Spark Frequency Research Articles

Long-term hypoxia modulates depolarization activation of BKCa currents in fetal sheep middle cerebral arterial myocytes

IntroductionPrevious evidence indicates that gestational hypoxia disrupts cerebrovascular development, increasing the risk of intracranial hemorrhage and stroke in the newborn. Due to the role of cytosolic Ca2+ in regulating vascular smooth muscle (VSM) tone and fetal cerebrovascular blood flow, understanding Ca2+ signals can offer insight into the pathophysiological disruptions taking place in hypoxia-related perinatal cerebrovascular disease. This study aimed to determine the extent to which gestational hypoxia disrupts local Ca2+ sparks and whole-cell Ca2+ signals and coupling with BKCa channel activity.MethodsConfocal imaging of cytosolic Ca2+ and recording BKCa currents of fetal sheep middle cerebral arterial (MCA) myocytes was performed. MCAs were isolated from term fetal sheep (∼140 days of gestation) from ewes held at low- (700 m) and high-altitude (3,801 m) hypoxia (LTH) for 100+ days of gestation. Arteries were depolarized with 30 mM KCl (30K), in the presence or absence of 10 μM ryanodine (Ry), to block RyR mediated Ca2+ release.ResultsMembrane depolarization increased Ry-sensitive Ca2+ spark frequency in normoxic and LTH groups along with BKCa activity. LTH reduced Ca2+ spark and whole-cell Ca2+ activity and induced a large leftward shift in the voltage-dependence of BKCa current activation. The influence of LTH on the spatial and temporal aspects of Ca2+ sparks and whole-cell Ca2+ responses varied.DiscussionOverall, LTH attenuates Ca2+ signaling while increasing the coupling of Ca2+ sparks to BKCa activity; a process that potentially helps maintain oxygen delivery to the developing brain.

The CNP analogue vosoritide reduces arrhythmia in diabetic cardiomyopathy via cGMP-dependent PDE2 stimulation on cellular and organ level

Abstract Background Detrimental arrhythmia occurs frequently in patients with diabetic cardiomyopathy (DiabCM), but safe and effective therapies are limited. Diabetes-associated neuropathological alterations increase the sympathetic tone, leading to hyperactivation of β-adrenoceptor-mediated cAMP signalling in cardiomyocytes (CM). Increased activity of deleterious cAMP-dependent kinases causes pro-arrhythmic dysregulation of intracellular Ca2+ homeostasis. Phosphodiesterases (PDE) can reduce cAMP levels. Exceptionally, the cAMP-hydrolysing activity of PDE2 can be cGMP-dependently increased by C-type natriuretic peptide (CNP). Thus, the CNP analogue vosoritide (approved for the treatment of achondroplasia) might mediate this cGMP/cAMP crosstalk, reducing arrhythmia. Purpose Here, we aim to study whether the cGMP-dependent activation of PDE2 by vosoritide (VO) is sufficient to reduce arrhythmia and arrhythmogenic triggers in DiabCM. Methods Diabetes was induced by 5 consecutive injections of streptozotocin (50 mg/kg) in control (WT) mice and mice with a CM-specific PDE2 knockout (KO). All experiments were performed 5 weeks later. Cardiac function was characterised by echocardiography. Primary ventricular CM were isolated to assess protein expression and phosphorylation levels by Western blot (WB) and cAMP levels by ELISA. Patch-clamp and Ca2+ imaging experiments were used to investigate pro-arrhythmic triggers. Arrhythmic events were quantified in ex vivo perfused hearts after ischaemia-reperfusion (I/R) injury. Results Compared to healthy controls, diabetic animals showed impaired systolic function with a ~9 % reduction in ejection fraction. The significant increase in E/E’ ratio by ~34 % indicated a diastolic dysfunction. Interestingly, the cardiac function was further worsened in diabetic PDE2 KO. WB analysis revealed upregulation of the CNP receptor NPR-B and PDE2 in diabetic CM and altered regulation of proteins involved in cAMP-dependent Ca2+ cycling such as protein kinase A, Ca2+/calmodulin-dependent kinase II and phospholamban. Interestingly, VO significantly reduced isoprenaline (Iso)-induced cAMP levels in diabetic CM. The effect of VO was prevented by specific PDE2 inhibition with BAY 60-7550 (BAY). Furthermore, VO was able to reduce the Iso-induced increase in L-type Ca2+ current, whereas BAY or genetic PDE2 deletion counteracted this effect. Accordingly, VO significantly decreased the Iso-triggered Ca2+ spark frequency by ~40 %. Concomitant PDE2 inhibition or PDE2 KO prevented this anti-arrhythmic effect. Finally, ECG analysis revealed that VO clearly reduced arrhythmia development after I/R in ex vivo perfused diabetic hearts via PDE2 activation. Conclusion Our data indicate that vosoritide-induced PDE2 stimulation may attenuate abnormal cardiac Ca2+ cycling and thereby reduce arrhythmia in diabetic hearts. Thus, vosoritide may be repurposed as a novel anti-arrhythmic drug in DiabCM.

