Sarco-endoplasmic Reticulum Calcium ATPase 2a Research Articles

Differential Downregulation of Subcellular Specific adrenoceptor Signaling in The Heart

Objective: Recent studies have shown that intracellular b1- adrenoceptors (b1ARs) are associated with the (Sarco)endoplasmic reticulum calcium ATPase 2a (SERCA2a) on the sarcoplasmic reticulum (SR) membrane. However, the mechanism of how this plasma membrane- and SR-associated pool of b1ARs signals downregulation under chronic sympathetic stimulation is not well understood. We aim to study these subcellular β1AR signals in different cellular compartments in a heart failure (HF) mouse model with chronic infusion of isoproterenol (ISO). Results and Methods: We investigated the association of b1AR with L-type calcium channel (LTCC), ryanodine receptor 2 (RyR2), and SERCA2a by utilizing proximity ligation assay (PLA) and confocal imaging in HF induced by chronic ISO infusion (30 mg/kg/day, 2 weeks). Chronic ISO infusion resulted in a downregulation of b1AR, and decreased receptor association with LTCC and RyR2 at the tubular membrane. In comparison, the b1AR association with SERCA2a was increased; however, the local cAMP-PKA mediated phospholamban phosphorylation is downregulated. Using Förster Resonance Energy Transfer (FRET) with genetically encoded biosensors to probe subcellular β1AR-PKA signaling, we observed reduced local ISO-induced PKA signaling at the RyR2 but not the SERCA2a nanodomains. Meanwhile, the expression of phosphodiesterases (PDEs) was altered including the downregulation of PDE4D and PDE3A and the upregulation of PDE2A, PDE4A, and PDE4B, which alters the local PKA activities in individual subcellular nanodomains. Interestingly, the localized PKA signal induced by endogenous norepinephrine at the SERCA2a nanodomain was impaired. Inhibition of monoamine oxidase A rescued the norepinephrine-induced local PKA signaling at the SERCA2a nanodomain and myocyte sarcomere shortening. Conclusion: We have uncovered distinct remodeling of b1AR signaling at the RyR2 and SERCA2a nanodomains in mouse HF after a chronic infusion of ISO. Our study offers strategies to restore the subcellular nanodomain cardiac b1AR signaling and contractile function in HF associated with chronic adrenergic stress, pointing toward potential therapeutic targets. National Institutes of Health grants R01-HL147263 and HL162825, Veteran Affair Merit grants IK6BX005753, 01BX002900 and BX005100. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Therapeutic Aspects of Prunus cerasus Extract in a Rabbit Model of Atherosclerosis-Associated Diastolic Dysfunction.

This study evaluates the potential therapeutic effects of anthocyanin-rich Prunus cerasus (sour cherry) extract (PCE) on atherosclerosis-associated cardiac dysfunction, described by the impairment of the NO-PKG (nitric oxide-protein kinase G) pathway and the antioxidant capacity. Initially, a rabbit model of atherosclerotic cardiovascular disease was established by administering a cholesterol-rich diet, enabling the examination of the impact of 9 g/kg PCE on the pre-existing compromised cardiovascular condition. After that, the animals were divided into four groups for 12 weeks: the (1) untreated control group; (2) PCE-administered healthy rabbits; (3) hypercholesterolemic (HC) group kept on an atherogenic diet; and (4) PCE-treated HC group. Dyslipidemia, impaired endothelial function, and signs of diastolic dysfunction were evident in hypercholesterolemic rabbits, accompanied by a reduced cardiac expression of eNOS (endothelial nitric oxide synthase), PKG, and SERCA2a (sarco/endoplasmic reticulum calcium ATPase 2a). Subsequent PCE treatment improved the lipid profile and the cardiac function. Additionally, PCE administration was associated with elevated myocardial levels of eNOS, PKG, and SERCA2a, while no significant changes in the vascular status were observed. Western blot analysis further revealed hypercholesterolemia-induced increase and PCE-associated reduction in heme oxygenase-1 expression. The observed effects of anthocyanins indicate their potential as a valuable addition to the treatment regimen for atherosclerosis-associated cardiac dysfunction.

