SERCA2a Activity Research Articles

A multiscale approach for bridging the gap between potency, efficacy, and safety of small molecules directed at membrane proteins

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting “undruggable” regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand–membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

Muscle-specific sirtuin 3 overexpression does not attenuate the pathological effects of high-fat/high-sucrose feeding but does enhance cardiac SERCA2a activity.

Obesity, type 2 diabetes, and heart disease are linked to an unhealthy diet. Sarco(endo)plasmic reticulum calcium (Ca2+) ATPase 2a (SERCA2a) controls cardiac function by transporting Ca2+ in cardiomyocytes. SERCA2a is altered by diet and acetylation, independently; however, it is unknown if diet alters cardiac SERCA2a acetylation. Sirtuin (SIRT) 3 is an enzyme that might preserve health under conditions of macronutrient excess by modulating metabolism via regulating deacetylation of target proteins. Our objectives were to determine if muscle‐specific SIRT3 overexpression attenuates the pathological effects of high fat‐high sucrose (HFHS) feeding and if HFHS feeding alters cardiac SERCA2a acetylation. We also determined if SIRT3 alters cardiac SERCA2a acetylation and regulates cardiac SERCA2a activity. C57BL/6J wild‐type (WT) mice and MCK‐mSIRT3‐M1‐Flag transgenic (SIRT3TG) mice, overexpressing SIRT3 in cardiac and skeletal muscle, were fed a standard‐diet or a HFHS‐diet for 4 months. SIRT3TG and WT mice developed obesity, glucose intolerance, cardiac dysfunction, and pathological cardiac remodeling after 4 months of HFHS feeding, indicating muscle‐specific SIRT3 overexpression does not attenuate the pathological effects of HFHS‐feeding. Overall cardiac lysine acetylation was increased by 63% in HFHS‐fed mice (p = 0.022), though HFHS feeding did not alter cardiac SERCA2a acetylation. Cardiac SERCA2a acetylation was not altered by SIRT3 overexpression, whereas SERCA2a V max was 21% higher in SIRT3TG (p = 0.039) than WT mice. This suggests that SIRT3 overexpression enhanced cardiac SERCA2a activity without direct SERCA2a deacetylation. Muscle‐specific SIRT3 overexpression may not prevent the complications associated with an unhealthy diet in mice, but it appears to enhance SERCA2a activity in the mouse heart.

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): This work was supported by CVie Therapeutics Limited (Taipei, Taiwan) and Windtree Therapeutics (Warrington, USA) Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the double property to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). The project aims to characterize istaroxime effects at a concentration (100 nM) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in a model of mild diabetes. Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, 7) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nM) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Abstract Figure.

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy.

Aims Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes.Methods and results Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed (i) marked DD not associated to cardiac fibrosis, (ii) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, (iii) reduced LV SERCA2 protein level and activity and (iv) slower SR Ca2+ uptake rate, (v) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, (vi) increased diastolic Ca2+, and (vii) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent.Conclusions SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment.

Bay 60-7550, a PDE2 inhibitor, exerts positive inotropic effect of rat heart by increasing PKA-mediated phosphorylation of phospholamban

This study investigated the hemodynamic effect of Bay 60-7550, a phosphodiesterase type 2 (PDE2) inhibitor, in healthy rat hearts both in vivo and ex vivo and its underlying mechanisms. In vivo rat left ventricular pressure-volume loop, Langendorff isolated rat heart, Ca2+ transient of left ventricular myocyte and Western blot experiments were used in this study. The results demonstrated that Bay 60-7550 (1.5 mg/kg, i. p.) increased the in vivo rat heart contractility by enhancing stroke work, cardiac output, stroke volume, end-diastolic volume, heart rate, and ejection fraction. The simultaneous aortic pressure recording indicated that the systolic blood pressure was increased and diastolic blood pressure was decreased by Bay 60-7550. Also, the arterial elastance which is proportional to the peripheral vessel resistance was significantly decreased. Bay 60-7550 (0.001, 0.01, 0.1, 1 μmol/l) also enhanced the left ventricular development pressure in non-paced and paced modes with a decrease of heart rate in non-paced model. Bay 60-7550 (1 μmol/l) increased SERCA2a activity and SR Ca2+ content and reduced SR Ca2+ leak rate. Furthermore, Bay 60-7550 (0.1 μmol/l) increased the phosphorylation of phospholamban at 16-serine without significantly changing the phosphorylation levels of phospholamban at 17-threonine and RyR2. Bay 60-7550 increased the rat heart contractility and reduced peripheral arterial resistance may be mediated by increasing the phosphorylation of phospholamban and dilating peripheral vessels. PDE2 inhibitors which result in a positive inotropic effect and a decrease in peripheral resistance might serve as a target for developing agents for the treatment of heart failure in clinical settings.

