Lars Cleemann

In vitro Modeling of Ryanodine Receptor 2 Dysfunction Using Human Induced Pluripotent Stem Cells

Background/Aims: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. Methods: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. Results: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca2+ release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca2+-induced Ca2+-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. Conclusion: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

Spatiotemporal Characteristics of Junctional and Nonjunctional Focal Ca2+ Release in Rat Atrial Myocytes

Abstract— Atrial myocytes have two functionally separate Ca2+ release sites: those in peripheral sarcoplasmic reticulum (SR) adjacent to the Ca2+ channels of surface membrane and those in central SR not associated with Ca2+ channels. Recently, we have reported on the gating of these two different Ca2+ release sites by Ca2+ current. In the present study, we report on the spatiotemporal properties of focal Ca2+ releases (sparks) occurring spontaneously in central and peripheral sites of voltage-clamped rat atrial myocytes, using rapid 2-dimensional (2-D) confocal Ca2+ imaging. Peripheral and central sparks were similar in size and release time (≈300 000 Ca2+ ions for ≅12 ms), but significantly larger and longer than ventricular sparks. Both sites were resistant to Cd2+ and inhibited by ryanodine. Peripheral sparks were brighter and flattened against surface membrane, had ≈5-fold higher frequency, ≈2 times faster diffusion coefficient, and dissipated abruptly. Central sparks, in contrast, occurred less frequently, were elongated along the cellular longitudinal axis, and dissipated slowly. Compound sparks (composed of 2 to 5 unitary focal releases) aligned longitudinally and occurred more frequently at the center. The diversity of peripheral and central sparks with respect to shape, frequency, and speed of spatial development and decay is consistent with regional ultrastructural heterogeneity of SR. The retarded dissipation of central atrial sparks, and high prevalence of compound sparks in cell center may be critical in facilitating the propagation of Ca2+ waves in atrial myocytes lacking t-tubular system and provide the atrial myocytes with functional Ca2+ signaling diversity. The full text of this article is available at http://www.circresaha.org.

Intracellular Ca2+ Oscillations, a Potential Pacemaking Mechanism in Early Embryonic Heart Cells

Early (E9.5–E11.5) embryonic heart cells beat spontaneously, even though the adult pacemaking mechanisms are not yet fully established. Here we show that in isolated murine early embryonic cardiomyocytes periodic oscillations of cytosolic Ca2+ occur and that these induce contractions. The Ca2+ oscillations originate from the sarcoplasmic reticulum and are dependent on the IP3 and the ryanodine receptor. The Ca2+ oscillations activate the Na+-Ca2+ exchanger, giving rise to subthreshold depolarizations of the membrane potential and/or action potentials. Although early embryonic heart cells are voltage-independent Ca2+ oscillators, the generation of action potentials provides synchronization of the electrical and mechanical signals. Thus, Ca2+ oscillations pace early embryonic heart cells and the ensuing activation of the Na+-Ca2+ exchanger evokes small membrane depolarizations or action potentials.

Ca2+ signaling in human induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) from normal and catecholaminergic polymorphic ventricular tachycardia (CPVT)-afflicted subjects.

Derivation of cardiomyocytes from induced pluripotent stem cells (iPS-CMs) allowed us to probe the Ca(2+)-signaling parameters of human iPS-CMs from healthy- and catecholaminergic polymorphic ventricular tachycardia (CPVT1)-afflicted individuals carrying a novel point mutation p.F2483I in ryanodine receptors (RyR2). iPS-CMs were dissociated on day 30-40 of differentiation and patch-clamped within 3-6 days. Calcium currents (ICa) averaged ∼8pA/pF in control and mutant iPS-CMs. ICa-induced Ca(2+)-transients in control and mutant cells had bell-shaped voltage-dependence similar to that of ICa, consistent with Ca(2+)-induced Ca(2+)-release (CICR) mechanism. The ratio of ICa-activated to caffeine-triggered Ca(2+)-transients was ∼0.3 in both cell types. Caffeine-induced Ca(2+)-transients generated significantly smaller Na(+)-Ca(2+) exchanger current (INCX) in mutant cells, reflecting their smaller Ca(2+)-stores. The gain of CICR was voltage-dependent as in adult cardiomyocytes. Adrenergic agonists enhanced ICa, but differentially altered the CICR gain, diastolic Ca(2+), and Ca(2+)-sparks in mutant cells. The mutant cells, when Ca(2+)-overloaded, showed longer and wandering Ca(2+)-sparks that activated adjoining release sites, had larger CICR gain at -30mV yet smaller Ca(2+)-stores. We conclude that control and mutant iPS-CMs express the adult cardiomyocyte Ca(2+)-signaling phenotype. RyR2 F2483I mutant myocytes have aberrant unitary Ca(2+)-signaling, smaller Ca(2+)-stores, higher CICR gains, and sensitized adrenergic regulation, consistent with functionally altered Ca(2+)-release profile of CPVT syndrome.

