Β-adrenergic Stimulation Research Articles

Genetic variants in calcium regulatory cardiac genes and their contribution to Takotsubo syndrome

Abstract Background and purpose Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction similar to a myocardial infarction (MI) in the absence of coronary artery stenosis. Patients show symptoms similar to the acute MI with increased biomarkers and blood serum catecholamines. Recently, we developed a patient-specific TTS stem cell model and identified a higher sensitivity to catecholamine-induced stress. Furthermore, familial TTS cases and genetic studies point to a genetic predisposition. The purpose of this study was to analyze a genetic predisposition by characterizing genetic variants in genes associated with cardiac pathologies and their impact on calcium homoeostasis in TTS. Methods and results Whole exome sequencing analysis of a TTS patient discovered 2 missense AHNAK variants in its C-terminal domain and in addition the missense variant F189L in the calcium buffering calsequestrin 2 gene (CASQ2). AHNAK is a 700kDa big nucleoprotein and is involved in the β-adrenergic regulation of the cardiac calcium channel Cav1.2. 3-month old TTS-iPSC-derived cardiomyocytes (CM) were generated and the variants were confirmed by sequencing. We found AHNAK higher expressed in TTS-iPSC-CMs compared to control, whereas no expression alteration was observed for Cav1.2. Since AHNAK is described to act as a repressor towards Cav1.2, which is relieved under β-adrenergic stimulation, we analyzed the effect of AHNAK variants on a potential co-localization and interaction between both proteins. AHNAK and Cav1.2 were shown to co-localize in the cytoplasm as well as the membranes and co-immunoprecipitation experiments confirmed an interaction of AHNAK and Cav1.2 in all tested control- and TTS-iPSC-CMs. On a functional level, we were able to show by patch clamp analysis that Cav1.2 calcium currents are significantly increased in TTS-iPSC-CMs compared to control. The influence of CASQ2-F189L on sarcomeric reticulum (SR) calcium load was analyzed by epifluorescence microscopy using FURA4 and caffeine-applications. We found significantly decreased SR calcium content with an increased fractional release during systole in TTS-iPSC-CMs. To test, whether these variants are the main reason for altered interaction of AHNAK and Cav1.2, calcium currents or SR calcium load in TTS need to be proven in the future by using CRISPR/Cas9-rescued AHNAK/CASQ2 lines. Conclusion Here we show the cardiac functional consequences of AHNAK and CASQ2 missense mutations in TTS-iPSC-CMs with regard to calcium currents and SR calcium load. These results show that AHNAK and CASQ2 variants may predispose to TTS and enable a new therapeutic option for TTS. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Else Kröner-Fresenius Foundation

RBM20-mutations induce disturbed splicing of calcium relevant genes in patient-specific stem cell models of cardiomyopathies

Abstract Background and aim Mutations in the splice factor RBM20 have been identified to account for ∼3% of cardiomyopathies. In particular, the highly conserved RS-domain is a hotspot for disease-associated mutations. Distinct mutations at position 634 in the RS-domain were already described to be associated to dilative cardiomyopathy (DCM) (R634W) or to left ventricular non-compaction cardiomyopathy (LVNC) (R634L), but the molecular mechanisms that govern the heterogenic entity of DCM and LVNC remain largely unknown. We aimed to analyze the molecular driver behind the RBM20 mutation-based DCM and LVNC in a patient-specific stem cell model. Methods Human somatic cells from 2 DCM- and 2 LVNC-patients harboring the RBM20-mutations R634W (DCM) or R634L (LVNC) were reprogrammed into induced pluripotent stem cells (iPSC) and differentiated into functional cardiomyocytes (CM). Gene expression, alternative splicing activity, sarcomeric regularity, cAMP level, kinase-specific phosphorylation of important Ca2+ players, and physiological cardiac functions as Ca2+ homeostasis were analyzed (Fluo3 and Fura4). Isogenic rescue lines were generated by CRISPR/Cas9 technology to analyze the direct impact of the RBM20 mutations to the cardiac phenotype. Results We investigated the role of RBM20 mutations in DCM and LVNC-iPSC-CMs RBM20-splicing and observed common splice defects in titin-isoform-switch or a 24bp insertion in the gene ryanodine receptor 2 (RYR2).. In contrast, the calcium-handling gene Camk2δ was predominantly mis-spliced in LVNC-CMs, whereas the structural gene LDB3 was mis-spliced in DCM-CMs. As a possible consequence of splice defects in sarcomeric genes both DCM and LVNC-CMs exhibited an irregular sarcomeric structure at the Z-disk and M-line. Interestingly, the LVNC-CMs showed faster Ca2+ transient decay time and weakened response to β-adrenergic stimulation. In contrast, the DCM-CMs did exhibit increased Ca2+-sparks and decreased systolic and diastolic Ca2+ highlighting that two distinct missense mutations can lead to different pathological Ca2+ phenotypes. Ca2+ kinetic defects in LVNC-iPSC-CMs were independent of cAMP, but in line with Camk2δ-dependent hyperphosphorylation of the specific target PLN. Isogenic WT-iPSC lines were generated using CRISPR/Cas9 technology and underscored the role of RBM20-mutations in cardiomyopathies as the sarcomeric defects, Ca2+ cycling and leakage were rescued for both LVNC-CMs and DCM-CMs. Conclusion We show the first iPSC-model of splice-defect-associated RBM20-dependent LVNC and DCM. Our data demonstrate that RBM20-R634L induce mis-splicing of Camk2δ leading to hyperphosphorylation of PLN-Thr17 along with increased Ca2+ kinetics in LVNC, whereas RBM20-R634W induced RYR2-dependent Ca2+ leak with disturbed systolic and diastolic Ca2+in DCM. Taken together these results suggest that the molecular aberrations in alternative splicing differ depending on the distinct missense mutation in RBM20. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): BMBF, DZHK German Center for Cardiovascular research

Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca2+ Dynamics.

Ca2+-induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction coupling after β-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

Adrenergic CaV1.2 Activation via Rad Phosphorylation Converges at α1C I-II Loop.

Changing activity of cardiac CaV1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic. The primary goal of this study was to determine how various factors converging at the CaV1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure. We generated transgenic mice with expression of CaV1.2 α1C subunits with (1) mutations ablating interaction between α1C and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of CaVβ to α1C I-II loop and eliminated β-adrenergic agonist stimulation of CaV1.2 current. In contrast, introduction of the exon 9* splice variant in the α1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β2B and Rad or transgenically expressed in cardiomyocytes. Ca2+ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α1C I-II loop. CaVβ binding to α1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca2+ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α1C.

Multisite phosphorylation of the cardiac ryanodine receptor: a random or coordinated event?

Many proteins are phosphorylated at more than one phosphorylation site to achieve precise tuning of protein function and/or integrate a multitude of signals into the activity of one protein. Increasing the number of phosphorylation sites significantly broadens the complexity of molecular mechanisms involved in processing multiple phosphorylation sites by one or more distinct kinases. The cardiac ryanodine receptor (RYR2) is a well-established multiple phospho-target of kinases activated in response to β-adrenergic stimulation because this Ca2+ channel is a critical component of Ca2+ handling machinery which is responsible for β-adrenergic enhancement of cardiac contractility. Our review presents a selective overview of the extensive, often conflicting, literature which focuses on identifying reliable lines of evidence to establish if multiple RYR2 phosphorylation is achieved randomly or in a specific sequence, and whether phosphorylation at individual sites is functionally specific and additive or similar and can therefore be substituted.

Distinguishing HF with reduced and preserved ejection fraction at the level of individual cardiomyocytes: implications for therapeutic development

Distinguishing HF with reduced and preserved ejection fraction at the level of individual cardiomyocytes: implications for therapeutic development

Effect of Hypothyroidism on Force and Kinetics in End-stage Failing Human Myocardium

Introduction The relationship between hypothyroidism and the occurrence and progression of heart failure (HF) had an increase interest over the past years. The low T3 syndrome, characterized by reduced T3 in the presence of normal thyroid stimulating hormone (TSH) and free T4 concentration, is a strong predictor of all-cause mortality in HF patients. Still, the impact of hypothyroidism on the contractility properties of failing human heart is unknown. Therefore, the goal of our study was to investigate the hypothyroidism in heart failure using ex-vivo force of contraction and kinetics assessments in left ventricular intact human myocardial muscle preparations. Methods Uniform linear trabeculae were dissected from three groups: (1) non-failing hearts (n=9), (2) failing hearts without underling thyroid dysfunction (n=9), and (3) failing hearts with hypothyroidism (n=9). These muscles were transferred into a custom-made setup where the three main physiological modulators that regulate the contractile strength, length-dependent activation, frequency-dependent activation, and β-adrenergic stimulation were assessed under near-physiological conditions. Clinical information for all patients with HF and hypothyroidism in our study revealed that these patients were receiving levothyroxine treatment. Results Hypothyroidism did not show any additional significant impact on the force of contraction and kinetics of contraction/relaxation different than the recognized alterations usually detected in such parameters in any end-stage failing heart without underlying thyroid dysfunction. Conclusions We suggest the usefulness of levothyroxine treatment in repressing the myocardial alterations of hypothyroidism and limiting them to those caused by HF syndrome itself in our study. Absence of statistical difference between TSH levels of patients with HF and hypothyroidism and TSH levels of patients with HF and no thyroid dysfunction reflects the efficiency and accuracy of the thyroid hormone doses used in treating these patients. More studies are still needed to explore the full potential of the therapeutic use of thyroid hormones in treating HF associated with hypothyroidism.