Oxidative stress in early metabolic syndrome impairs cardiac RyR2 and SERCA2a activity and modifies the interplay of these proteins during Ca2+ waves

We investigated how oxidative stress (OS) alters Ca2+ handling in ventricular myocytes in early metabolic syndrome (MetS) in sucrose-fed rats. The effects of N-acetyl cysteine (NAC) or dl-Dithiothreitol (DTT) on systolic Ca2+ transients (SCaTs), diastolic Ca2+ sparks (CaS) and Ca2+ waves (CaW), recorded by confocal techniques, and L-type Ca2+ current (ICa), assessed by whole-cell patch clamp, were evaluated in MetS and Control cells. MetS myocytes exhibited decreased SCaTs and CaS frequency but unaffected CaW propagation. In Control cells, NAC/DTT reduced RyR2/SERCA2a activity blunting SCaTs, CaS frequency and CaW propagation, suggesting that basal ROS optimised Ca2+ signalling by maintaining RyR2/SERCA2a function and that these proteins facilitate CaW propagation. Conversely, NAC/DTT in MetS recovered RyR2/SERCA2a function, improving SCaTs and CaS frequency, but unexpectedly decreasing CaW propagation. We hypothesised that OS decreases RyR2/SERCA2a activity at early MetS, and while decreased SERCA2a favours CaW propagation, diminished RyR2 restrains it.

Ophiopogonin D Increases SERCA2a Interaction with Phospholamban by Promoting CYP2J3 Upregulation.

Ophiopogonin D (OPD), a compound from the Chinese herb Radix Ophiopogonis, reportedly induces increased levels of cytochrome P450 2J3 (CYP2J3)/epoxyeicosatrienoic acids (EETs) and Ca2+ in rat cardiomyocytes. Little is known regarding the specific mechanism between CYP2J3 and Ca2+ homeostasis. Here, we investigated whether CYP2J3 is involved in the protective action of OPD on the myocardium by activating the Ca2+ homeostasis-related protein complex (SERCA2a and PLB) in H9c2 rat cardiomyoblast cells. The interaction between SERCA2a and PLB was measured using fluorescence resonance energy transfer. OPD attenuated heart failure and catalyzed the active transport of Ca2+ into the sarcoplasmic reticulum by inducing the phosphorylation of PLB and promoting the SERCA2a activity. These beneficial effects of OPD on heart failure were abolished after knockdown of CYP2J3 in a model of heart failure. Together, our results identify CYP2J3 as a critical intracellular target for OPD and unravel a mechanism of CYP2J3-dependent regulation of intracellular Ca2+.

Phospholamban and sarcolipin prevent thermal inactivation of sarco(endo)plasmic reticulum Ca2+-ATPases

Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25-35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.

Atrial fibrillation impairs ventricular function by altering excitation-contraction coupling in the human heart

