SERCA Function Research Articles

Restoring Atrial T-Tubules Augments Systolic Ca Upon Recovery From Heart Failure.

Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca2+. HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patch clamp, Ca2+, and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure. Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca2+ release in the cell interior. Systolic Ca2+, ICa-L, sarcoplasmic reticulum Ca2+ content, and sarcoendoplasmic reticulum Ca2+ ATPase function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca2+ transient, the rate of Ca2+ removal, and the peak L-type Ca2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria. We show that recovery from HF restores atrial t-tubules, and this promotes recovery of ICa-L, sarcoplasmic reticulum Ca2+ content, and systolic Ca2+. We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy.

Inhibition of ER stress using tauroursodeoxycholic acid rescues obesity-evoked cardiac remodeling and contractile anomalies through regulation of ferroptosis

Interrupted ER homeostasis contributes to the etiology of obesity cardiomyopathy although it remains elusive how ER stress evokes cardiac anomalies in obesity. Our study evaluated the impact of ER stress inhibition on cardiac anomalies in obesity. Lean and ob/ob obese mice received chemical ER chaperone tauroursodeoxycholic acid (TUDCA, 50 mg/kg/d, p.o.) for 35 days prior to evaluation of glucose sensitivity, echocardiographic, myocardial geometric, cardiomyocyte mechanical and subcellular Ca2+ property, mitochondrial integrity, oxidative stress, apoptosis, and ferroptosis. Intracellular Ca2+ governing domains including sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) were monitored by45Ca2+uptake and immunoblotting. Our results noted that TUDCA alleviated myocardial remodeling (fibrosis, hypertrophy, enlarged LVESD), echocardiographic anomalies (compromised fractional shortening and ejection fraction), cardiomyocyte contractile dysfunction (amplitude and velocity of cell shortening, relengthening time) and intracellular Ca2+ anomalies (compromised subcellular Ca2+ release, clearance and SERCA function), mitochondrial damage (collapsed membrane potential, downregulated mitochondrial elements and ultrastructural alteration), ER stress (GRP78, eIF2α and ATF4), oxidative stress, apoptosis and ferroptosis [downregulated SLC7A11, GPx4 and upregulated transferrin receptor (TFRC)] without affecting global glucose sensitivity and serum Fe2+ in obese mice. Obesity-evoked change in HSP90, phospholamban and Na+-Ca2+ exchanger was spared by the chemical ER chaperone. Moreover, in vitro results noted that TUDCA, PERK inhibitor GSK2606414, TFRC neutralizing antibody and ferroptosis inhibitor LIP1 mitigated palmitic acid-elicited changes in lipid peroxidation and mechanical function. Our findings favored a role for ferroptosis in obesity cardiomyopathy downstream of ER stress.

Abstract P2077: FoxO3 Transcription Factor Regulates Myocyte Contractility And Calcium Homeostasis In Diabetic Cardiomyopathy

Diabetes is a risk factor for heart failure with preserved ejection fractions (HFpEF). Calcium (Ca 2+ ) mishandling and diastolic abnormalities are the characteristics of diabetic cardiomyopathy (DCM), but the molecular mechanisms of these are elusive. FoxO transcription factors (FoxOs) are suppressed by insulin and this study evaluated the role of FoxO3 in myocardial contractility and Ca 2+ handling in the heart of a mouse model of type 1 diabetes. We show that cardiomyocyte-restricted deletion of FoxO3 (H-FoxO3 KO) prevented reduction in the peak rate of left ventricular (LV) pressure (dP/dt max) and prevented the increase in the minimum rate of fall in LV pressure (dP/dt min) that was evident after 4-7 weeks of Streptozotocin (STZ) diabetes. Time for pressure decay of LV while relaxation (Tau) was partially increased in STZ hearts and tended to decrease in STZ H-FoxO3 KO. Echocardiography showed LVEF was unchanged in control and H-FoxO3 KO animals treated with/without STZ. Ca 2+ imaging in perfused hearts found that Ca 2+ transient amplitude was decreased and decay kinetics prolonged (indicates decreased SERCA activity) in STZ hearts, but these changes were prevented in STZ H-FoxO3 KO. Phosphorylation of SERCA at Thr484 by SPEG (Striated muscle preferentially expressed protein kinase) controls Ca 2+ re-uptake and cardiac contractility. We show that STZ diabetes decreases SPEG protein and pSERCA2a Thr484 , but levels of each were normalized or increased in STZ H-FoxO3 KO. Mechanistically, overexpression of FoxO3 in neonatal cardiomyocytes increases high-molecular weight SPEG adducts when proteasome degradation was inhibited with MG-132, suggesting that ubiquitin-mediated degradation of SPEG is a mechanism by which FoxO3 controls Ca 2+ handling via SERCA phosphorylation. SERCA function requires ATP, thus, we evaluated mitochondrial energetics in these animals. Respiratory capacity in saponin-permeabilized cardiac fibers was unchanged in hearts from controls and H-FoxO3 KO treated with/without STZ. ATP generation was decreased in STZ hearts but was not normalized in STZ-H FoxO3 KO. These data indicate FoxO3 regulates myocyte contractility via SPEG degradation to control intracellular Ca 2+ via pSERCA2a Thr484 in the heart in a type 1 diabetes model.

