loss of the targeted gene. In mRNA microarrays, 35% of the 34,760 examined genes were significantly regulated in MCM(+/0) compared with wild type. In MCM(+/0), we found a correlation between tamoxifen dose and degree of gene regulation. Comparing one and four intraperitoneal injections of 40 mg·kg(-1)·day(-1) tamoxifen, regulated genes were reduced to 1/5 in the single injection group. Pronounced alteration in protein abundance and acute cardiomyopathy were observed after the four-injection protocols but not the one-injection protocol. For verification of gene inactivation following one injection of tamoxifen, this protocol was applied to MCM(+/0)/Serca2(fl/fl). Serca2 mRNA levels and protein abundance followed the same pattern of decline with one and four tamoxifen injections. The presence of the MCM transgene induced major alterations of gene expression while administration of tamoxifen induced additional but less gene regulation. Thus nonfloxed MCM(+/0) should be considered as controls for mice that carry both a floxed gene of interest and the MCM transgene. One single tamoxifen injection administered to MCM(+/0)/Serca2(fl/fl) was sufficient for target gene inactivation, without acute cardiomyopathy, allowing acute studies subsequent to gene inactivation.

Endothelin downregulates SERCA2 gene and protein expression in adult rat ventricular myocytes: regulation by pertussis toxin-sensitive Giprotein and cAMP

Downregulation of the sarcoplasmic reticulum calcium ATPase (SERCA2) is associated with diastolic dysfunction in the failing heart. Elevated plasma endothelin-1 (ET) levels are correlated with congestive heart failure suggesting that ET may play a pathophysiological role. We have investigated the ability of ET to regulate SERCA2 gene expression in isolated adult rat ventricular myocytes. We find that ET enhances net protein synthesis by approximately 40% but significantly downregulates SERCA2 mRNA expression, time dependently, by approximately 30-50%, and the expression of SERCA2 protein by approximately 50%. In myoyctes, ET binds to ET(A) receptor that couples to G(q) and G(i) proteins. Inhibition of G(q)-PLC-induced phosphoinositide (PI) hydrolysis with U73122 (1 muM) or inhibition of G(i) protein with pertussis toxin (PTX) abolishes the ability of ET to downregulate SERCA2 mRNA gene expression. Further investigation suggests that ET coupling to PTX-sensitive G(i) with consequent lowering of cAMP is required for downregulation of SERCA2 mRNA levels. Increasing intracellular cAMP quantity using cAMP-specific PDE inhibitor Ro20-1724 or cAMP analog dibutyryl-cAMP reverses ET-induced downregulation of SERCA2 mRNA levels. The data indicate that, in adult myocytes, ET downregulates SERCA2 mRNA and protein levels, and the effect requires cross-talk between G(q) and PTX-sensitive G(i) pathways.

Prostaglandin F2.ALPHA. Inhibits SERCA2 Gene Transcription Through an Induction of Egr-1 in Cultured Neonatal Rat Cardiac Myocytes

Prostaglandin F(2alpha) (PGF(2alpha)) stimulates hypertrophic growth of neonatal rat cardiac myocytes, a feature of which includes downregulation of the Ca(2+)-ATPase (SERCA2), a major Ca(2+) transport protein in SR. The molecular mechanisms by which PGF(2alpha) inhibits SERCA2 gene expression remain unknown. We determined the cis-regulatory elements responsible for the regulation of the SERCA2 gene expression in cultured neonatal rat cardiac myocytes exposed to PGF(2alpha). The role of Egr-1 was evaluated by transient transfection of its expression vector and antisense oligonucleotide. Signaling pathways were determined by using the pharmacological inhibitors or cDNA expression plasmids coding for dominant negative forms of Ras and Rac. PGF(2alpha) reduced the SERCA2 mRNA levels in a time- and dose-dependent manner in cultured rat cardiac myocytes. Transient transfection analyses showed that PGF(2alpha) -responsive elements are located between -284 and -72 of the SERCA2 promoter, which contains G+C-rich sequences homologous to Sp1, Egr-1 and AP2-binding sites. PGF(2alpha) significantly increased Egr-1 expression, and overexpression of Egr-1 largely reduced the transcription of the SERCA2 gene. Egr-1 antisense oligonucleotides blocked the PGF(2alpha) -mediated decrease in SERCA2 mRNA expression. Furthermore, inhibitors for either genistein-sensitive tyrosine kinase or p38 MAPK, and dominant negative forms of either Ras or Rac, prevented PGF(2alpha) -induced repression of SERCA2 mRNA levels. These results suggest that Egr-1, as well as Ras, Rac, and p38 MAPK, plays a crucial role in the repression of SERCA2 gene expression during PGF(2alpha) -induced cardiac hypertrophy.