Deubiquitinase JOSD2 improves calcium handling and attenuates cardiac hypertrophy and dysfunction by stabilizing SERCA2a in cardiomyocytes.

Cardiac hypertrophy leads to myocardial dysfunction and represents a serious threat to global public health security. Deubiquitinating enzymes (DUBs) mainly maintain the stability of substrate proteins and are essential to cardiac pathophysiology. Here, we explored the role and regulating mechanism of a DUB, Josephin domain-containing protein 2 (JOSD2), in cardiac hypertrophy. We found that JOSD2 expression was significantly upregulated in hypertrophic myocardium. Josd2 gene knockout aggravated cardiac dysfunction and hypertrophy in mice, whereas cardiac overexpression of JOSD2 mediated by the AAV9 vector prevented angiotensin II-induced cardiac hypertrophy. A comprehensive proteome-wide quantitative analysis identified sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) as a key substrate of JOSD2. Mechanistically, JOSD2 mediates SERCA2a deubiquitination, enhancing the stability of SERCA2a. By regulating SERCA2a, JOSD2 deficiency impairs calcium handling and promotes hypertrophy in primary cardiomyocytes. Our findings highlight the promise of JOSD2 as a beneficial therapeutic target for hypertrophic cardiomyopathy and provide an additional strategy for SERCA2a-targeted therapy.

Hydrogen sulfide regulates SERCA2a SUMOylation by S-Sulfhydration of SENP1 to ameliorate cardiac systole-diastole function in diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is a serious complication of diabetes mellitus that eventually progresses to heart failure. The sarco(endo)plasmic reticulum calcium ATPase 2a (SERCA2a), an important calcium pump in cardiomyocytes, is closely related to myocardial systolic-diastolic function. In mammalian cells, hydrogen sulfide (H2S), as a second messenger, antioxidant, and sulfurizing agent, is involved in diverse biological processes. Despite the importance of H2S for protection against DCM, the mechanisms remain poorly understood. The aim of the present study was to determine whether H2S regulates intracellular calcium homeostasis by acting on SERCA2a to reduce cardiomyocyte apoptosis during DCM. Db/db mice were injected with NaHS for 18 weeks. Neonatal rat cardiomyocytes (NRCMs) were treated with high glucose, palmitate, oleate, and NaHS for 48 h. Compared to the NaHS-treated groups, in vivo and in vitro type 2 diabetic models both showed reduced intracellular H2S content, reduced cystathionine γ-lyase (CSE) expression, impaired cardiac function, decreased SERCA2a expression and decreased SERCA2a activity, reduced SUMOylation of SERCA2a, increased sentrin-specific protease 1 (SENP1) expression, and disruption of calcium homeostasis leading to activation of the mitochondrial apoptosis pathway. Compared to the NaHS-treated type 2 diabetes cellular model, overexpression of SENP1 C683A reduced the S-sulfhydration of SENP1, reduced the SUMOylation of SERCA2a, reduced the increased expression and activity of SERCA2a, and induced mitochondrial apoptosis in cardiomyocytes. These results suggested that exogenous H2S elevates SENP1 S-sulfhydration to increase SERCA2a SUMOylation, improve myocardial systolic-diastolic function, and decrease cardiomyocyte apoptosis in DCM.

Probing spatiotemporal PKA activity at the ryanodine receptor and SERCA2a nanodomains in cardomyocytes

Spatiotemporal regulation of subcellular protein kinase A (PKA) activity for precise substrate phosphorylation is essential for cellular responses to hormonal stimulation. Ryanodine receptor 2 (RyR2) and (sarco)endoplasmic reticulum calcium ATPase 2a (SERCA2a) represent two critical targets of β adrenoceptor (βAR) signaling on the sarcoplasmic reticulum membrane for cardiac excitation and contraction coupling. Using novel biosensors, we show that cardiac β1AR signals to both RyR2 and SERCA2a nanodomains in cardiomyocytes from mice, rats, and rabbits, whereas the β2AR signaling is restricted from these nanodomains. Phosphodiesterase 4 (PDE4) and PDE3 control the baseline PKA activity and prevent β2AR signaling from reaching the RyR2 and SERCA2a nanodomains. Moreover, blocking inhibitory G protein allows β2AR signaling to the RyR2 but not the SERCA2a nanodomains. This study provides evidence for the differential roles of inhibitory G protein and PDEs in controlling the adrenergic subtype signaling at the RyR2 and SERCA2a nanodomains in cardiomyocytes.6RLYcPB_fJ7pnT18c_tdPoVideo abstract

Chronic intermittent hypoxia accelerates cardiac dysfunction and cardiac remodeling during cardiac pressure overload in mice and can be alleviated by PHD3 overexpression.