Autonomous initiation of pacemaking by L-type Cav1.3 calcium channels in mouse sino-atrial myocytes

Cardiac pacemaking relies on the spontaneous electrical activity in the right atrium of sino-atrial myocytes (SANCs). Automaticity in SANCs results from a robust interplay of membrane ion channels activity and intracellular calcium dynamics. However, only a fraction of isolated SANCs exhibit rhythmic firing, whereas most SANCs show irregular (dysrhythmic) firing or remain dormant. To study the capability of L-type Cav1.3 calcium channels to initiate automaticity in dormant SANCs under β-adrenergic stimulation we used a knock-in mouse strain in which the sensitivity of Cav1.2 α1 subunits to dihydropyridines (DHP) was inactivated (Cav1.2DHP-/-). We performed current-clamp recordings on isolated SANCs under isoprenaline (ISO, 100 nM) and in the absence or presence of the DHP blocker Nifedipine (Nife, 3 μM). In rhythmic SANCs, Nife significantly reduced the spontaneous firing under ISO perfusion (ISO: 447 ± 12, ISO + Nife: 233 ± 25 bpm). In dysrhythmic SANCs, Nife significantly reduced the spontaneous rate under ISO in 60% of the cells (ISO: 386 ± 12, ISO + Nife: 188 ± 47 bpm) and completely stopped it in 40% (295 ± 29 bpm to 0). On 25 dormant SANCs, 50% started firing after ISO perfusion (0 to 320 ± 46 bpm). Strikingly, in 75% of them, Nife totally blocked this ISO-induced firing. Moreover, dormant SANCs showed a statistically significant increase in action potential (AP) threshold under ISO compared to dysrhythmic and rhythmic SANCs (dormant: −30.1 ± 2.5, dysrhythmic: −43.3 ± 2.3, rhythmic: −41.2 ± 2.1 mV). No significant difference was observed in the other AP parameters between dormant, dysrhythmic and rhythmic SANCs under ISO. Our results indicate that Cav1.3 channels can generate pacemaker activity autonomously, at least in a particular subpopulation of SANCs, while it is responsible of about half of the automaticity in the others.

Moderate FKBP12.6 overexpression mitigates β-adrenergic-associated pro-arrhythmogenic Ca2+ events, but a higher expression level leads to a cardiomyopathic phenotype

Cardiac ryanodine receptors RyR2 have a crucial role in excitation-contraction coupling by releasing Ca2+ from the sarcoplasmic reticulum (SR). In cardiomyocytes, two FK506 binding proteins (FKBP) of 12 and 12.6 kDa bind and stabilize RyR2 in a closed state. Despite its lower concentration, FKBP12.6 has a stronger affinity for RyR2 than FKBP12. In heart failure (HF), the expression of both isoforms decreases, associated with a high incidence of arrhythmias. By contrast, cardiac FKBP12.6 overexpression protects against β-adrenergic-activated arrhythmias, but the underlying molecular mechanism remains elusive. We wanted to understand the anti-arrhythmic mechanism of FKBP12.6 overexpression, with the hypothesis of dose-effect anti-adrenergic properties. Accordingly, we developed two transgenic mouse lines with cardiac-specific moderate- ( × 10, TG1) and high- ( × 40, TG2) FKBP12.6 overexpression. We characterized cardiac function, [Ca2 + ]i cycling, and its response to sympathetic stimulation in both mouse lines. TG2 mice died prematurely and exhibited marked cardiac hypertrophy with nonsignificant pulmonary congestion, whereas TG1 mice had a normal phenotype. At 2-3 months, TG1 mice had a normal cardiac function, whereas TG2 had a collapsed heart rate and a dilated and hypertrophied left ventricle with unaltered fractional shortening. TG1 cardiomyocytes had higher [Ca2 + ]i transient amplitude and less pro-arrhythmogenic Ca2+ release events than WT mice, without modification of the SR Ca2+ load, and an attenuated response to β-adrenergic stimulation. In contrast, TG2 mice showed [Ca2 + ]i handling characteristics similar to HF with slower [Ca2 + ]i transient relaxation, although pro-arrhythmogenic Ca2+ release events were reduced. These results confirm that FKBP12.6 overexpression interferes with pro-arrhythmic sympathetic signaling, and demonstrate that high level of FKBP12.6 has a deleterious cardiac effect.