Abstract Atrial fibrillation (AF) often co-exists in patients with heart failure (HF). Recent clinical evidence suggests that the arrhythmic component of AF alone may contribute to ventricular dysfunction. However, the pathophysiological effects of a non-tachycardic AF on the human ventricle are unknown. To investigate the effects of normofrequent AF on the human ventricle we investigated ventricular myocardium from patients with preserved ejection fraction with sinus rhythm (SR) or AF in the absence of HF (compensated hypertrophy, EF>50%, matched clinical characteristics). In histological analysis we detected no difference between SR (n=9) vs. AF (n=6) regarding the amount and distribution of fibrosis. For functional investigation, Ca-handling was studied (Fura-2 AM). While systolic Ca-transient amplitude was in trend reduced in isolated human ventricular AF cardiomyocytes, we found a significantly prolonged Ca-elimination time (n=17–22 cells/4 pat.). Using caffeine application, a decreased SR Ca-load in AF was detected, which may be explained by a significant decrease in SERCA2a activity (ksys-kCaff, n=10–12/4 pat.). Patch-clamp experiments revealed a prolonged action potential duration in AF cardiomyocytes (n=5/15 cells). For the standardized evaluation of the mechanisms of persistent normofrequent arrhythmia, we simulated AF in vitro by using arrhythmic (1 Hz, 40% R-R-variability) or rhythmic (1 Hz) field stimulation. We performed contractility experiments using in-toto isolated human ventricular trabeculae from explanted human hearts. After 8h of pacing, arrhythmically stimulated human trabeculae showed a significantly reduced systolic force, an increase in diastolic tension and a prolonged relaxation (n=11–12 trabeculae/11 pat.). For studying the cellular effects of persistent normofrequent arrhythmia in a model suitable for chronic pacing (up to 7 days), we utilized human iPSC cardiomyocytes (iPSC-CM) from healthy donors (n=6). After 7 days, arrhythmic paced iPSC-CM showed a significantly reduced systolic Ca-transient amplitude, a prolonged Ca-elimination time (n=35/45 cells) as well as a reduced SR Ca-load and a trend towards a lower SERCA2a activity compared to control (n=11 cells). Confocal line-scans (Fluo-4 AM) showed an increased diastolic SR Ca-release, which might also explain the reduced SR Ca-content (n=45/35 cells). Moreover, in irregularly paced iPSC-CM we found significant increased levels of cytosolic Na (n=69 cells each) and in patch-clamp experiments a significantly prolonged action potential duration (n=14/11 cells/3 diff.). This study demonstrates that a normofrequent arrhythmic ventricular excitation as it occurs in AF impairs human ventricular myocardial function by altering cardiomyocyte excitation-contraction coupling. Thus, this study provides the first translational mechanistic characterization and the potential negative impact of isolated AF in the absence of tachycardia on the human ventricle. Funding Acknowledgement Type of funding source: None

Novel in vivo and in vitro mechanisms of positive inotropic effect of atractylodin.

This study was to investigate the inotropic effect of atractylodin and its underlying mechanism. The cardiac pressure-volume loop (P-V loop), Langendroff-perfused isolated rat heart, patch-clamp, Ca2+ transient and western blot techniques were used. The results demonstrated that atractylodin (3mg/kg, ip) remarkably increased the left ventricular stroke work, cardiac output, stroke volume, heart rate, ejection fraction, end-systolic pressure, peak rates of rise and fall of left ventricular pressures (+dP/dtmax , -dP/dtmax ), the slopes of end-systolic pressure-volume relationship (also named as end-systolic elastance, Ees) and reducing end-systolic volume and end-diastolic volume in the in vivo rat study. Also, atractylodin (3mg/kg, ip) significantly decreased diastolic blood pressure and the arterial elastance (Ea) without significant systolic blood pressure change. In addition, atractylodin (0.1, 1, 10µmol/L) also increased the isolated rat heart left ventricular developed pressure which is the difference between the systolic and diastolic pressure in non-pacing and pacing modes. Furthermore, JMV-2959 (1μmol/L), a ghrelin receptor unbiased antagonist, blocked the increased left ventricular developed pressure of atractylodin in isolated rat hearts. Finally, atractylodin (5µmol/L) increased the amplitude of Ca2+ transient by enhancing SERCA2a activity, the sarcoplasmic reticulum Ca2+ content and the phosphorylation of phospholamban at 16-serine. These results demonstrated that atractylodin had a positive inotropic effect by enhancing SERCA2a activity which might be mediated by acting ghrelin receptor in myocardium. In conclusion, atractylodin which had the positive inotropic effect and decreased diastolic blood pressure might serve as an agent for the treatment of heart failure in clinical settings.

