SERCA Activity Research Articles

Contribution of SERCA Activity to Contractile Properties of the Rat Soleus Muscle during One-Week Unloading

Contribution of SERCA Activity to Contractile Properties of the Rat Soleus Muscle during One-Week Unloading

296-LB: Loss of SERCA2 Induces Mitochondrial Dysfunction, Increases ß-Cell Senescence, and Accelerates Type 1 Diabetes Development

Type 1 diabetes (T1D) results from immune-mediated destruction of pancreatic β-cells. Senescence and activation of a senescence-associated secretory phenotype (SASP) in β-cells may contribute to T1D pathogenesis; however, the mechanisms responsible for this phenotype are not well understood. We hypothesized that loss of endoplasmic reticulum (ER) Ca2+ induces mitochondrial dysfunction and drives the development of β-cell senescence during T1D. To test this hypothesis, we generated sarco/ER Ca2+-ATPase 2 (SERCA2) haploinsufficient mice on the NOD background (NOD-S2+/-). NOD-S2+/- mice displayed accelerated T1D onset compared to NOD-WT mice (14 wks vs 18 wks; p≤ 0.0001 female and 21 wks vs 24.5 wks male; p<0.05). As expected, basal ER Ca2+ levels were reduced in NOD-S2+/- islets, but basal mitochondrial Ca2+ levels were elevated compared to NOD-WT islets. Basal cytoplasmic Ca2+ levels remained unchanged. Single cell RNA sequencing showed an increase in immune cell markers in the NOD-S2+/- islets at 6 wks of age, with signatures of ER stress and mitochondrial dysfunction at both 6 and 10 wks of age. Islets from prediabetic female NOD-S2+/- mice showed decreased glucose-stimulated O2 consumption, reduced ATP production, and decreased mitochondrial membrane potential. Additionally, protein expression of ATP synthase and Complex III was decreased in female NOD-S2+/- islets, but mitochondrial content was unchanged. There was an increased expression of multiple markers of senescence and SASP in NOD-S2+/- islets between 6 and 12 wks of age compared to NOD-WT islets. Lastly, in vivo allosteric activation of SERCA decreased insulitis and delayed diabetes onset in WT NOD mice. Our results show that SERCA2 deficiency leads to mitochondrial dysfunction, β-cell senescence, and accelerated T1D development. In future studies, we will test whether senolytic agents can delay diabetes onset and rescue mitochondrial health in models of T1D and SERCA2 deficiency. Disclosure S. A. Weaver: Employee; Eli Lilly and Company. R. N. Bone: None. D. J. Acri: None. J. Kim: None. T. Kono: None. R. Dahl: None. D. L. Eizirik: None. F. Syed: None. C. Evans-molina: Advisory Panel; Provention Bio, Inc., DiogenX, Avotres Inc., Neurodon, MaiCell Therapeutics, Other Relationship; Isla Technology, Bristol-Myers Squibb Company, Nimbus Therapeutics, Research Support; Lilly, Astellas Pharma Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (F31DK134168, R01DK093954, R01DK127308, R01DK127236, UC4DK104166, U01DK127786); U.S. Department of Veterans Affairs (I01BX001733, 2I01BX001733-05); JDRF (3-PDF-2017-385-A-N, 3-IND-2022-1235-I-X); National Center for Advancing Translational Sciences (UL1TR002529)

High-sensitivity calcium biosensor on the mitochondrial surface reveals that IP3R channels participate in the reticular Ca2+ leak towards mitochondria.

Genetically encoded biosensors based on fluorescent proteins (FPs) are widely used to monitor dynamics and sub-cellular spatial distribution of calcium ion (Ca2+) fluxes and their role in intracellular signaling pathways. The development of different mutations in the Ca2+-sensitive elements of the cameleon probes has allowed sensitive range of Ca2+ measurements in almost all cellular compartments. Region of the endoplasmic reticulum (ER) tethered to mitochondria, named as the mitochondrial-associated membranes (MAMs), has received an extended attention since the last 5 years. Indeed, as MAMs are essential for calcium homeostasis and mitochondrial function, molecular tools have been developed to assess quantitatively Ca2+ levels in the MAMs. However, sensitivity of the first generation Ca2+ biosensors on the surface of the outer-mitochondrial membrane (OMM) do not allow to measure μM or sub-μM changes in Ca2+ concentration which prevents to measure the native activity (unstimulated exogenously) of endogenous channels. In this study, we assembled a new ratiometric highly sensitive Ca2+ biosensor expressed on the surface of the outer-mitochondrial membrane (OMM). It allows the detection of smaller differences than the previous biosensor in or at proximity of the MAMs. Noteworthy, we demonstrated that IP3-receptors have an endogenous activity which participate to the Ca2+ leak channel on the surface of the OMM during hypoxia or when SERCA activity is blocked.

