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

Abstract

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.

Keywords: CICR gain; CPVT; Calcium signaling; Mutation in RyR2 gene; Pluripotent stem cells.

Fig. 1

Automated analysis of Ca²⁺ sparks based on TIRF-imaging. (A) Equalization of the diastolic fluorescence intensity. The image (ROI) shows a single frame with superimposed regions of interest that were marked in a semi-transparent manner in red (cell) and blue (surroundings). The fluorescence intensities in these regions were plotted versus time (red and blue curves), and were approximated throughout the diastolic intervals by a black curve and a line that together determined a scale factor that varied with time and was used to compensate for the slow decline in fluorescence that often occurred in the intervals between beats. (B) Ratiometric images. The distribution of fluorescence intensity in each frame (e.g. #38) was divided by an average fluorescence intensity calculated based on multiple selected fluorescence images without noticeable Ca²⁺-release activity. (C) A sequence of partial ratiometric images where the development and decay of local Ca²⁺ release is shown in bright colors on a dark blue mottled background representing the resting Ca²⁺ activity. (D) Determination of unitary properties of Ca²⁺ release based on a Gaussian approximation. The two sample images (#29 and #34) are shown with superimposed white circles that mark the location and standard deviations of Gaussian approximations. The curves above and to the left of the images show how well horizontal and vertical mid sections through the images were approximated by Gaussian curves. The graph at the bottom of Panel D quantifies the horizontal shift in center of the Gaussian approximations as seen also in the curves above the sample images. The shown partial frames correspond to 30 × 30 pixels or 9.4 μm × 9.4 μm.

Fig. 2

Distribution of calcium currents (I_Ca) density and average time constant (tau) in control (IMR-C8) and mutant (NP0014-C1) iPS-CM. Cells were cultured for 3–6 days. I_Ca was recorded from a holding potential −40 mV with step depolarization to 0 mV. The number at the bottom of each bar indicates the capacitance of individual cells. (Panels A and B) Distribution of I_Ca density in control and mutant iPS-CM recorded with [Na⁺]_i at 5 or 15 mM Na⁺_i. C: Average fast (tau f) and slow (tau s) time constants of inactivation of I_Ca in control and mutant iPS-CM recorded with 5 mM Na⁺ in the pipette solution. (D) Average I_Ca density of control and mutant iPS-CM and representative I_Ca traces from each group. The average membrane capacitance values were: Panel A, IMR-C8, 32.7 ± 2.3 pF, NP0014, 43.6 ± 3.7 pF, and Panel B, NP0014, 45.8 ± 4.2 pF.

Fig. 3

Voltage-dependence and subcellular distribution of I_Ca -activated Ca²⁺ -transients. Representative current–voltage (I–V) relations normalized relative to the membrane capacitance and the corresponding fluorescence (Fluo-4) Ca²⁺ signal recorded from control (C5, Panel A) and mutant (NP0014, Panel B) iPS-CM. Currents were recorded with a 250 ms step depolarizations from holding potential of −50 mV in 10 mV steps to +60 mV. The middle panel shows the I–V curves for I_Ca and the corresponding Ca²⁺ Fluo-4 signal. The internal solution contained 5 mM Na⁺, and was Ca²⁺-buffered with 0.1 mM Fluo-4, 0.2 mM EGTA, and 0.1 mM Ca²⁺. The right panel shows the I_Ca and fluorescence traces at +60 or +70 mV activating rises in Ca²⁺ on repolarization. (C) Confocal image of baseline fluorescence (Fluo-4, F₀). (D) Color-coded regions of interest (ROI) corresponding to nuclei (n), patch electrode (e) and cytoplasmic regions with increasing distances from the cell membrane (red, orange, green). (E) Differences in the time course of the normalized Ca²⁺-dependent fluorescence (ΔF/F₀). The color coded traces corresponds to the ROI in Panel D. The 2-D confocal fluorescence images were recorded at 120 Hz using a focal plane intersecting the nuclei. The initial response is shown on an expanded time scale. The voltage-clamp pulse (from −60 to −30 mV) and the resulting (Na⁺ and Ca²⁺) membrane currents are shown at the top. (F) Confocal image of immunofluorescence labeled RyR2 (green) in a small cluster of control iPS-CM with DAPI-labeled nuclei (blue).

Fig. 4

Fractional Ca²⁺ release in control and mutant iPS-CM. Fractional release was calculated by dividing the I_Ca -triggered Ca_i -transient by that generated by application of caffeine (ΔF_(ICa)/ΔF_(Caff)). Cells were voltage-clamped from −50 to 0 mV or were held at −50 mV while subjecting them to 0.5 s long 3 mM caffeine pulses. (A and B) Distribution of the value of efficiency in control (A) and mutant iPS-CM (B). The numbers at the bottom of each bar indicate the membrane capacitance of each cell. (C) Average fractional Ca²⁺ release with 5 and 15 mM Na⁺ in the pipette solution. (D) Representative Ca²⁺ signals triggered by I_Ca or caffeine in control (a) and mutant (b) iPS-CM.