Indoxyl sulfate deteriorates cardiac electrophysiological function and increases the likelihood of arrhythmias in human ventricular myocardial cells

Abstract Background Approximately 10% of the world population is suffering from chronic kidney disease (CKD). In these patients, an increased mortality and morbidity, predominantly derived from cardiovascular diseases, can be observed. Purpose The impact of CKD on myocardial cell mechanisms is only partially understood. This study aims to investigate the impact of the uremic toxin indoxyl sulfate (IS) on human ventricular myocardial cells. Methods Human left ventricular myocardium was obtained from patients with severe aortic stenosis who underwent surgical valve replacement. In addition, human ventricular induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) obtained from donors without any known cardiovascular disease were used. Before measurements were conducted, isolated single primary human cardiomyocytes were incubated for 15 minutes with an IS concentration of 120 mg/l (highest measured IS-level in dialysis patients). Further, iPSC-CM were incubated for one week with IS 45 mg/l and IS 120 mg/l, with IS 45 mg/l representing the average dialysis patients IS-level. Epifluorescence and confocal microscopy were performed to investigate Ca2+hemostasis and patch clamp measurements assessed cardiac action potentials and the occurrence of arrythmias. Results Experiments were conducted using human ventricular myocardium from 10 patients with a mean left ventricular ejection fraction of 51.5±12% and a mean glomerular filtration rate of 66.3±22.7 ml/min. Compared to the control group, higher systolic Ca2+ transient amplitudes were observed after acute incubation with IS 120 mg/l in single primary human cardiomyocytes. Measurements after 7 days of IS incubation (IS 45 mg/l and 120 mg/l) in iPSC-CM confirmed these previous results by showing higher Ca2+ transient amplitudes (Figure 1). Additionally, cells showed a diminished diastolic Ca2+ level compared to controls. An increased Ca2+ spark frequency in IS-treated iPSC-CM versus the control group was unveiled by confocal microscopy measurements. While an increased number of delayed afterdepolarizations could be observed, IS seemed to have no effect on cardiac action potential duration in iPSC-CM. Conclusion For the first time, we could reveal the impact of IS on cellular electrophysiology of primary human myocardium cells and iPSC-CM and identify IS as a proarrhythmogenic trigger. Therefore, IS may be of relevance in explaining the high cardiovascular mortality in end-stage CKD patients. To improve the outcome of these patients, new therapeutic approaches will be necessary since IS, a protein-bound uremic toxin, is only insufficiently removed by dialysis.

Calcium handling abnormalities increase arrhythmia susceptibility in DMSXL myotonic dystrophy type 1 mice

BackgroundMyotonic dystrophy type 1 (DM1) is a multiorgan disorder with significant cardiac involvement. ECG abnormalities, including arrhythmias, occur in 80 % of DM1 patients and are the second-most common cause of death after respiratory complications; however, the mechanisms underlying the arrhythmogenesis remain unclear. The objective of this study was to investigate the basis of the electrophysiological abnormalities in DM1 using the DMSXL mouse model. MethodsECG parameters were evaluated at baseline and post flecainide challenge. Calcium transient and action potential parameters were evaluated in Langendorff-perfused hearts using fluorescence optical mapping. Calcium transient/sparks were evaluated in ventricular myocytes via confocal microscopy. Protein and mRNA levels for calcium handling proteins were evaluated using western blot and RT-qPCR, respectively. ResultsDMSXL mice showed arrhythmic events on ECG including premature ventricular contractions and sinus block. DMSXL mice showed increased calcium transient time to peak without any change to voltage parameters. Calcium alternans and both sustained and non-sustained ventricular tachyarrhythmias were readily inducible in DMSXL mice. The confocal experiments also showed calcium transient alternans and increased frequency of calcium sparks in DMSXL cardiomyocytes. These calcium abnormalities were correlated with increased RyR2 phosphorylation without changes to the other calcium handling proteins. ConclusionsThe DMSXL mouse model of DM1 exhibited enhanced arrhythmogenicity associated with abnormal intracellular calcium handling due to hyperphosphorylation of RyR2, pointing to RyR2 as a potential new therapeutic target in DM1 treatment.

