Showing papers by "Donald M. Bers published in 2021"

Intracellular β1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility

Abstract: Rationale: β1ARs (β1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intra...

CaMKII Serine 280 O-GlcNAcylation Links Diabetic Hyperglycemia to Proarrhythmia.

Abstract: Rationale: Diabetic hyperglycemia is associated with cardiac dysfunction and increased arrhythmia risk, and calcium/calmodulin-dependent protein kinase II (CaMKII) function has been implicated. CaMKII activity is promoted by both oxidation and O linked β-N-acetylglucosamine (O GlcNAc) of known CaMKII sites. Objective: To investigate which post-translational modifications occur in human diabetic hearts and how they alter electrophysiological and Ca2+ handling properties in hyperglycemia. Methods and Results: We assessed echocardiography, electrophysiology, Ca2+-handling, and protein expression in site-specific CaMKII mutant mice (O GlcNAc-resistant S280A and oxidation-resistant MM281/2VV knock-ins, and global and cardiac-specific knockouts), in myocytes subjected to acute hyperglycemia and angiotensin II (Ang-II) and mice after streptozotocin injections (to induce diabetes). Human patients with diabetes exhibit elevated CaMKII O GlcNAcylation but not oxidation. In mice, acute hyperglycemia increased spontaneous diastolic Ca2+ sparks and waves and arrhythmogenic action potential changes (prolongation, alternans and delayed afterdepolarizations), all of which required CaMKII-S280 O GlcNAcylation. Ang-II effects were dependent on NADPH oxidase 2 (NOX2)-mediated CaMKII MM281/2 oxidation. Diabetes led to much greater Ca2+ leak, RyR2 S2814 phosphorylation, electrophysiological remodeling, and increased susceptibility to in vivo arrhythmias, requiring CaMKII activation, predominantly via S280 O GlcNAcylation and less via MM281/2 oxidation. These effects were present in myocytes at normal glucose, but were exacerbated with the in-vivo high circulating glucose. Phospholamban (PLB) O-GlcNAcylation was increased and coincided with reduced PLB S16 phosphorylation in diabetes. Dantrolene, that reverses CaMKII-dependent proarrhythmic RyR-mediated Ca2+ leak, also prevented hyperglycemia-induced APD prolongation and delayed afterdepolarizations. Conclusions: We found that CaMKII-S280 O GlcNAcylation is required for increased arrhythmia susceptibility in diabetic hyperglycemia, which can be worsened by an additional angiotensin II-NOX2-CaMKII MM281/2 oxidation pathway. CaMKII-dependent RyR2 S2814 phosphorylation markedly increases proarrhythmic Ca2+ leak and PLB O-GlcNAcylation may limit SR Ca2+ reuptake, leading to impaired excitation-contraction coupling and arrhythmogenesis in diabetic hyperglycemia.

Cardiomyocyte Na+ and Ca2+ mishandling drives vicious cycle involving CaMKII, ROS, and ryanodine receptors.

Abstract: Cardiomyocyte Na+ and Ca2+ mishandling, upregulated Ca2+/calmodulin-dependent kinase II (CaMKII), and increased reactive oxygen species (ROS) are characteristics of various heart diseases, including heart failure (HF), long QT (LQT) syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). These changes may form a vicious cycle of positive feedback to promote cardiac dysfunction and arrhythmias. In HF rabbit cardiomyocytes investigated in this study, the inhibition of CaMKII, late Na+ current (INaL), and leaky ryanodine receptors (RyRs) all attenuated the prolongation and increased short-term variability (STV) of action potential duration (APD), but in age-matched controls these inhibitors had no or minimal effects. In control cardiomyocytes, we enhanced RyR leak (by low [caffeine] plus isoproterenol mimicking CPVT) which markedly increased STV and delayed afterdepolarizations (DADs). These proarrhythmic changes were significantly attenuated by both CaMKII inhibition and mitochondrial ROS scavenging, with a slight synergy with INaL inhibition. Inducing LQT by elevating INaL (by Anemone toxin II, ATX-II) caused markedly prolonged APD, increased STV, and early afterdepolarizations (EADs). Those proarrhythmic ATX-II effects were largely attenuated by mitochondrial ROS scavenging, and partially reduced by inhibition of CaMKII and pathological leaky RyRs using dantrolene. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) bearing LQT3 mutation SCN5A N406K, dantrolene significantly attenuated cell arrhythmias and APD prolongation. Targeting critical components of the Na+-Ca2+-CaMKII-ROS-INaL arrhythmogenic vicious cycle may exhibit important on-target and also trans-target effects (e.g., INaL and RyR inhibition can alter INaL-mediated LQT3 effects). Incorporating this vicious cycle into therapeutic strategies provides novel integrated insight for treating cardiac arrhythmias and diseases.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