Obstructive sleep apnea (OSA) accelerates the progression of chronic heart failure (CHF). OSA is characterized by chronic intermittent hypoxia (CIH), and CIH exposure accelerates cardiac systolic dysfunction and cardiac remodeling in a cardiac afterload stress mouse model. Mechanistic experiments showed that long-term CIH exposure activated hypoxia-inducible factor 1α (HIF-1α) expression in the mouse heart and upregulated miR-29c expression and that both HIF-1α and miR-29c simultaneously inhibited sarco-/endoplasmic reticulum calcium ATPase 2a (SERCA2a) expression in the mouse heart. Cardiac HIF-1α activation promoted cardiomyocyte hypertrophy. SERCA2a expression was suppressed in mouse heart in middle- and late-stage cardiac afterload stress, and CIH exposure further downregulated SERCA2a expression and accelerated cardiac systolic dysfunction. Prolyl hydroxylases (PHDs) are physiological inhibitors of HIF-1α, and PHD3 is most highly expressed in the heart. Overexpression of PHD3 inhibited CIH-induced HIF-1α activation in the mouse heart while decreasing miR-29c expression, stabilizing the level of SERCA2a. Although PHD3 overexpression did not reduce mortality in mice, it alleviated cardiac systolic dysfunction and cardiac remodeling induced by CIH exposure.

Liposomal Resveratrol and/or Carvedilol Attenuate Doxorubicin-Induced Cardiotoxicity by Modulating Inflammation, Oxidative Stress and S100A1 in Rats.

Doxorubicin (DOX) is a cytotoxic anthracycline antibiotic and one of the important chemotherapeutic agents for different types of cancers. DOX treatment is associated with adverse effects, particularly cardiac dysfunction. This study examined the cardioprotective effects of carvedilol (CAR) and/or resveratrol (RES) and liposomal RES (LIPO-RES) against DOX-induced cardiomyopathy, pointing to their modulatory effect on oxidative stress, inflammation, S100A1 and sarco/endoplasmic reticulum calcium ATPase2a (SERCA2a). Rats received CAR (30 mg/kg) and/or RES (20 mg/kg) or LIPO-RES (20 mg/kg) for 6 weeks and were challenged with DOX (2 mg/kg) twice per week from week 2 to week 6. DOX-administered rats exhibited a significant increase in serum creatine kinase-MB (CK-MB), troponin-I and lactate dehydrogenase (LDH) along with histological alterations, reflecting cardiac cell injury. Cardiac toll-like receptor 4 (TLR-4), inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α and interleukin (IL)-6 protein expression were up-regulated, and lipid peroxidation was increased in DOX-administered rats. Treatment with CAR, RES or LIPO-RES as well as their alternative combinations ameliorated all observed biochemical and histological alterations with the most potent effect exerted by CAR/LIPO-RES. All treatments increased cardiac antioxidants, and the expression of S100A1 and SERCA2a. In conclusion, the present study conferred new evidence on the protective effects of CAR and its combination with either RES or LIPO-RES on DOX-induced inflammation, oxidative stress and calcium dysregulation.

