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Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling.

ABSTRACT: There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca(2+)Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca(2+)current (I(CaL)) induces a global Ca(2+) release (CICR), depleting the Ca(2+) content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca(2+)release of multiple LCRs throughout the cell. The LCRs generate an inward Na(+)/Ca(2+)exchanger (NCX) current (absent in Na(+)-free solution) that ignites the next AP. Interfering with SR Ca(2+) cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na(+)-free solution), or I(CaL) (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca(2+) loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic "pacemaker-like" cells. SR Ca(2+) loading and LCR activity could be also increased with a selective activation of SR Ca(2+) pumping by a phospholamban antibody. We conclude that SR Ca(2+) loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I(CaL) synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca(2+) release and NCX current. Rhythmic ESC automaticity can be achieved by boosting "coupling" factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma.

SUBMITTER: Zahanich I

PROVIDER: S-EPMC3018669 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling.

Zahanich Ihor I Sirenko Syevda G SG Maltseva Larissa A LA Tarasova Yelena S YS Spurgeon Harold A HA Boheler Kenneth R KR Stern Michael D MD Lakatta Edward G EG Maltsev Victor A VA

Journal of molecular and cellular cardiology 20101015 1

There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs gen ...[more]

PMID: 20920509

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Project description:Purkinje cells (Pcell) are characterized by different electrophysiological properties and Ca(2+) cycling processes than ventricular myocytes (Vcell) and are frequently involved in ventricular arrhythmias. Yet, the mechanistic basis for their arrhythmic vulnerability is not completely understood. The objectives were to: (1) characterize Pcell electrophysiology, Ca(2+) cycling, and their rate dependence; (2) investigate mechanisms underlying Pcell arrhythmogenicity; and compare Pcell and Vcell electrophysiology, Ca(2+) cycling, and arrhythmic properties. We developed a new mathematical model of Pcell. The Ca(2+) subsystem includes spatial organization and receptors distribution unique to Pcell. Results were: (1) in Pcell and Vcell, Na(+) accumulation via its augmentation of repolarizing I(NaK) dominates action potential duration adaptation and, in Pcell, I(NaL) contributes additional action potential duration shortening at short cycle length; (2) steep Pcell restitution is attributable to slow recovery of I(NaL); (3) biphasic Ca(2+) transients of Pcell reflect the delay between Ca(2+) release from junctional sarcoplasmic reticulum and corbular sarcoplasmic reticulum; (4) Pcell Ca(2+) alternans, unlike Vcell, can develop without inducing action potential alternans; (5) Pcell action potential alternans develops at a shorter cycle length than Vcell, with increased subcellular heterogeneity of Ca(2+) cycling attributable to refractoriness of Ca(2+) release from corbular sarcoplasmic reticulum and junctional sarcoplasmic reticulum; (6) greater Pcell vulnerability to delayed afterdepolarizations is attributable to higher sarcoplasmic reticulum Ca(2+) content and ionic currents that reduce excitation threshold and promote triggered activity; and (7) early after depolarizations generation in Pcell is mostly attributable to reactivation of I(NaL2), whereas I(CaL) plays this role in Vcell. Steeper rate dependence of action potential and Ca(2+) transients, central peripheral heterogeneity of Ca(2+) cycling, and distinct ion channel profile underlie greater arrhythmic vulnerability of Pcell compared to Vcell.

Project description:Oscillatory behavior of mitochondrial inner membrane potential (ΔΨm) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨm depolarization as well as synchronized limit cycle oscillations of ΔΨm in the network. The functional impact of ΔΨm instability on cardiac electrophysiology, Ca(2+) handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨm oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10(-2) ± 0.98 × 10(-2) s(-1)) and pyruvate (7.49 × 10(-2) ± 1.65 × 10(-2) s(-1)) than lactate (4.83 × 10(-2) ± 1.25 × 10(-2) s(-1)) or β-hydroxybutyrate (4.11 × 10(-2) ± 0.62 × 10(-2) s(-1)). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨm instability, ultimately scaling to produce organ level dysfunction.

Project description:BackgroundCardiac optical mapping is an imaging technique that measures fluorescent signals across a cardiac preparation. Dual optical imaging of voltage-sensitive and calcium-sensitive probes allows for simultaneous recordings of cardiac action potentials and intracellular calcium transients with high spatiotemporal resolution. The analysis of these complex optical datasets is both time intensive and technically challenging; as such, we have developed a software package for semi-automated image processing and analysis. Herein, we report an updated version of our software package (KairoSight-3.0) with features to enhance the characterization of cardiac parameters using optical signals.MethodsTo test software validity and applicability, we used Langendorff-perfused heart preparations to record transmembrane voltage and intracellular calcium signals from the epicardial surface. Isolated hearts from guinea pigs and rats were loaded with a potentiometric dye (RH237) and/or calcium indicator dye (Rhod-2AM) and fluorescent signals were acquired. We used Python 3.8.5 programming language to develop the KairoSight-3.0 software. Cardiac maps were validated with a user-specified manual mapping approach.ResultsManual maps of action potential duration (30 or 80 % repolarization), calcium transient duration (30 or 80 % reuptake), action potential and calcium transient alternans were constituted to validate the accuracy of software-generated maps. Manual and software maps had high accuracy, with >97 % of manual and software values falling within 10 ms of each other and >75 % within 5 ms for action potential duration and calcium transient duration measurements (n = 1000-2000 pixels). Further, our software package includes additional measurement tools to analyze signal-to-noise ratio, conduction velocity, action potential and calcium transient alternans, and action potential-calcium transient coupling time to produce physiologically meaningful optical maps.ConclusionsKairoSight-3.0 has enhanced capabilities to perform measurements of cardiac electrophysiology, calcium handling, alternans, and the excitation-contraction coupling with satisfactory accuracy.

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Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling.

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Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling.

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