Ryanodine Receptor Type 2: A Molecular Target for Dichlorodiphenyltrichloroethane- and Dichlorodiphenyldichloroethylene-Mediated Cardiotoxicity.

DDT/DDE interactions with RyR2 alter cardiomyocyte Ca2+ dynamics that may contribute to adverse cardiovascular outcomes associated with exposures.

Roles of sarcoplasmic reticulum Ca2+ ATPase pump in the impairments of lymphatic contractile activity in a metabolic syndrome rat model

The intrinsic lymphatic contractile activity is necessary for proper lymph transport. Mesenteric lymphatic vessels from high-fructose diet-induced metabolic syndrome (MetSyn) rats exhibited impairments in its intrinsic phasic contractile activity; however, the molecular mechanisms responsible for the weaker lymphatic pumping activity in MetSyn conditions are unknown. Several metabolic disease models have shown that dysregulation of sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump is one of the key determinants of the phenotypes seen in various muscle tissues. Hence, we hypothesized that a decrease in SERCA pump expression and/or activity in lymphatic muscle influences the diminished lymphatic vessel contractions in MetSyn animals. Results demonstrated that SERCA inhibitor, thapsigargin, significantly reduced lymphatic phasic contractile frequency and amplitude in control vessels, whereas, the reduced MetSyn lymphatic contractile activity was not further diminished by thapsigargin. While SERCA2a expression was significantly decreased in MetSyn lymphatic vessels, myosin light chain 20, MLC20 phosphorylation was increased in these vessels. Additionally, insulin resistant lymphatic muscle cells exhibited elevated intracellular calcium and decreased SERCA2a expression and activity. The SERCA activator, CDN 1163 partially restored lymphatic contractile activity in MetSyn lymphatic vessel by increasing phasic contractile frequency. Thus, our data provide the first evidence that SERCA2a modulates the lymphatic pumping activity by regulating phasic contractile amplitude and frequency, but not the lymphatic tone. Diminished lymphatic contractile activity in the vessels from the MetSyn animal is associated with the decreased SERCA2a expression and impaired SERCA2 activity in lymphatic muscle.

Diabetes mellitus exacerbates post-myocardial infarction heart failure by reducing sarcolipin promoter methylation.

AimsSarcolipin (SLN) is a key regulator of sarcoplasmic reticulum calcium‐ATPase (SERCA)2a, which handles intracellular calcium re‐uptake. This study was aimed to investigate the involvement of SLN in post‐myocardial infarction (MI) heart failure (HF) in diabetes.Methods and resultsDiabetes/MI rat models were established. Altered SLN expression in diabetic hearts was screened out by microarray. A myocardiotropic viral vector was used to deliver siRNA to silence SLN. DNA methylation was evaluated by bisulfite sequencing. Cardiac functions were evaluated by invasive haemodynamic examinations. The SERCA2a activity, cytoplasmic calcium concentration ([Ca2+]i), calcium spark, and myocyte contraction were detected. Correlation between HF and diabetes was analysed in a cohort consisted of 101 ST‐segment elevated myocardial infarction (STEMI) patients between 2017 and 2019 [53.54 ± 4.64 years old; 61.4% male gender; HbA1c% 6.15 ± 2.00; and left ventricular ejection fraction (LVEF%) 40.64 ± 3.20%]. SLN expression was evaluated in left ventricular tissue sample from six STEMI patients complicated with diabetes and six STEMI patients without diabetes. Expressions of DNA methyltransferase 1a and DNA methyltransferase 3 were reduced in diabetic hearts, leading to down‐regulation of SLN promoter methylation, resulting in increased SLN expression in rats. Impaired heart systolic functions were found in experimental diabetic MI rats, which were attenuated by SLN silencing. SERCA2a activity reduction and [Ca2+]i elevation were attenuated by SLN silencing in diabetic animal hearts and high‐glucose incubated primary myocytes. SLN silencing suppressed calcium sparks and improved contraction and sarcoplasmic reticulum calcium re‐uptake in high‐glucose incubated primary myocytes. Expression of SLN was up‐regulated in LV sampled from STEMI patients complicated with diabetes compared with non‐diabetic ones (P < 0.05). LVEF% was reduced in STEMI patients complicated with diabetes compared with non‐diabetic ones (P < 0.01). HbA1c% and LVEF% was related (r = −0.218, P = 0.028). Increased HbA1c% was correlated with reduced LVEF% after adjustment for age, sex, body mass index, cigarette smoking, creatinine, UA, low density lipoprotein, K+, Na+, and troponin I (adjusted odds ration = 0.75, 95% confidence interval 0.62–0.90, P = 0.002).ConclusionsDiabetes increases the vulnerability of STEMI patients to post‐MI HF by down‐regulating SLN promoter methylation, which further regulates SERCA2a activity via increasing cardiac SLN expression.