Activation of the androgen receptor alters the intracellular calcium response to glutamate in primary hippocampal neurons and modulates sarco/endoplasmic reticulum calcium ATPase 2 transcription

Androgens have been shown to have a number of effects on hippocampal function. Although androgen receptors (AR) are found at high levels in hippocampal neurons, the intracellular mechanisms responsible for androgen’s actions are unknown. If androgens were capable of altering internal calcium concentration ([Ca 2+] i), they could influence a variety of intracellular signaling pathways, maintain neuronal homeostasis and Ca 2+ induced excitotoxicity. In the present study, calcium imaging was used to measure the [Ca 2+] i in rat primary hippocampal neurons treated with either the AR agonist dihydrotestosterone (DHT), DHT+flutamide (AR antagonist), flutamide alone, or vehicle for 24 h and subsequently presented with an excitatory glutamate stimulus. In the absence of glutamate stimulation, DHT treatment caused a significant upward shift in baseline [Ca 2+] i when compared with neurons from all other groups. Glutamate had a greater effect on [Ca 2+] i in DHT-treated neurons and DHT-treated neurons returned to baseline levels significantly faster than all other groups. Cyclopiazonic acid, an inhibitor of sarco/endoplasmic reticulum calcium ATPase (SERCA) had a larger response in DHT-treated neurons compared with controls, suggesting increased Ca 2+ stores in DHT-treated neurons. In all cases the effects of DHT were blocked by treatment with flutamide indicating an AR-mediated mechanism. To determine a possible mechanism by which AR activation could be influencing [Ca 2+] i, SERCA2 mRNA levels were measured in primary hippocampal neurons. SERCA2 is inserted into the endoplasmic reticulum (ER) membrane and functions to rapidly pump [Ca 2+] i into the ER. Following treatment of primary hippocampal neurons with DHT, SERCA2 mRNA was increased, an effect that was blocked in the presence of flutamide. Taken together these results indicate that DHT, working through AR, causes an up-regulation of SERCA2, which increases the sequestering of [Ca 2+] i in the endoplasmic reticulum of hippocampal neurons. Such changes may allow the neurons to respond more robustly to a stimulus and recover more quickly following a highly stimulatory challenge.

Mechanism of reduced colonic contractility in experimental colitis: role of sarcoplasmic reticulum pump isoform-2

Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis are inflammatory disorders associated with decreased colonic contractility. Here we show that, in experimental colitis in rat induced by trinitrobenzenesulfonic acid, there is a decrease in contraction in response to carbamoylcholine and the sarco/endoplasmic reticulum Ca(+2) (SERCA) pump inhibitor thapsigargin. However, the decrease in contractility may occur due to decrease in the SERCA pump levels or their inactivation. Therefore, we examined the protein and mRNA levels for SERCA2 isoform, which is predominant isoform in colonic smooth muscle. There was a decrease in the levels of SERCA2 protein and mRNA levels in inflamed colonic muscle. These findings suggest that decreased SERCA pump levels is responsible for a decrease in the Ca(+2) stores in the sarco/endoplasmic reticulum that causes a decrease in the contractility in colonic smooth muscle leading to poor bowel movements.