Glutathione is a cofactor for H2O2-mediated stimulation of Ca2+-induced Ca2+ release in cardiac myocytes.

Reactive oxygen species are known to cause attenuation of cardiac muscle contraction. This attenuation is usually preceded by transient augmentation of twitch amplitude as well as cytosolic Ca2+. The present study examines the role of an endogenous antioxidant, glutathione in the mechanism of H2O2-mediated augmentation of Ca2+ release from the sarcoplasmic reticulum. Whole-cell patch-clamped single rat ventricular myocytes were dialyzed with the Cs+-rich internal solution containing 200 microM fura-2 and 2 mM glutathione (reduced form). After equilibration of the myocyte with intracellular dialyzing solution, Ca2+ current-induced Ca2+ release from the sarcoplasmic reticulum was monitored. Rapid perfusion with H2O2 (100 microM or 1 mM) for 20 s inhibited Ca2+ current, but enhanced the intracellular Ca2+ transients for 3-4 min. Thus, the efficacy of Ca2+-induced Ca2+ release mechanism was augmented in 71% of myocytes (n = 7). This enhancement ranged between 1.5- to threefold as the concentrations of H2O2 were raised from 100 microM to 1 mM. If glutathione were excluded from the patch pipette or replaced with glutathione disulfide, the enhancement of Ca2+-induced Ca2+ release was seen in only a minority (20%) of the myocytes. H2O2 exposure did not increase the basal intracellular Ca2+ levels, suggesting that the mechanism of H2O2 action was not mediated by inhibition of the sarcoplasmic reticulum Ca2+ uptake or activation of passive Ca2+ leak pathway. H2O2-mediated stimulation of Ca2+-induced Ca2+ release was also observed in myocytes dialyzed with dithiothreitol (0.5 mM). Therefore, reduced thiols support the action of H2O2 to enhance the efficacy of Ca2+-induced Ca2+ release, suggesting that redox reactions might regulate Ca2+ channel-gated Ca2+ release by the ryanodine receptor.

Ca2+ current-gated focal and local Ca2+ release in rat atrial myocytes: evidence from rapid 2-D confocal imaging

In atrial myocytes immunocytochemistry has shown two groups of ryanodine receptors (RyRs): those at the periphery colocalized with dihydropyridine receptors (DHPRs) and those at the cell interior not associated with DHPRs. The extent to which the two sets of RyRs are controlled by Ca2+ current (ICa) or Ca2+ diffusion remains to be determined. Here, using rapid (240 Hz) two‐dimensional confocal Ca2+ imaging in rat atrial myocytes, we examine directly the role of ICa on the two‐dimensional patterns of local and focal Ca2+ releases. ICa evoked peripheral Ca2+ release within 1–4 ms, causing a rapid monophasic local rise of Ca2+, which then propagated into the cell interior along sarcomeric lines (∼2 μm) with a velocity of ∼230 μm s−1, even though we found no evidence for organized t‐tubules using di‐8‐ANEPPS staining. ICa‐triggered Ca2+ release in the cell centre, on the other hand, had both a rapid (≤12 ms) and slower delayed components (12–50 ms). The voltage dependence of peripheral Ca2+ release and the two components of central release was bell shaped, and the magnitude of each release component was linearly related to ICa. Premature termination (2–10 ms) of ICa was equally effective in abbreviating both the peripheral and slow central Ca2+ release. High concentration of Ca2+ buffers (2–5 mm EGTA plus 1 mm fluo‐3) completely abolished the ICa‐gated propagation wave and the slow delayed component of Ca2+ release, but had little or no effect on the rapid component of central release. The efficacy of ICa to trigger Ca2+ release in periphery of the myocyte was ∼5 times higher than in the centre, consistent with the smaller measured central Ca2+ release. The quantification of central Ca2+ release as a function of peripheral release suggests a cooperative gating mechanism(s) for central release. These findings indicate that both ICa and diffusion of Ca2+ from the peripheral sites contribute to the gating of Ca2+ release from central SR. How in fact the ICa‐dependent fast component of central release is activated remains to be determined.