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.

Low-dose lithium chloride supplementation protects cardiac SERCA function during heat stress

Background: The sarco(endo)plasmic reticulum calcium (Ca2+)-ATPase (SERCA) is responsible for ~70% of intracellular Ca2+ removal in cardiac muscle contributing to the regulation of cardiac relaxation and contractility. SERCA is susceptible to heightened oxidative stress that is often observed with aging and disease resulting in reduced activity and cardiac dysfunction. Under normal physiological conditions, the inhibition of the enzyme glycogen synthase kinase 3 (GSK3) via low-dose lithium chloride (LiCl) supplementation (10 mg/kg/day) improved SERCA function in murine cardiac muscle via an increase in SERCA and decrease in its regulator, phospholamban. However, GSK3 also represses the transcription of the heat shock protein 70 (Hsp70), which can bind to SERCA and protect it from oxidative damage. Here, we sought to determine whether lithium treatment could protect murine cardiac SERCA function against heat stress by increasing Hsp70 content. Methods: Young (3-6 month old) male C57BL6 mice were provided water with or without low-dose (10 mg/kg/day) LiCl for 6-12 weeks ad libitum. High frequency ultrasound was done to assess cardiac morphology and contractility. Left ventricles (LV) were collected and SERCA activity assays were performed at baseline, and 30 and 60 min after heat treatment (40˚C incubation). Western blots were performed on non-heated and heated (60 min) samples for SERCA2, PLN, Hsp70 and GSK3 phosphorylation status. Results: Assessment of cardiac function indicated functional eccentric hypertrophy after 12 weeks of LiCl treatment with greater LV internal diameter during systole and diastole (Ctrl vs LiCl: 2.74 vs. 2.99mm, p<0.05; 3.99 vs. 4.32mm, p<0.01, respectively) as well as increases in end-diastolic volume and stroke volume (Ctrl vs LiCl: 70.1 vs. 84.4μL, p<0.05; 41.8 vs 49.1μL, p<0.05, respectively). This was associated with an increase in Ser9 inhibitory phosphorylation of GSK3β in the LiCl-treated mice. With respect to SERCA, after 6 weeks of treatment, LiCl mitigated reductions in absolute maximal SERCA activity at 30 and 60 minutes of heat stress (Ctrl vs LiCl: 46.2% vs 66.4% and 30.6% vs 43%, respectively; p<0.05). However, markers of oxidative stress (protein nitration and nitrosylation) and Hsp70 levels were not different between control and LiCl hearts. Conclusions: Low-dose LiCl supplementation can improve cardiac function potentially through its improvements in SERCA function in both physiological and heat stress conditions. The exact mechanisms leading to the apparent protection against heat stress are still under investigation, with future directions including measurements of total and oxidized glutathione levels. SIH holds a Vanier Canada Graduate Scholarship. VAF 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.

PO-01-209 THE MECHANISMS OF IMPAIRED CALCIUM HANDLING UNDERLYING THE INCREASED SUSCEPTIBILITY TO ATRIAL FIBRILLATION IN METABOLIC SYNDROME

