Search

Persistent over-activation of CaMKII (Calcium/Calmodulin-dependent protein Kinase II) in the heart is implicated in arrhythmias, heart failure, pathological remodeling, and other cardiovascular diseases. Several post-translational modifications (PTMs)—including autophosphorylation, oxidation, S-nitrosylation, and O-GlcNAcylation—have been shown to trap CaMKII in an autonomously active state. The molecular mechanisms by which these PTMs regulate calmodulin (CaM) binding to CaMKIIδ—the primary cardiac isoform—has not been well-studied particularly in its native myocyte environment. Typically, CaMKII activates upon Ca-CaM binding during locally elevated [Ca]free and deactivates upon Ca-CaM dissociation when [Ca]free returns to basal levels. To assess the effects of CaMKIIδ PTMs on CaM binding, we developed a novel FRET (Forster resonance energy transfer) approach to directly measure CaM binding to and dissociation from CaMKIIδ in live cardiac myocytes. We demonstrate that autophosphorylation of CaMKIIδ increases affinity for CaM in its native environment and that this increase is dependent on [Ca]free. This leads to a 3-fold slowing of CaM dissociation from CaMKIIδ (time constant slows from ~0.5 to 1.5 s) when [Ca]free is reduced with physiological kinetics. Moreover, oxidation further slows CaM dissociation from CaMKIIδ T287D (phosphomimetic) upon rapid [Ca]free chelation and increases FRET between CaM and CaMKIIδ T287A (phosphoresistant). The CaM dissociation kinetics–measured here in myocytes–are similar to the interval between heartbeats, and integrative memory would be expected as a function of heart rate. Furthermore, the PTM-induced slowing of dissociation between beats would greatly promote persistent CaMKIIδ activity in the heart. Together, these findings suggest a significant role of PTM-induced changes in CaMKIIδ affinity for CaM and memory under physiological and pathophysiological processes in the heart.

Aims Diabetic hyperglycaemia is associated with increased arrhythmia risk. We aimed to investigate whether hyperglycaemia 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 hyperglycaemia 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. Hyperglycaemia 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 hyperglycaemia, consistent with elevated INaL in hyperglycaemia. 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 hypokalaemia, acute hyperglycaemia 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, hyperglycaemia significantly prolonged APD, increased STV and promoted alternans. Moreover, acute hyperglycaemia markedly prolonged APD and further enhanced STV in failing rabbit ventricular myocytes. Conclusion We conclude that even though hyperglycaemia alone can enhance cellular proarrhythmic mechanisms, a second hit which reduces the repolarization reserve or stimulates G protein-coupled receptor signalling greatly exacerbates cardiac arrhythmogenesis in diabetic hyperglycaemia.

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca2+ release during excitation–contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose–response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation–contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation–contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.

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.

A key therapeutic target for heart failure and arrhythmia is the deleterious leak through sarcoplasmic reticulum (SR) ryanodine receptor 2 (RyR2) calcium release channels. We have previously developed methods to detect the pathologically leaky state of RyR2 in adult cardiomyocytes by monitoring RyR2 binding to either calmodulin (CaM) or a biosensor peptide (DPc10). Here, we test whether these complementary binding measurements are effective as high-throughput screening (HTS) assays to discover small molecules that target leaky RyR2. Using FRET, we developed and validated HTS procedures under conditions that mimic a pathological state, to screen the library of 1280 pharmaceutically active compounds (LOPAC) for modulators of RyR2 in cardiac SR membrane preparations. Complementary FRET assays with acceptor-labeled CaM and DPc10 were used for Hit prioritization based on the opposing binding properties of CaM vs. DPc10. This approach narrowed the Hit list to one compound, Ro 90-7501, which altered FRET to suggest increased RyR2-CaM binding and decreased DPc10 binding. Follow-up studies revealed that Ro 90-7501 does not detrimentally affect myocyte Ca

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.

Supplemental Digital Content is available in the text., Rationale: Diabetic hyperglycemia is associated with cardiac dysfunction and increased arrhythmia risk, and CaMKII (calcium/calmodulin-dependent protein kinase II) 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 posttranslational 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 Ang II (angiotensin 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 NOX2 (NADPH oxidase 2)-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. PLB (phospholamban) O-GlcNAcylation was increased and coincided with reduced PLB S16 phosphorylation in diabetes. Dantrolene, which 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 Ang II-NOX2-CaMKII MM281/2 oxidation pathway. CaMKII-dependent RyR2 S2814 phosphorylation markedly increases proarrhythmic Ca2+ leak and PLB O-GlcNAcylation may limit sarcoplasmic reticulum Ca2+ reuptake, leading to impaired excitation-contraction coupling and arrhythmogenesis in diabetic hyperglycemia.