Maintaining serum response factor activity in the older heart equal to that of the young adult is associated with better cardiac response to isoproterenol stress

To understand the effect of transcription regulation in modulating cardiac aging, we sought to study the role of serum response factor (SRF), a key transcription factor in the heart that is normally increased with senescence and also in congestive heart failure. A Tet-Off gene expression system was used for cardiac-specific over-expression of a mutant SRF protein. In these binary transgenic mice, there is no age-related increase in SRF protein expression; in fact, there appeared to be a mild reduction of SRF protein (Mild-R SRF Tg). The older, middle-aged (15 mo) Mild-R SRF Tg mice appeared healthier and were better able to maintain their left ventricular systolic pressure (LVSP) in response to moderate â-adrenergic stimulation compared with age-matched Non-Tg mice, which demonstrated a negative ionotropic response. The Mild-R SRF Tg hearts had lower mRNA expression of BNP (p < 0.05), and the sodium calcium exchanger (p < 0.05), compared to Non-Tg. Mild-R SRF Tg had higher mRNA levels of SERCA2 (p < 0.05) and ryanodine receptor 2 (p < 0.05) compared to Non-Tg hearts. These findings suggest that preventing the age-associated increase in SRF is associated with better preserved intracellular calcium handling and functional response to stress; it might be advantageous for the older adult heart. This mouse model could be helpful in elucidating the molecular mechanisms underlying certain age-related changes in cardiac reserve capacity and response to stress.

Reduced myocardial expression of calcium handling protein in patients with severe chronic mitral regurgitation☆☆☆

Left ventricle (LV) function was shown to be a principal determinant of morbidity and mortality in both uncorrected and surgically corrected mitral regurgitation (MR). However, the cellular mechanisms that develop in the LV remodeling secondary to volume overload in chronic severe MR is still not well defined. In single ventricular myocyte, a reduced contraction and slowed relaxation have been mainly attributed to defective intracellular Ca2+ currents. Between several Ca2+ handling proteins, sarcoplasmic reticulum Ca2+-ATPase 2 (SERCA2) expression and activity determines not only the extent and rate of relaxation, but also the rate and amplitude of contraction. The aim of the study was to determine whether modifications of SERCA2 gene expression occurs in LV wall remodeling process secondary to chronic severe MR. The LV samples were obtained from 12 patients presented LV wall remodeling (LV: diastolic/systolic diameter-70+/-7 mm vs 46+/-10 mm; diastolic/systolic volume-260+/-65 ml vs 102+/-68 ml) due to chronic, severe MR. Expressions of SERCA2 isoforms-SERCA2a and 2b mRNAs were estimated by semiquantitative RT-PCR and normalized to GAPDH. The protein levels of SERCA2 were determined by Western blot after normalization to actin. Results were compared with samples from non-failing human hearts (NFH). On SERCA2 mRNA levels, important reduction on both SERCA isoforms SERCA2a (-40%) and SERCA2b (-49%) compared to NFH, together with significant correlation between isoforms (r = 0.89; p = 0.01) were observed. SERCA2 protein levels were decreased (-38%) in MR compared to NFH. Also significant correlations between SERCA2a/2b and SERCA2 protein expression (r = 0.83, p = 0.017; r = 0.68, p = 0.05, respectively) were observed. Moreover, a negative correlation between protein levels of SERCA2 (r = -0.64, p = 0.053) and left ventricular diastolic diameter was observed. In chronic volume overload the down-regulation of SERCA2a and 2b at the mRNA and SERCA2 protein levels exist. Moreover, protein levels of SERCA2 tend to correlate to the grade of left ventricular diastolic dilatation and suggest an important role LV remodeling.