Fig. 5

Caffeine-releasable Ca²⁺ stores. (A–E) Simultaneous measurements of caffeine-induced I_NCX current and Ca²⁺ -dependent fluorescence (ΔF/F₀) in control (IMR-C8) and mutant (NP0014) iPS-CM dialyzed with 5 mM Na⁺ and superfused with standard Tyrode’s solution. Cells were voltage-clamped to −50 mV and exposed rapidly to 3 mM caffeine for 500 ms. (A and B) Representative caffeine-induced NXC currents and corresponding fluorescence Ca²⁺ signal from control (a) and mutant iPS-CM (b). (C and D) Distribution of I_NCX values in control and mutant iPS-CM. (E) Average values of caffeine-activated Ca²⁺ signals (top) I_NCX currents (bottom) in each group. Stars indicate significance levels (*p < 0.05, **p < 0.01). (F and G) Effect of intracellular Na⁺ on the amount of Ca²⁺ released from SR in response to application of caffeine. Average values of Ca²⁺ release from SR calculated from the integral of the caffeine-induced I_NCX in control and mutant iPS-CM with 5 or 15 mM Na⁺ in the internal solution. (G) Average valves of gain factor and I_Ca density at −30 and 0 mV. The gain factor is plotted in units corresponding to the fraction in % of the caffeine-induced Ca²⁺ release that is release by a Ca²⁺ current with a density of 1 pA/pF.

Fig. 6

Effects of isoproterenol. (A–E) Caffeine-induced I_NCX current in control iPS-CM (IMR-C5) recorded with 5 mM Na⁺_i before and after exposure to isoproterenol. (A and B) Representative I_NCX currents and corresponding fluorescence Ca²⁺ signal before (A) and after (B) exposure to 75 nM isoproterenol. (C and D) Distribution of Ca_i -transients (ΔF/F₀) and I_NCX values before (C) and after (D) exposure to isoproterenol. (E) Comparison of average values of ΔF/F₀ and I_NCX in each group (*p < 0.05, **p < 0.01). (F and G) Effects of isoproterenol on spontaneous Ca²⁺ release activity in regularly depolarized (0.2 Hz) dialyzed with pipette solutions containing 5 or 15 mM Na⁺. (F) Changes in Ca²⁺ transients (Fluo-4), I_Ca and I_NCX at the onset of exposure to 100 nM isoproterenol in mutant iPS-CM (NP0014-C1) dialyzed with 5 (top) or 15 mM Na⁺ (bottom). (G and H) Effects of isoproterenol on baseline Ca²⁺ (F₀, Panel G) and the fraction (in %) of cells with spontaneous Ca²⁺ releases (intervening between the I_Ca -triggered transients; Panel H) of control (WT) and mutant (NP0014) biPS-CM) dialyzed with 5 or 15 mM Na⁺ before and after exposure to isoproterenol. The numbers of examined cells are shown in parentheses.

Fig. 7

Ca²⁺ sparks in control (A and D) and mutant (B and E) iPS-CM. (A and B) From left to right the panels show: (1) Images of average diastolic fluorescence distributions, (2) color coded regions of interests corresponding to locations of Ca²⁺ sparks, and (3) the time course of the normalized fluorescence intensity at these locations. The histogram in Panel C shows the distributions of the duration of Ca²⁺ sparks measured at half peak amplitude for control cells (black, average = 40.4 ± 3.5 ms, n = 76) and mutant cells (red, average = 89 ± 7.5 ms, n = 50). (D and E) Image sequences showing the evolution of Ca²⁺ sparks. The colored labels correspond to the traces in Panels A and B.

Fig. 8

Effects of adrenergic stimulation on I_Ca, Ca_i -transients, and gain factor in control and mutant iPS-CM. (A and B) I_Ca traces and the corresponding Ca²⁺ fluorescence in control and mutant iPS-CM before (lack) and after (red) treatment with Isoproterenol. Cells were depolarized from −40 mV to 0 mV. (C and D) Average values of peak I_Ca and ΔF/F₀ in each group before and after exposure to isoproterenol. (D and E) Effects of isoproterenol on the gain factor at −30 and 0 mV. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 9

TIRF imaging of Ca²⁺ sparks in mutant iPS-CM before (A–D) and after (E–H) 3 min exposure to 100 μM dBcAMP. From top to bottom the matched panels show: The time course of cellular Ca²⁺ transients (A and E), Ca²⁺ sparks at selected color coded sites (during the diastolic interval showed in gray above, B and F), and maps of the locations of Ca²⁺ sparks superimposed on sample ratiometric images of Ca²⁺ sparks (C and G) or the onset of Ca²⁺ release (D and H at the times indicated by *s in A and E). Connecting lines show movements of the center of release from one frame to the next.