Atrial fibrillation (AF) is becoming increasingly common partly due to the prevalence of metabolic syndrome (MetS). Altered Ca2+-handling proteins on the atrial cellular membrane as well as across the sarcoplasmic reticulum (SR) network and axial tubules due to this condition can precipitate AF through elevation of Ca2+ in junctional clefts, triggering Ca2+ sparks. Through the stochastic recruitment of these sparks, a Ca2+ wave can manifest to activate neighbouring ryanodine receptors (RyRs), stimulating Na+/Ca2+ exchangers (INaCa) into potentially triggering arrhythmogenic spontaneous action potentials (SAPs). However, the mechanisms by which MetS can disrupt Ca2+ handling and trigger SAPs is not well understood. To investigate the mechanisms of impaired calcium handling underlying the increased susceptibility to AF in MetS. We utilized a 3D model of subcellular Ca2+ signalling by Zhang et al. 2022, which compromises of a network of Ca2+-release units (CRUs) and ion channel and exchanger formulations from the Grandi et al. common pool model. The Zhang et al. model far more accurately captures the processes by which Ca2+ sparks can occur and terminate through the inclusion of crucial membrane ion channels/transporters within the extensive network of axial tubules interwoven across the CRUs (Fig A). Based on experimental studies, MetS meditated functional change in Ca2+-handling proteins was replicated by increasing the conductivity of SERCA and RyR and reducing ICaL and INaCa. To evaluate the contribution of each individual remodeled Ca2+-handling component to the membrane potential and Ca2+ sparks, a series of simulations was conducted where we first incorporated each individual ionic remodeling into the control model, then from the MetS model, we reversed each remodeled Ca2+-handling component separately. We have demonstrated that SAPs were attributed to a decrease in INaCa and an increase in SERCA. For the former, this also corresponded to a much greater incidence of Ca2+ sparks compared to other cases. However, for the latter, increasing SERCA surprisingly had a negligible impact on the genesis of these sparks (Fig B). This suggests that an alternate pathway can potentially exist by which upregulated SERCA function can induce SAPs. Increased Ca2+ spark incidence due to functional changes in Ca2+-handling proteins is not the sole mechanism to SAP genesis. Our study therefore highlights that MetS-mediated AF can occur through different pathways.

Insights from an electro-mechanical heart failure cell model: Role of SERCA enhancement on arrhythmogenesis and myocyte contraction

Background and objectiveStructural and electrical remodeling in heart failure predisposes the heart to ventricular arrhythmias. Computer modeling approaches, used to complement experimental results, can provide a more mechanistic knowledge of the biophysical phenomena underlying cardiac pathologies. Indeed, previous in-silico studies have improved the understanding of the electrical correlates of heart failure involved in arrhythmogenesis; however, information on the crosstalk between electrical activity, intracellular Ca2+ and contraction is still incomplete. This study aims to investigate the electro-mechanical behavior of virtual failing human ventricular myocytes to help in the development of therapies, which should ideally target pump failure and arrhythmias at the same time. MethodsWe implemented characteristic remodeling of heart failure with reduced ejection fraction by including reported changes in ionic conductances, sarcomere function and cell structure (e.g. T-tubules disarray). Model parametrization was based on published experimental data and the outcome of simulations was validated against experimentally observed patterns. We focused on two aspects of myocardial dysfunction central in heart failure: altered force-frequency relationship and susceptibility to arrhythmogenic early afterdepolarizations. Because biological variability is a major problem in the generalization of in-silico findings based on a unique set of model parameters, we generated and evaluated a population of models. ResultsThe population-based approach is crucial in robust identification of parameters at the core of abnormalities and in generalizing the outcome of their correction. As compared to non-failing ones, failing myocytes had prolonged repolarization, a higher incidence of early afterdepolarizations, reduced contraction and a shallower force-frequency relationship, all features peculiar of heart failure. Component analysis applied to the model population identified reduced SERCA function as a relevant contributor to most of these derangements, which were largely reverted or diminished by restoration of SERCA function alone. ConclusionsThese simulated results encourage the development of strategies comprising SERCA stimulation and highlight the need to evaluate both electrical and mechanical outcomes.

Intracellular Calcium Localization Mediates the Activity of Venetoclax in Targeting Acute Myeloid Leukemia Stem Cells