Live-Cell Cardiac-Specific High-Throughput Screening Platform for Drug-Like Molecules that Enhance Ca2+ Transport.

We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development.

Astragaloside IV attenuates chronic intermittent hypoxia-induced myocardial injury by modulating Ca2+ homeostasis.

Obstructive sleep apnea syndrome (OSAS) is an important consequence of chronic intermittent hypoxia (CIH). Astragaloside IV (AS-IV) exerts multiple protective effects in diverse diseases. However, whether AS-IV can attenuate CIH-induced myocardial injury is unclear. In this study, rats exposed to CIH were established and treated with AS-IV for 4 weeks. In vitro, H9C2 cardiomyocytes subjected to CIH exposure were treated with AS-IV for 48 hours. Then the cardiac function, morphology, fibrosis, apoptosis and Ca2+ homeostasis were determined to assess cardiac damage. Results showed that AS-IV attenuated cardiac dysfunction and histological lesions in CIH rats. The increased TUNEL-positive cells and activated apoptotic proteins in CIH rats were reduced by AS-IV. We also noticed that AS-IV reversed the accumulation of Ca2+ and altered expressions of Ca2+ handling proteins (decreases of SERCA2a and RYR2, and increases of p-CaMKII and NCX1) under CIH exposure. Furthermore, CIH-induced reduction of SERCA2a activity was increased by AS-IV in rats. Similar results were also observed in H9C2 cells. Altogether, these findings indicate that AS-IV modulates Ca2+ homeostasis to inhibit apoptosis, protecting against CIH-induced myocardial injury eventually, suggesting it may be a potential agent for cardiac damage of OSAS patients. SIGNIFICANCE OF THE STUDY: Chronic intermittent hypoxia (CIH) is a great contributor of OSAS, which is closely associated with cardiovascular diseases. It is necessary for developing a promising drug to attenuate CIH-induced myocardial injury. This work suggests that AS-IV can attenuate myocardial apoptosis and calcium disruption, thus protecting against CIH-induced myocardial injury. It may represent a novel therapeutic for cardiac damage of OSAS.

Structure and Topology of the SERCA Regulator DWORF in Lipid Bilayers by Oriented Sample Solid-State NMR

Calcium (Ca2+) dysregulation is a hallmark of heart failure and is characterized by impaired Ca2+ sequestration into the sarcoplasmic reticulum (SR) by the SR-Ca2+-ATPase (SERCA). Regulins, single-pass membrane proteins, bind and inhibit SERCA by allosterically modulating the affinity of its Ca2+ binding sites. DWarf Open Reading Frame (DWORF) is a 35 amino acid regulin that enhances SERCA2a activity in cardiomyocytes. As a first step to understanding DWORF regulation of SERCA, we used Oriented-Sample Solid-State NMR spectroscopy (OS-ssNMR) and computational methods to determine the structure and topology of DWORF in liquid crystalline lipid bilayers. We found that DWORF adopts a helical structure, with a pronounced kink at the N-terminus. Restrained molecular dynamics calculations using backbone dipolar couplings and chemical shift anisotropy show the dynamic topology of DWORF, with the N-terminal helix spanning tilt angles ranging from 50-60 degrees and C-terminal helix ranging from 30-40 degrees. The two helical domains are anchored to both membrane leaflets by polar residues. This topology differs from that of phospholamban, a main regulator of SERCA, and might explain the opposite effects on the ATPase's apparent affinity for Ca2+ ions of these two regulins.