SERCA2 phosphorylation at serine 663 is a key regulator of Ca2+ homeostasis in heart diseases

Despite advances in cardioprotection, new therapeutic strategies capable of preventing ischemia-reperfusion injury of patients are still needed. Here, we discover that sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2) phosphorylation at serine 663 is a clinical and pathophysiological event of cardiac function. Indeed, the phosphorylation level of SERCA2 at serine 663 is increased in ischemic hearts of patients and mouse. Analyses on different human cell lines indicate that preventing serine 663 phosphorylation significantly increases SERCA2 activity and protects against cell death, by counteracting cytosolic and mitochondrial Ca2+ overload. By identifying the phosphorylation level of SERCA2 at serine 663 as an essential regulator of SERCA2 activity, Ca2+ homeostasis and infarct size, these data contribute to a more comprehensive understanding of the excitation/contraction coupling of cardiomyocytes and establish the pathophysiological role and the therapeutic potential of SERCA2 modulation in acute myocardial infarction, based on the hotspot phosphorylation level of SERCA2 at serine 663 residue.

RANKL Inhibition Reduces Cardiac Hypertrophy in mdx Mice and Possibly in Children with Duchenne Muscular Dystrophy.

Cardiomyopathy has become one of the leading causes of death in patients with Duchenne muscular dystrophy (DMD). We recently reported that the inhibition of the interaction between the receptor activator of nuclear factor κB ligand (RANKL) and receptor activator of nuclear factor κB (RANK) significantly improves muscle and bone functions in dystrophin-deficient mdx mice. RANKL and RANK are also expressed in cardiac muscle. Here, we investigate whether anti-RANKL treatment prevents cardiac hypertrophy and dysfunction in dystrophic mdx mice. Anti-RANKL treatment significantly reduced LV hypertrophy and heart mass, and maintained cardiac function in mdx mice. Anti-RANKL treatment also inhibited NFκB and PI3K, two mediators implicated in cardiac hypertrophy. Furthermore, anti-RANKL treatment increased SERCA activity and the expression of RyR, FKBP12, and SERCA2a, leading possibly to an improved Ca2+ homeostasis in dystrophic hearts. Interestingly, preliminary post hoc analyses suggest that denosumab, a human anti-RANKL, reduced left ventricular hypertrophy in two patients with DMD. Taken together, our results indicate that anti-RANKL treatment prevents the worsening of cardiac hypertrophy in mdx mice and could potentially maintain cardiac function in teenage or adult patients with DMD.

Alterations of the Ca2+ clearing mechanisms by type 2 diabetes in aortic smooth muscle cells of Zucker diabetic fatty rat.

Type 2 Diabetes Mellitus (T2DM) is a rapidly rising disease with cardiovascular complications constituting the most common cause of death among diabetic patients. Chronic hyperglycemia can induce vascular dysfunction through damage of the components of the vascular wall, such as vascular smooth muscle cells (VSMCs), which regulate vascular tone and contribute to vascular repair and remodeling. These functions are dependent on intracellular Ca2+ changes. The mechanisms by which T2DM affects Ca2+ handling in VSMCs still remain poorly understood. Therefore, the objective of this study was to determine whether and how T2DM affects Ca2+ homeostasis in VSMCs. We evaluated intracellular Ca2+ signaling in VSMCs from Zucker Diabetic Fatty rats using Ca2+ imaging with Fura-2/AM. Our results indicate that T2DM decreases Ca2+ release from the sarcoplasmic reticulum (SR) and increases the activity of store-operated channels (SOCs). Moreover, we were able to identify an enhancement of the activity of the main Ca2+ extrusion mechanisms (SERCA, PMCA and NCX) during the early stage of the decay of the ATP-induced Ca2+ transient. In addition, we found an increase in Ca2+ entry through the reverse mode of NCX and a decrease in SERCA and PMCA activity during the late stage of the signal decay. These effects were appreciated as a shortening of ATP-induced Ca2+ transient during the early stage of the decay, as well as an increase in the amplitude of the following plateau. Enhanced cytosolic Ca2+ activity in VSMCs could contribute to vascular dysfunction associated with T2DM.