CRNK Overexpression Alters Hypertrophic Gene Expression In Cultured Neonatal Rat Ventricular Myocytes

We previously showed that overexpression of PYK2, a Ca2+-dependent nonreceptor protein tyrosine kinase, activated the stress-activated protein kinases JNK1/2 and p38MAPK, and down-regulated SERCA2 gene transcription in neonatal rat ventricular myocytes (NRVM) (Heidkamp et al., AJP:Cell 289:C471-C482, 2005). In the present study, we examined the effects of adenovirally (Adv)-mediated overexpression of a C-terminal, dominant-negative inhibitor of PYK2, known as CRNK (Cell adhesion kinase-β Related NonKinase) on cellular growth and hypertrophic gene expression in similarly cultured NRVM. NRVM were maintained in serum-free culture medium (UI), or infected with either Adv-CRNK or control Adv expressing green fluorescent protein (Adv-GFP; 5 moi, 24–48h). Cell extracts were analyzed for JNK and p38MAPK phosphorylation by Western blotting, for total protein and DNA content by spectrophotometry and fluorometry, and for SERCA2 and ANF mRNA levels by real-time RT-PCR. CRNK overexpression had no effect on basal JNK or p38MAPK, but blocked the H2O2-induced increase in JNK1/2 (but not p38MAPK) phosphorylation. CRNK overexpression also did not affect basal total protein/DNA ratio, and did not prevent the increase in total protein/DNA ratio produced by treatment with phorbol myristate acetate (200nM, 24h). However, CRNK overexpression significantly (P<0.05) increased SERCA2 mRNA levels 2.1±0.5-fold as compared to UI or GFP-expressing NRVM. In contrast, ANF mRNA levels were significantly decreased to 36±14% of UI by CRNK overexpression. These data indicate that PYK2 is necessary to elicit specific aspects of the hypertrophic phenotype in cultured NRVM. Supported by NIH RO1 HL34328 and the Dr. Ralph and Marian Falk Medical Research Trust.

PYK2 regulates SERCA2 gene expression in neonatal rat ventricular myocytes

The nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (PYK2) has been implicated in cell signaling pathways involved in left ventricular hypertrophy and heart failure, but its exact role has not been elucidated. In this study, replication-defective adenoviruses (Adv) encoding green fluorescent protein (GFP)-tagged, wild-type (WT), and mutant forms of PYK2 were used to determine whether PYK2 overexpression activates MAPKs, and downregulates SERCA2 mRNA levels in neonatal rat ventricular myocytes (NRVM). PYK2 overexpression significantly decreased SERCA2 mRNA (as determined by Northern blot analysis and real-time RT-PCR) to 54 +/- 4% of Adv-GFP-infected cells 48 h after Adv infection. Adv-encoding kinase-deficient (KD) and Y(402)F phosphorylation-deficient mutants of PYK2 also significantly reduced SERCA2 mRNA (WT>KD>Y(402)F). Conversely, the PTK inhibitor PP2 (which blocks PYK2 phosphorylation by Src-family PTKs) significantly increased SERCA2 mRNA levels. PYK2 overexpression had no effect on ERK1/2, but increased JNK1/2 and p38(MAPK) phosphorylation from fourfold to eightfold compared with GFP overexpression. Activation of both "stress-activated" protein kinase cascades appeared necessary to reduce SERCA2 mRNA levels. Adv-mediated overexpression of constitutively active (ca)MKK6 or caMKK7, which activated only p38(MAPK) or JNKs, respectively, was not sufficient, whereas combined infection with both Adv reduced SERCA2 mRNA levels to 45 +/- 12% of control. WTPYK2 overexpression also significantly reduced SERCA2 promoter activity, as determined by transient transfection of a 3.8-kb SERCA2 promoter-luciferase construct. Thus a PYK2-dependent signaling cascade may have a role in abnormal cardiac Ca(2+) handling in left ventricular hypertrophy and heart failure via downregulation of SERCA2 gene transcription.

