Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor

Abstract

Cardiac ryanodine receptor (RyR2) is a homotetramer of 560 kDa polypeptides regulated by calmodulin (CaM), which decreases its open probability at diastolic and systolic Ca(2+) concentrations. Point mutations in the CaM-binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2(ADA)) in mice result in severe cardiac hypertrophy, poor left ventricle contraction and death by postnatal day 16, suggesting that CaM inhibition of RyR2 is required for normal cardiac function. Here, we report on Ca(2+) signalling properties of enzymatically isolated, Fluo-4 dialysed whole cell clamped cardiac myocytes from 10-15-day-old wild-type (WT) and homozygous Ryr2(ADA/ADA) mice. Spontaneously occurring Ca(2+) spark frequency, measured at -80 mV, was 14-fold lower in mutant compared to WT myocytes. ICa, though significantly smaller in mutant myocytes, triggered Ca(2+) transients that were of comparable size to those of WT myocytes, but with slower activation and decay kinetics. Caffeine-triggered Ca(2+) transients were about three times larger in mutant myocytes, generating three- to four-fold bigger Na(+)-Ca(2+) exchanger NCX currents (INCX). Mutant myocytes often exhibited Ca(2+) transients of variable size and duration that were accompanied by similarly alternating and slowly activating INCX. The data suggest that RyR2(ADA) mutation produces significant reduction in ICa density and ICa-triggered Ca(2+) release gain, longer but infrequently occurring Ca(2+) sparks, larger sarcoplasmic reticulum Ca(2+) loads, and spontaneous Ca(2+) releases accompanied by activation of large and potentially arrhythmogenic inward INCX.

Figure 1. CaMBD of RyR2 (A) causes cardiac hypertrophy (B) and disorganized cellular structure (C)

A, location of the CaMBD in the context of binding domains for FKBP12.6 and Ca²⁺, phosphorylation sites and hot spots for mutations causing catecholaminergic polymorphic ventricular tachycardia such as the F2431I mutation (Li & Chen, 2001; Lanner et al. 2010; Fatima et al. 2011). B, hypertrophy of hearts from HOM as seen at day 11 compared to WT. C, distributions of RyR2, NCX and Mem staining with voltage-sensitive dye (di-ANEPQ) in freshly isolated WT and HOM cardiomyocytes showing sarcomeric distributions with differing regularity and predominant localization of NCX at the surface membrane. It is noticeable that the ultrastructure of HOM cells is much less organized. Arrows in the right panel suggest that HOM cells have more prominent longitudinal tubules. Nuclei are marked with Ns and/or blue staining (top). CaMBD, calmodulin-binding domain; HOM, homozygous mutant mice; LA, left atria; LV, left ventricle; Mem, membrane; NCX, Na⁺-Ca²⁺ exchanger; RA, right atria; RV, right ventricle; RyR2, type 2 ryanodine receptor; WT, wild-type.

Figure 2. Ca²⁺ transients and membrane currents in single voltage-clamped cardiomyocytes from WT (black traces) and HOM (grey traces) mice

A, calcium transients (Fluo-4, ΔF/F₀, bottom) evoked by activation of I_Ca (top) during 400 ms depolarizations to 0 mV. B, ratiometric confocal images (ΔF(x,y,t)/F₀(x,y)) of Fluo-4 fluorescence measured at 30 Hz at the onset of the depolarization. The first frame in each sequence was recorded just before depolarization and shows a fairly uniform coloration over the entire cell with some blue/green mottle corresponding to the noise of the recordings. In the next frame, the WT cell shows an abrupt increase in fluorescence as a transition to warmer colours (See colour scale) yellow, half way through the 30 ms scan from top to bottom. In comparison, the second frame from the cardiomyocyte from the HOM mouse shows a delayed and gradual increase in fluorescence that starts at the edges, increases slowly towards the end of the scan and is still rising in the following two frames. C, I_NCX (top) and Ca²⁺ transients evoked by a 2 s application of 10 mm caffeine. D, ratiometric confocal fluorescence images indicate that Ca²⁺ signals evoked by caffeine start at the ends of the cells and rise more slowly than those triggered by I_Ca, especially in cells from the HOM mouse. E, ratio of the Ca²⁺ releases was calculated as the magnitude of depolarization-induced Ca²⁺ transients compared to those evoked by caffeine. This efficiency of I_Ca-gated Ca²⁺ release (fractional release) was significantly larger for WT (82%) than for HOM (30%) mice. The cells were dialysed with an internal solution containing 0.5 mm K₅Fluo-4 and 0.2 mm CaCl₂. HOM, homozygous mutant mice; WT, wild-type.