Abstract: Background: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates a2+-reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates a2+-release from SR and triggers contraction. a2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulates activities of SERCA2 through phosphorylation of phospholamban (PLN) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local a2+-signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. Methods: A role for A-kinase anchoring protein 18δ (AKAP18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull-downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR-vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adeno-associated virus injection, structural modeling, surface plasmon resonance and alpha screen technology. Results: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via two distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to natural CaMKII inhibitor peptide and Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent a2+-threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster a2+-reuptake by SERCA2 and a2+-release through RYR, AKAP18δ-N had opposite effects. We propose a model where the two unique AKAP18δ regions fine-tune a2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. Conclusions: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

Two-hit mechanism of cardiac arrhythmias in diabetic hyperglycemia: reduced repolarization reserve, neurohormonal stimulation and heart failure exacerbate susceptibility.

Abstract: Aim Diabetic hyperglycemia is associated with increased arrhythmia risk. We aimed to investigate whether hyperglycemia alone can be accountable for arrhythmias or whether it requires the presence of additional pathological factors. Methods and results Action potentials (APs) and arrhythmogenic spontaneous diastolic activities were measured in isolated murine ventricular, rabbit atrial and ventricular myocytes acutely exposed to high glucose. Acute hyperglycemia increased the short-term variability (STV) of action potential duration (APD), enhanced delayed afterdepolarizations and the inducibility of APD alternans during tachypacing in both murine and rabbit atrial and ventricular myocytes. Hyperglycemia also prolonged APD in mice and rabbit atrial cells but not in rabbit ventricular myocytes. However, rabbit ventricular APD was more strongly depressed by block of late Na+ current (INaL) during hyperglycemia, consistent with elevated INaL in hyperglycemia. All the above proarrhythmic glucose effects were Ca2+-dependent and abolished by CaMKII inhibition. Importantly, when the repolarization reserve was reduced by pharmacological inhibition of K+ channels (either Ito, IKr, IKs or IK1) or hypokalemia, acute hyperglycemia further prolonged APD and further increased STV and alternans in rabbit ventricular myocytes. Likewise, when rabbit ventricular myocytes were pretreated with isoproterenol or angiotensin II, hyperglycemia significantly prolonged APD, increased STV and promoted alternans. Moreover, acute hyperglycemia markedly prolonged APD and further enhanced STV in failing rabbit ventricular myocytes. Conclusion We conclude that even though hyperglycemia alone can enhance cellular proarrhythmic mechanisms, a second hit which reduces the repolarization reserve or stimulates G protein-coupled receptor signaling greatly exacerbates cardiac arrhythmogenesis in diabetic hyperglycemia.

JNK2, a Newly-Identified SERCA2 Enhancer, Augments an Arrhythmic [Ca2+]SR Leak-Load Relationship

Abstract: Rationale: We recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic re...

Ketone Ester D-β-Hydroxybutyrate-(R)-1,3 Butanediol Prevents Decline in Cardiac Function in Type 2 Diabetic Mice.

Abstract: Background Heart failure is responsible for approximately 65% of deaths in patients with type 2 diabetes mellitus. However, existing therapeutics for type 2 diabetes mellitus have limited success o...

CaMKII and PKA-dependent phosphorylation co-regulate nuclear localization of HDAC4 in adult cardiomyocytes

Abstract: Nuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII-but not PKA, PKC or PKD-raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (FNuc/FCyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca2+], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising FNuc/FCyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.

Inositol Trisphosphate Receptors and Nuclear Calcium in Atrial Fibrillation.

Abstract: Rationale: The mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca2+ regulates gene expression, but the nature and significa...

Increasing SERCA function promotes initiation of calcium sparks and breakup of calcium waves