Spontaneous calcium (Ca) waves in cardiac myocytes underlie delayed afterdepolarizations (DADs) that trigger cardiac arrhythmias. How these subcellular/cellular events overcome source-sink factors in cardiac tissue to generate DADs of sufficient amplitude to trigger action potentials is not fully understood. Here, we evaluate quantitatively how factors at the subcellular scale (number of Ca wave initiation sites), cellular scale (sarcoplasmic reticulum (SR) Ca load), and tissue scale (synchrony of Ca release in populations of myocytes) determine DAD features in cardiac tissue using a combined experimental and computational modeling approach. Isolated patch-clamped rabbit ventricular myocytes loaded with Fluo-4 to image intracellular Ca were rapidly paced during exposure to elevated extracellular Ca (2.7 mmol/L) and isoproterenol (0.25 μmol/L) to induce diastolic Ca waves and subthreshold DADs. As the number of paced beats increased from 1 to 5, SR Ca content (assessed with caffeine pulses) increased, the number of Ca wave initiation sites increased, integrated Ca transients and DADs became larger and shorter in duration, and the latency period to the onset of Ca waves shortened with reduced variance. In silico analysis using a computer model of ventricular tissue incorporating these experimental measurements revealed that whereas all of these factors promoted larger DADs with higher probability of generating triggered activity, the latency period variance and SR Ca load had the greatest influences. Therefore, incorporating quantitative experimental data into tissue level simulations reveals that increased intracellular Ca promotes DAD-mediated triggered activity in tissue predominantly by increasing both the synchrony (decreasing latency variance) of Ca waves in nearby myocytes and SR Ca load, whereas the number of Ca wave initiation sites per myocyte is less important.

Ventricular myocytes are excitable cells whose voltage threshold for action potential (AP) excitation is ∼-60mV at which INa is activated to give rise to a fast upstroke. Therefore, for a short stimulus pulse to elicit an AP, a stronger stimulus is needed if the resting potential lies further away from the INa threshold, such as in hypokalemia. However, for an AP elicited by a long duration stimulus or a diastolic spontaneous calcium release, we observed that the stimulus needed was lower in hypokalemia than in normokalemia in both computer simulations and experiments of rabbit ventricular myocytes. This observation provides insight into why hypokalemia promotes calcium-mediated triggered activity, despite the resting potential lying further away from the INa threshold. To understand the underlying mechanisms, we performed bifurcation analyses and demonstrated that there is a dynamical threshold, resulting from a saddle-node bifurcation mainly determined by IK1 and INCX. This threshold is close to the voltage at which IK1 is maximum, and lower than the INa threshold. After exceeding this dynamical threshold, the membrane voltage will automatically depolarize above the INa threshold due to the large negative slope of the IK1-V curve. This dynamical threshold becomes much lower in hypokalemia, especially with respect to calcium, as predicted by our theory. Because of the saddle-node bifurcation, the system can automatically depolarize even in the absence of INa to voltages higher than the ICa,L threshold, allowing for triggered APs in single myocytes with complete INa block. However, because INa is important for AP propagation in tissue, blocking INa can still suppress premature ventricular excitations in cardiac tissue caused by calcium-mediated triggered activity. This suppression is more effective in normokalemia than in hypokalemia due to the difference in dynamical thresholds.

Hypertension is a risk factor for sudden cardiac death caused by ventricular tachycardia and fibrillation (VT/VF). We hypothesized that, in early hypertension, the susceptibility to stress-induced VT/VF increases. We compared the susceptibility of 5- to 6-month-old male spontaneously hypertensive rats (SHR) and age/sex-matched normotensive rats (NR) to VT/VF during challenge with oxidative stress (H2 O2 ; 0.15 mmol l(-1) ). We found that only SHR hearts exhibited left ventricular fibrosis and hypertrophy. H2 O2 promoted VT in all 30 SHR but none of the NR hearts. In 33% of SHR cases, focal VT degenerated to VF within 3 s. Simultaneous voltage-calcium optical mapping of Langendorff-perfused SHR hearts revealed that H2 O2 -induced VT/VF arose spontaneously from focal activations at the base and mid left ventricular epicardium. Microelectrode recording of SHR hearts showed that VT was initiated by early afterdepolarization (EAD)-mediated triggered activity. However, despite the increased susceptibility of SHR hearts to VT/VF, patch clamped isolated SHR ventricular myocytes developed EADs and triggered activity to the same extent as NR ventricular myocytes, except with larger EAD amplitude. During the early stages of hypertension, when challenged with oxidative stress, SHR hearts showed an increased ventricular arrhythmogenicity that stems primarily from tissue remodelling (hypertrophy, fibrosis) rather than cellular electrophysiological changes. Our findings highlight the need for early hypertension treatment to minimize myocardial fibrosis, ventricular hypertrophy, and arrhythmias.