The antiapoptotic protein HAX-1 mediates half of phospholamban's inhibitory activity on calcium cycling and contractility in the heart

The antiapoptotic protein HAX-1 (HS-associated protein X-1) localizes to sarcoplasmic reticulum (SR) in the heart and interacts with the small membrane protein phospholamban (PLN), inhibiting the cardiac sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) in the regulation of overall calcium handling and heart muscle contractility. However, because global HAX-1 deletion causes early lethality, how much endogenous HAX-1 contributes to PLN's inhibitory activity on calcium cycling is unknown. We therefore generated a cardiac-specific and inducible knock-out mouse model. HAX-1 ablation in the adult heart significantly increased contractile parameters and calcium kinetics, associated with increased SR calcium load. These changes occurred without any changes in the protein expression of SERCA2a, PLN, and ryanodine receptor or in the PLN phosphorylation status. The enhanced calcium cycling in the HAX-1-depleted heart was mediated through increases in the calcium affinity of SERCA2a and reduced PLN-SERCA2a binding. Comparison of the HAX-1 deletion-induced stimulatory effects with those elicited by PLN ablation indicated that HAX-1 mediates ∼50% of the PLN-associated inhibitory effects in the heart. Stimulation with the inotropic and lusitropic agent isoproterenol eliminated the differences among wild-type, HAX-1-deficient, and PLN-deficient hearts, and maximally stimulated contractile and calcium kinetic parameters were similar among these three groups. Furthermore, PLN overexpression in the HAX-1-null cardiomyocytes did not elicit any inhibitory effects, indicating that HAX-1 may limit PLN activity. These findings suggest that HAX-1 is a major mediator of PLN's inhibitory activity and a critical gatekeeper of SR calcium cycling and contractility in the heart.

Overexpression of Sarcoendoplasmic Reticulum Calcium ATPase 2a Promotes Cardiac Sympathetic Neurotransmission via Abnormal Endoplasmic Reticulum and Mitochondria Ca2+ Regulation.

Reduced cardiomyocyte excitation-contraction coupling and downregulation of the SERCA2a (sarcoendoplasmic reticulum calcium ATPase 2a) is associated with heart failure. This has led to viral transgene upregulation of SERCA2a in cardiomyocytes as a treatment. We hypothesized that SERCA2a gene therapy expressed under a similar promiscuous cytomegalovirus promoter could also affect the cardiac sympathetic neural axis and promote sympathoexcitation. Stellate neurons were isolated from 90 to 120 g male, Sprague-Dawley, Wistar Kyoto, and spontaneously hypertensive rats. Neurons were infected with Ad-mCherry or Ad-mCherry-hATP2Aa (SERCA2a). Intracellular Ca2+ changes were measured using fura-2AM in response to KCl, caffeine, thapsigargin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazine to mobilize intracellular Ca2+ stores. The effect of SERCA2a on neurotransmitter release was measured using [3H]-norepinephrine overflow from 340 to 360 g Sprague-Dawley rat atria in response to right stellate ganglia stimulation. Upregulation of SERCA2a resulted in greater neurotransmitter release in response to stellate stimulation compared with control (empty: 98.7±20.5 cpm, n=7; SERCA: 186.5±28.41 cpm, n=8; P<0.05). In isolated Sprague-Dawley rat stellate neurons, SERCA2a overexpression facilitated greater depolarization-induced Ca2+ transients (empty: 0.64±0.03 au, n=57; SERCA: 0.75±0.03 au, n=68; P<0.05), along with increased endoplasmic reticulum and mitochondria Ca2+ load. Similar results were observed in Wistar Kyoto and age-matched spontaneously hypertensive rats, despite no further increase in endoplasmic reticulum load being observed in the spontaneously hypertensive rat (spontaneously hypertensive rats: empty, 0.16±0.04 au, n=18; SERCA: 0.17±0.02 au, n=25). In conclusion, SERCA2a upregulation in cardiac sympathetic neurons resulted in increased neurotransmission and increased Ca2+ loading into intracellular stores. Whether the increased Ca2+ transient and neurotransmission after SERCA2A overexpression contributes to enhanced sympathoexcitation in heart failure patients remains to be determined.

Ablation of Matrix Metalloproteinase-9 Prevents Cardiomyocytes Contractile Dysfunction in Diabetics.