Role of spark energy and frequency on wire-EDM performances of Al-2Mg alloy and Al-2Mg/20 Al3Fe composite: a comparative study

ABSTRACT The performance of wire-electric discharge machining (WEDM) method is inevitably affected by the electric spark. A comparative study is presented related to the influences of spark energy (SE, 1.68–8.40 mJ) and spark frequency (SF, 12.82–15.15 kHz) on machining responses of Al-2Mg/20 vol.% in-situ Al3Fe composite with respect to Al-2Mg alloy. Such comparison is scanty in the literature. Machining performances were measured concerning material removal rate (MRR), cutting speed (CS), and surface roughness (SR) apart from the morphological evolution of machined surfaces. Irrespective of machining condition (for instance, at SE of 5.04 mJ), composite exhibited lower CS (1.27 mm/min), MRR (4.20 mm3/min), and SR (4.42 µm) due to the insulating effect of reinforcement than alloy (i.e. 1.70 mm/min, 5.81 mm3/min, and 4.82 µm, respectively). For both alloy and composite, all three machining performances increased linearly with rising the SE and SF, although the influence of SE was more pronounced over SF. With increasing SE, the difference in the magnitudes of CS and MRR between alloy and composite widened, while the same for SR narrowed. Formations of micro-cracks, lumps of debris, craters, and re-solidified layers were observed more on alloy machining surface than composite; these features enlarged with increasing SE and SF.

Abstract Or102: Macrophage-Mediated Interleukin-6 Signaling Drives Ryanodine Receptor-2 Calcium Leak in Postoperative Atrial Fibrillation

Postoperative atrial fibrillation (poAF) is self-limited AF that occurs days after cardiac surgery in one-third of patients. The degree of perioperative increase in systemic inflammation, particularly involving interleukin (IL)-6, correlates with poAF risk. However, no studies to-date have identified the cell types involved nor mechanistically linked IL-6 signaling to fundamental arrhythmia mechanisms. To study poAF, we developed a mouse model consisting of atrial pericardiectomy and transient aortic cross-clamping followed by intracardiac burst pacing on postoperative day three to assess poAF inducibility. Single-cell RNA sequencing of atrial non-myocytes revealed macrophages to be the most prominently altered atrial cell type – increased 2.4-fold ( P <0.001) in mice with versus without poAF. Pseudotime trajectory analyses revealed IL-6 to be the top cytokine altered in macrophages, which we confirmed by western blot demonstrating a 1.7-fold ( P =0.012) increase in atrial IL-6 protein in mice with versus without poAF. Indeed, both macrophage depletion and conditional knockout of IL-6Ra in macrophages decreased poAF inducibility by 5.2-fold ( P =0.048) and 5.5-fold ( P =0.027), respectively. Downstream inhibition using an FDA-approved STAT3 inhibitor also rescued poAF (6.0-fold decrease, P =0.022). At the single atrial cardiomyocyte (ACM) level, IL-6 challenge led to a 6.3-fold ( P <0.001) increase in calcium (Ca 2+ ) spark frequency after normalization to sarcoplasmic reticulum Ca 2+ load, indicative of ryanodine receptor-2 (RyR2) dysfunction. Indeed, RyR2 phosphorylation at Ser2814, the target of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), was increased in the atria of mice with poAF, and CaMKII inhibition was sufficient to rescue arrhythmogenic Ca 2+ sparks in ACMs from mice with poAF. We corroborated our findings in human ACMs and atrial tissue to demonstrate translational relevance. Taken together, we use an integrated approach incorporating mouse and human biochemical, functional, and single-cell sequencing data to demonstrate that infiltrating atrial macrophages, specifically IL-6Ra from macrophages, are fundamentally required for poAF development ( Fig. 1 ). Molecularly, enhanced atrial IL-6 signaling led to arrhythmogenic RyR2 dysfunction through CaMKII. Our findings portend therapeutic utility by providing mechanistic evidence that targeting the IL-6 axis may be used for poAF prevention and treatment in a clinically amenable timeframe.