A bioelectrochemical approach based on a solid supported membrane to evaluate the effect of natural products on Ca2+-ATPase: The case of 6-gingerol

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) plays an essential role in maintaining the low cytosolic Ca2+ level that enables a variety of cellular processes. SERCA couples ATP hydrolysis to the transport of two Ca2+ ions against their electrochemical potential gradient from the cytoplasm into the lumen of the sarco/endoplasmic reticulum (SR/ER). Because of its central role in regulating cytoplasmic Ca2+ concentration, SERCA dysfunction has been associated with several pathological conditions. Stimulation of SERCA activity may represent a potential therapeutic strategy in various disease states connected with dysfunctional SERCA. The natural phenolic compound 6-gingerol, the most abundant and the major biologically active compound of ginger, was reported to activate the SERCA enzyme. The present study aimed at investigating the effect of 6-gingerol on SERCA transport activity using a bioelectrochemical approach based on a solid supported membrane (SSM). We first performed a voltammetric characterization of 6-gingerol to better understand its electrochemical behavior. We then studied the interaction of 6-gingerol with SR vesicles containing SERCA adsorbed on the SSM electrode. The measured current signals indicated that ATP-dependent Ca2+ translocation by SERCA was remarkably increased in the presence of 6-gingerol at low micromolar concentration. We also found that 6-gingerol has a rather high affinity for SERCA (EC50 of 1.8 ± 0.3 µM), and SERCA activation by 6-gingerol is reversible. The observed stimulatory effect of 6-gingerol on SERCA Ca2+-translocating activity may be beneficial in the prevention and/or treatment of pathological conditions related to SERCA dysfunction.

The effects of doxorubicin on calcium handling and contractility in sheep ventricular myocytes; role of oxidative stress

Anthracyclines such as doxorubicin (DOX) are effective chemotherapeutics and contribute to improved cancer survival rates in children and adults. However, anthracyclines exhibit acute and chronic cardiotoxicity which can produce heart failure in cancer survivors. While the cellular basis remains unclear, previous studies show anthracyclines perturb aspects of excitation-contraction coupling (ECC), though to our knowledge this has yet to be demonstrated in a large animal model. Furthermore, the extent to which altered ECC is dependent on anthracycline-induced reactive oxygen species (ROS) production / oxidative stress remains ambiguous. This is important given the key role of oxidative stress in other cardiotoxic processes. Therefore, we aimed to (1) characterise the effects of DOX on Ca handling and contractility in sheep ventricular myocytes and (2) quantify the dependence of any effect on oxidative stress.Sheep myocytes were enzymatically isolated in accordance with the Animals (Scientific Procedures) Act, UK, 1986. Cells were loaded with fura-2 (0.1 μM) then field stimulated at 0.5 Hz. Fura-2 was excited sequentially at 340 nm and 380 nm (200 Hz) and changes of cytoplasmic Ca inferred from the ratio of emitted light. Sarcomere length was measured simultaneously using a Myocam-S CCD camera and SarcLen acquisition module (Ion Optix, USA). Changes to sarcoplasmic reticulum (SR) Ca content were estimated by rapid application of caffeine (10 mM). Two-way ANOVA was used for statistical analysis.1 nM DOX reduced the amplitude of systolic Ca and shortening by 39 ± 3 % (n=49, p<0.001) and 42 ± 4 % (n=45, p<0.001) respectively. The amplitude of the caffeine-evoked Ca transient decreased by 33 ± 5 % (n=24, p<0.001). The rate of decay of the systolic Ca transient ( k sys ) represents the combined activity of SERCA and NCX and was reduced by 17 ± 3 % (n=46, p<0.001). The rate of decay of the caffeine-evoked Ca transient ( k caff ) represents NCX alone and was increased by 64 ± 26 % (n=23, p=0.002).Subtraction of k caff from k sys indicates SERCA activity ( k SERCA ), which was decreased by 34 ± 8 % (n=20, p<0.001). When the experiment was repeated in the presence of the antioxidant N-acetylcysteine (NAC), the effect of DOX on systolic Ca, systolic shortening and the caffeine-evoked Ca transient was attenuated by 52 % (n=29, p<0.001), 58 % (n=29, p<0.001) and 45 % (n=15, p=0.003) respectively. Furthermore, NAC prevented the effect of DOX on k sys , k caff and k SERCA .These data suggest DOX increases NCX activity and reduces SERCA activity which decreases SR Ca content thence systolic Ca and contractility. The effect of DOX on NCX and SERCA appears to be entirely ROS / oxidative stress dependent. However, other non-oxidative mechanisms likely contribute to net DOX-mediated negative cellular inotropy. Our next experiments will investigate this and the sources of anthracycline-induced oxidative stress. This work was funded by Kidscan childrens cancer research, UK. This is the full abstract presented at the American Physiology Summit 2023 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.