Elevated expression and activity of matrix metalloproteinase-9 (MMP9) and decreased contractility of cardiomyocytes are documented in diabetic hearts. However, it is unclear whether MMP is involved in the regulation of contractility of cardiomyocytes in diabetic hearts. In the present study, we tested the hypothesis that MMP9 regulates contractility of cardiomyocytes in diabetic hearts, and ablation of MMP9 prevents impaired contractility of cardiomyocytes in diabetic hearts. To determine the specific role of MMP9 in cardiomyocyte contractility, we used 12–14 week male WT (normoglycemic sibling of Akita), Akita, and Ins2+∕−/MMP9−∕− (DKO) mice. DKO mice were generated by cross-breeding male Ins2+∕− Akita (T1D) with female MMP9 knockout (MMP9−∕−) mice. We isolated cardiomyocytes from the heart of the above three groups of mice and measured their contractility and calcium transients. Moreover, we determined mRNA and protein levels of sarco-endoplasmic reticulum calcium ATPase-2a (SERCA-2a), which is involved in calcium handling during contractility of cardiomyocytes in WT, Akita, and DKO hearts using QPCR, Western blotting and immunoprecipitation, respectively. Our results revealed that in Akita hearts where increased expression and activity of MMP9 is reported, the rates of shortening and re-lengthening (±dL/dt) of cardiomyocytes were decreased, time to 90% peak height and baseline during contractility was increased, rate of calcium decay was increased, and calcium transient was decreased as compared to WT cardiomyocytes. However, these changes in Akita were blunted in DKO cardiomyocytes. The molecular analyses of SERCA-2a in the hearts showed that it was downregulated in Akita as compared to WT but was comparatively upregulated in DKO. These results suggest that abrogation of MMP9 gene prevents contractility of cardiomyocytes, possibly by increasing SERCA-2a and calcium transients. We conclude that MMP9 plays a crucial role in the regulation of contractility of cardiomyocytes in diabetic hearts.

Short-term exercise training attenuates acute doxorubicin cardiotoxicity.

Doxorubicin (DOX) is a potent and widely used antineoplastic agent. Despite the efficacy of DOX, its clinical use is limited by a dose-dependent cardiotoxicity. Chronic exercise training has been shown to protect against DOX-induced cardiotoxicity. It is less clear whether short-term exercise can attenuate DOX-induced dysfunction. The purposes of this study were to determine if short-term wheel running and treadmill exercise training can attenuate the cardiac dysfunction that accompanies DOX treatment and to investigate possible mechanisms that may be involved with any protective effects of exercise. Male Sprague-Dawley rats engaged in a short-term 5-day voluntary wheel running (WR) or treadmill exercise (TM) regimen. Following the exercise preconditioning period, animals received either 10 or 15 mg/kg of DOX or an equivalent volume of saline (SAL). Five days after DOX/SAL exposure, cardiac function was examined. Western immunoblotting was used to quantify left ventricular sarcoendoplasmic reticulum calcium-ATPase 2a (SERCA2a) protein expression. Exercise preconditioning attenuated in vivo and ex vivo cardiac dysfunction observed with DOX treatment alone. Specifically, short-term treadmill exercise (TM + DOX10, 56 ± 4%; TM + DOX15, 48 ± 5%) and voluntary wheel running (WR + DOX10, 51 ± 5%; WR + DOX15, 45 ± 3%) consistently preserved fractional shortening when compared to sedentary (SED) animals treated with DOX (SED + DOX10, 48 ± 4%; SED + DOX15, 39 ± 6%). Likewise, both exercise protocols preserved left ventricular developed pressure (TM + DOX10, 115 ± 6 mmHg; TM + DOX15, 85 ± 5 mmHg; WR + DOX10, 92 ± 12 mmHg; WR + DOX15, 91 ± 8 mmHg) when compared to SED animals treated with DOX (SED + DOX10, 79 ± 6 mmHg; SED + DOX15, 69 ± 7 mmHg). SERCA2a expression was also preserved in TM + DOX and WR + DOX. These findings suggest that short-term exercise prior to DOX treatment may be a valuable adjuvant therapy to offset acute cardiotoxicities and that maintaining calcium handling in cardiomyocytes may be responsible, in part, for the preservation in cardiac function.