Complex Arrhythmia Syndrome in a Knock-In Mouse Model Carrier of the N98S Calm1 Mutation.

Calmodulin mutations are associated with arrhythmia syndromes in humans. Exome sequencing previously identified a de novo mutation in CALM1 resulting in a p.N98S substitution in a patient with sinus bradycardia and stress-induced bidirectional ventricular ectopy. The objectives of the present study were to determine if mice carrying the N98S mutation knocked into Calm1 replicate the human arrhythmia phenotype and to examine arrhythmia mechanisms. Mouse lines heterozygous for the Calm1N98S allele (Calm1N98S/+) were generated using CRISPR/Cas9 technology. Adult mutant mice and their wildtype littermates (Calm1+/+) underwent electrocardiographic monitoring. Ventricular de- and repolarization was assessed in isolated hearts using optical voltage mapping. Action potentials and whole-cell currents and [Ca2+]i, as well, were measured in single ventricular myocytes using the patch-clamp technique and fluorescence microscopy, respectively. The microelectrode technique was used for in situ membrane voltage monitoring of ventricular conduction fibers. Two biologically independent knock-in mouse lines heterozygous for the Calm1N98S allele were generated. Calm1N98S/+ mice of either sex and line exhibited sinus bradycardia, QTc interval prolongation, and catecholaminergic bidirectional ventricular tachycardia. Male mutant mice also showed QRS widening. Pharmacological blockade and activation of β-adrenergic receptors rescued and exacerbated, respectively, the long-QT phenotype of Calm1N98S/+ mice. Optical and electric assessment of membrane potential in isolated hearts and single left ventricular myocytes, respectively, revealed β-adrenergically induced delay of repolarization. β-Adrenergic stimulation increased peak density, slowed inactivation, and left-shifted the activation curve of ICa.L significantly more in Calm1N98S/+ versus Calm1+/+ ventricular myocytes, increasing late ICa.L in the former. Rapidly paced Calm1N98S/+ ventricular myocytes showed increased propensity to delayed afterdepolarization-induced triggered activity, whereas in situ His-Purkinje fibers exhibited increased susceptibility for pause-dependent early afterdepolarizations. Epicardial mapping of Calm1N98S/+ hearts showed that both reentry and focal mechanisms contribute to arrhythmogenesis. Heterozygosity for the Calm1N98S mutation is causative of an arrhythmia syndrome characterized by sinus bradycardia, QRS widening, adrenergically mediated QTc interval prolongation, and bidirectional ventricular tachycardia. β-Adrenergically induced ICa.L dysregulation contributes to the long-QT phenotype. Pause-dependent early afterdepolarizations and tachycardia-induced delayed afterdepolarizations originating in the His-Purkinje network and ventricular myocytes, respectively, constitute potential sources of arrhythmia in Calm1N98S/+ hearts.

Optimizing the Direction and Order of the Motion Unveiled the Ability of Conventional Monolayers of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Show Frequency-Dependent Enhancement of Contraction and Relaxation Motion.

