Cardiac alternans has been linked to the onset of ventricular fibrillation and ventricular tachycardia, leading to life-threatening arrhythmias. Here, we investigated the effects of stretch-activated currents (ISAC) on alternans using a physiologically detailed model of the ventricular myocyte. We found that increasing ISAC suppresses alternans if the voltage-Ca coupling is positive or the alternans is voltage driven. However, for electromechanically discordant alternans, which occurs when the alternans is Ca driven with negative voltage-Ca coupling, increasing ISAC promotes Ca alternans. In addition, if action potential duration-Ca transients show quasiperiodicity, we observe a biphasic effect of ISAC, i.e., suppressing quasiperiodic oscillation at small stretch but promoting electromechanically discordant alternans at larger stretch. Our results demonstrate how ISAC interacts with coupled voltage-Ca dynamical systems with respect to alternans.

The sodium/potassium ATPase (NKA) is essential for establishing the normal intracellular [Na⁺] and [K⁺] and transmembrane gradients that are essential for many cellular functions, including cardiac electrophysiology and contractility. Different NKA isoforms exhibit differential expression levels, cellular localization, and function in different tissues and species. Prior work has indicated that the NKA-α1 isoform is quantitatively predominant in cardiac myocytes, but that the α2 isoform is preferentially concentrated in the transverse tubules (TT), possibly at junctions with the sarcoplasmic reticulum (SR) where α2 may preferentially modulate cardiac contractility. Here we measured subcellular localization of NKA-α1 and α2 using super-resolution microscopy (STED and STORM) and isoform-selective antibodies in mouse ventricular myocytes. We confirm the preferential localization of NKA-α2 in TT vs. surface sarcolemma, but also show that α2 is relatively excluded from longitudinal TT elements. In contrast NKA-α1 is relatively uniformly expressed in all three sarcolemmal regions. We also tested the hypothesis that NKA-α2 (vs. α1) is preferentially concentrated at SR junctional sites near ryanodine receptors (RyR2). The results refute this hypothesis, in that NKA-α1 and α2 were equally close to RyR2 at the TT, with no preferential NKA isoform localization near RyR2. We conclude that in contrast to relatively uniform NKA-α1 distribution, NKA-α2 is preferentially concentrated in the truly transverse (and not longitudinal) TT elements. However, NKA-α2 does not preferentially cluster at RyR2 junctions, so the TT NKA-α2 concentration may suffice for preferential effects of NKA-α2 inhibition on cardiac contractility.

Ryanodine receptors (RyRs) mediate calcium (Ca)-induced Ca release and intracellular Ca homeostasis. In a cardiac myocyte, RyRs group into clusters of variable size from a few to several hundred RyRs, creating a spatially nonuniform intracellular distribution. It is unclear how heterogeneity of RyR cluster size alters spontaneous sarcoplasmic reticulum (SR) Ca releases (Ca sparks) and arrhythmogenic Ca waves. Here, we tested the impact of heterogeneous RyR cluster size on the initiation of Ca waves. Experimentally, we measured RyR cluster sizes at Ca spark sites in rat ventricular myocytes and further tested functional impacts using a physiologically detailed computational model with spatial and stochastic intracellular Ca dynamics. We found that the spark frequency and amplitude increase nonlinearly with the size of RyR clusters. Larger RyR clusters have lower SR Ca release threshold for local Ca spark initiation and exhibit steeper SR Ca release versus SR Ca load relationship. However, larger RyR clusters tend to lower SR Ca load because of the higher Ca leak rate. Conversely, smaller clusters have a higher threshold and a lower leak, which tends to increase SR Ca load. At the myocyte level, homogeneously large or small RyR clusters limit Ca waves (because of low load for large clusters but low excitability for small clusters). Mixtures of large and small RyR clusters potentiates Ca waves because the enhanced SR Ca load driven by smaller clusters enables Ca wave initiation and propagation from larger RyR clusters. Our study suggests that a spatially heterogeneous distribution of RyR cluster size under pathological conditions may potentiate Ca waves and thus afterdepolarizations and triggered arrhythmias.

Background

Ryanodine receptors (RyR) mediate sarcoplasmic reticulum calcium (Ca²⁺) release and influence myocyte Ca²⁺ homeostasis and arrhythmias. In cardiac myocytes, RyRs are found in clusters of various sizes and shapes, and RyR cluster size may critically influence normal and arrhythmogenic Ca²⁺ spark and wave formation. However, the actual RyR cluster sizes at specific Ca²⁺ spark sites have never been measured in the physiological setting.