Summary Despite over a decade of intense research, the identity and differentiation potential of human adult cardiac progenitor cells (aCPC) remains controversial. Cardiospheres have been proposed as a means to expand aCPCs in vitro, but the identity of the progenitor cell within these 3D structures is unknown. We show that clones derived from cardiospheres could be subdivided based on expression of thymocyte differentiation antigen 1 (THY-1/CD90) into two distinct populations that exhibit divergent cardiac differentiation potential. One population, which is CD90+, expressed markers consistent with a mesenchymal/myofibroblast cell. The second clone type was CD90− and could form mature, functional myocytes with sarcomeres albeit at a very low rate. These two populations of cardiogenic clones displayed distinct cell surface markers and unique transcriptomes. Our study suggests that a rare aCPC exists in cardiospheres along with a mesenchymal/myofibroblast cell, which demonstrates incomplete cardiac myocyte differentiation., Highlights • The majority of human adult CS-derived cardiomyocytes do not form sarcomeres • There is little overlap between the C-KIT and NKX2.5 cell populations in human CS • CD90 distinguishes two populations that exhibit divergent cardiogenic potential • CD90neg CS cells can differentiate into mature cardiomyocytes at a very low rate, Cardiospheres (CSs) have been reported to contain an adult cardiac progenitor cell (aCPC) with multilineage differentiation potential. MacLellan and colleagues show that CS-derived cells differentiate robustly into endothelial and smooth muscle cells but predominantly form immature cardiomyocytes. CD90 expression distinguishes CS-derived cells with a mesenchymal cell phenotype with incomplete cardiomyocyte differentiation (CD90+) and a CD90− population that could give rise to mature cardiomyocytes, although infrequently.

Calcium (Ca(2+)) waves generating oscillatory Ca(2+) signals are widely observed in biological cells. Experimental studies have shown that under certain conditions, initiation of Ca(2+) waves is random in space and time, while under other conditions, waves occur repetitively from preferred locations (pacemaker sites) from which they entrain the whole cell. In this study, we use computer simulations to investigate the self-organization of Ca(2+) sparks into pacemaker sites generating Ca(2+) oscillations. In both ventricular myocyte experiments and computer simulations of a heterogeneous Ca(2+) release unit (CRU) network model, we show that Ca(2+) waves occur randomly in space and time when the Ca(2+) level is low, but as the Ca(2+) level increases, waves occur repetitively from the same sites. Our analysis indicates that this transition to entrainment can be attributed to the fact that random Ca(2+) sparks self-organize into Ca(2+) oscillations differently at low and high Ca(2+) levels. At low Ca(2+), the whole cell Ca(2+) oscillation frequency of the coupled CRU system is much slower than that of an isolated single CRU. Compared to a single CRU, the distribution of interspike intervals (ISIs) of the coupled CRU network exhibits a greater variation, and its ISI distribution is asymmetric with respect to the peak, exhibiting a fat tail. At high Ca(2+), however, the coupled CRU network has a faster frequency and lesser ISI variation compared to an individual CRU. The ISI distribution of the coupled network no longer exhibits a fat tail and is well-approximated by a Gaussian distribution. This same Ca(2+) oscillation behaviour can also be achieved by varying the number of ryanodine receptors per CRU or the distance between CRUs. Using these results, we develop a theory for the entrainment of random oscillators which provides a unified explanation for the experimental observations underlying the emergence of pacemaker sites and Ca(2+) oscillations.

Hypertension is a risk factor for sudden cardiac death caused by ventricular tachycardia and fibrillation (VT/VF). We hypothesized that during the early stages of uncontrolled hypertension, the susceptibility to otherwise benign stress promotes VT/VF. To test this hypothesis we compared the susceptibility of 5-6-month-old male spontaneously hypertensive rats (SHR) and age/gender-matched normotensive rats (NR) to VT/VF during challenge with mild oxidative stress (H2O2, 0.15 mmol/L). H2O2 initiated spontaneous VT in all 30 SHR but none of the NR hearts. In 33% of SHR cases, focal VT degenerated to VF within 3 s. Simultaneous voltage-Ca optical mapping of Langendorff-perfused SHR hearts revealed that H2O2-induced VT/VF arose spontaneously from focal activations at the base and mid left ventricular epicardium. Microelectrode recording of SHR hearts showed that VT was initiated by early afterdepolarization (EAD)-mediated triggered activity. However, despite the increased susceptibility of SHR hearts to VT/VF, patch-clamped isolated SHR ventricular myocytes developed EADs and triggered activity to the same extent as NR ventricular myocytes, except that SHR myocytes had a relatively larger EAD amplitude. Histopathological and echocardiographic studies showed that compared to NR, SHR hearts exhibited increased left ventricular fibrosis and mild hypertrophy. We conclude that during the early stages of hypertension (i.e., prior to the development of overt heart failure) SHR hearts manifest increased susceptibility to VT/VF when challenged with mild oxidative stress that appears to be related to tissue remodeling (i.e., hypertrophy, fibrosis) moreso than cellular electrophysiological remodeling. Our findings highlight the need for aggressive treatment during the stages of early hypertension to minimize myocardial fibrosis, ventricular hypertrophy, and arrhythmias.