Abstract Tu049: Stabilization of RyR2 with dantrolene treatment ameliorates left ventricular remodeling and ventricular tachycardia after myocardial infarction

Background: Dantrolene has been reported to suppress diastolic Ca 2+ leakage through the cardiac ryanodine receptor (RyR2) by restoring defective inter-subunit interactions within RyR2 and enhancing the binding affinity of calmodulin (CaM) to RyR2. Here, we investigated whether chronic administration of dantrolene which was started after myocardial infarction (MI) inhibits ventricular tachycardia (VT) and alleviates left ventricular remodeling. Methods: We developed MI model by left anterior descending coronary artery (LAD) ligation in mice. Wild type mice were divided into 4 groups; sham-operated mice (WT-Sham), sham-operated mice treated with dantrolene (WT-Sham-DAN, 20mg/kg/day, i.p.), MI mice (WT-MI) and MI mice treated with dantrolene (WT-MI-DAN). Then, they were followed up for 12 weeks. Results: The 12-week survival rate was significantly higher in MI-DAN group (71 %) than in WT-MI (40%). Serial echocardiography showed that adverse LV remodeling was ameliorated in WT-MI-DAN group compared to WT-MI group (WT-MI 6.3 ± 0.25 mm, WT-MI-DAN 5.5 ± 0.14 mm, P<0.01). VT test by epinephrine (2mg/kg, i.p.) showed that VT was induced in all WT-MI group, although it was significantly inhibited in WT-MI-DAN group. An increase in diastolic Ca 2+ spark frequency and a decrease in sarcoplasmic reticulum Ca 2+ content were observed in isolated cardiomyocytes from WT-MI group whereas both were significantly ameliorated in WT-MI-DAN group. Conclusions: Dantrolene improves the diastolic Ca 2+ leakage from sarcoplasmic reticulum of damaged cardiomyocytes. Chronic administration of dantrolene after MI inhibits VT and ameliorates LV remodeling, leading to improved prognosis after MI. RyR2-targeting therapy could be a novel strategy for the treatment of post MI.

Abstract Tu072: O-GlcNAcylation underlies the activation of sodium-glucose cotransporter 1 in diabetic hearts

Rationale: Type-2 diabetes (T2D) greatly increases the risk for developing heart failure. Recent evidence indicates that the cardiac expression and function of the sodium-glucose cotransporter 1 (SGLT1) is elevated in disease states, including T2D, leading to structural and functional remodeling of the heart. The mechanisms underlying SGLT1 activation in the diabetic heart are currently unknown. Objective: To investigate if SGLT1 undergoes the O-linked attachment of β- N -acetylglucosamine (O-GlcNAcylation), a post-translational modification that is exacerbated in T2D, and its effect on SGLT1 activity in diabetic hearts. Methods/Results: SGLT1 co-immunoprecipitates with O-GlcNAc in heart homogenates and HL-1 cardiomyocyte lysates. Enhancing global protein O-GlcNAcylation in HL-1 cells via si-RNA-mediated silencing of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, resulted in increased amounts of O-GlcNAc that precipitate with SGLT1. SGLT1 function, measured as the SGLT1-mediated Na + influx, was significantly increased in HL-1 cells with silenced OGA. These data suggest that SGLT1 undergoes O-GlcNAcylation and this modification results in SGLT1 activation. Using rats that express human amylin in the pancreatic β-cells (HIP rats) as a model of late-onset T2D and their wild-type littermates as controls, we found that a larger fraction of SGLT1 co-immunoprecipitates with O-GlcNAc in hearts from T2D rats compared to controls. The SGLT1-mediated Na + influx was larger in myocytes from T2D vs . control rats. Increasing total O-GlcNAcylation with Thiamet-G in control myocytes resulted in larger Na + influx. In reverse experiments, inhibition of O-GlcNAcylation with 6-diazo-5-oxo-L-norleucine crystalline reduced SGLT1-mediated Na + uptake in myocytes from T2D rats. Inhibition of O-GlcNAcylation in T2D myocytes reduced Ca 2+ spark frequency and this effect was significantly less pronounced with SGLT1 blocked. These results suggest that O-GlcNAcylation affects myocyte Na + regulation by activating SGLT1, which contributes to myocyte Na + overload and its downstream effects on sarcoplasmic reticulum Ca 2+ leak in T2D. Conclusion: Exacerbated O-GlcNAcylation underlies SGLT1 activation in diabetic hearts. Preventing O-GlcNAcylation may limit myocyte Na + overload and the frequency of pro-arrhythmogenic Ca 2+ sparks in myocytes from T2D rats.

The inotropic and arrhythmogenic effects of acutely increased late INa are associated with elevated ROS but not oxidation of PKARIα.