Methods and results

Here we measured RyR cluster size and Ca²⁺ sparks simultaneously to assess how RyR cluster size influences Ca²⁺ sparks and sarcoplasmic reticulum Ca²⁺ leak. For small RyR cluster sizes (<50), Ca²⁺ spark frequency is very low but then increases dramatically at larger cluster sizes. In contrast, Ca²⁺ spark amplitude is nearly maximal even at relatively small RyR cluster size (≈10) and changes little at larger cluster size. These properties agreed with computational simulations of RyR gating within clusters.

Conclusions

Our study explains how this combination of properties may limit arrhythmogenic Ca²⁺ sparks and wave propagation (at many junctions) while preserving the efficacy and spatial synchronization of Ca²⁺-induced Ca²⁺-release during normal excitation-contraction coupling. However, variations in RyR cluster size among individual junctions and RyR sensitivity could exacerbate heterogeneity of local sarcoplasmic reticulum Ca²⁺ release and arrhythmogenesis under pathological conditions.

Cardiac β-adrenergic receptors (β-AR) and Ca²⁺-Calmodulin dependent protein kinase (CaMKII) regulate both physiological and pathophysiological Ca²⁺ signaling. Elevated diastolic Ca²⁺ leak from the sarcoplasmic reticulum (SR) contributes to contractile dysfunction in heart failure and to arrhythmogenesis. β-AR activation is known to increase SR Ca²⁺ leak via CaMKII-dependent phosphorylation of the ryanodine receptor. Two independent and reportedly parallel pathways have been implicated in this β-AR-CaMKII cascade, one involving exchange protein directly activated by cAMP (Epac2) and another involving nitric oxide synthase 1 (NOS1). Here we tested whether Epac and NOS function in a single series pathway to increase β-AR induced and CaMKII-dependent SR Ca²⁺ leak. Leak was measured as both Ca²⁺ spark frequency and tetracaine-induced shifts in SR Ca²⁺, in mouse and rabbit ventricular myocytes. Direct Epac activation by 8-CPT (8-(4-chlorophenylthio)-2'-O-methyl-cAMP) mimicked β-AR-induced SR Ca²⁺ leak, and both were blocked by NOS inhibition. The same was true for myocyte CaMKII activation (assessed via a FRET-based reporter) and ryanodine receptor phosphorylation. Inhibitor and phosphorylation studies also implicated phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) downstream of Epac and above NOS activation in this pathway. We conclude that these two independently characterized parallel pathways function mainly via a single series arrangement (β-AR-cAMP-Epac-PI3K-Akt-NOS1-CaMKII) to mediate increased SR Ca²⁺ leak. Thus, for β-AR activation the cAMP-PKA branch effects inotropy and lusitropy (by effects on Ca²⁺ current and SR Ca²⁺-ATPase), this cAMP-Epac-NOS pathway increases pathological diastolic SR Ca²⁺leak. This pathway distinction may allow novel SR Ca²⁺ leak therapeutic targeting in treatment of arrhythmias in heart failure that spare the inotropic and lusitropic effects of the PKA branch.

Calcium-calmodulin dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac electrophysiology, calcium (Ca) balance, contraction, transcription, arrhythmias and progression to heart failure. CaMKII is readily activated at mouths of dyadic cleft Ca channels, but because of its low Ca-calmodulin affinity and presumed immobility it is less clear how CaMKII gets activated near other known, extra-dyad targets. CaMKII is typically considered to be anchored in cardiomyocytes, but while untested, mobility of active CaMKII could provide a mechanism for broader target phosphorylation in cardiomyocytes. We therefore tested CaMKII mobility and how this is affected by kinase activation in adult rabbit cardiomyocytes. We measured translocation of both endogenous and fluorescence-tagged CaMKII using immunocytochemistry, fluorescence recovery after photobleach (FRAP) and photoactivation of fluorescence. In contrast to the prevailing view that CaMKII is anchored near its myocyte targets, we found CaMKII to be highly mobile in resting myocytes, which was slowed by Ca chelation and accelerated by pacing. At low [Ca], CaMKII was concentrated at Z-lines near the dyad but spread throughout the sarcomere upon pacing. Nuclear exchange of CaMKII was also enhanced upon pacing- and heart failure-induced chronic activation. This mobilization of active CaMKII and its intrinsic memory may allow CaMKII to be activated in high [Ca] regions and then move towards more distant myocyte target sites.