Spontaneous calcium (Ca) release causes delayed afterdepolarizations (DADs) which can result in triggered activity (TA) if the DAD amplitude reaches a critical threshold. It is known that increasing Na-Ca exchange (NCX) or deceasing IK1, which both occur in heart failure remodeling, increases the diastolic Ca-voltage coupling gain potentiating TA. However, the relative influence of NCX, IK1, and the Ca release amplitude on the Ca-voltage coupling gain and threshold for TA are not well understood. Using computer simulation and stability analysis, we show that elevation of the membrane voltage to the TA threshold is governed by non-linear dynamics, consistent with experiments in rabbit ventricular myocytes. This results in the Ca-voltage coupling gain being a nonlinear function of NCX, IK1, and peak Ca. For example, in the UCLA rabbit action potential model, a clamped submembrane Ca peak of 0.4 μM corresponds to a 3.5 mV subthreshold DAD (below the threshold for TA). For this DAD, the NCX conductance had to be increased by a factor of 4 to elicit TA, but decreasing IK1 conductance even to zero failed to elicit TA. For a clamped Ca peak of 0.6 μM corresponding to a 7.5 mV subthreshold DAD, NCX conductance had to be increased by a factor of 2 to elicit TA, and now reducing IK1 by a factor of 4 was able to cause TA. When the peak Ca was increased to 1.1 μM, TA occurred with the unchanged control conductance values of NCX and IK1. Analysis of the underlying nonlinear dynamics provides the theoretical basis for the differential and nonlinear dependence of TA threshold and Ca-voltage coupling gain on peak Ca, NCX, and IK1.

Calcium (Ca) is a ubiquitous second messenger that regulates many biological functions. The elementary events of local Ca signaling are Ca sparks, which occur randomly in time and space, and integrate to produce global signaling events such as intra- and intercellular Ca waves and whole-cell Ca oscillations. Despite extensive experimental characterization in many systems, the transition from local random to global synchronous events is still poorly understood. Here we show that criticality, a ubiquitous dynamical phenomenon in nature, is responsible for the transition from local to global Ca signaling. We demonstrate this first in a computational model of Ca signaling in a cardiac myocyte and then experimentally in mouse ventricular myocytes, complemented by a theoretical agent-based model to delineate the underlying dynamics. We show that the interaction between the Ca release units via Ca-induced Ca release causes self-organization of Ca spark clusters. When the coupling between Ca release units is weak, the cluster-size distribution is exponential. As the interactions become strong, the cluster-size distribution changes to a power-law distribution, which is characteristic of criticality in thermodynamic and complex nonlinear systems, and facilitates the formation and propagation of Ca waves and whole-cell Ca oscillations. Our findings illustrate how criticality is harnessed by a biological cell to regulate Ca signaling via self-organization of random subcellular events into cellular-scale oscillations, and provide a general theoretical framework for the transition from local Ca signaling to global Ca signaling in biological cells.

Both Rb and p130 are required for the recruitment of heterochromatin proteins that mediate silencing of proliferation genes in adult cardiac myocytes., The mammalian heart loses its regenerative potential soon after birth. Adult cardiac myocytes (ACMs) permanently exit the cell cycle, and E2F-dependent genes are stably silenced, although the underlying mechanism is unclear. Heterochromatin, which silences genes in many biological contexts, accumulates with cardiac differentiation. H3K9me3, a histone methylation characteristic of heterochromatin, also increases in ACMs and at E2F-dependent promoters. We hypothesize that genes relevant for cardiac proliferation are targeted to heterochromatin by retinoblastoma (Rb) family members interacting with E2F transcription factors and recruiting heterochromatin protein 1 (HP1) proteins. To test this hypothesis, we created cardiac-specific Rb and p130 inducible double knockout (IDKO) mice. IDKO ACMs showed a decrease in total heterochromatin, and cell cycle genes were derepressed, leading to proliferation of ACMs. Although Rb/p130 deficiency had no effect on total H3K9me3 levels, recruitment of HP1-γ to promoters was lost. Depleting HP1-γ up-regulated proliferation-promoting genes in ACMs. Thus, Rb and p130 have overlapping roles in maintaining the postmitotic state of ACMs through their interaction with HP1-γ to direct heterochromatin formation and silencing of proliferation-promoting genes.

Duchenne muscular dystrophy is caused by dystrophin mutations that lead to structural instability of the sarcolemma membrane, myofiber degeneration/regeneration and progressive muscle wasting. Here we show that myogenic Akt signaling in mouse models of dystrophy promotes increased expression of utrophin, which replaces the function of dystrophin thereby preventing sarcolemma damage and muscle wasting. In contrast to previous suggestions that increased Akt in dystrophy was a secondary consequence of pathology, our findings demonstrate a pivotal role for this signaling pathway such that modulation of Akt can significantly affect disease outcome by amplification of existing, physiological compensatory mechanisms.