Acute stimulation of the late sodium current (INaL) as pharmacologically induced by Anemonia toxin II (ATX-II) results in Na+-dependent Ca2+ overload and enhanced formation of reactive oxygen species (ROS). This is accompanied by an acute increase in the amplitude of the systolic Ca2+ transient. Ca2+ transient amplitude is determined by L-type Ca2+-mediated transsarcolemmal Ca2+ influx (ICa) into the cytosol and by systolic Ca2+ release from the sarcoplasmic reticulum (SR). Type-1 protein kinase A (PKARIα) becomes activated upon increased ROS and is capable of stimulating ICa, thereby sustaining the amplitude of the systolic Ca2+ transient upon oxidative stress. We aimed to investigate whether the increase of the systolic Ca2+ transient as acutely induced by INaL (by ATX-II) may involve stimulation of ICa through oxidized PKARIα. We used a transgenic mouse model in which PKARIα was made resistant to oxidative activation by homozygous knock-in replacement of redox-sensitive Cysteine 17 with Serine within the regulatory subunits of PKARIα (KI). ATX-II (at 1 nmol/L) was used to acutely enhance INaL in freshly isolated ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence and confocal imaging were used to assess intracellular Ca2+ handling and ROS formation. A ruptured-patch whole-cell voltage-clamp was used to measure INaL and ICa. The impact of acutely enhanced INaL on RIα dimer formation and PKA target structures was studied using Western blot analysis. ATX-II increased INaL to a similar extent in KI and WT cells, which was associated with significant cytosolic and mitochondrial ROS formation in both genotypes. Acutely activated Ca2+ handling in terms of increased Ca2+ transient amplitudes and elevated SR Ca2+ load was equally present in KI and WT cells. Likewise, cellular arrhythmias as approximated by non-triggered Ca2+ elevations during Ca2+ transient decay and by diastolic SR Ca2+-spark frequency occurred in a comparable manner in both genotypes. Most importantly and in contrast to our initial hypothesis, ATX-II did not alter the magnitude or inactivation kinetics of ICa in neither WT nor KI cells and did not result in PKARIα dimerization (i.e., oxidation) despite a clear prooxidant intracellular environment. The inotropic and arrhythmogenic effects of acutely increased INaL are associated with elevated ROS, but do not involve oxidation of PKARIα.

Ca2+/calmodulin-dependent kinase IIδC-induced chronic heart failure does not depend on sarcoplasmic reticulum Ca2+ leak.

Hyperactivity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) has emerged as a central cause of pathologic remodelling in heart failure. It has been suggested that CaMKII-induced hyperphosphorylation of the ryanodine receptor 2 (RyR2) and consequently increased diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) is a crucial mechanism by which increased CaMKII activity leads to contractile dysfunction. We aim to evaluate the relevance of CaMKII-dependent RyR2 phosphorylation for CaMKII-induced heart failure development in vivo. We crossbred CaMKIIδC overexpressing [transgenic (TG)] mice with RyR2-S2814A knock-in mice that are resistant to CaMKII-dependent RyR2 phosphorylation. Ca2+-spark measurements on isolated ventricular myocytes confirmed the severe diastolic SR Ca2+ leak previously reported in CaMKIIδC TG [4.65±0.73 mF/F0 vs. 1.88±0.30 mF/F0 in wild type (WT)]. Crossing in the S2814A mutation completely prevented SR Ca2+-leak induction in the CaMKIIδC TG, both regarding Ca2+-spark size and frequency, demonstrating that the CaMKIIδC-induced SR Ca2+ leak entirely depends on the CaMKII-specific RyR2-S2814 phosphorylation. Yet, the RyR2-S2814A mutation did not affect the massive contractile dysfunction (ejection fraction=12.17±2.05% vs. 45.15±3.46% in WT), cardiac hypertrophy (heart weight/tibia length=24.84±3.00 vs. 9.81±0.50mg/mm in WT), or severe premature mortality (median survival of 12weeks) associated with cardiac CaMKIIδC overexpression. In the face of a prevented SR Ca2+ leak, the phosphorylation status of other critical CaMKII downstream targets that can drive heart failure, including transcriptional regulator histone deacetylase 4, as well as markers of pathological gene expression including Xirp2, Il6, and Col1a1, was equally increased in hearts from CaMKIIδC TG on a RyR WT and S2814A background. S2814 phosphoresistance of RyR2 prevents the CaMKII-dependent SR Ca2+ leak induction but does not prevent the cardiomyopathic phenotype caused by enhanced CaMKIIδC activity. Our data indicate that additional mechanisms-independent of SR Ca2+ leak-are critical for the maladaptive effects of chronically increased CaMKIIδC activity with respect to heart failure.

A dual-targeted drug inhibits cardiac ryanodine receptor Ca2+ leak but activates SERCA2a Ca2+ uptake.