Exendin-4 improves cardiac function in mice overexpressing monocyte chemoattractant protein-1 in cardiomyocytes

The incretin hormone glucagon-like peptide-1 (Glp1) is cardioprotective in models of ischemia-reperfusion injury, myocardial infarction and gluco/lipotoxicity. Inflammation is a factor in these models, yet it is unknown whether Glp1 receptor (Glp1r) agonists are protective against cardiac inflammation. We tested the hypothesis that the Glp1r agonist Exendin-4 (Ex4) is cardioprotective in mice with cardiac-specific monocyte chemoattractant protein-1 overexpression. These MHC-MCP1 mice exhibit increased cardiac monocyte infiltration, endoplasmic reticulum (ER) stress, apoptosis, fibrosis and left ventricular dysfunction. Ex4 treatment for 8weeks improved cardiac function and reduced monocyte infiltration, fibrosis and apoptosis in MHC-MCP1 mice. Ex4 enhanced expression of the ER chaperone glucose-regulated protein-78 (GRP78), decreased expression of the pro-apoptotic ER stress marker CCAAT/-enhancer-binding protein homologous protein (CHOP) and increased expression of the ER calcium regulator Sarco/Endoplasmic Reticulum Calcium ATPase-2a (SERCA2a). These findings suggest that the Glp1r is a viable target for treating cardiomyopathies associated with stimulation of pro-inflammatory factors.

SERCA2a gene therapy can improve symptomatic heart failure in δ-sarcoglycan-deficient animals.

The loss of dystrophin or its associated proteins results in the development of muscle wasting frequently associated with cardiomyopathy. Contractile cardiac tissue is injured and replaced by fibrous tissue or fatty infiltrates, leading to a progressive decrease of the contractile force and finally to end-stage heart failure. At the time symptoms appear, restoration of a functional allele of the causative gene might not be sufficient to prevent disease progression. Alterations in Ca(2+) transport and intracellular calcium levels have been implicated in many types of pathological processes, especially in heart disease. On the basis of a gene transfer strategy, we analyzed the therapeutic efficacy of primary gene correction in a δ-sarcoglycan (δ-SG)-deficient animal model versus gene transfer of the Ca(2+) pump hSERCA2a (human sarco-endoplasmic reticulum calcium ATPase 2a), at a symptomatic stage of heart disease. Our results strongly suggest that restoration of δ-SG at this stage of disease will not lead to improved clinical outcome. However, restoration of proper Ca(2+) handling by means of amplifying SERCA2a expression in the myocardium can lead to functional improvement. Abnormalities in Ca(2+) handling play an important role in disease progression toward heart failure, and increased SERCA2a levels appear to significantly improve cardiac contraction and relaxation. Beneficial effects persist at least over a period of 6 months, and the evolution of cardiac functional parameters paralleled those of normal controls. Furthermore, we demonstrate that a plasmid formulation based on amphiphilic block copolymers can provide a safe and efficient platform for myocardial gene therapies. The use of synthetic formulations for myocardial gene transfer might thus overcome one of the major hurdles linked to viral vectors, that is, repeat administrations.

Sarcoendoplasmic Reticulum Calcium Transport ATPase 2a

The development of cardiac-specific adeno-associated viral vectors capable of long-term transgenic expression has opened new avenues in the therapeutic approach to heart failure. Failing cardiomyocytes demonstrate altered calcium-handling secondary to depressed expression and activity of myocardial sarcoendoplasmic reticulum calcium ATPase 2a (SERCA2a). This observation has led to a large body of research investigating the therapeutic utility of SERCA2a gene therapy in heart failure.

Effects of sarcoplasmic reticulum Ca(2+)-ATPase gene transfer in a minipig model of chronic ischemic heart failure