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

Carvedilol is a β-adrenoceptor blocker and a potent antioxidant that improves cardiac function in patients with heart failure. The restoration of sarcoplasmic reticulum Ca 2+-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 β-receptor inactive metabolite, BM910228, attenuated the hydrogen peroxide-mediated decrease in SERCA2 mRNA and protein levels, while metoprolol, a pure β-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 Antidiabetic Agent Rosiglitazone Upregulates SERCA2 and Enhances TNF-α- and LPS-Induced NF-κB-Dependent Transcription and TNF-α-Induced IL-6 Secretion in Ventricular Myocytes

Positive hemodynamic effects of the antidiabetic agent rosiglitazone on perfused whole hearts have recently been described, but the mechanisms regulating these effects are not well understood. This study reports the effects of rosiglitazone on calcium regulation in isolated neonatal rat ventricular myocytes by measurement of Ca²⁺ transient decay rates and SERCA2 gene expression, and shows that rosiglitazone enhances known cardioprotective signaling pathways. Myocyte treatment with 10 μmol/L rosiglitazone accelerated Ca²⁺ transient decay rates by ñ30%, enhanced SERCA2 mRNA levels by ñ1.5-fold and SERCA2 production by ñ3-fold. Rosiglitazone treatment (1, 5, and 10 μmol/L) also led to a dose-dependent increase (ñ1.2-1.5-fold) in SERCA2 promoter activity. Comparable levels of cardiac SERCA overexpression have been associated with physiologically relevant and compensatory effects in vivo. These data link thiazolidinedione-induced improvement in cardiac myocyte function to an upregulation of SERCA2 gene expression. Since NF-κB-dependent pathways, including the upregulation of IL-6 secretion, were shown to protect neonatal rat ventricular myocytes from apoptosis upon TNFα stimulation, additional experiments were designed to determine whether rosiglitazone enhances TNFα-induced NF-κB-dependent transcription and IL-6 secretion. Because the endotoxin stress response in ventricular myocytes involves the upregulation of TNFα, and the activation of NF-κB, the effects of rosiglitazone on lipopolysaccharide-induced NF-κB-dependent transcription were also investigated. Treatment of neonatal rat ventricular myocytes with 10 μmol/L rosiglitazone enhanced TNF-α- and lipopolysaccharide-induced NF-κB-dependent transcription by ñ1.8- and ñ1.4-fold respectively, and TNF-α-induced IL-6 secretion by ñ1.5-fold. Rosiglitazone had no significant effects on basal levels of NF-κB-dependent transcription and IL-6 secretion. Thus, cardioprotective effects of rosiglitazone may be partly mediated by NF-κB.

Increased expression of SERCA2 mRNA in hearts of genetically obese Zucker rats after a moderate treadmill running

Aim. – Analyze the effects of exercise training on cardiac sarco endoplasmic reticulum calcium ATPase (SERCA) 2 gene expression of genetically obese Zucker rats, an animal model of non-insulin dependent diabetes mellitus (NIDDM). Methods. – Twenty male and 20 female Zucker fatty rats ran 30–50 min/d at 35–40% VO 2max, 6 d/week for 13 weeks. Semi-quantitative RT-PCR associated with specific restriction fragment analysis was used to detect and quantify the relative expression of SERCA mRNA isoforms. Results. – Fatty rats had an enhanced expression of SERCA2 and phospholamban (PLB) mRNA levels in ventricles. Moreover, fatty rats exposed to a moderate treadmill running exercise for 13 weeks exhibited a significant increase in ventricles SERCA2 mRNA levels, whereas, no change occurred in control lean rats. Exercise training produced a significant induction of α-myosin heavy chain mRNA levels only in fatty rats. Conclusion. – We hypothesize that exercise training may improve the cardiac function in diabetic rats by increasing the mRNA expression of SERCA2 since it is known that NIDDM rats have a reduced left ventricular diastolic compliance, and that exercise training improves the ventricular function in diabetic rats.