Figure 3. Voltage dependence of I_Ca and I_Ca-activated Ca²⁺ transients in cardiomyocytes from WT (A and C) and HOM (B and D)

A and B, representative traces where I_Ca was normalized relative to the membrane capacitance and changes in the Ca²⁺-dependent fluorescence (ΔF/F₀) was measured ratiometrically with Fura-2. The recordings were obtained with 200 ms depolarizing pulses from a holding potential of −50 mV in 10 mV steps to +70 mV. C and D, voltage dependencies of peak I_Ca and ΔF/F₀. The internal solution contained 5 mm Na⁺, and was Ca²⁺ buffered with 0.5 mm Fura-2, and 0.5 mm Ca²⁺. HOM, homozygous mutant mice; WT, wild-type.

Figure 4. Average values of the rate of rise and decay of Fura-2-detected cytosolic Ca²⁺ transients measured during 400 ms depolarizations from −50 to 0 mV

A, rate of rise (ΔF/ms) was significantly slower in HOM than in the WT mice (P < 0.01). B, rate of decay showed considerable variation in the HOM mice, but on average was not significantly different from the WT. Ca²⁺-sensitive fluorescence was measured using Fura2 dialysed cells (0.5 mm Fura2 and 0.5 mm Ca²⁺. HOM, homozygous mutant mice; WT, wild-type.

Figure 5. Distribution of calcium currents (I_Ca) density and average cell capacitance (C_m) in WT and HOM mice

I_Ca was recorded from a holding potential −50 mV with step depolarization to 0 mV. A and B, distribution of I_Ca densities in 37 cardiomyocytes from seven WT mice (A) compared to those in 52 cardiomyocytes from eight HOM mice (B). C, average I_Ca density was larger in WT than in HOM mice. D, average membrane capacitance of the cardiomyocytes was 60 ± 6 pF in WT myocytes compared to 92 ± 8 pF in the HOM myocytes. s.e.m. and thereby P values, in C and D are based conservatively on the numbers of experimental animals (seven and eight). HOM, homozygous mutant mice; WT, wild-type.

Figure 6. Average values of I_Ca, I_NCX, Ca_i transients (Fura-2 signals), and gain factor in WT and HOM mice myocytes

A and C, average Ca²⁺ signals (A) evoked I_Ca at 0 mV (C) in each group. B and D, average values of caffeine-activated Ca²⁺ signals (B) and the accompanying I_NCX (D) in each group. E, average valves of gain factor at −30 and 0 mV. The gain factor is plotted in units corresponding to the fraction (in %) of the caffeine-induced Ca²⁺ release, which is released by an I_Ca with a density of 1 pA/pF (WT black, HOM grey). Ca²⁺ transients were measured with Fura-2. HOM, homozygous mutant mice; WT, wild-type.

Figure 7. Calcium sparks in Fluo-4-dialysed voltage-clamped cardiomyocytes from WT and HOM mice

A, ratiometric recording of Ca²⁺ sparks in whole cells at 30 Hz. From top to bottom each panel shows: (1) images of the average fluorescence distributions in frames without Ca²⁺ sparks (F₀(x,y)); (2) colour-coded regions of interests corresponding to locations of Ca²⁺ sparks (middle panel); (3) examples of sparks as seen in single normalized frames (ΔF(x,y)/F₀(x,y)), lower panel; and (4) the time course of the normalized fluorescence intensity at the chosen locations. B, number of sparks per cell per second was reduced from 3.62 ± 0.54per s in WT to 0.26 ± 0.17 per s in HOM cells. C, time course of Ca²⁺ sparks measured at 240 frames per s in portions of cells. At the top are shown colour-coded locations of Ca²⁺ sparks and sample frames with sparks at these locations. The traces below show the time course of ΔF/F₀ in colour-coded traces corresponding to the chosen locations. For illustration purposes, we chose a rare mutant cell with several sparks that nevertheless was characteristic in having long durations. D and E, amplitude of the Ca²⁺ sparks are comparable in WT and HOM, but the duration is significantly longer in the HOM (FWHA). F, distribution of the sparks in the WT and the HOM according to ΔF/F₀ and FWHA. FWHA, full width at half amplitude; HOM, homozygous mutant mice; WT, wild-type.

Figure 8. Spontaneous Ca²⁺ releases and associated I_NCX in cardiomyocytes from homozygous mutant mice

From top to bottom each panel shows voltage clamp depolarizations, membrane currents and cellular Ca²⁺ signals measured ratio metrically with Fura-2. A, six consecutive depolarizations at 0.2 Hz with alternating spontaneous large Ca²⁺ releases and I_NCX. B–E, cells with spontaneous Ca²⁺ releases and I_NCX with variable magnitudes and delays. F and G, cumulative effects of repeated depolarizations leading to large delayed Ca²⁺ releases before (F) and after (G) exposure to forskolin.