Abstract: KEY POINTS Increasing sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump activity enhances sarcoplasmic reticulum calcium (Ca) load, which increases both ryanodine receptor opening and driving force of Ca release flux. Both of these effects promote Ca spark formation and wave propagation. However, increasing SERCA activity also accelerates local cytosolic Ca decay as the wave front travels to the next cluster, which limits wave propagation. As a result, increasing SERCA pump activity has a biphasic effect on the propensity of arrhythmogenic Ca waves, but a monotonic effect to increase Ca spark frequency and amplitude. ABSTRACT Waves of sarcoplasmic reticulum (SR) calcium (Ca) release can cause arrhythmogenic afterdepolarizations in cardiac myocytes. Ca waves propagate when Ca sparks at one Ca release unit (CRU) recruit new Ca sparks in neighbouring CRUs. Under normal conditions, Ca sparks are too small to recruit neighbouring Ca sparks where Ca sensitivity is also low. However, under pathological conditions such as a Ca overload or ryanodine receptor (RyR) sensitization, Ca sparks can be larger and propagate more readily as macro-sparks or full Ca waves. Increasing SERCA pump activity promotes SR Ca load, which promotes RyR opening and increases driving force of the Ca release flux from SR to cytosol, promoting Ca waves. However, high sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) activity can also decrease local cytosolic [Ca] as it approaches the next CRU, thereby reducing wave appearance and propagation. In this study, we use a physiologically detailed model of subcellular Ca cycling and experiments in phospholamban-knockout mice, to show how Ca waves are initiated and propagate and how different conditions contribute to the generation and propagation of Ca waves. We show that reducing diffusive coupling between Ca sparks by increasing SERCA activity prevents Ca waves by reducing [Ca] at the next CRU, as do Ca buffers, low intra-SR Ca diffusion and distance between CRUs. Increasing SR Ca uptake rate has a biphasic effect on Ca wave propagation; initially it enhances Ca spark probability and amplitude and CRU coupling, thereby promoting arrhythmogenic Ca wave propagation, but at higher levels SR Ca uptake can abort those arrhythmogenic Ca waves.

Quantitative cross-species translators of cardiac myocyte electrophysiology: Model training, experimental validation, and applications.

Abstract: Animal experimentation is key in the evaluation of cardiac efficacy and safety of novel therapeutic compounds. However, interspecies differences in the mechanisms regulating excitation-contraction ...

Mechanoelectric coupling and arrhythmogenesis in cardiomyocytes contracting under mechanical afterload in a 3D viscoelastic hydrogel.

Abstract: The heart pumps blood against the mechanical afterload from arterial resistance, and increased afterload may alter cardiac electrophysiology and contribute to life-threatening arrhythmias. However, the cellular and molecular mechanisms underlying mechanoelectric coupling in cardiomyocytes remain unclear. We developed an innovative patch-clamp-in-gel technology to embed cardiomyocytes in a three-dimensional (3D) viscoelastic hydrogel that imposes an afterload during regular myocyte contraction. Here, we investigated how afterload affects action potentials, ionic currents, intracellular Ca2+ transients, and cell contraction of adult rabbit ventricular cardiomyocytes. We found that afterload prolonged action potential duration (APD), increased transient outward K+ current, decreased inward rectifier K+ current, and increased L-type Ca2+ current. Increased Ca2+ entry caused enhanced Ca2+ transients and contractility. Moreover, elevated afterload led to discordant alternans in APD and Ca2+ transient. Ca2+ alternans persisted under action potential clamp, indicating that the alternans was Ca2+ dependent. Furthermore, all these afterload effects were significantly attenuated by inhibiting nitric oxide synthase 1 (NOS1). Taken together, our data reveal a mechano-chemo-electrotransduction (MCET) mechanism that acutely transduces afterload through NOS1-nitric oxide signaling to modulate the action potential, Ca2+ transient, and contractility. The MCET pathway provides a feedback loop in excitation-Ca2+ signaling-contraction coupling, enabling autoregulation of contractility in cardiomyocytes in response to afterload. This MCET mechanism is integral to the individual cardiomyocyte (and thus the heart) to intrinsically enhance its contractility in response to the load against which it has to do work. While this MCET is largely compensatory for physiological load changes, it may also increase susceptibility to arrhythmias under excessive pathological loading.

Role of Reduced Sarco-Endoplasmic Reticulum Ca2+-ATPase Function on Sarcoplasmic Reticulum Ca2+ Alternans in the Intact Rabbit Heart

Abstract: Sarcoplasmic reticulum (SR) Ca2+ cycling is tightly regulated by ryanodine receptor (RyR) Ca2+ release and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ uptake during each excitation-contraction coupling cycle. We previously showed that RyR refractoriness plays a key role in the onset of SR Ca2+ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca2+ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (Vm) and SR Ca2+ was performed in isolated rabbit hearts (n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca2+ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid (CPA; 1-10 μM). SERCA inhibition (1, 5, and 10 μM of CPA) resulted in dose-dependent slowing of SR Ca2+ reuptake, with the time constant (tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively (p < 0.05 vs. baseline for all doses). At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca2+ and APD alternans, most strongly at 10 μM (pacing cycle length = 220 ms: SR Ca2+ alternans magnitude: 57.1 ± 4.7 vs. 13.4 ± 8.9 AU; APD alternans magnitude 3.8 ± 1.9 vs. 0.2 ± 0.19 AU; p < 0.05 10 μM of CPA vs. baseline for both). SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca2+ release occurred prior to alternation of diastolic SR Ca2+ load as pacing frequency increased. Simultaneous optical mapping of SR Ca2+ and Vm in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca2+ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca2+ release alternans always occurred before the onset of SR Ca2+ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca2+ release governs the onset of intracellular Ca2+ alternans.