Intracellular calcium mishandling leads to cardiac dysfunction and ventricular arrhythmias in a mouse model of propionic acidemia

Propionic acidemia (PA) is a rare metabolic disease associated with mutations in genes encoding the α and β subunits of the enzyme propionyl-CoA carboxylase. The accumulation of toxic metabolites results in mitochondrial dysfunction, increased reactive oxygen species production and oxidative damage, which have been associated with the disease pathophysiology. Clinical symptoms are heterogeneous and include cardiac complications, mainly cardiac dysfunction and arrhythmias, which are recognized as one of the major life-threatening manifestations in patients. We aimed to investigate the molecular mechanisms underlying the cardiac phenotype using a hypomorphic mouse model (Pcca−/−(A138T)) that recapitulates some biochemical and clinical characteristics of PA. We demonstrate that Pcca−/−(A138T) mice present with depressed cardiac function along with impaired cell contractility when compared to the wild-type mice. Cardiac dysfunction in Pcca−/−(A138T) mice was associated with lower systolic Ca2+ release ([Ca2+]i transients), impairment in the sarcoplasmic reticulum (SR) Ca2+ load and decreased Ca2+ re-uptake by SR-Ca2+ ATPase (SERCA2a). These functional changes correlated well with the depressed activity of SERCA2a, the elevated ROS levels and SERCA2a oxidation rate in cardiomyocytes isolated from Pcca−/−(A138T) mice. In addition, decreased SR-Ca2+ load in Pcca−/−(A138T) cardiomyocytes was associated with increased diastolic Ca2+ release. The increase in Ca2+ sparks, Ca2+ waves and spontaneous [Ca2+]i transients in Pcca−/−(A138T) cardiomyocytes could be responsible for the induction of ventricular arrhythmias detected in these mice. Overall, our results uncover the role of impaired Ca2+ handling in arrhythmias and cardiac dysfunction in PA, and identify new targets for the development of therapeutic approaches for this devastating metabolic disease.

Cisd2 is essential to delaying cardiac aging and to maintaining heart functions.

CDGSH iron-sulfur domain-containing protein 2 (Cisd2) is pivotal to mitochondrial integrity and intracellular Ca2+ homeostasis. In the heart of Cisd2 knockout mice, Cisd2 deficiency causes intercalated disc defects and leads to degeneration of the mitochondria and sarcomeres, thereby impairing its electromechanical functioning. Furthermore, Cisd2 deficiency disrupts Ca2+ homeostasis via dysregulation of sarco/endoplasmic reticulum Ca2+-ATPase (Serca2a) activity, resulting in an increased level of basal cytosolic Ca2+ and mitochondrial Ca2+ overload in cardiomyocytes. Most strikingly, in Cisd2 transgenic mice, a persistently high level of Cisd2 is sufficient to delay cardiac aging and attenuate age-related structural defects and functional decline. In addition, it results in a younger cardiac transcriptome pattern during old age. Our findings indicate that Cisd2 plays an essential role in cardiac aging and in the heart’s electromechanical functioning. They highlight Cisd2 as a novel drug target when developing therapies to delay cardiac aging and ameliorate age-related cardiac dysfunction.