Contractility of the human heart increases as its beating rate is elevated, so-called positive force-frequency relationship; however, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been reported to exert a negative force-frequency relationship. We tested the hypothesis that the regulation of motion directions by electrical pacing and/or oxygen supply may improve the electro-mechanical properties of hiPSC-CMs monolayers. To better evaluate the spatial and temporal relationship between electrical excitation and contractile motion, we simultaneously observed the field potential and motion vector of hiPSC-CMs sheets. Under spontaneous contraction, although an electrical excitation originating from a region propagated unidirectionally over the cell sheet, contraction wave started from multiple sites, and relaxation wave was initiated from a geometric center of hiPSC-CMs sheet. During electrical pacing, contraction and relaxation waves were propagated from the stimulated site. Interestingly, the maximum contraction speed was more increased when the hiPSC-CMs sheet was stimulated at an area relaxation initiated under spontaneous condition. Furthermore, motion vector analysis demonstrated that “positive contraction velocity-frequency relationship” in contraction and “frequency-dependent enhancement of relaxation” were produced in the cell sheet by optimizing the direction and order of the contractile motion with pacing at the relaxation-initiating area. A close analysis of motion vectors along with field potential recording demonstrated that relaxation process consists of fast and slow phases, and suggest that intracellular Ca2+ dynamics may be closely related to functions of Ca2+-ATPase pump and Na+-Ca2+ exchangers. Namely, the slow relaxation phase occurred after the second peak of field potential, suggesting that the slow phase may be associated with extrusion of Ca2+ by Na+-Ca2+ exchangers during repolarization. Increase of oxygen concentration from 20 to 95% as well as β-adrenergic stimulation with isoproterenol accelerated the fast relaxation, suggesting that it could depend on Ca2+ uptake via Ca2+-ATPase during the depolarization phase. The ratio of maximum contraction speed to field potential duration was increased by the β-adrenergic stimulation, indicating the elevated contraction efficiency per Ca2+-influx. Thus, these findings revealed potential ability of conventional monolayers of hiPSC-CMs, which will help apply them to translational study filling the gap between physiological as well as pharmacological studies and clinical practice.

Effects of β-adrenergic stimulation on fetal heart rate, heart rate variability, and T-wave elevation during brief umbilical cord occlusions in fetal sheep.

Circulating catecholamines are critical for fetal adaptation to hypoxia by regulating fetal heart rate (FHR) and promoting myocardial contractility and peripheral vasoconstriction. They have been hypothesized to contribute to changes in FHR variability (FHRV) and T-wave morphology, clinical indexes of fetal well-being during labor. β-Adrenergic blockade with propranolol does not affect FHRV during labor-like hypoxemia and only attenuated the increase in T-wave height between the episodes of hypoxemia. To further investigate the potential role of catecholamines, we investigated whether pharmacological β-adrenergic stimulation could increase FHRV and T-wave elevation during intermittent labor-like hypoxemia. Nineteen chronically instrumented fetal sheep at 0.85 of gestation received isoprenaline hydrochloride (n = 7) or saline (control, n = 12), followed by three 1-min complete umbilical cord occlusions (UCOs) separated by 4-min reperfusion periods. Before the UCOs, infusion of isoprenaline increased FHR (P < 0.001), absolute-T/QRS ratio (P < 0.001), and one measure of FHRV [root-mean-square of successive RR interval differences (RMSSD), P < 0.05]. UCOs triggered deep FHR decelerations. During UCOs, isoprenaline was associated with increased FHR (P < 0.001) and absolute-T/QRS ratio (P < 0.05), but no effect on T/QRS ratio was observed when normalized to baseline before UCOs (normalized-T/QRS ratio). Between UCOs, isoprenaline increased FHR (P < 0.001) and absolute-T/QRS ratio (P < 0.05) but did not affect normalized-T/QRS ratio or any measures of FHRV. Arterial pressure was not affected by isoprenaline at any point. Our findings indicate that circulating catecholamines regulate FHR but not FHRV during labor-like hypoxemia and promote T-wave elevation between but not during intermittent fetal hypoxemia.

14-3-3 binding creates a memory of kinase action by stabilizing the modified state of phospholamban.

The cardiac membrane protein phospholamban (PLN) is targeted by protein kinase A (PKA) at Ser16 and by Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr17 β-Adrenergic stimulation and PKA-dependent phosphorylation of Ser16 acutely stimulate the sarcoplasmic reticulum calcium pump (SERCA) by relieving its inhibition by PLN. CaMKII-dependent phosphorylation may lead to longer-lasting SERCA stimulation and may sustain maladaptive Ca2+ handling. Here, we demonstrated that phosphorylation at either Ser16 or Thr17 converted PLN into a target for the phosphoadaptor protein 14-3-3 with different affinities. 14-3-3 proteins were localized within nanometers of PLN and endogenous 14-3-3 coimmunoprecipitated with pentameric PLN from cardiac membranes. Molecular dynamics simulations predicted different molecular contacts for peptides phosphorylated at Ser16 or Thr17 with the binding groove of 14-3-3, resulting in varied binding affinities. 14-3-3 binding protected either PLN phosphosite from dephosphorylation. β-Adrenergic stimulation of isolated adult cardiomyocytes resulted in the membrane recruitment of endogenous 14-3-3. The exogenous addition of 14-3-3 to β-adrenergic-stimulated cardiomyocytes led to prolonged SERCA activation, presumably because 14-3-3 protected PLN pentamers from dephosphorylation. Phosphorylation of Ser16 was disrupted by the cardiomyopathy-associated ∆Arg14 mutation, implying that phosphorylation of Thr17 by CaMKII may become crucial for 14-3-3 recruitment to ∆Arg14 PLN. Consistent with PLN acting as a dynamic hub in the control of Ca2+ handling, our results identify 14-3-3 binding to PLN as a contractility-augmenting mechanism.