Venetoclax (BCL2 inhibitor) based regimens have significantly improved outcomes for elderly patients with acute myeloid leukemia (AML). While most patients initially respond to venetoclax-based regimens, upfront resistance or relapse on therapy occurs in many. Therefore, elucidating the mechanistic link between BCL2 inhibition and AML cell death is crucial to finding novel therapeutic strategies to combat venetoclax resistance and to discover predictors of venetoclax response. Previous studies have shown BCL2 has a non-canonical function in modulating oxidative phosphorylation (OXPHOS) activity of AML stem cells (LSCs). However, the mechanistic link between BCL2 and OXPHOS has not yet been established. BCL2 has been shown to regulate intracellular calcium localization by modulating the activity of calcium channels at the ER and mitochondria. Optimal mitochondrial calcium concentrations are crucial for cell metabolism as calcium overload or decrease can inhibit OXPHOS activity. In preliminary studies, we established venetoclax increases mitochondrial calcium levels (Fig. 1A) leading to a subsequent decrease in OXPHOS activity and changes in the activity of key calcium dependent TCA cycle enzymes in primary human AML LSCs. Genetic inhibition of BCL2 in LSCs phenocopied the effects of venetoclax treatment. Based on these data, we hypothesized that BCL2 exerts its effect on LSC metabolism by modulating calcium localization between the ER and mitochondria. Furthermore, we sought to explore whether dysregulation of calcium localization by venetoclax is a mechanistic component of drug activity that leads to suppression of OXPHOS. To determine how BCL2 may be influencing mitochondrial calcium levels, we assessed the relationship between BCL2 and SERCA3, a key ion channel responsible for calcium uptake into the ER. Venetoclax disrupted the physical interaction between BCL2 and SERCA3. Mechanistically, venetoclax perturbed this interaction by decreasing SERCA3 protein levels. These findings were recapitulated by genetic inhibition of BCL2 in LSCs. Functional analysis of SERCA3 via siRNA-mediated knock-down in primary AML specimens demonstrated increased mitochondrial calcium levels, decreased OXPHOS activity and decreased engraftment of immune deficient NSG-S mice. Based on these data, we propose a model in which venetoclax disrupts SERCA function leading to less calcium uptake at the ER and more calcium sequestered into the mitochondria leading to overload, decreased OXPHOS activity and decreased LSC function. Next, we evaluated how calcium pathways were impacted in venetoclax resistant AML patient specimens. Intriguingly, venetoclax did not affect mitochondrial calcium levels (Fig. 1B) or the relationship between BCL2 and SERCA3. Therefore, we tested whether inhibiting the activity of MCU, the channel responsible for calcium uptake into the mitochondrial matrix, would target venetoclax resistant LSCs as it was overexpressed in this population. MCU inhibition led to decreased mitochondrial calcium levels, decreased OXPHOS activity and decreased LSC engraftment. These data indicate that venetoclax-resistant AML cells are no longer subject to changes in calcium localization induced by drug treatment but do retain an overall reliance on calcium regulation for LSC function/survival. Finally, we assessed whether our findings could be leveraged to predict venetoclax sensitivity. Analysis of single cell RNA-seq data from venetoclax sensitive versus resistant AML patient specimens (n=19) revealed multiple calcium pathway gene sets were enriched in venetoclax resistant LSCs. Additionally, levels of certain genes including SERCA3 and MCU were differentially expressed in primitive versus monocytic clusters between sensitive and resistant specimens. Taken together, our data demonstrate an unexpected and critical link between BCL2 and calcium signaling in human LSCs. A surprising aspect of our work is that venetoclax, a BH3 mimetic, acts to modulate the interaction with SERCA3. Lastly, our findings suggest that targeting intracellular calcium dynamics may represent a novel therapeutic strategy in the context of venetoclax resistant AML. In addition, we show that there are significant differences in calcium gene signatures between venetoclax sensitive vs resistant AML specimens that may be useful in predicting patient response to venetoclax based regimens. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Regulation of the regulator: Endopeptidase cleavage controls the expression of a micropeptide that regulates SERCA.

Micropeptides regulate cellular calcium handling by modulating the function of the calcium transporter SERCA. In a recent Nature Communications paper [4] authors Schiemann et al. describe regulation of an invertebrate SERCA-active micropeptide, sarcolamban, by an endopeptidase called neprilysin 4 (NEP4). NEP4 activity limits sarcolamban expression by cleavage of luminal residues near the micropeptide's c-terminus. This cleavage event liberates sarcolamban from the membrane, reduces its oligomerization, and prevents it from inhibiting SERCA. The study reveals a novel mechanism for "regulation of the regulator" that may be a general feature of micropeptide/SERCA physiology.