Loss of CASK Accelerates Heart Failure Development.

Abstract: Rationale: Increased myocardial activity of Ca/calmodulin-dependent kinase II (CaMKII) leads to heart failure (HF) and arrhythmias. In Drosophila neurons, interaction of CaMKII with Ca/CaM-dependent serine protein kinase (CASK) has been shown to inhibit CaMKII activity, but the consequences of this regulation for HF and ventricular arrhythmias are unknown. Objective: We hypothesize that CASK associates with CaMKII in human and mouse hearts thereby limiting CaMKII activity, and that altering CASK expression in mice changes CaMKII activity accordingly, with functional consequences for contractile function and arrhythmias. Methods and Results: Immunoprecipitation revealed that CASK associates with CaMKII in human hearts. CASK expression is unaltered in HF but increased in patients with aortic stenosis. In mice, cardiomyocyte-specific knockout of CASK (CASK-KO) increased CaMKII auto-phosphorylation at the stimulatory T287 site, but reduced phosphorylation at the inhibitory T305/306 site. CASK-KO mice showed increased CaMKII-dependent sarcoplasmic reticulum (SR) Ca leak, reduced SR Ca-content, increased susceptibility to ventricular arrhythmias, greater loss of ejection fraction, and increased mortality after transverse aortic constriction. Intriguingly, stimulation of the cardiac glucagon-like peptide 1-receptor with exenatide increased CASK expression resulting in increased inhibitory CaMKII T305 phosphorylation, reduced CaMKII activity, and reduced SR Ca leak in WT but not CASK KO. Conclusions: CASK associates with CaMKII in the human heart. CASK-KO in mice increases CaMKII activity, leading to contractile dysfunction and arrhythmias. Increasing CASK expression reduces CaMKII activity, improves Ca handling and contractile function.

PK11195 Protects From Cell Death Only When Applied During Reperfusion: Succinate-Mediated Mechanism of Action.

Abstract: Aim: Reperfusion after myocardial ischemia causes cellular injury, in part due to changes in mitochondrial Ca2+ handling, oxidative stress, and myocyte energetics. We have previously shown that the 18-kDa translocator protein of the outer mitochondrial membrane (TSPO) can modulate Ca2+ handling. Here, we aim to evaluate the role of the TSPO in ischemia/reperfusion (I/R) injury. Methods: Rabbit ventricular myocytes underwent simulated acute ischemia (20 min) and reperfusion (at 15 min, 1 h, and 3 h) in the absence and presence of 50 μM PK11195, a TSPO inhibitor. Cell death was measured by lactate dehydrogenase (LDH) assay, while changes in mitochondrial Ca2+, membrane potential (ΔΨm), and reactive oxygen species (ROS) generation were monitored using confocal microscopy in combination with fluorescent indicators. Substrate utilization was measured with Biolog mitochondrial plates. Results: Cell death was increased by ~200% following I/R compared to control untreated ventricular myocytes. Incubation with 50 μM PK11195 during both ischemia and reperfusion did not reduce cell death but increased mitochondrial Ca2+ uptake and ROS generation. However, application of 50 μM PK11195 only at the onset and during reperfusion effectively protected against cell death. The large-scale oscillations in ΔΨm observed after ~1 h of reperfusion were significantly delayed by 1 μM cyclosporin A and almost completely prevented by 50 μM PK11195 applied during 3 h of reperfusion. After an initial increase, mitochondrial Ca2+, measured with Myticam, rapidly declined during 3 h of reperfusion after the initial transient increase. This decline was prevented by application of PK11195 at the onset and during reperfusion. PK11195 prevented a significant increase in succinate utilization following I/R and succinate-induced forward-mode ROS generation. Treatment with PK11195 was also associated with a significant increase in glutamate and a decrease in leucine utilization. Conclusion: PK11195 administered specifically at the moment of reperfusion limited ROS-induced ROS release and cell death, likely in part, by a shift from succinate to glutamate utilization. These data demonstrate a unique mechanism to limit cardiac injury after I/R.