Evidence for mechano-electrical coupling in isolated rat hearts: Effects of loading and β-adrenergic receptor stimulation on repolarization indices

Evidence for mechano-electrical coupling in isolated rat hearts: Effects of loading and β-adrenergic receptor stimulation on repolarization indices

Autonomous activation of CaMKII exacerbates diastolic calcium leak during beta-adrenergic stimulation in cardiomyocytes of metabolic syndrome rats

Autonomous Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation induces abnormal diastolic Ca2+ leak, which leads to triggered arrhythmias in a wide range of cardiovascular diseases, including diabetic cardiomyopathy. In hyperglycemia, Ca2+ handling alterations can be aggravated under stress conditions via the β-adrenergic signaling pathway, which also involves CaMKII activation. However, little is known about intracellular Ca2+ handling disturbances under β-adrenergic stimulation in cardiomyocytes of the prediabetic metabolic syndrome (MetS) model with obesity, and the participation of CaMKII in these alterations.MetS was induced in male Wistar rats by administering 30 % sucrose in drinking water for 16 weeks. Fluo 3-loaded MetS cardiomyocytes exhibited augmented diastolic Ca2+ leak (in the form of spontaneous Ca2+ waves) under basal conditions and that Ca2+ leakage was exacerbated by isoproterenol (ISO, 100 nM). At the molecular level, [3H]-ryanodine binding and basal phosphorylation of cardiac ryanodine receptor (RyR2) at Ser2814, a CaMKII site, were increased in heart homogenates of MetS rats with no changes in RyR2 expression. These alterations were not further augmented by Isoproterenol. SERCA pump activity was augmented 48 % in MetS hearts before β-adrenergic stimuli, which is associated to augmented PLN phosphorylation at T17, a target of CaMKII. In MetS hearts. CaMKII auto-phosphorylation (T287) was increased by 80 %. The augmented diastolic Ca2+ leak was prevented by CaMKII inhibition with AIP. In conclusion, CaMKII autonomous activation in cardiomyocytes of MetS rats with central obesity significantly contributes to abnormal diastolic Ca2+ leak, increasing the propensity for β-adrenergic receptor-driven lethal arrhythmias.

The effect of temperature acclimation on the force-frequency relationship and adrenergic sensitivity of the ventricle of two populations of juvenile sockeye salmon.

We tested the hypothesis that cardiorespiratory differences known to exist among adult sockeye salmon populations also exist in the juveniles. To test this hypothesis, we compared cardiac contractility and adrenergic responsiveness of juvenile sockeye salmon from two geographically isolated populations that were reared from eggs under common garden conditions and at two acclimation temperatures (5°C and 14°C). However, we found no substantive differences in the force-frequency response (FFR) and the cardiac pumping capacity of juveniles from Weaver Creek and Chilko River populations, even when we considered wild-reared juveniles from one of the populations. An unexpected discovery for all fish groups at 5°C was a rather flat FFR during tonic β-adrenergic stimulation (βAR) stimulation. Curiously, while active tension nearly doubled with maximum βAR stimulation at low pacing frequencies for all fish groups, a negative FFR with maximum βAR stimulation meant that this inotropic benefit was lost at the highest pacing frequency (0.8Hz). Active tension with tonic βAR stimulation was similar at 14°C, but maximum pacing frequency doubled and all fish groups displayed a modest negative FFR. Maximum βAR stimulation again doubled active tension and this benefit was retained even at the highest pacing frequency (1.6Hz) at 14°C. Even though subtle population differences were apparent for the FFR and pumping capacity, their biological significance is unclear. What is clear, however, is that the cardiac pumping capacity of juvenile sockeye would benefit more from βAR stimulation swimming at 15°C than when swimming at 5°C.