GSK3-inhibition improves maximal SERCA activity in a murine model of Arrhythmogenic cardiomyopathy

•Arrhythmogenic cardiomyopathy is characterized by cardiac arrhythmias, myocardial remodelling, and exercise-induced premature death.•Recent work in a homozygous Desmoglein-2 mutant (Dsg2mut/mut) mouse model of ACM has identified pathogenic roles of increased glycogen synthase kinase-3 (GSK3) activity, calcium (Ca2+) overload, and mitochondrial dysfunction, which ultimately contributes to apoptosis and myocardial remodelling.•GSK3 negatively regulates the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump, a key regulator of Ca2+ uptake in the heart.•Here, our results demonstrate that the benefits of GSK3 inhibition, previously observed in Dsg2mut/mut mice, could be partly attributed to improvements in cardiac SERCA function.

Sarco(endo)plasmic reticulum Ca2+-ATPase function is impaired in skeletal and cardiac muscles from young DBA/2J mdx mice

SummaryThe DBA/2J (D2) mdx mouse is a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. Both D2 and C57 mdx mice exhibited signs of impaired Ca2+ uptake in the gastrocnemius, diaphragm, and left ventricle; however, the level of impairment was more severe in D2 mdx mice. Reductions in maximal SERCA activity were also more prominent in the D2 mdx gastrocnemius and diaphragm when compared to those from C57 mdx mice; however, there were no differences detected in the left ventricle. Across all muscles, D2 mdx mice had the highest levels of oxidative stress as indicated by protein nitrosylation and/or nitration. In conclusion, our study shows that SERCA function is more impaired in young D2 mdx mice compared with age-matched C57 mdx mice.

A kink in DWORF helical structure controls the activation of the sarcoplasmic reticulum Ca2+-ATPase

SERCA is a P-type ATPase embedded in the sarcoplasmic reticulum and plays a central role in muscle relaxation. SERCA's function is regulated by single-pass membrane proteins called regulins. Unlike other regulins, dwarf open reading frame (DWORF) expressed in cardiac muscle has a unique activating effect. Here, we determine the structure and topology of DWORF in lipid bilayers using a combination of oriented sample solid-state NMR spectroscopy and replica-averaged orientationally restrained molecular dynamics. We found that DWORF's structural topology consists of a dynamic N-terminal domain, an amphipathic juxtamembrane helix that crosses the lipid groups at an angle of 64°, and a transmembrane C-terminal helix with an angle of 32°. A kink induced by Pro15, unique to DWORF, separates the two helical domains. A single Pro15Ala mutant significantly decreases the kink and eliminates DWORF's activating effect on SERCA. Overall, our findings directly link DWORF's structural topology to its activating effect on SERCA.

Sirtuin 3 overexpression preserves maximal sarco(endo)plasmic reticulum calcium ATPase activity in the skeletal muscle of mice subjected to high fat - high sucrose feeding.

Sarco(endo)plasmic reticulum calcium (Ca2+) ATPase (SERCA) transports Ca2+ in muscle. Impaired SERCA activity may contribute to diabetic myopathy. Sirtuin (SIRT) 3 regulates muscle metabolism and function; however, it is unknown if SIRT3 regulates muscle SERCA activity or acetylation. We determined if SIRT3 overexpression enhances SERCA activity in mouse gastrocnemius muscle and if SIRT3 overexpression preserves gastrocnemius SERCA activity in a model of type 2 diabetes, induced by high fat - high sucrose (HFHS) feeding. We also determined if the acetylation status of SERCA proteins in mouse gastrocnemius is altered by SIRT3 overexpression or HFHS feeding. Wild-type (WT) and SIRT3 transgenic (SIRT3TG) mice, overexpressing SIRT3 in skeletal muscle, were fed a standard or HFHS diet for 4 months. SIRT3TG and WT mice developed obesity and glucose intolerance after 4 months of HFHS feeding. SERCA Vmax was higher in gastrocnemius of SIRT3TG mice compared with WT mice. HFHS-fed mice had lower SERCA1a protein levels and lower SERCA Vmax in their gastrocnemius than control-fed mice. The decrease in SERCA Vmax in gastrocnemius muscle due to HFHS feeding was attenuated by SIRT3 overexpression in HFHS-fed SIRT3TG mice. SERCA1a and SERCA2a acetylation in mouse gastrocnemius was not altered by genotype or diet. These findings suggest SIRT3 overexpression improves SERCA function in mouse skeletal muscle.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

The Role of GSK3β in SERCA Dysfunction and the Development of Arrhythmogenic Cardiomyopathy