Chronic myocardial ischemia (CMI) has become the most important cause of heart failure (HF) all over the world. The aim of the current study was to investigate the effects of Sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) gene transfer on cardiac function and endoplasmic reticulum stress (ERS) associated myocardial apoptosis in a minipig HF animal model induced by CMI. HF was induced in minipigs by implantation of ameroid constrictor in the initial segment of left anterior descending (LAD) branch of coronary artery. After confirmation of myocardial perfusion defects and cardiac function impairment by myocardial perfusion imaging and echocardiography, animals were divided into 4 groups (n = 4 each): HF group, HF + enhanced green fluorescent protein (EGFP) group, HF + SERCA2a group, and shamed animals as control group. A total amount of 1 × 10(12) v.g. of rAAV1-EGFP or rAAV1-SERCA2a were injected intramyocardially to each animal of HF + EGFP and HF + SERCA2a groups. Sixty days after gene transfer, protein level and activity of SERCA2a were examined, cardiac functions and changes of serum inflammatory and neuro-hormonal factors were determined. Apoptotic index of the ischemic myocardium, protein levels of ER stress marker glucose regulated protein 78 (GRP 78) and ER stress specific apoptotic marker caspase-12 were also assayed. At the study end, echocardiographic and hemo dynamic measurements indicated a significant improvement of both cardiac systolic and diastolic function in HF + SERCA2a group compared with HF/HF + EGFP groups [LVEF (60.2 ± 8.6)% vs (44.2 ± 7.1)% and (46.8 ± 6.7)%, Ev/Av 1.28 ± 0.24 vs 0.77 ± 0.17 and 0.80 ± 0.21, +dp/dt(max) (2713.9 ± 434.0) mm Hg/s (1 mm Hg = 0.133 kPa) vs (1892.3 ± 434.2) mm Hg/s and (1931.2 ± 397.4) mm Hg/s, -dp/dt(max) (1422.1 ± 334.4) mm Hg/s vs (848.3 ± 308.3) mm Hg/s and (849.5 ± 278.3) mm Hg/s, P < 0.05], along with increase in both SERCA2a protein level (1.13 ± 0.26 vs 0.73 ± 0.17 and 0.64 ± 0.18, P < 0.05) and activity [(16.2 ± 5.5) IU/ml vs (7.9 ± 3.1) IU/ml and (7.5 ± 2.8) IU/ml, P < 0.05] compared with HF/HF + EGFP groups. Serum concentrations of inflammatory factor tumor necrotic factor α [(382.3 ± 114.4) ng/L vs (732.3 ± 201.4) ng/L and (689.8 ± 192.5) ng/L, P < 0.05], neural-hormonal factors brain natriuretic peptide [(142.6 ± 45.3) ng/L vs (422.3 ± 113.6) ng/L and (393.7 ± 103.3) ng/L, P < 0.01], endothelin-1 [(111.4 ± 37.5) ng/L vs (193.5 ± 54.3) ng/L and (201.0 ± 72.1) ng/L, P < 0.05] and angiotensin II [(189.7 ± 65.2) µg/L vs (538.3 ± 135.2) µg/L and (525.5 ± 144.1) µg/L, P < 0.01] were also significantly decreased in HF + SERCA2a group compared with HF/HF + EGFP groups. The apoptotic index [(12.71 ± 4.11)% vs (23.22 ± 7.23)% and (24.31 ± 6.38)%, P < 0.05], protein levels of GRP 78 (1.27 ± 0.33 vs 3.23 ± 1.14 and 4.18 ± 1.13, P < 0.05) and protein level ratios of cleaved caspase-12 to total caspase-12 [(4.62 ± 1.93)% vs (9.71 ± 2.70)% and (10.14 ± 2.81)%, P < 0.05] were also significantly reduced in the ischemic myocardium of HF + SERCA2a group compared with the HF/HF + EGFP groups. Overexpression of SERCA2a significantly improved cardiac systolic and diastolic function in this HF model partly through attenuation of ER stress related myocardial apoptosis, suggesting its therapeutic potential for CMI related heart failure.

α-Kinase Anchoring Protein αKAP Interacts with SERCA2A to Spatially Position Ca2+/Calmodulin-dependent Protein Kinase II and Modulate Phospholamban Phosphorylation

The sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) is critical for sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR) and regulating cardiac muscle relaxation. Protein-protein interactions indicated that it exists in complex with Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and its anchoring protein alphaKAP. Confocal imaging of isolated cardiomyocytes revealed the colocalization of CAMKII and alphaKAP with SERCA2a at the SR. Deletion analysis indicated that SERCA2a and CaMKII bind to different regions in the association domain of alphaKAP but not with each other. Although deletion of the putative N-terminal hydrophobic amino acid stretch in alphaKAP prevented its membrane targeting, it did not influence binding to SERCA2a or CaMKII. Both CaMKIIdelta(C) and the novel CaMKIIbeta(4) isoforms were found to exist in complex with alphaKAP and SERCA2a at the SR and were able to phosphorylate Thr-17 on phospholamban (PLN), an accessory subunit and known regulator of SERCA2a activity. Interestingly, the presence of alphaKAP was also found to significantly modulate the Ca(2+)/calmodulin-dependent phosphorylation of Thr-17 on PLN. These data demonstrate that alphaKAP exhibits a novel interaction with SERCA2a and may serve to spatially position CaMKII isoforms at the SR and to uniquely modulate the phosphorylation of PLN.

Phosphorylation and binding of AUF1 to the 3′-untranslated region of cardiomyocyte SERCA2a mRNA

Experimental animals and patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum calcium ATPase 2a (SERCA2a), the cardiomyocyte sarcoplasmic reticulum Ca2+ pump. We previously showed that SERCA2a downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the hypertrophic agonist phorbol myristate acetate (PMA) or by overexpression of the novel protein kinase C (PKC) isoenzymes PKCdelta and PKCepsilon. PKC activation, in turn, decreased SERCA2a promoter activity and destabilized the SERCA2a mRNA. Here we demonstrate by using an RSV beta-galactosidase reporter system that a 609-nt fragment of the SERCA2a mRNA 3'-untranslated region (UTR), containing five adenylate-uridylate (AU)-rich regions, may be responsible for destabilizing the message following PMA treatment. UV cross-linking analysis demonstrated that several proteins found in the NRVM cell extracts bind to the 609-nt fragment. In addition, protein binding was transiently increased in response to PMA stimulation. 3'-UTR mRNA pull-down assays and Western blot analysis indicated that the AU binding protein AUF1 interacted with the SERCA2a 3'-UTR. AUF1 binding activity was predominantly found in the nuclear fraction, and PMA-induced AUF1 binding was associated with increased threonine phosphorylation of AUF1. These data suggest that the phosphorylation, binding, and location of AUF1 affect the posttranscriptional regulation of the SERCA2a message in NRVM.

Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular cardiomyocytes.

Downregulation of sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) expression is a critical marker of pathological myocardial hypertrophy. The effects of calcium-dependent signaling and of contractile activity on the regulation of myocardial SERCA2a expression remain unclear. The present study dissociates effects of calcium-dependent signaling through calcineurin (CN) and calmodulin dependent protein kinase-II (CAMK-II), from effects of contractile activity in spontaneously contracting rat neonatal ventricular cardiomyocytes (NVCM) using 2,3-butanedione monoxime (BDM), which arrests contractions but maintains calcium fluxes. SERCA2a mRNA expression was analysed using Northern hybridisation in spontaneously contracting NVCM (control) and in NVCM treated with either BDM, L-type Ca2+-channel blocker (verapamil), CN-blocker (cyclosporin A; CsA), CAMK-II blocker (KN-93), or combinations thereof. Transient transfection of the CN-dependent transcription factor nuclear factor of activated T-lymphocytes (NFATc), coupled to GFP, was used to detect NFAT nuclear translocation. The effects of CN/CAMK-II-dependent signaling were further dissected into effects of the transcription factors NFATc4 and myocyte enhancer factor 2c (MEF2c) on the activity of various SERCA2a promoter fragments using transient transfection assays. Treatment with BDM induced a 2.5-fold rise in SERCA2a mRNA, which was abolished by addition of verapamil and was reduced by addition of CsA (-40%) and KN-93 (-20%). NFAT nuclear translocation was similar in control and BDM-treated NVCM. SERCA2a promoter activity was stimulated by NFATc4 and MEF2c, but only when both factors were co-transfected. Following contractile arrest with BDM, upregulation of SERCA2a mRNA expression by CN/CAMK-II signaling becomes evident. This upregulation is likely the result of synergistic stimulation of SERCA2a promoter activity by NFATc4 and MEF2c. Contractile activity opposes this upregulation through distinct and independent pathways.