In the heart, genetic or acquired mishandling of diastolic [Ca2+] by ryanodine receptor type 2 (RyR2) overactivity correlates with risks of arrhythmia and sudden cardiac death. Strategies to avoid these risks include decrease of Ca2+ release by drugs modulating RyR2 activity or increase in Ca2+ uptake by drugs modulating SR Ca2+ ATPase (SERCA2a) activity. Here, we combine these strategies by developing experimental compounds that act simultaneously on both processes. Our screening efforts identified the new 1,4-benzothiazepine derivative GM1869 as a promising compound. Consequently, we comparatively studied the effects of the known RyR2 modulators Dantrolene and S36 together with GM1869 on RyR2 and SERCA2a activity in cardiomyocytes from wild type and arrhythmia-susceptible RyR2R2474S/+ mice by confocal live-cell imaging. All drugs reduced RyR2-mediated Ca2+ spark frequency but only GM1869 accelerated SERCA2a-mediated decay of Ca2+ transients in murine and human cardiomyocytes. Our data indicate that S36 and GM1869 are more suitable than dantrolene to directly modulate RyR2 activity, especially in RyR2R2474S/+ mice. Remarkably, GM1869 may represent a new dual-acting lead compound for maintenance of diastolic [Ca2+].

Nitric Oxide Modulates Ca2+ Leak and Arrhythmias via S-Nitrosylation of CaMKII.

Nitric oxide (NO) has been identified as a signaling molecule generated during β-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during β-adrenergic receptor stimulation. We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (200 μM), and β-adrenergic agonist isoproterenol (100 nmol/L). Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent β-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain β-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Investigation of the WEDM Parameters’ Influence on the Recast Layer Thickness of Spark Plasma Sintered SiC-TiB2-TiC Ceramic

The influence of WEDM parameters (Spark gap voltage, Pulse-on time, Spark frequency, and Wire speed) on the recast layer thickness and surface roughness of Spark Plasma Sintered SiC-TiB2-TiC ceramic composite was investigated. For this, an orthogonal L9 Taguchi design was used, and grey relational analysis was carried out for multi-response WEDM parameter optimization in order to determine the minimum RLT and SR. It was noticed that for RLT, the Pulse-on time was observed as the most significant process parameter, followed by Spark gap voltage. On the other hand, Spark frequency and Wire speed had no significance for RLT. Moreover, Spark frequency was observed as the most significant process parameter, followed by Pulse-on time and Spark gap voltage, while Wire speed had a negligible effect on SR. It was found that at optimal process parameters (U = 48V; Ton = 1.0 µs; f = 10 kHz; q = 8 m/min), we obtained an RLT of 3.16 µm and an SR of Ra = 0.847 µm. The confirmation test showed a decrease in RLT and SR by 43.67% and 7.12%, respectively, in comparison to the initial machining conditions.

Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in isolated mouse ventricular myocytes.

The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.

Cardiomyocytes from female compared to male mice have larger ryanodine receptor clusters and higher calcium spark frequency.

Sex differences in cardiac physiology are receiving increased attention as it has become clear that men and women have different aetiologies of cardiac disease and require different treatments. There are experimental data suggesting that male cardiomyocytes exhibit larger Ca2+ transients due to larger Ca2+ sparks and a higher excitation-contraction coupling gain; in addition, they exhibit a larger response to adrenergic stimulation with isoprenaline (ISO). Here, we studied whether there are sex differences relating to structural organization of the transverse tubular network and ryanodine receptors (RyRs). Surprisingly, we found that female cardiomyocytes exhibited a higher spark frequency in a range of spark magnitudes. While overall RyR expression and phosphorylation were the same, female cardiomyocytes had larger but fewer RyR clusters. The density of transverse t-tubules was the same, but male cardiomyocytes had more longitudinal t-tubules. The Ca2+ transients were similar in male and female cardiomyocytes under control conditions and in the presence of ISO. The synchrony of the Ca2+ transients was similar between sexes as well. Overall, our data suggest subtle sex differences in the Ca2+ influx and efflux pathways and their response to ISO, but these differences are balanced, resulting in similar Ca2+ transients in field-stimulated male and female cardiomyocytes. The higher spark frequency in female cardiomyocytes is related to the organization of RyRs into larger, but fewer clusters. KEY POINTS: During a heartbeat, the force of contraction depends on the amplitude of the calcium transient, which in turn depends on the amount of calcium released as calcium sparks through ryanodine receptors in the sarcoplasmic reticulum. Previous studies suggest that cardiomyocytes from male compared to female mice exhibit larger calcium sparks, larger sarcoplasmic reticulum calcium release and greater response to adrenergic stimulation triggering a fight-or-flight response. In contrast, we show that cardiomyocytes from female mice have a higher spark frequency during adrenergic stimulation and similar spark morphology. The higher spark frequency is related to the organization of ryanodine receptors into fewer, but larger clusters in female compared to male mouse cardiomyocytes. Despite subtle sex differences in cardiomyocyte structure and calcium fluxes, the differences are balanced, leading to similar calcium transients in cardiomyocytes from male and female mice.