Background— Hypokalemia is known to promote ventricular arrhythmias, especially in combination with class III antiarrhythmic drugs like dofetilide. Here, we evaluated the underlying molecular mechanisms. Methods and Results— Arrhythmias were recorded in isolated rabbit and rat hearts or patch-clamped ventricular myocytes exposed to hypokalemia (1.0–3.5 mmol/L) in the absence or presence of dofetilide (1 μmol/L). Spontaneous early afterdepolarizations (EADs) and ventricular tachycardia/fibrillation occurred in 50% of hearts at 2.7 mmol/L [K] in the absence of dofetilide and 3.3 mmol/L [K] in its presence. Pretreatment with the Ca-calmodulin kinase II (CaMKII) inhibitor KN-93, but not its inactive analogue KN-92, abolished EADs and hypokalemia-induced ventricular tachycardia/fibrillation, as did the selective late Na current ( I Na ) blocker GS-967. In intact hearts, moderate hypokalemia (2.7 mmol/L) significantly increased tissue CaMKII activity. Computer modeling revealed that EAD generation by hypokalemia (with or without dofetilide) required Na-K pump inhibition to induce intracellular Na and Ca overload with consequent CaMKII activation enhancing late I Na and the L-type Ca current. K current suppression by hypokalemia and dofetilide alone in the absence of CaMKII activation were ineffective at causing EADs. Conclusions— We conclude that Na-K pump inhibition by even moderate hypokalemia plays a critical role in promoting EAD-mediated arrhythmias by inducing a positive feedback cycle activating CaMKII and enhancing late I Na . Class III antiarrhythmic drugs like dofetilide sensitize the heart to this positive feedback loop.

Phospholamban (PLB) inhibits the activity of cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a). Phosphorylation of PLB during sympathetic activation reverses SERCA2a inhibition, increasing SR Ca(2+) uptake. However, sympathetic activation also modulates multiple other intracellular targets in ventricular myocytes (VMs), making it impossible to determine the specific effects of the reversal of PLB inhibition on the spontaneous SR Ca(2+) release. Therefore, it remains unclear how PLB regulates rhythmic activity in VMs. Here, we used the Fab fragment of 2D12, a monoclonal anti-PLB antibody, to test how acute reversal of PLB inhibition affects the spontaneous SR Ca(2+) release in normal VMs. Ca(2+) sparks and spontaneous Ca(2+) waves (SCWs) were recorded in the line-scan mode of confocal microscopy using the Ca(2+) fluorescent dye Fluo-4 in isolated permeabilized mouse VMs. Fab, which reverses PLB inhibition, significantly increased the frequency, amplitude, and spatial/temporal spread of Ca(2+) sparks in VMs exposed to 50 nM free [Ca(2+)]. At physiological diastolic free [Ca(2+)] (100-200 nM), Fab facilitated the formation of whole-cell propagating SCWs. At higher free [Ca(2+)], Fab increased the frequency and velocity, but decreased the decay time of the SCWs. cAMP had little additional effect on the frequency or morphology of Ca(2+) sparks or SCWs after Fab addition. These findings were complemented by computer simulations. In conclusion, acute reversal of PLB inhibition alone significantly increased the spontaneous SR Ca(2+) release, leading to the facilitation and organization of whole-cell propagating SCWs in normal VMs. PLB thus plays a key role in subcellular Ca(2+) dynamics and rhythmic activity of VMs.

© 2015 Biophysical Society. Early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) are voltage oscillations known to cause cardiac arrhythmias. EADs are mainly driven by voltage oscillations in the repolarizing phase of the action potential (AP), while DADs are driven by spontaneous calcium (Ca) release during diastole. Because voltage and Ca are bidirectionally coupled, they modulate each other's behaviors, and new AP and Ca cycling dynamics can emerge from this coupling. In this study, we performed computer simulations using an AP model with detailed spatiotemporal Ca cycling incorporating stochastic openings of Ca channels and ryanodine receptors to investigate the effects of Ca-voltage coupling on EAD and DAD dynamics. Simulations were complemented by experiments in mouse ventricular myocytes. We show that: 1) alteration of the Ca transient due to increased ryanodine receptor leakiness and/or sarco/endoplasmic reticulum Ca ATPase activity can either promote or suppress EADs due to the complex effects of Ca on ionic current properties; 2) spontaneous Ca waves also exhibit complex effects on EADs, but cannot induce EADs of significant amplitude without the participation of ICa,L; 3) lengthening AP duration and the occurrence of EADs promote DADs by increasing intracellular Ca loading, and two mechanisms of DADs are identified, i.e., Ca-wave-dependent and Ca-wave-independent; and 4) Ca-voltage coupling promotes complex EAD patterns such as EAD alternans that are not observed for solely voltage-driven EADs. In conclusion, Ca-voltage coupling combined with the nonlinear dynamical behaviors of voltage and Ca cycling play a key role in generating complex EAD and DAD dynamics observed experimentally in cardiac myocytes, whose mechanisms are complex but analyzable.