Modulation of focal and global Ca2+ release in calsequestrin-overexpressing mouse cardiomyocytes

1 Focal and global Ca2+ releases were monitored in voltage‐clamped control and hypertrophied calsequestrin (CSQ)‐overexpressing mouse cardiomyocytes, dialysed with fluo‐3, using rapid (120‐240 frames s−1) two‐dimensional confocal imaging. 2 Spontaneous focal Ca2+ releases (Ca2+ sparks) were absent or significantly reduced in frequency in hypertrophied myocytes of CSQ‐overexpressing mice compared to their age‐matched controls. Sporadic Ca2+ sparks seen in CSQ‐overexpressing myocytes had intensities and durations similar to those of controls although quantitative analysis showed a trend towards more diffuse focal releases. 3 Activation of Ca2+ current (ICa) failed to produce the typical sarcomeric Ca2+ striping pattern consistently seen in control myocytes. Instead, focal Ca2+ releases appeared as a disorganized patchwork of diffuse or ‘woolly’ fluorescence signals, resulting in slowly developing and reduced global Ca2+ transients. 4 Although the density of ICa in CSQ‐overexpressing myocytes was only slightly smaller than that of controls, the inactivation kinetics of the current were greatly reduced, consistent with the much smaller rate of rise of cytosolic Ca2+. 5 Enhancement of ICa by elevation of [Ca2+]o from 2 to 10 mM or addition of 3 μM isoproterenol (isoprenaline) failed to normalize the frequency of spontaneous Ca2+ sparks at rest or the pattern and the magnitude of ICa‐gated Ca2+ transients. Isoproterenol was somewhat more effective than elevation of [Ca2+]o. 6 In sharp contrast, low (0·5 mM) caffeine concentrations that produced no measurable effects on ICa or Ca2+ transients in control myocytes, re‐established spontaneous focal Ca2+ releases in CSQ‐overexpressing cells, triggered large ICa‐gated cellular Ca2+ transients, and strongly enhanced the kinetics of inactivation of ICa. 7 Our data suggest that impaired Ca2+ signalling in CSQ‐overexpressing myocytes results from reduced co‐ordination and decreased frequency of Ca2+ sparks. The impaired Ca2+ signalling could not be restored by procedures that increased ICa, but was mostly restored in the presence of caffeine, which may alter the Ca2+ sensitivity of the ryanodine receptor.

Diversity of atrial local Ca2+ signalling: evidence from 2‐D confocal imaging in Ca2+‐buffered rat atrial myocytes

Atrial myocytes, lacking t‐tubules, have two functionally separate groups of ryanodine receptors (RyRs): those at the periphery colocalized with dihydropyridine receptors (DHPRs), and those at the cell interior not associated with DHPRs. We have previously shown that the Ca2+ current (ICa)‐gated central Ca2+ release has a fast component that is followed by a slower and delayed rising phase. The mechanisms that regulate the central Ca2+ releases remain poorly understood. The fast central release component is highly resistant to dialysed Ca2+ buffers, while the slower, delayed component is completely suppressed by such exogenous buffers. Here we used dialysis of Ca2+ buffers (EGTA) into voltage‐clamped rat atrial myocytes to isolate the fast component of central Ca2+ release and examine its properties using rapid (240 Hz) two‐dimensional confocal Ca2+ imaging. We found two populations of rat atrial myocytes with respect to the ratio of central to peripheral Ca2+ release (Rc/p). In one population (‘group 1’, ∼60% of cells), Rc/p converged on 0.2, while in another population (‘group 2’, ∼40%), Rc/p had a Gaussian distribution with a mean value of 0.625. The fast central release component of group 2 cells appeared to result from in‐focus Ca2+ sparks on activation of ICa. In group 1 cells intracellular membranes associated with t‐tubular structures were never seen using short exposures to membrane dyes. In most of the group 2 cells, a faint intracellular membrane staining was observed. Quantification of caffeine‐releasable Ca2+ pools consistently showed larger central Ca2+ stores in group 2 and larger peripheral stores in group 1 cells. The Rc/p was larger at more positive and negative voltages in group 1 cells. In contrast, in group 2 cells, the Rc/p was constant at all voltages. In group 1 cells the gain of peripheral Ca2+ release sites (Δ[Ca2+]/ICa) was larger at −30 than at +20 mV, but significantly dampened at the central sites. On the other hand, the gains of peripheral and central Ca2+ releases in group 2 cells showed no voltage dependence. Surprisingly, the voltage dependence of the fast central release component was bell‐shaped and similar to that of ICa in both cell groups. Removal of extracellular Ca2+ or application of Ni2+ (5 mm) suppressed equally ICa and Ca2+ release from the central release sites at +60 mV. Depolarization to +100 mV, where ICa is absent and the Na+–Ca2+ exchanger (NCX) acts in reverse mode, did not trigger the fast central Ca2+ releases in either group, but brief reduction of [Na+]o to levels equivalent to [Na+]i facilitated fast peripheral and central Ca2+ releases in group 2 myocytes, but not in group 1 myocytes. In group 2 cells, long‐lasting (> 1 min) exposures to caffeine (10 mm) or ryanodine (20 μm) significantly suppressed ICa‐triggered central and peripheral Ca2+ releases. Our data suggest significant diversity of local Ca2+ signalling in rat atrial myocytes. In one group, ICa‐triggered peripheral Ca2+ release propagates into the interior triggering central Ca2+ release with significant delay. In a second group of myocytes ICa triggers a significant number of central sites as rapidly and effectively as the peripheral sites, thereby producing more synchronized Ca2+ releases throughout the myocytes. The possible presence of vestigial t‐tubules and larger Ca2+ content of central sarcoplasmic reticulum (SR) in group 2 cells may be responsible for the rapid and strong activation of central release of Ca2+ in this subset of atrial myocytes.