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump catalyzes the active transport of Ca2+ into the sarcoplasmic reticulum and is responsible for over 70% of Ca2+ turnover after muscle contraction. Phospholamban (PLN) is a well-known SERCA regulator, and impaired SERCA function due to reductions in SERCA and/or enhanced inhibitory PLN action has been linked to the development of various cardiomyopathies. Arrhythmogenic cardiomyopathy (ACM) is an inherited, non-ischemic heart disease presenting with ventricular arrhythmias, hypo/dyskinesia, and fibrotic remodeling. Over 60% of ACM cases are caused by pathogenic variants in genes encoding the cardiac desmosome, where Desmoglein-2 (DSG2) is the second most prevalent desmosomal gene variant in the ACM patient population. Using Dsg2 mutant (Dsg2mut/mut) mice, previous studies showed that the enzyme glycogen synthase kinase-3β (GSK3β), plays a major role in ACM pathogenesis. Inhibiting GSK3β in these mice improved cardiac contractility and ACM pathological phenotypes. We have shown that GSK3β inhibition in cardiomyocytes can also improve SERCA function in wild-type (WT) mice; however, the role of GSK3β activity on SERCA function in Dsg2mut/mut mice is currently unknown. Here, we sought to examine the effects of GSK3β inhibition on SERCA function using a GSK3β-specific inhibitor, SB216763 (SB2), in Dsg2mut/mut mice. At 3 weeks of age, both WT and Dsg2mut/mut mice were treated with SB2 (2.5 mg/kg/day) or equivalent volume/kg/day of vehicle (DMSO) for 13 weeks via intraperitoneal injection. After treatment, mice were sacrificed and hearts were collected and homogenized. Our results show that cardiomyocytes obtained from vehicle treated Dsg2mut/mut mice had significantly lower SERCA2a expression (-52%, p<0.01) compared to vehicle treated WT mice, leading to reduced SERCA2a:PLN (-65%, p=0.04). Maximal SERCA activity was also significantly lower in vehicle treated Dsg2mut/mut mice (-65%, p=0.001). However, in myocardium from SB2 treated Dsg2mut/mut mice, maximal SERCA activity, SERCA2a expression, and SERCA2a:PLN ratio were all restored to near WT levels. We also examined the effects of heterozygous and homozygous GSK3β S9A mutation in the Dsg2mut/mut mice, which prevents phosphorylation and maintains GSK3β in its constitutively active form. When comparing WT with Dsg2mut/mutheterozygous and homozygous GSK3β S9A mutants, we saw a significant but non-progressive decrease in maximal SERCA activity (-60%, p=0.02 and -58%, p=0.03 respectively). In conclusion, these results show that SERCA function is negatively impacted in Dsg2mut/mut cardiomyocytes. Conversely, GSK3β inhibition with SB2 treatment improved SERCA content and activity to healthy WT levels. These results further illuminate the role of GSK3β in ACM pathogenesis and support the use of GSK3β inhibitors in its treatment, as our new promising results indicate this therapeutic strategy can also improve Ca2+ handling.

Low-dose lithium feeding increases the SERCA2a-to-phospholamban ratio, improving SERCA function in murine left ventricles.

What is the central question of this study? Inhibition of glycogen synthase kinase-3 (GSK3) has been shown to improve cardiac SERCA2a function. Lithium can inhibit GSK3, but therapeutic doses used in treating bipolar disorder can have toxic effects. It has not been determined whether subtherapeutic doses of lithium can improve cardiac SERCA function. What is the main finding and its importance? Using left ventricles from wild-type mice, we found that subtherapeutic lithium feeding for 6weeks decreased GSK3 activity and increased cardiac SERCA function compared with control-fed mice. These findings warrant the investigation of low-dose lithium feeding in preclinical models of cardiomyopathy and heart failure to determine the therapeutic benefit of GSK3 inhibition. The sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) pump is responsible for regulating calcium (Ca2+ ) within myocytes, with SERCA2a being the dominant isoform in cardiomyocytes. Its inhibitor, phospholamban (PLN), acts by decreasing the affinity of SERCA for Ca2+ . Changes in the SERCA2a:PLN ratio can cause Ca2+ dysregulation often seen in patients with dilated cardiomyopathy and heart failure. The enzyme glycogen synthase kinase-3 (GSK3) is known to downregulate SERCA function by decreasing the SERCA2a:PLN ratio. In this study, we sought to determine whether feeding mice low-dose lithium, a natural GSK3 inhibitor, would improve left ventricular SERCA function by altering the SERCA2a:PLN ratio. To this end, male wild-type C57BL/6J mice were fed low-dose lithium via drinking water (10mgkg-1 day-1 LiCl for 6weeks) and left ventricles were harvested. GSK3 activity was significantly reduced in LiCl-fed versus control-fed mice. The apparent affinity of SERCA for Ca2+ was also increased (pCa50 ; control, 6.09±0.03 versus LiCl, 6.26±0.04, P<0.0001) along with a 2.0-fold increase in SERCA2a:PLN ratio in LiCl-fed versus control-fed mice. These findings suggest that low-dose lithium supplementation can improve SERCA function by increasing the SERCA2a:PLN ratio. Future studies in murine preclinical models will determine whether GSK3 inhibition via low-dose lithium could be a potential therapeutic strategy for dilated cardiomyopathy and heart failure.