Subcellular Calcium (Ca) cycling plays fundamental roles in normal heart dynamics. In cardiac myocytes, the elementary Ca cycling events are Ca sparks: random discretized Ca release events due to random and collective openings of the ryanodine receptor (RyR) channels clustered in Ca release units (CRUs). A typical cardiac myocyte includes about 10,000 to 20,000 CRUs, and the spatial arrangement of CRUs varies widely across myocyte type and changes in diseased conditions. Dysfunction of the CRU network leaves cells prone to subcellular Ca waves, notorious triggers of highly arrhythmogenic delayed afterdepolarizations. Recent experimental studies have isolated three timescales involved in the formation of Ca waves: rate of sarcoplasmic reticulum (SR) Ca reuptake, intrinsic RyR refractoriness, and a so-called “idle” period. Here we use a physiologically detailed computational model of a spatial and stochastic CRU network to study the variables that contribute to the aforementioned timescales, and identify how the relative dominance of each affects the morphology of Ca waves. We show that the “idle” period is far from idle, as it emerges out of complex Ca mediated CRU-to-CRU interactions in both the myoplasm and SR. We also find that while reduced refractory period and increased SR Ca diffusion enhance the local initiation of waves, they hinder propagation, resulting in fractionated wave events. Furthermore, at very short refractory periods the system degenerates into spiral waves and chaos. Pinpointing the mechanisms underlying the variety of observed wave morphologies is an important step in our understanding of diseased states, as each may play a different role in arrhythmogenesis.

Please cite this article in press as: Song et al., Calcium-Voltage Coupling in the Genesis of Early and Delayed Afterdepolarizations in Cardiac Myocytes, Bio- physical Journal (2015), http://dx.doi.org/10.1016/j.bpj.2015.03.011 Biophysical Journal Volume 108 April 2015 1–14 Article Calcium-Voltage Coupling in the Genesis of Early and Delayed Afterdepolarizations in Cardiac Myocytes Zhen Song, 1,2 Christopher Y. Ko, 1,2 Michael Nivala, 1,2 James N. Weiss, 1,2,3 and Zhilin Qu 1,2, * Cardiovascular Research Laboratory, 2 Department of Medicine (Cardiology), and 3 Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, California ABSTRACT Early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) are voltage oscillations known to cause cardiac arrhythmias. EADs are mainly driven by voltage oscillations in the repolarizing phase of the action potential (AP), while DADs are driven by spontaneous calcium (Ca) release during diastole. Because voltage and Ca are bidirectionally coupled, they modulate each other’s behaviors, and new AP and Ca cycling dynamics can emerge from this coupling. In this study, we performed computer simulations using an AP model with detailed spatiotemporal Ca cycling incorporating stochastic openings of Ca channels and ryanodine receptors to investigate the effects of Ca-voltage coupling on EAD and DAD dynamics. Simulations were complemented by experiments in mouse ventricular myocytes. We show that: 1) alteration of the Ca transient due to increased ryanodine receptor leakiness and/or sarco/endoplasmic reticulum Ca ATPase activity can either promote or suppress EADs due to the complex effects of Ca on ionic current properties; 2) spontaneous Ca waves also exhibit complex effects on EADs, but cannot induce EADs of significant amplitude without the participation of I Ca,L ; 3) lengthening AP duration and the occurrence of EADs promote DADs by increasing intracellular Ca loading, and two mechanisms of DADs are identified, i.e., calcium-wave-dependent and a calcium-wave-independent; and 4) Ca-voltage coupling promotes complex EAD patterns such as EAD alternans that are not observed for solely voltage-driven EADs. In conclusion, Ca-voltage coupling combined with the nonlinear dynamical behaviors of voltage and Ca cycling play a key role in generating complex EAD and DAD dynamics observed experimentally in cardiac myocytes, whose mechanisms are complex but analyzable. INTRODUCTION Early afterdepolarizations (EADs) and delayed afterdepola- rizations (DADs) are known to cause arrhythmias in a vari- ety of cardiac diseases, including long QT syndromes (1–3), catecholaminergic polymorphic ventricular tachycardia (CPVT) (4,5), and heart failure (6,7). EADs are voltage oscillations that occur during the repolarizing phases of the action potential (AP). By prolonging the AP duration (APD) regionally, EADs can increase dispersion of refracto- riness, forming a tissue substrate vulnerable to reentry. Furthermore, if EADs reach the threshold to propagate out this region as premature ventricular contractions, they can serve as triggers to induce reentry. DADs, on the other hand, are voltage oscillations during diastole. By depolariz- ing the resting membrane potential regionally, DADs can cause conduction block (8), and, if they reach the threshold for sodium (Na) channel activation, can generate triggered activity (TA) manifesting as premature ventricular contrac- tions that induce reentry. EADs occur in the setting of reduced repolarization reserve, such as long QT syndromes, and have classically been attributed to voltage-driven oscillations in the repola- rizing phase of the AP, although spontaneous sarcoplasmic Submitted October 29, 2014, and accepted for publication March 10, 2015. *Correspondence: zqu@mednet.ucla.edu reticulum (SR) calcium (Ca) release has also been proposed as an important mechanism. DADs, on the other hand, are promoted by Ca cycling disorders, such as CPVT and digi- talis toxicity, which promote Ca-driven oscillations. Heart failure represents a combination of reduced repolarization reserve and abnormal Ca cycling resulting from electrical and excitation-contraction coupling remodeling processes driven by both genetic transcriptional and posttranslational signaling components. In all of these settings, it is common for EADs and DADs to coexist and influence each other. This is because membrane voltage is strongly affected by Ca-sensitive ionic currents, and, conversely, cellular Ca loading is strongly influenced by voltage-dependent ionic currents, referred to as bidirectional Ca-voltage coupling. Ca-voltage coupling can promote complex AP dynamics in the heart (9). Analyzing the interactions between EADs and DADs (and voltage and Ca-cycling coupling dynamics in general), however, has been challenging, because the Ca cycling dynamics (e.g., Ca waves and oscillations) result from a spatially distributed heterogeneous network of Ca release units (CRUs) in the cell. The Ca waves emanating from various subcellular regions integrate to depolarize dia- stolic membrane voltage by activating Ca-sensitive inward currents including the Na-Ca exchange (NCX) current (I NCX ) and Ca-activated nonselective cation channels (I ns(Ca) ), generating DADs. Similarly, spontaneous SR Ca Editor: Randall Rasmusson. O 2015 by the Biophysical Society http://dx.doi.org/10.1016/j.bpj.2015.03.011 BPJ 6468