Abstract P3134: Impaired Calcium Homeostasis Underlies Lamin A/C Mutation R331Q Associated Atrial Fibrillation

Background: Atrial fibrillation (AF) is one of the first cardiac manifestations in patients harboring mutations in nucleoskeletal protein lamin A/C (LMNA). However, the underlying arrhythmogenic mechanisms are unclear. Objective: We hypothesize that impaired intracellular Ca 2+ -homeostasis contribute to LMNA mutation-associated atrial arrhythmogenesis. Methods: LMNA mutation R331Q was introduced in a healthy control induced pluripotent stem cell (iPSC) line using CRISPR/Cas9 and cells were differentiated into atrial iPSC-derived cardiomyocytes (iPSC-CMs). L-type Ca 2+ current and intracellular Ca 2+ concentration were measured using patch-clamp technique and epifluorescence microscopy (Fluo-3), respectively. Results: R331Q iPSC-CMs demonstrated increased L-type Ca 2+ current density followed by increased amplitude of systolic Ca 2+ transient (CaT) compared to isogenic control (Fig. A). Sarcoplasmic reticulum (SR) Ca 2+ content was evaluated from caffeine-induced CaT amplitude and integrated Na + -Ca 2+ exchange current, and found to be greater in R331Q iPSC-CMs (Fig. B). Confocal line scan analysis revealed an increased Ca 2+ spark frequency in R331Q iPSC-CMs (Fig. C). In addition, by applying a stepwise increase in stimulation frequency (1-5 Hz), R331Q iPSC-CMs developed Ca 2+ alternans at lower threshold frequency (Fig. D). Conclusions: LMNA mutation R331Q is associated with disturbed intracellular Ca 2+ cycling, promoting occurrence of diastolic Ca 2+ sparks and arrhythmogenic Ca 2+ alternans. Collectively, our findings suggest that impaired Ca 2+ homeostasis may contribute to atrial arrhythmogenesis in patients harboring LMNA mutations.

Distinct Effects of Mitochondrial Na+/Ca2+ Exchanger Inhibition and Ca2+ Uniporter Activation on Ca2+ Sparks and Arrhythmogenesis in Diabetic Rats.

Background Mitochondrial dysfunction contributes to the cardiac remodeling triggered by type 2 diabetes (T2D). Mitochondrial Ca2+ concentration ([Ca2+]m) modulates the oxidative state and cytosolic Ca2+ regulation. Thus, we investigated how T2D affects mitochondrial Ca2+ fluxes, the downstream consequences on myocyte function, and the effects of normalizing mitochondrial Ca2+ transport. Methods and Results We compared myocytes/hearts from transgenic rats with late-onset T2D (rats that develop late-onset T2D due to heterozygous expression of human amylin in the pancreatic β-cells [HIP] model) and their nondiabetic wild-type (WT) littermates. [Ca2+]m was significantly lower in myocytes from diabetic HIP rats compared with WT cells. Ca2+ extrusion through the mitochondrial Na+/Ca2+ exchanger (mitoNCX) was elevated in HIP versus WT myocytes, particularly at moderate and high [Ca2+]m, while mitochondrial Ca2+ uptake was diminished. Mitochondrial Na+ concentration was comparable in WT and HIP rat myocytes and remained remarkably stable while manipulating mitoNCX activity. Lower [Ca2+]m was associated with oxidative stress, increased sarcoplasmic reticulum Ca2+ leak in the form of Ca2+ sparks, and mitochondrial dysfunction in T2D hearts. MitoNCX inhibition with CGP-37157 reduced oxidative stress, Ca2+ spark frequency, and stress-induced arrhythmias in HIP rat hearts while having no significant effect in WT rats. In contrast, activation of the mitochondrial Ca2+ uniporter with SB-202190 enhanced spontaneous sarcoplasmic reticulum Ca2+ release and had no significant effect on arrhythmias in both WT and HIP rat hearts. Conclusions [Ca2+]m is reduced in myocytes from rats with T2D due to a combination of exacerbated mitochondrial Ca2+ extrusion through mitoNCX and impaired mitochondrial Ca2+ uptake. Partial mitoNCX inhibition limits sarcoplasmic reticulum Ca2+ leak and arrhythmias in T2D hearts, whereas mitochondrial Ca2+ uniporter activation does not.