Voluntary wheel running and lithium supplementation improves muscle force production without altering SERCA function in the D2 mdx mouse

Background: Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disease that, with no cure, leads to early mortality (30-40 years of age). Previous studies have shown that voluntary wheel running can mitigate disease pathology and improve muscle contractility in the preclinical mdx mouse model. We have shown that treating wild-type (WT) mice with a low dose of lithium, a natural inhibitor of glycogen synthase kinase 3, enhanced soleus (SOL) and extensor digitorum longus (EDL) muscle contractility. Our objectives were to test our hypothesis of whether combining VWR with low dose lithium supplementation could provide further benefits to muscle contractility in DBA/2J mdx mice – a model that displays far worse disease severity compared with the traditional C57BL/10 mdx mouse. We also examined if this combined intervention could improve muscle calcium regulation in muscle by assessing sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) function as we have recently shown that SERCA function is drastically impaired in D2 mdx mice, which could also contribute to deficits in muscle force production. Methods: Young (5-6 week old) male D2 WT and mdx mice were ordered from Jackson Laboratory. D2 mdx mice were assigned into each of the following conditions: sedentary ( mdxSED), voluntary wheel running ( mdxVWR), or VWR+lithium chloride ( mdxVWR+Li). The VWR groups were given access to a cagewheel for a total of 6 weeks, and the mdxVWR+Li group were provided a low dose of LiCl (50 mg/kg/day) via drinking water for the entire duration of the study. After 6 weeks, all mice were euthanized and the SOL and EDL muscles were subjected to force-frequency curve analysis (1-160 Hz). SERCA function was assessed by performing SERCA activity assays and a Ca 2+ uptake assay. Results: In the SOL, the combined effect of lithium and VWR improved specific force production to near WT levels across submaximal and maximal frequencies. Specific force production in the mdxVWR+Li group was significantly greater compared to the mdxSED group at all stimulation frequencies. Rate of SOL force development was significantly lower in all mdx groups compared to WT. However, rate of relaxation was only significantly reduced (vs. WT) within the mdxSED group, suggesting that VWR and VWR+Li improved relaxation. This could not be explained by any changes in SERCA function. In the EDL, specific force was significantly (p<0.001) reduced across mdx groups compared to WT; however, of all mdx groups, the mdxVWR+Li produced the most force and was significantly different compared to mdxVWR at 80 and 160 Hz. Rates of force development and relaxation were all slower in all mdx groups compared to WT, and there were no differences in SERCA activity. Conclusions: Combining lithium supplementation with VWR improved SOL and EDL force production in the D2 mdx mouse without any changes to SERCA function. V.A.F is a Tier 2 Canada Research Chair in Muscle Plasticity and Tissue-Remodelling. This is the full abstract presented at the American Physiology Summit 2023 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.