Calcium (Ca(2+)) waves generating oscillatory Ca(2+) signals are widely observed in biological cells. Experimental studies have shown that under certain conditions, initiation of Ca(2+) waves is random in space and time, while under other conditions, waves occur repetitively from preferred locations (pacemaker sites) from which they entrain the whole cell. In this study, we use computer simulations to investigate the self-organization of Ca(2+) sparks into pacemaker sites generating Ca(2+) oscillations. In both ventricular myocyte experiments and computer simulations of a heterogeneous Ca(2+) release unit (CRU) network model, we show that Ca(2+) waves occur randomly in space and time when the Ca(2+) level is low, but as the Ca(2+) level increases, waves occur repetitively from the same sites. Our analysis indicates that this transition to entrainment can be attributed to the fact that random Ca(2+) sparks self-organize into Ca(2+) oscillations differently at low and high Ca(2+) levels. At low Ca(2+), the whole cell Ca(2+) oscillation frequency of the coupled CRU system is much slower than that of an isolated single CRU. Compared to a single CRU, the distribution of interspike intervals (ISIs) of the coupled CRU network exhibits a greater variation, and its ISI distribution is asymmetric with respect to the peak, exhibiting a fat tail. At high Ca(2+), however, the coupled CRU network has a faster frequency and lesser ISI variation compared to an individual CRU. The ISI distribution of the coupled network no longer exhibits a fat tail and is well-approximated by a Gaussian distribution. This same Ca(2+) oscillation behaviour can also be achieved by varying the number of ryanodine receptors per CRU or the distance between CRUs. Using these results, we develop a theory for the entrainment of random oscillators which provides a unified explanation for the experimental observations underlying the emergence of pacemaker sites and Ca(2+) oscillations.

Calcium (Ca) is a ubiquitous second messenger regulating many biological functions. The elementary events of local Ca signaling are Ca sparks, which occur randomly in time and space, and integrate to produce global signaling events including intracellular and intercellular Ca waves and whole-cell Ca oscillations. In a recent study using a computational model of Ca signaling in a cardiac myocyte and then experimentally in mouse ventricular myocytes, we demonstrated that criticality is the underlying theoretical mechanism that governs the transition from local Ca sparks to global Ca waves, analogous to a second-order phase transition in thermodynamics. Theoretically, criticality predicts that wave initiation sites should occur randomly and uniformly in space. While this has been demonstrated experimentally at relatively low Ca loads, recent experiments suggest that at higher Ca loads certain regions of the cell dominate the wave initiation process, acting as pacemakers to entrain whole cell oscillations. Here we show that the formation of these pacemaking sites is still governed by the theory of criticality, however, heterogeneities of the ryanodine receptor clusters result in different firing frequencies, with the fastest regions tending to entrain the whole cell. We demonstrate that there is a critical size and relative degree of heterogeneity that must be reached for entrainment to occur, with the novel finding that the degree of entrainment depends on the overall Ca load of the cell. Furthermore, we show that the stochastic nature of the ryanodine receptor channel is crucial to the wave generation process.

