Yatong Li

Stability and instability of regulation of intracellular calcium

[Ca2+]i is used as a signal in many tissues. In this review we discuss the mechanisms that regulate [Ca2+]i and, importantly, what determines their stability. Brief mention is made of the effects of feedback gain and delays on stability. The control of cytoplasmic Ca concentration is shown to be generally stable as Ca pumping is essentially an instantaneous function of [Ca2+]i. In contrast, regulation of the Ca content of intracellular stores may be less stable. One example of this is instability in the control of sarcoplasmic reticulum (SR) Ca content in cardiac muscle. An increase of SR Ca content increases the systolic Ca transient amplitude. This in turn decreases Ca influx into the cell and increases efflux, thereby restoring SR Ca to control levels. This feedback system has an inherent delay and is potentially unstable if the gain is increased beyond a certain level. This instability produces Ca transients of alternating amplitude and may contribute to the clinical syndrome of pulsus alternans.

The effects of membrane potential, SR Ca2+ content and RyR responsiveness on systolic Ca2+ alternans in rat ventricular myocytes

Previous work has shown that small depolarizing pulses produce a beat to beat alternation in the amplitude of the systolic Ca2+ transient in ventricular myocytes. The aim of the present work was to investigate the role of changes of SR Ca2+ content and L‐type Ca2+ current in this alternans. As the amplitude of the depolarizing pulse was increased from 10 to 30 mV the magnitude of alternans decreased. Confocal linescan studies showed that this was accompanied by an increase in the number of sites from which Ca2+ waves propagated. A sudden decrease in the depolarisation amplitude resulted in three classes of behaviour: (1) a gradual decrease in Ca2+ transient amplitude before alternans developed accompanied by a loss of SR Ca2+, (2) a gradual increase in Ca2+ transient amplitude before alternans accompanied by a gain of SR Ca2+, and (3) immediate development of alternans with no change of SR content. We conclude that alternans develops if the combination of decreased opening of L‐type channels and change of SR Ca2+ content results in spatially fragmented release from the SR as long as there is sufficient Ca2+ in the SR to sustain wave propagation. Potentiation of the opening of the ryanodine receptor (RyR) by low concentrations of caffeine (100 μm) abolished alternans for a few pulses but the alternans then redeveloped once SR Ca2+ content fell to the new threshold for wave propagation. Finally we show evidence that inhibiting L‐type Ca2+ current with 200 μm Cd2+ produces alternans by means of a similar fragmentation of the Ca2+ release profile and propagation of mini‐waves of Ca2+ release.

Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak

Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay. In the presence of β‐adrenergic stimulation, RyR‐mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase. Two forms of Ca leak have been studied, Ca‐sensitising (induced by caffeine) and non‐sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient. Only Ca‐sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine. Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient.

Pak1 Is Required to Maintain Ventricular Ca2+ Homeostasis and Electrophysiological Stability through SERCA2a Regulation in Mice

Background—Impaired sarcoplasmic reticular Ca 2+ uptake resulting from decreased sarcoplasmic reticulum Ca 2+ -ATPase type 2a (SERCA2a) expression or activity is a characteristic of heart failure with its associated ventricular arrhythmias. Recent attempts at gene therapy of these conditions explored strategies enhancing SERCA2a expression and the activity as novel approaches to heart failure management. We here explore the role of Pak1 in maintaining ventricular Ca 2+ homeostasis and electrophysiological stability under both normal physiological and acute and chronic &bgr;-adrenergic stress conditions. Methods and Results—Mice with a cardiomyocyte-specific Pak1 deletion (Pak1 cko ), but not controls (Pak1 f/f ), showed high incidences of ventricular arrhythmias and electrophysiological instability during either acute &bgr;-adrenergic or chronic &bgr;-adrenergic stress leading to hypertrophy, induced by isoproterenol. Isolated Pak1 cko ventricular myocytes correspondingly showed aberrant cellular Ca 2+ homeostasis. Pak1 cko hearts showed an associated impairment of SERCA2a function and downregulation of SERCA2a mRNA and protein expression. Further explorations of the mechanisms underlying the altered transcriptional regulation demonstrated that exposure to control Ad-shC2 virus infection increased SERCA2a protein and mRNA levels after phenylephrine stress in cultured neonatal rat cardiomyocytes. This was abolished by the Pak1-knockdown in Ad-shPak1–infected neonatal rat cardiomyocytes and increased by constitutive overexpression of active Pak1 (Ad-CAPak1). We then implicated activation of serum response factor, a transcriptional factor well known for its vital role in the regulation of cardiogenesis genes in the Pak1-dependent regulation of SERCA2a. Conclusions—These findings indicate that Pak1 is required to maintain ventricular Ca 2+ homeostasis and electrophysiological stability and implicate Pak1 as a novel regulator of cardiac SERCA2a through a transcriptional mechanism.

Do calcium waves propagate between cells and synchronize alternating calcium release in rat ventricular myocytes

•  In cell pairs isolated from rat ventricular muscle, spontaneous waves of Ca2+‐induced Ca2+ release are much more likely to propagate between cells connected side to side than end to end. •  Investigation of this difference using fluorescence techniques shows that the end‐to‐end connection probably contains the intercalated disc. It is this structure that accounts for the greater distance between sarcoplasmic reticulum Ca2+ release units being so large (about 2.3 μm). At side‐to‐side connections, this distance is much less (about 1.6 μm). This variation may be the cause of the difference in propagation properties. •  In cell pairs isolated from rat ventricular muscle, we have been able to induce systolic Ca2+ alternans in‐phase between the cells in a pair. This is not due to propagation of Ca2+ waves between cells. In some cases, wave propagation can interfere with synchrony.