Differentiating the effects of β-adrenergic stimulation and stretch on calcium and force dynamics using a novel electromechanical cardiomyocyte model.

Cardiac electrophysiology and mechanics are strongly interconnected. Calcium is crucial in this complex interplay through its role in cellular electrophysiology and sarcomere contraction. We aim to differentiate the effects of acute β-adrenergic stimulation (β-ARS) and cardiomyocyte stretch (increased sarcomere length) on calcium-transient dynamics and force generation, using a novel computational model of cardiac electromechanics. We implemented a bidirectional coupling between the O'Hara-Rudy model of human ventricular electrophysiology and the MechChem model of sarcomere mechanics through the buffering of calcium by troponin. The coupled model was validated using experimental data from large mammals or human samples. Calcium transient and force were simulated for various degrees of β-ARS and initial sarcomere lengths. The model reproduced force-frequency, quick-release, and isotonic contraction experiments, validating the bidirectional electromechanical interactions. An increase in β-ARS increased the amplitudes of force (augmented inotropy) and calcium transient, and shortened both force and calcium-transient duration (lusitropy). An increase in sarcomere length increased force amplitude even more, but decreased calcium-transient amplitude and increased both force and calcium-transient duration. Finally, a gradient in relaxation along the thin filament may explain the nonmonotonic decay in cytosolic calcium observed with high tension. Using a novel coupled human electromechanical model, we identified differential effects of β-ARS and stretch on calcium and force. Stretch mostly contributed to increased force amplitude and β-ARS to the reduction of calcium and force duration. We showed that their combination, rather than individual contributions, is key to ensure force generation, rapid relaxation, and low diastolic calcium levels.NEW & NOTEWORTHY This work identifies the contribution of electrical and mechanical alterations to regulation of calcium and force under exercise-like conditions using a novel human electromechanical model integrating ventricular electrophysiology and sarcomere mechanics. By better understanding their individual and combined effects, this can uncover arrhythmogenic mechanisms in exercise-like situations. This publicly available model is a crucial step toward understanding the complex interplay between cardiac electrophysiology and mechanics to improve arrhythmia risk prediction and treatment.

Abstract 506: Enhanced Mitochondrial Calcium Uptake During Chronic β-adrenergic Stimulation Causes Maladaptive Remodeling and Impaired Left Ventricular Function Independent of Cyclophilin D-mediated Mitochondrial Permeability Transition

The mitochondrial calcium uniporter (MCU) forms the pore of the mitochondrial calcium uniporter channel (mtCU) and is required for rapid mitochondrial Ca 2+ ( m Ca 2+ ) uptake. MCU is necessary to increase cardiac energetics to fuel an increase in cardiac contractility during acute sympathetic stimulation. However, little is known about how MCU-dependent Ca 2+ flux may contribute to the heart’s adaptations to chronic stress. We therefore compared mice with adult cardiomyocyte (ACM)-specific loss ( Mcu fl/fl ; Mcu-cKO) or gain (CAG-CAT-MCU; MCU-Tg) of MCU function to examine the role of MCU-dependent m Ca 2+ uptake in a model of chronic catecholamine overload. In vitro characterization of ACMs confirmed that MCU overexpression enhanced and Mcu deletion inhibited acute m Ca 2+ uptake. Neither loss nor gain of MCU function altered baseline contractile function in vivo . In αMHC-MCM control mice, fractional shortening was transiently increased after 2 days of isoproterenol infusion. This initial increase in contractility was attenuated in MCU-cKO mice. In contrast, MCU-Tg mice exhibited decreased fractional shortening at 7 and 14 days of isoproterenol infusion. This detrimental effect on contractile function was associated with increased LV dilation, HW/BW ratio, and lung edema. MCU-Tg cardiomyocytes in vitro exhibited increased ROS production and a trend towards increased cell death upon elevation of cytosolic Ca 2+ with ionomycin. These data prompted us to test the hypothesis that isoproterenol-induced contractile dysfunction in MCU-Tg hearts is caused by cardiomyocyte dropout due to m Ca 2+ overload and mitochondrial permeability transition. However, genetic deletion of the mPTP component cyclophilin D did not prevent the decline in contractile function, diminish cardiomyocyte death, or attenuate LV remodeling in MCU-Tg animals during chronic isoproterenol infusion. We conclude that although mtCU-dependent m Ca 2+ uptake is essential for early energetic adaptations to high adrenergic load, under conditions of chronic adrenergic stress it is maladaptive and predisposes to heart failure. Our data suggest that this maladaptive response occurs via mechanisms independent of cyclophilin D-mediated permeability transition.