4967Effects of atrial fibrillation on the human ventricle

Abstract Background The consequence of normofrequent atrial fibrillation (AF) on the ventricle remains largely unknown. Methods and results To elucidate the effects of arrhythmic excitation on human ventricular myocardium we performed contractility experiments using ventricular trabecula from patients with heart failure (HF). Normofrequent AF was simulated using arrhythmic (60 bpm, 40% R-R interval variability) or rhythmic field stimulation (60 bpm). Within 8h of arrhythmic stimulation, human specimen showed an impaired systolic force, while diastolic tension increased pathologically (n=5–7 each/7 HF patients, Fig. 1). The characterization of the ventricular (in-vivo) phenotype in patients with AF was performed by utilizing ventricular myocardium from patients with sinus rhythm (SR) and from patients with AF in the absence of HF (compensated hypertrophy, EF&gt;50%, matched clinical characteristics, LV myocardium obtained from aortic valve replacement surgery). Histological investigation showed increased levels of interstitial fibrosis in myocardium from patients with AF compared to SR (n=10 patients each). Studies of cellular Ca-homeostasis (epifluorescence microscopy, Fura-2) were performed using isolated human ventricular cardiomyocytes. While systolic Ca-transient amplitude (0.5 Hz) was preserved in ventricular cardiomyocytes from patients with AF, we found a significantly prolonged Ca-elimination time (RT80) by 22.0±7.7% and a trend towards increased diastolic Ca-levels (n=17–23 cells/4 patients each). This finding may be explained by a decrease in SERCA2a activity (ksys-kCaff, n=10–12/4 each) and an enhanced phospholamban expression in Western Blot experiments (n=5 patients each). For the standardized investigation of the involved targets/mechanisms mediating the pathological changes upon arrhythmic excitation, we utilized human induced pluripotent stem cell cardiomyocytes (iPSC-CM) from healthy donors for chronic arrhythmic culture stimulation (24h). Arrhythmic paced iPSC-CM showed no changes in systolic Ca-transient amplitude (0.5 Hz), whereas diastolic Ca-levels were increased, which fits nicely to the finding of disturbed trabeculae diastolic function (n=15 cells each). In patch clamp experiments, arrhythmic paced cells showed no alterations of resting membrane potential, upstroke velocity, action-potential amplitude or -duration (n=7–9 cells each). Protein expression levels of key Ca-handling proteins in iPSC-CM as well as regulated genes are already under investigation. Conclusion This study demonstrates that arrhythmic ventricular excitation deteriorates human myocardial contractility early in HF. In biopsies from patients with preserved EF, chronic AF was associated with increased levels of interstitial fibrosis and pathological diastolic Ca-handling, which could be causally confirmed in chronically arrhythmic paced iPSC-CM. Therefore, this study provides first mechanistic characterization of AF mediated effects on the human ventricle.

P6348Phospholamban antisense oligonucleotides drive the reversal of cardiac dysfunction and multiple heart failure parameters during murine dilated cardiomyopathy

Abstract Background Mice lacking muscle LIM protein (Mlp/Cspr3 −/−) develop dilated cardiomyopathy (DCM). Previous work established this model to be amenable to improvements in cardiac function by genetic ablation of phospholamban (PLN). Purpose To test the hypothesis that therapeutic reductions of PLN would similarly improve cardiac function, Mlp KO mice were administered an antisense oligonucleotide (ASO) targeting PLN. Methods Echocardiography measurements of ejection fraction (EF), end-diastolic volume (EDV) and end-systolic volume (ESV) were performed before and after treatment. In addition, global transcriptome profiling using 3'RNA-seq was performed to identify gene expression changes in diseased Mlp KO mice and following PLN ASO treatments. Mlp KO mice with ejection fraction (EF%) of less than 45% (median, 37.6%; interquartile range, 32.2–42.0%) were treated with vehicle (n=10) or PLN ASO (n=9) for 4 weeks. Results Three subcutaneous injections of PLN ASO were administered to Mlp KO mice resulting in 50–70% PLN reductions. Echocardiography performed at study end revealed improvements of EF (60±8 vs. 46±12%), ESV (31±11 vs. 56±21μl) and EDV (79±22 vs. 100±25μl) with PLN ASO treatment. Corrected for baseline values, PLN ASO treatment improved all echocardiographic measurements (p&lt;0.001). Transcriptional analyses revealed that PLN ASO treatment reduced expression of heart failure related markers, such as Myh7 (−70%), Nppa (−72%), Nppb (−71%), Acta1 (−84%) and Ankrd1 (−40%), p&lt;0.05 vs. vehicle. In addition, genes not previously known to be dysregulated in this model, Edn3 and Xirp2, were identified and shown to be reduced following PLN ASO treatment by 71% and 67%, respectively (p&lt;0.001). Bioinformatic analysis suggested improvement of known and novel heart failure associated pathways by PLN inhibition in this model. In conclusion, antisense inhibition of PLN reduced functional and transcriptional indices of heart failure in a DCM model. In view of the failed CUPID trials, a gene therapy approach to improve SERCA2a activity, targeting PLN with ASO may be advantageous due to a likely more robust pharmacological profile.