Sarcolipin ablation decreases whole body VO2 in young female nice

The sarco(endo)plasmic reticulum calcium (Ca 2+ ) ATPase (SERCA) pump is a major regulator of cytosolic Ca 2+ in striated muscle. Sarcolipin (SLN) reduces SERCA-mediated Ca 2+ pumping efficiency and is a key regulator of muscle metabolism and contractile function. With SERCA activity comprising approximately 12% of whole-body metabolic rate, the inefficient SERCA Ca 2+ cycling promoted by SLN would be predicted to increase metabolic rate. Although SLN is involved in adaptive diet-induced thermogenesis and overexpression of SLN increases metabolic rate and protects against diet-induced obesity, we found previously that SLN ablation had no effect on whole body metabolic rate or body composition in male mice that were fed a standard chow diet. However, unpublished work from our lab indicates that SLN expression in mouse soleus is significantly higher in females compared with males. Therefore, the purpose of this study was to investigate the effects of SLN ablation on body composition and metabolic rate of female mice. Adult (4-6mo) female wild type (WT) and SLN knockout (KO) mice (n=4 per group) were used to collect anthropometric measures (adiposity and body weight) and were housed in a comprehensive lab animal monitoring system (CLAMS) over a 48-hour period to assess daily whole body VO2 and total cage activity. With SLN ablation, total whole body VO 2 (ml/kg/hr) was lower (p<0.05) in the KO females (3534 ± 95.1) compared to WT females (5080 ± 98.8), with no differences (p>0.05) found for total cage activity (counts) (WT, 14935.42 ± 1799.6 vs. KO, 11860.83 ± 679.48), body weight (grams) (WT, 20.97 ± 0.76 vs. KO, 22.18 ± 0.80), or adiposity (arbitrary units) (WT, 5.18 ± 1.71 vs. KO, 2.12 ± 0.36). These results showing that high levels of endogenous muscle SLN expression in female mice influence whole-body metabolism even under chow-fed conditions, demonstrate a potential important sex difference in whole body physiology and metabolism. Natural Sciences and Engineering Research Council of Canada (NSERC) [grant number 311922-05 (to A.R.T.)] This is the full abstract presented at the American Physiology Summit 2023 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.

Pomiferin targets SERCA, mTOR, and P-gp to induce autophagic cell death in apoptosis-resistant cancer cells, and reverses the MDR phenotype in cisplatin-resistant tumors in vivo

Drug resistance in cancer has been classified as innate resistance or acquired resistance, which were characterized by apoptotic defects and ABC transporters overexpression respectively. Therefore, to preclude or reverse these resistance mechanisms could be a promising strategy to improve chemotherapeutic outcomes. In this study, a natural product from Osage Orange, pomiferin, was identified as a novel autophagy activator that circumvents innate resistance by triggering autophagic cell death via SERCA inhibition and activation of the CaMKKβ-AMPK-mTOR signaling cascade. In addition, pomiferin also directly inhibited the P-gp (MDR1/ABCB1) efflux and reversed acquired resistance by potentiating the accumulation and efficacy of the chemotherapeutic agent, cisplatin. In vivo study demonstrated that pomiferin triggered calcium-mediated tumor suppression and exhibited an anti-metastatic effect in the LLC-1 lung cancer-bearing mouse model. Moreover, as an adjuvant, pomiferin potentiated the anti-tumor effect of the chemotherapeutic agent, cisplatin, in RM-1 drug-resistant prostate cancer-bearing mouse model by specially attenuating ABCB1-mediated drug efflux, but not ABCC5, thereby promoting the accumulation of cisplatin in tumors. Collectively, pomiferin may serve as a novel effective agent for circumventing drug resistance in clinical applications.

Cadmium Disrupted ER Ca2+ Homeostasis by Inhibiting SERCA2 Expression and Activity to Induce Apoptosis in Renal Proximal Tubular Cells