BAY K 8644 modifies Ca2+cross signaling between DHP and ryanodine receptors in rat ventricular myocytes

The amplification factor of dihydropyridine (DHP)/ryanodine receptors was defined as the amount of Ca2+ released from the sarcoplasmic reticulum (SR) relative to the influx of Ca2+ through L-type Ca2+ channels in rat ventricular myocytes. The amplification factor showed steep voltage dependence at potentials negative to -10 mV but was less dependent on voltage at potentials positive to this value. In cells dialyzed with 0.2 mM cAMP in addition to 2 mM fura 2, the Ca2+-channel agonist (-)-BAY K 8644 enhanced Ca2+-channel current ( I Ca), shifted the activation curve by -10 mV, and significantly delayed its inactivation. Surprisingly, BAY K 8644 reduced the amplification factor by 50% at all potentials, even though the caffeine-releasable Ca2+ stores were mostly intact at holding potentials of -90 mV. In contrast, brief elevation of extracellular Ca2+ activity from 2 to 10 mM enhanced both I Ca and intracellular Ca2+ transients in the absence or presence of BAY K 8644 but had no significant effect on the amplification factor. BAY K 8644 abolished the direct dependence of the rate of inactivation of I Ca on the release of Ca2+ from the SR. These findings suggest that the gain of the Ca2+-induced Ca2+ release in cardiac myocytes is regulated by the gating kinetics of cardiac L-type Ca2+ channels via local exchange of Ca2+ signals between DHP and ryanodine receptors and that BAY K 8644 suppresses the amplification factor through attenuation of the Ca2+-dependent inactivation of Ca2+ channels.

Molecular determinants of cAMP‐mediated regulation of the Na+–Ca2+ exchanger expressed in human cell lines

The cardiac Na+–Ca2+ exchanger (NCX1) is one of the major sarcolemmal Ca2+ transporters of cardiomyocytes. Structure–function studies suggest that β‐adrenergic inhibition of NCX1, as reported for frog, but not mammalian hearts, may be associated with a unique splice variant of frog cardiac NCX1 where insertion of an extra exon completes the coding of a nucleotide binding P‐loop. To test the involvement of the P‐loop in cAMP‐mediated regulation of NCX1 we used four stably transfected human cell lines (a previously established line of baby hamster kidney (BHK) cells and three new lines of human embryonic kidney (HEK) cells) expressing: (1) wild‐type dog NCX1 (dog NCX1); (2) wild‐type frog NCX1 (frog NCX1); (3) chimeric frog‐dog NCX1 incorporating the completed P‐loop from the frog NCX1 into the dog NCX1 sequence (frog/dog NCX1); and (4) a mutated frog NCX1 where a putative protein kinase A (PKA) site was disrupted by substitution of a single serine residue with glycine (S374G frog NCX1). Structural expression of these NCX1 constructs was confirmed using Western blot analysis of extracted proteins and immunofluorescence imaging. The NCX1‐generated current (INa–Ca) was reliably measured in cells expressing dog (2.0 ± 0.15 pA pF−1), frog (0.6 ± 0.1 pA pF−1) and frog/dog (0.6 ± 0.1 pA pF−1) NCX1, but less so in those expressing S374G frog NCX1 (0.3 ± 0.1 pA pF−1). Addition of 100 μm 8‐bromoadenosine 3′,5′ cyclic monophosphate (8‐Br‐cAMP) suppressed INa–Ca of frog and frog/dog NCX1 by 60–80 %. The suppression of INa–Ca was smaller and transient in cells expressing S374G frog NCX1, and absent in cells expressing dog NCX1. Intracellular Ca2+ (Ca2+1)‐transients, activated by rapid withdrawal of Na+, were also downregulated in the frog and frog/dog NCX1 and to a smaller and transient extent in S374G frog NCX1. Our findings suggest that the suppressive effect of β‐adrenergic agonists requires the presence of the P‐loop domain of the frog NCX1, and provide evidence that the putative PKA site, present in both dog and frog NCX1, might also be critical in the cAMP‐mediated regulation of the exchanger.