216 Identifying a novel role for pmca1 (atp2b1) in heart rhythm instability

Arrhythmias continue to be a leading cause of death and disability across the world, and genetics are one of the mechanisms that are known to increase susceptibility. By identifying new genetic influences and further understanding the pathways involved in heart rhythm control we can begin to tackle some of the main challenges facing treatment development. Here we aim to identify a new role for a gene linked to several features of heart failure Atp2b1 (Plasma membrane calcium ATPase 1, PMCA1). Along with its role in hypertension and other aspects of cardiac physiology, we believe PMCA1 may also influence heart rhythm stability and consequently the development of arrhythmias. To investigate the role of PMCA1 in cardiac rhythm, cardiomyocyte-specific knockout mice (PMCA1CKOÃ,Â) were generated. In vivo electrocardiography showed PMCA1CKO displayed signs of cardiac repolarisation dysfunction related to prolonged QT and JT intervals. Supplementary analysis using Langendorff-perfused hearts revealed PMCA1CKO hearts have prolonged action potential duration compared to controls. Additionally using the methods highlighted above, PMCA1CKO mice were shown to have an increase arrhythmia susceptibility to both in vivo and ex vivo programmed electrical stimulation. Further echocardiography and histological analysis showed these heart rhythm abnormalities occur in the absence of detectable structural heart disease with PMCA1CKO cardiac structure and function being comparable to controls. Our findings suggest a novel role for PMCA1 in heart rhythm stability, distinct from other cardiac disease. Furthermore, alterations in expression of Atp2b1 could influence an individuals susceptibility to developing arrhythmias.

Stress-Activated Kinase MKK7 Governs Epigenetics of Cardiac Repolarization for Arrhythmia Prevention

Background: Ventricular arrhythmia is a leading cause of cardiac mortality. Most antiarrhythmics present paradoxical proarrhythmic side effects, culminating in a greater risk of sudden death. Methods: We describe a new regulatory mechanism linking mitogen-activated kinase kinase-7 deficiency with increased arrhythmia vulnerability in hypertrophied and failing hearts using mouse models harboring mitogen-activated kinase kinase-7 knockout or overexpression. The human relevance of this arrhythmogenic mechanism is evaluated in human-induced pluripotent stem cell–derived cardiomyocytes. Therapeutic potentials by targeting this mechanism are explored in the mouse models and human-induced pluripotent stem cell–derived cardiomyocytes. Results: Mechanistically, hypertrophic stress dampens expression and phosphorylation of mitogen-activated kinase kinase-7. Such mitogen-activated kinase kinase-7 deficiency leaves histone deacetylase-2 unphosphorylated and filamin-A accumulated in the nucleus to form a complex with Krüppel-like factor-4. This complex leads to Krüppel-like factor-4 disassociation from the promoter regions of multiple key potassium channel genes (Kv4.2, KChIP2, Kv1.5, ERG1, and Kir6.2) and reduction of their transcript levels. Consequent repolarization delays result in ventricular arrhythmias. Therapeutically, targeting the repressive function of the Krüppel-like factor-4/histone deacetylase-2/filamin-A complex with the histone deacetylase-2 inhibitor valproic acid restores K+ channel expression and alleviates ventricular arrhythmias in pathologically remodeled hearts. Conclusions: Our findings unveil this new gene regulatory avenue as a new antiarrhythmic target where repurposing of the antiepileptic drug valproic acid as an antiarrhythmic is supported.

Effect of reduction in ryanodine receptor calcium leak on arrhythmogenesis in rat ventricular myocytes

Abstract Background Cardiomyocytes in heart failure have increased ryanodine receptor (RyR) open probability (po) leading to calcium leak from the sarcoplasmic reticulum. This process results in calcium waves, delayed after-depolarisations, and subsequent ventricular arrhythmias. However, this leak will decrease the calcium content of the sarcoplasmic reticulum which will in turn decrease the occurrence of waves. We therefore hypothesised that with extreme RyR leak (such as in end-stage heart failure) the calcium content of the sarcoplasmic reticulum might be too low to sustain calcium waves and that part correction of severe leak could lead to a resurgence of calcium waves. Methods We used single rat ventricular myocytes, voltage clamped using the perforated patch clamp technique. Cells were stimulated at 0·5 Hz with 100 ms duration depolarising pulses from −40 to 0 mV. Cytosolic calcium concentration was measured with the Fluo-4 AM indicator. Calcium waves were induced by the application of 1 μmol/L isoproterenol in raised external calcium concentration (2 or 3 mM). Severe RyR leak was induced with caffeine (1–2 mM), and then tetracaine (50–100 μM) was co-applied to reduce RyR po. Findings Isoproterenol increased the amplitude of the systolic calcium transient in all the myocytes studied and induced calcium waves in 26 (87%) of 30 myocytes. Application of 1 mM caffeine abolished calcium waves in 11 (65%) of 17 of these myocytes and reduced the frequency of waves in the remaining six cells. Application of 2 mM caffeine abolished calcium waves in all nine myocytes. Subsequent addition of tetracaine (50 μM) led to a re-emergence or increased frequency of waves in nine (53%) of 17 myocytes whereas 100 μM tetracaine re-established calcium waves in 14 (93%) of 15 myocytes (50 vs 100 μM tetracaine, Fishers exact test p=0·02). Interpretation Induction of severe calcium leak depleted the calcium content of the sarcoplasmic reticulum below the level required for calcium waves. Inhibition of RyR leak could be a useful therapeutic strategy in heart failure to improve systolic contraction as well as relaxation. However, this strategy might also be pro-arrhythmogenic since part correction of leak can replenish the calcium content of the sarcoplasmic reticulum above the level required for initiation of calcium waves. Funding British Heart Foundation.