Spontaneous calcium (Ca) release in cardiac myocytes can produce delayed afterdepolarizations (DADs) that promote arrhythmias. In these studies, we combined experimental and computational approaches to investigate quantitatively how Ca release features at subcellular, cellular, and tissue scales influence DADs. Spontaneous Ca release events and corresponding DADs were measured experimentally from isolated rabbit ventricular myocytes exposed to elevated extracellular Ca (2.7 mM) and isoproterenol (0.25 μM), and were compared between lower and higher total intracellular Ca levels following 1 or 5 paced beats at 400 ms intervals, respectively. At the subcellular scale, Ca waves emerged simultaneously from an average of 1.36 (low) and 2.5 (high) sites per Ca release event. Consistent with the criticality-based theory of Ca wave emergence, Ca wave numbers were proportional to sarcoplasmic reticulum (SR) Ca levels that produced INCX with −71.1 pA/pF×ms (low) and −106.0 pA/pF×ms (high) integrated current densities upon rapid exposure to caffeine (10 mM). At the cellular scale, whole-cell calcium transients had peak amplitudes of 0.462 F/Fo (low) and 0.717 F/Fo (high) and full widths at half maximum (FWHMs) of 309.0 ms (low) and 180.0 ms (high). Resultant DADs had peak amplitudes of 1.41 mV (low) and 6.03 mV (high) and FWHMs of 302.3 ms (low) and 140.3 ms (high). Ca wave latencies in single myocytes, which determine Ca release synchrony in cardiac tissue, were 0.996 s (low) and 0.416 s (high). Computer simulation analyses implementing various combinations of the experimentally measured factors suggest that greater numbers of Ca waves, increased SR Ca release, and greater synchrony are associated with larger DAD amplitudes in cardiac tissue. Simulations also suggest that the subcellular number of Ca waves has the largest impact on DAD amplitude in tissue.

In cardiac myocytes, calcium (Ca)-induced Ca release can give rise to propagating Ca waves that promote cardiac arrhythmias. To understand the mechanism underlying the transition from localized Ca sparks to global Ca waves, we developed a mathematical model of a spatially-distributed network of diffusively coupled Ca release units (CRUs) comprised of junctional/longitudinal sarcoplasmic reticulum and dyadic/cytoplasmic spaces with physiologically realistic dimensions. As total Ca was increased in the model, Ca spark cluster sizes initially exhibited an exponential distribution, which transitioned to a scale-free power-law (f-β) distribution near the threshold at which random Ca sparks transitioned to Ca waves. The power-law relationship indicates that Ca release patterns in the CRU network are governed by a dynamical mechanism called self-organized criticality, the same process underlying many natural world phenomena such as avalanches and earthquakes. We tested this prediction experimentally in saponin-permeabilized cardiac myocytes. As free Ca, buffered with 0.5 mM EGTA, was increased, Ca spark cluster size transitioned from an exponential distribution to a power-law distribution at a free Ca concentration of 400 nM. Consistent with self-organized criticality, this Ca concentration was near the threshold for spark-to-wave transition since fully propagating waves were observed above 400 nM Ca. Below this concentration, Ca was released mostly as individual sparks, and spark cluster sizes followed an exponential rather than power-law distribution. In conclusion, our findings provide both theoretical and experimental evidence that the transition from Ca sparks to arrhythmogenic Ca waves in cardiac myocytes is mediated by the dynamical process of self-organized criticality, common to many natural phenomena. This provides a theoretical framework for developing interventions which modulate the Ca spark-to-wave transition threshold as a potential therapeutic strategy for preventing arrhythmias.

Intracellular calcium (Ca) waves in cardiac myocytes can cause delayed afterdepolarizations (DADs), which are known triggers of cardiac arrhythmias. How these Ca waves are modulated by diffusive Ca-mediated coupling among Ca release units (CRUs) and promote DADs is not fully understood. Here, we hypothesized that myocytes are most susceptible to DADs due to periodic Ca wave activity at intermediate levels of Ca overload. To test this hypothesis, we strengthened CRU coupling by progressively raising intracellular free [Ca] and studied the transition from Ca sparks to waves using both confocal Ca imaging experiments in permeabilized mouse ventricular myocytes and computer simulations of a homogeneous 3D array (100x20x10) of diffusively-coupled CRUs. As free Ca was increased in experiments from 100 nM to 1,500 nM, intracellular Ca release activity evolved through four stages: Stage 1- random sparks and macrosparks arising from multiple sites; Stage 2- irregular aborted Ca waves arising from multiple sites; Stage 3- periodic full Ca waves arising from a small number of sites mostly near cell borders; Stage 4- high frequency “fibrillatory” Ca waves exhibiting mixed focal and reentrant features. Raising virtual intracellular Ca in computer simulations reproduced Stages 1-3 but not Stage 4, which may require spatial heterogeneities to occur. In both experiments and simulations, Stage 3 produced the largest whole-cell Ca transients and most synchronous Ca release, making this intermediate stage of Ca overload the most likely to generate DADs of sufficient amplitude to trigger arrhythmias. High frequency “fibrillatory” waves under severe Ca overload in Stage 4 diminished Ca transient amplitudes and reduced Ca release synchrony. In conclusion, our findings suggest that ventricular myocytes are most susceptible to DADs when intracellular Ca overload is intermediate rather than mild or severe.