Sex-specific IKAS activation in rabbit ventricles with drug-induced QT prolongation

Female sex is a known risk factor for drug-induced long QT syndrome (diLQTS). We recently demonstrated a sex difference in apamin-sensitive small-conductance Ca2+-activated K+ current (IKAS) activation during β-adrenergic stimulation. The purpose of this study was to test the hypothesis that there is a sex difference in IKAS in the rabbit models of diLQTS. We evaluated the sex difference in ventricular repolarization in 15 male and 22 female Langendorff-perfused rabbit hearts with optical mapping techniques during atrial pacing. HMR1556 (slowly activating delayed rectifier K+ current [IKs] blocker), E4031 (rapidly activating delayed rectifier K+ current [IKr] blocker) and sea anemone toxin (ATX-II, late Na+ current [INaL] activator) were used to simulate types 1-3 long QT syndrome, respectively. Apamin, an IKAS blocker, was then added to determine the magnitude of further QT prolongation. HMR1556, E4031, and ATX-II led to the prolongation of action potential duration at 80% repolarization (APD80) in both male and female ventricles at pacing cycle lengths of 300-400 ms. Apamin further prolonged APD80 (pacing cycle length 350 ms) from 187.8±4.3 to 206.9±7.1 (P=.014) in HMR1556-treated, from 209.9±7.8 to 224.9±7.8 (P=.003) in E4031-treated, and from 174.3±3.3 to 188.1±3.0 (P=.0002) in ATX-II-treated female hearts. Apamin did not further prolong the APD80 in male hearts. The Cai transient duration (CaiTD) was significantly longer in diLQTS than baseline but without sex differences. Apamin did not change CaiTD. We conclude that IKAS is abundantly increased in female but not in male ventricles with diLQTS. Increased IKAS helps preserve the repolarization reserve in female ventricles treated with IKs and IKr blockers or INaL activators.

14-3-3ζ mediates an alternative, non-thermogenic mechanism in male mice to reduce heat loss and improve cold tolerance

ObjectiveAdaptive thermogenesis, which is partly mediated by sympathetic input on brown adipose tissue (BAT), is a mechanism of heat production that confers protection against prolonged cold exposure. Various endogenous stimuli, for example, norepinephrine and FGF-21, can also promote the conversion of inguinal white adipocytes to beige adipocytes, which may represent a secondary cell type that contributes to adaptive thermogenesis. We previously identified an essential role of the molecular scaffold 14-3-3ζ in adipogenesis, but one of the earliest, identified functions of 14-3-3ζ is its regulatory effects on the activity of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of norepinephrine. Herein, we examined whether 14-3-3ζ could influence adaptive thermogenesis via actions on BAT activation or the beiging of white adipocytes. MethodsTransgenic mice over-expressing a TAP-tagged human 14-3-3ζ molecule or heterozygous mice without one allele of Ywhaz, the gene encoding 14-3-3ζ, were used to explore the contribution of 14-3-3ζ to acute (3 h) and prolonged (3 days) cold (4 °C) exposure. Metabolic caging experiments, PET-CT imaging, and laser Doppler imaging were used to determine the effect of 14-3-3ζ over-expression on thermogenic and vasoconstrictive mechanisms in response to cold. ResultsTransgenic over-expression of 14-3-3ζ (TAP) in male mice significantly improved tolerance to acute and prolonged cold. In response to cold, body temperatures in TAP mice did not decrease to the same extent when compared to wildtype (WT) mice, and this was associated with increased UCP1 expression in beige inguinal white tissue (iWAT) and BAT. Of note was the paradoxical finding that cold-induced changes in body temperatures of TAP mice were associated with significantly decreased energy expenditure. The marked improvements in tolerance to prolonged cold were not due to changes in sensitivity to β-adrenergic stimulation or BAT or iWAT oxidative metabolism; instead, over-expression of 14-3-3ζ significantly decreased thermal conductance and heat loss in mice via increased peripheral vasoconstriction. ConclusionsDespite being associated with elevations in cold-induced UCP1 expression in brown or beige adipocytes, these findings suggest that 14-3-3ζ regulates an alternative, non-thermogenic mechanism via vasoconstriction to minimize heat loss during cold exposure.