High-Throughput FRET Screening in Living Cells Based on Lifetime Detection to Identify Small-Molecule Effectors of Serca

The development of an effective high-throughput screening (HTS) paradigm using FRET biosensors in living cells has enabled us to identify multiple classes of small-molecules that alter the activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). We aim to discover small-molecule activators that improve SERCA's function for the treatment of heart failure. We have validated an intramolecular SERCA FRET biosensor (red and green fluorescent proteins fused to distinct SERCA domains) by screening a small test library using novel plate readers (PRs) that detect the fluorescence lifetime (FLT) (Schaaf et al, 2017). Here we describe an FLT-PR that scans a 1536-well plate in ≤2.5 min. This instrument is equipped with multichannel FLT detection at two emission wavelengths, used to filter out fluorescent compounds that would be falsely identified as hits. False hits were further eliminated using FRET biosensors based on non-specific peptide linkers, and using spectral analysis. FRET biosensors were developed and tested based on two different isoforms of SERCA: the SERC2a cardiac-specific isoform and SERCA2b non-muscle isoform were used to discover compounds with specificity toward SERCA2a. Concentration-response studies were performed to determine potency of compounds. Hits were assessed by two separate functional assays to determine how they alter ATPase activity and calcium-uptake. The majority were found to be inhibitors, but some were activators, and some compounds affected the coupling of SERCA's ATPase and calcium transport activities Our future focus is to evaluate the structure-function relationships of the newly discovered SERCA activators, using medicinal chemistry to optimize drug-like properties. These new HTS methods are being applied to a wide range of drug targets.

Myocardial Redox Hormesis Protects the Heart of Female Mice in Sepsis.

Mice challenged with lipopolysaccharide develop cardiomyopathy in a sex and redox-dependent fashion. Here we extended these studies to the cecal ligation and puncture (CLP) model.We compared male and female FVB mice (wild type, WT) and transgenic littermates overexpressing myocardial catalase (CAT). CLP induced 100% mortality within 4 days, with similar mortality rates in male and female WT and CAT mice. 24 h after CLP, isolated (Langendorff) perfused hearts showed depressed contractility in WT male mice, but not in male CAT or female WT and CAT mice. In WT male mice, CLP induced a depression of cardiomyocyte sarcomere shortening (ΔSS) and calcium transients (ΔCai), and the inhibition of the sarcoplasmic reticulum Ca ATPase (SERCA). These deficits were associated with overexpression of NADPH-dependent oxidase (NOX)-1, NOX-2, and cyclooxygenase 2 (COX-2), and were partially prevented in male CAT mice. Female WT mice showed unchanged ΔSS, ΔCai, and SERCA function after CLP. At baseline, female WT mice showed partially depressed ΔSS, ΔCai, and SERCA function, as compared with male WT mice, which were associated with NOX-1 overexpression and were prevented in CAT female mice.In conclusion, in male WT mice, septic shock induces myocardial NOX-1, NOX-2, and COX-2, and redox-dependent dysregulation of myocardial Ca transporters. Female WT mice are resistant to CLP-induced cardiomyopathy, despite increased NOX-1 and COX-2 expression, suggesting increased antioxidant capacity. Female resistance occurred in association with NOX-1 overexpression and signs of increased oxidative signaling at baseline, indicating the presence of a protective myocardial redox hormesis mechanism.

Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples.

In skeletal muscle, the Na/K ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle. Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize "background noise." We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 μM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized "background noise" (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively. The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

A calcium transport mechanism for atrial fibrillation in Tbx5-mutant mice.

Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific Tbx5-mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5-deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca2+ sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca2+-dependent pathway for AF risk.