Molecular and Functional Relevance of NaV1.8-Induced Atrial Arrhythmogenic Triggers in a Human SCN10A Knock-Out Stem Cell Model.

In heart failure and atrial fibrillation, a persistent Na+ current (INaL) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have recently shown that NaV1.8 contributes to arrhythmogenesis by inducing a INaL. Genome-wide association studies indicate that mutations in the SCN10A gene (NaV1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden cardiac death. However, the mediation of these NaV1.8-related effects, whether through cardiac ganglia or cardiomyocytes, is still a subject of controversial discussion. We used CRISPR/Cas9 technology to generate homozygous atrial SCN10A-KO-iPSC-CMs. Ruptured-patch whole-cell patch-clamp was used to measure the INaL and action potential duration. Ca2+ measurements (Fluo 4-AM) were performed to analyze proarrhythmogenic diastolic SR Ca2+ leak. The INaL was significantly reduced in atrial SCN10A KO CMs as well as after specific pharmacological inhibition of NaV1.8. No effects on atrial APD90 were detected in any groups. Both SCN10A KO and specific blockers of NaV1.8 led to decreased Ca2+ spark frequency and a significant reduction of arrhythmogenic Ca2+ waves. Our experiments demonstrate that NaV1.8 contributes to INaL formation in human atrial CMs and that NaV1.8 inhibition modulates proarrhythmogenic triggers in human atrial CMs and therefore NaV1.8 could be a new target for antiarrhythmic strategies.

Cardiac Protein Kinase D1 ablation alters the myocytes β-adrenergic response

β-adrenergic (β-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute β-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte β-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal β-adrenergic responses in Ca2+ handling.

BK channel dysfunction underlies small vessel disease of the brain in both hypertension and Alzheimer's disease

Alzheimer's disease (AD) and hypertension-related vascular dementia share many common clinical and radiological features including cerebral small vessel disease (cSVD) of the brain, thought to be the mechanism behind the reduction in cerebral blood flow (CBF). The aim of this study was to determine if there are common mechanisms underlying a reduced CBF in dementia. CBF is determined by depolarisation induced contraction of arterial smooth muscle cells (SMCs). This contraction is buffered by the activation of the large-conductance Ca 2+ -activated K + (BK) channels on the plasma membrane (PM), by transient localised Ca 2+ release from ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR), known as Ca 2+ sparks.Hypothesis: Defects in BK channel activation leads to the cSVD phenotype in these diseases.8-month old male spontaneously hypertensive (BPH/2) mice and normotensive controls (BPN/3) were used to study hypertension-induced cSVD. To study AD related cSVD we used 18–20 month old male APP23 mice. We used a range of physiological techniques to investigate vascular ion channel function within the cerebral microvasculature. Laser Doppler flowmetry and behavioural assessments were performed on the BPH/2 and BPN/3 mice to initially determine if they are a model of hypertension-related vascular dementia.BPH/2 mice displayed reduced CBF and cognitive impairment. Pressure-induced constriction from cerebral pial arteries was significantly greater in both the BPH/2 and APP23 animals compared to controls. This was the result of reduced BK channel activity, as confirmed by reduced constriction to the BK channel blocker paxilline. Electrophysiology experiments showed a decrease in spontaneous transient outward currents, which represent BK channel activation by Ca 2+ sparks. In the APP23 model, this attenuated BK channel activation was due to reduced Ca 2+ spark frequency. However, in the BPH/2 mice, Ca 2+ spark frequency was equal between hypertensive and normotensive mice. Furthermore, there were no differences in single channel BK channel activity over a range of [Ca 2+ ]. Live cell imaging of isolated SMCs labelled with membrane dyes, showed a reduced interaction between the SR and the PM. Proximity ligation assay was used on fixed SMCs to determine if BK channel and RyR are within 40 nm of each other. SMCs from BPH/2 cerebral arteries has significantly less puncta suggesting a physical separation between Ca 2+ sparks and BK channel, rendering the [Ca 2+ ] local to the BK channel insufficient for activation.Our data indicate that vascular BK channel dysfunction is a common mechanism underpinning reduced CBF in SVD. Therapies aimed at improving vascular BK channel function could provide new therapeutic targets for both AD and hypertension-induced vascular dementia. British Heart Foundation 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.