Cadmium (Cd2+) exposure induces chronic kidney disease and renal cancers, which originate from injury and cancerization of renal tubular cells. Previous studies have shown that Cd2+ induced cytotoxicity by disrupting the intracellular Ca2+ homeostasis that is physically regulated by the endoplasmic reticulum (ER) Ca2+ store. However, the molecular mechanism of ER Ca2+ homeostasis in Cd2+-induced nephrotoxicity remains unclear. In this study, our results firstly revealed that the activation of calcium-sensing receptor (CaSR) by NPS R-467 could protect against Cd2+ exposure-induced cytotoxicity of mouse renal tubular cells (mRTEC) by restoring ER Ca2+ homeostasis through the ER Ca2+ reuptake channel sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Cd2+-induced ER stress and cell apoptosis were effectively abrogated by SERCA agonist CDN1163 and SERCA2 overexpression. In addition, in vivo, and in vitro results proved that Cd2+ reduced the expressions of SERCA2 and its activity regulator phosphorylation phospholamban (p-PLB) in renal tubular cells. Cd2+-induced SERCA2 degradation was suppressed by the treatment of proteasome inhibitor MG132, which suggested that Cd2+ reduced SERCA2 protein stability by promoting the proteasomal protein degradation pathway. These results suggested that SERCA2 played pivotal roles in Cd2+-induced ER Ca2+ imbalance and stress to contribute to apoptosis of renal tubular cells, and the proteasomal pathway was involved in regulating SERCA2 stability. Our results proposed a new therapeutic approach targeting SERCA2 and associated proteasome that might protect against Cd2+-induced cytotoxicity and renal injury.

Disruption of Phosphodiesterase 3A Binding to SERCA2 Increases SERCA2 Activity and Reduces Mortality in Mice With Chronic Heart Failure.

Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca2+ ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators. Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays. PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding. Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.

The mechanism of dwarf open reading frame (DWORF) activation of the sarcoplasmic reticulum calcium pump SERCA.

The sarcoplasmic reticulum calcium pump SERCA plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitory transmembrane peptide phospholamban. A new transmembrane peptide regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. We have shown that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban (1). Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake, while phospholamban decreases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium, while phospholamban has a higher affinity for SERCA in the absence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium. Mutagenesis and structure-function analysis of DWORF reveals three important features required for SERCA activation: Pro15 promotes a helix-linker-helix structure of DWORF; the N-terminal amphipathic helix of DWORF perturbs the membrane bilayer; and the short transmembrane helix of DWORF contributes to hydrophobic mismatch and positions the amphipathic helix at the surface of the membrane bilayer. Our current understanding of the mechanism of SERCA activation by DWORF will be presented.

Complementary in vitro functional studies and in silico alchemical free energy simulations of the sarco-endoplasmic reticulum calcium pump (SERCA) and phospholamban (PLN) complex.

SERCA is ubiquitously expressed in all eukaryotic cells and is responsible for actively transporting calcium ions from the cytoplasm into the SR/ER lumen. PLN is a small transmembrane peptide that regulates SERCA's calcium transport activity in cardiac muscle. Human genetic variants of PLN have been implicated in cardiomyopathies. PLN is a 52 amino acid peptide with an N-terminal cytoplasmic helix (residues 1-17), a short linker region (residues 18-27) and a C-terminal transmembrane helix (residues 28-52). Previous structural studies have identified the binding groove for PLN formed by transmembrane segments M2, M6, and M9 of SERCA (PDB: 4KYT). The structure of the SERCA-PLN complex consists of SERCA in a calcium-free, E1-like state and includes the linker region and transmembrane helix of PLN (residues 20-52). Here, we carry out in vitro functional studies and in silico free energy calculations to examine the effects of alanine scanning mutations in PLN (residues 20-52) on SERCA regulation. From the functional studies, changes in kinetics parameters such as calcium affinity (KCa) and maximal activity (VMax) of SERCA are determined for each of the alanine substitutions. From the in silico alchemical free energy simulations (using Compute Canada supercomputing resources), each of the residues in PLN are alchemically transformed to alanine to determine the change in Gibbs free energy (Δ;Δ;G) relative to the wild-type SERCA-PLN complex. Together, the complementary in vitro and in silico mutational studies provide novel insights into a membrane protein-peptide complex. The PLN variants are quantified in terms of their physiological significance – gain-of-function, loss-of-function, or neutral – and the molecular determinants that control binding specificity.

Regulation of beige adipocyte thermogenesis by the cold-repressed ER protein NNAT

ObjectiveCold stimuli trigger the conversion of white adipose tissue into beige adipose tissue, which is capable of non-shivering thermogenesis. However, what process drives this activation of thermogenesis in beige fat is not well understood. Here, we examine the ER protein NNAT as a regulator of thermogenesis in adipose tissue. MethodsWe investigated the regulation of adipose tissue NNAT expression in response to changes in ambient temperature. We also evaluated the functional role of NNAT in thermogenic regulation using Nnat null mice and primary adipocytes that lack or overexpress NNAT. ResultsCold exposure or treatment with a β3-adrenergic agonist reduces the expression of adipose tissue NNAT in mice. Genetic disruption of Nnat in mice enhances inguinal adipose tissue thermogenesis. Nnat null mice exhibit improved cold tolerance both in the presence and absence of UCP1. Gain-of-function studies indicate that ectopic expression of Nnat abolishes adrenergic receptor-mediated respiration in beige adipocytes. NNAT physically interacts with the ER Ca2+-ATPase (SERCA) in adipocytes and inhibits its activity, impairing Ca2+ transport and heat dissipation. We further demonstrate that NHLRC1, an E3 ubiquitin protein ligase implicated in proteasomal degradation of NNAT, is induced by cold exposure or β3-adrenergic stimulation, thus providing regulatory control at the protein level. This serves to link cold stimuli to NNAT degradation in adipose tissue, which in turn leads to enhanced SERCA activity. ConclusionsOur study implicates NNAT in the regulation of adipocyte thermogenesis.

Suppression of lusitropy as a disease mechanism in cardiomyopathies.

In cardiac muscle the action of adrenaline on β1 receptors of heart muscle cells is essential to adjust cardiac output to the body's needs. Adrenergic activation leads to enhanced contractility (inotropy), faster heart rate (chronotropy) and faster relaxation (lusitropy), mainly through activation of protein kinase A (PKA). Efficient enhancement of heart output under stress requires all of these responses to work together. Lusitropy is essential for shortening the heartbeat when heart rate increases. It therefore follows that, if the lusitropic response is not present, heart function under stress will be compromised. Current literature suggests that lusitropy is primarily achieved due to PKA phosphorylation of troponin I (TnI) and phospholamban (PLB). It has been well documented that PKA-induced phosphorylation of TnI releases Ca2+ from troponin C faster and increases the rate of cardiac muscle relaxation, while phosphorylation of PLB increases SERCA activity, speeding up Ca2+ removal from the cytoplasm. In this review we consider the current scientific evidences for the connection between suppression of lusitropy and cardiac dysfunction in the context of mutations in phospholamban and thin filament proteins that are associated with cardiomyopathies. We will discuss what advances have been made into understanding the physiological mechanism of lusitropy due to TnI and PLB phosphorylation and its suppression by mutations and we will evaluate the evidence whether lack of lusitropy is sufficient to cause cardiomyopathy, and under what circumstances, and consider the range of pathologies associated with loss of lusitropy. Finally, we will discuss whether suppressed lusitropy due to mutations in thin filament proteins can be therapeutically restored.

InsP3R-RyR Ca2+ channel crosstalk facilitates arrhythmias in the failing human ventricle.

Dysregulated intracellular Ca2+ handling involving altered Ca2+ release from intracellular stores via RyR channels underlies both arrhythmias and reduced function in heart failure (HF). Mechanisms linking RyR dysregulation and disease are not fully established. Studies in animals support a role for InsP3 receptor Ca2+ channels (InsP3R) in pathological alterations in cardiomyocyte Ca2+ handling but whether these findings translate to the divergent physiology of human cardiomyocytes during heart failure is not determined. Using electrophysiological and Ca2+ recordings in human ventricular cardiomyocytes, we uncovered that Ca2+ release via InsP3Rs facilitated Ca2+ release from RyR and induced arrhythmogenic delayed after depolarisations and action potentials. InsP3R-RyR crosstalk was particularly increased in HF at RyR clusters isolated from the T-tubular network. Reduced SERCA activity in HF further facilitated the action of InsP3. Nanoscale imaging revealed co-localisation of InsP3Rs with RyRs in the dyad, which was increased in HF, providing a mechanism for augmented Ca2+ channel crosstalk. Notably, arrhythmogenic activity dependent on InsP3Rs was increased in tissue wedges from failing hearts perfused with AngII to promote InsP3 generation. These data indicate a central role for InsP3R-RyR Ca2+ signalling crosstalk in the pro-arrhythmic action of GPCR agonists elevated in HF and the potential for their therapeutic targeting.

Alterations of sarcoplasmic reticulum-mediated Ca2+ uptake in a model of premature ventricular contraction (PVC)-induced cardiomyopathy.

Premature ventricular contractions (PVCs) are the most frequent ventricular arrhythmias in the overall population. PVCs are known to acutely enhance contractility by the post-extrasystolic potentiation phenomenon, but over time persistent PVCs promote PVC-induced cardiomyopathy (PVC-CM), characterized by a reduction of the left ventricular (LV) ejection fraction. Ca2+ cycling in myocytes commands muscle contraction and in this process, SERCA2 leads the Ca2+ reuptake into the sarcoplasmic reticulum (SR) shaping cytosolic Ca2+ signal decay and muscle relaxation. Altered Ca2+ reuptake can contribute to the contractile dysfunction observed in PVC-CM. To better understand Ca2+ handling using our PVC-CM model (canines with 50% PVC burden for 12weeks), SR-Ca2+ reuptake was investigated by measuring Ca2+ dynamics and analyzing protein expression. Kinetic analysis of Ca2+ reuptake in electrically paced myocytes showed a ~ 21ms delay in PVC-CM compared to Sham in intact isolated myocytes, along with a ~ 13% reduction in SERCA2 activity assessed in permeabilized myocytes. Although these trends were not statistically significant between groups using hierarchical statistics, relaxation of myocytes following contraction was significantly slower in PVC-CM vs Sham myocytes. Western blot analyses indicate a 22% reduction in SERCA2 expression, a 23% increase in phospholamban (PLN) expression, and a 50% reduction in PLN phosphorylation in PVC-CM samples vs Sham. Computational analysis simulating a 20% decrease in SR-Ca2+ reuptake resulted in a ~ 22ms delay in Ca2+ signal decay, consistent with the experimental result described above. In conclusion, SERCA2 and PLB alterations described above have a modest contribution to functional adaptations observed in PVC-CM.

Abstract 12531: Electrophysiological Mechanisms of Tachycardia-Induced Cardiomyopathy in Human Myocardium

Introduction: Tachycardia-induced cardiomyopathy (TCM) is a reversible form of heart failure. However, the effects of persistent tachycardia leading to TCM in the human are less understood limiting appropriate management of patients with this underrecognized condition. This study investigated the electrophysiological mechanisms of TCM. Methods and Results: Tachycardic in vitro electrical stimulation (120 bpm versus 60 bpm control) of human specimen was performed. In human twitching ventricular trabeculae from patients with preexistent heart failure, tachycardia for 8h compromised systolic force, while diastolic tension and relaxation time were increased (n=6/8 patients each). Human iPSC-cardiomyocytes (iPSC-CM) of healthy donors were utilized as human model suitable for extended electrical pacing. After 24h of tachycardia, systolic Ca 2+ -transient amplitude of iPSC-CM was reduced, while diastolic Ca 2+ levels and cytosolic Ca 2+ elimination were not changed (Fura-2, n tachycardia/control=49/44 cells). 7d of tachycardia resulted in a progressive decline of Ca 2+ -transient amplitude together with an impaired Ca 2+ elimination (n=73/66) thereby demonstrating the progression of TCM. Sarcoplasmic reticulum (SR) Ca 2+ load (caffeine application) and SERCA activity (K sys -K caff ) were reduced after 7d of tachycardia (n=13/13). Besides an impaired SERCA activity, we could identify an increased diastolic SR Ca 2+ spark frequency after 7d of tachycardia (n=76/79) as another mechanism of reduced SR Ca 2+ load. In voltage clamp studies, I CaL density was reduced after 7d of tachycardia (n=11/12), which may further contribute to impaired systolic Ca 2+ -handling. Conversely, action potential duration was prolonged after 7d of tachycardia compared to control (n=19/21). As a potential explanation for prolonged action potentials however, late sodium current (I NaL ) was enhanced after 7d of tachycardia (n=26/19). Conclusions: This study demonstrates for the first time in human myocardium that persistent tachycardia induces early adverse electrophysiological remodeling that may underlie the progression of TCM. These findings may help to understand the pathophysiology of an underrated but prevalent disease