Protein protein interactions between triadin and calsequestrin are involved in modulation of sarcoplasmic reticulum calcium release in cardiac myocytes

Abstract

In cardiac muscle, intracellular Ca2+ release is controlled by a number of proteins including the ryanodine receptor (RyR2), calsequestrin (CASQ2), triadin-1 (Trd) and junctin (Jn) which form a complex in the junctional sarcoplasmic reticulum (SR) membrane. Within this complex, Trd appears to link CASQ2 to RyR2 although the functional significance of this interaction is unclear. In this study, we explored the functional importance of Trd-CASQ2 interactions for intracellular Ca2+ handling in rat ventricular myocytes. A peptide encompassing the homologous CASQ2 binding domain of Trd (residues 206-230 in the rat; TrdPt) was expressed in the lumen of the SR to disrupt Trd-CASQ2 interactions. Myocytes expressing TrdPt exhibited increased responsiveness of SR Ca2+ release to activation by ICa as manifested by flattened and broadened voltage dependency of the amplitude of cytosolic Ca2+ transients. Rhythmically paced, TrdPt-expressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca2+ and membrane potential that was not seen in control cells. In addition, the frequency of spontaneous Ca2+ sparks and Ca2+ waves was significantly increased in TrdPt-expressing, permeabilized myocytes. These alterations in SR Ca2+ release were accompanied by a significant decrease in basal free intra-SR[Ca2+] and total SR Ca2+ content in TrdPt-expressing cells. At the same time a synthetic peptide corresponding to the CASQ2 binding domain of Trd produced no direct effects on the activity of single RyR2 channels incorporated into lipid bilayers while interfering with the ability of CASQ2 to inhibit the RyR2 channel. These results suggest that CASQ2 stabilizes SR Ca2+ release by inhibiting the RyR2 channel through interaction with Trd. They also show that intracellular Ca2+ cycling in the heart relies on coordinated interactions between proteins of the RyR2 channel complex and that disruption of these interactions may represent a molecular mechanism for cardiac disease.

Figure 1

SR localization in ventricular myocytes of adenovirally expressed TrdPt CASQ2 and TrdPt were coimmunostained with CASQ2 and Myc Tag-specific antibodies using green and red fluorescent tagged secondary antibodies, respectively. Images were recorded simultaneously with an Olympus Fluoview-1000 spectral confocal system. Alexa-Fluo 488 was excited with the 488 nm argon laser line and emission was collected at 500–530 nm, while Alexa Fluo-546 was excited at 543 nm and emission recorded at > 570 nm. Measurements were performed 48–56 h after infection.

Figure 2

Loss of voltage dependency of Ca²⁺ release in cells expressing TrdPt A, traces of I_Ca (lower traces) and intracellular Ca²⁺ transients (upper traces) evoked by depolarization steps from −50 mV HP to different voltages in myocytes infected with Ad-TrdPt and Ad-Control vectors. B and C, voltage dependencies of Ca²⁺ transients (B) and I_Ca (C) in myocytes infected with Ad-Control (▪), and Ad-TrdPt (•) vectors (n from 6 to12). Average rise times and maximal rates of rise of Ca²⁺ transients evoked by depolarization to 0 mV were not significantly different for control and TrdPt expressing myocytes (32 ± 4 ms and 0.49 ± 0.12 ms⁻¹ (n = 6) versus 24 ± 4 ms and 0.49 ± 0.16 ms⁻¹ (n = 5), respectively).

Figure 3

Arrhythmogenic disturbances in Ca²⁺ cycling in myocytes expressing TrdPt Whole cell current clamp recordings of membrane potential (upper traces), line-scan images of Ca²⁺ transients (middle) and time dependent profiles (lower traces) in the presence of 1 μm isoproterenol. Stimulation frequency was 2 Hz.

Figure 4

Expression of TrdPt leads to increased frequency of spontaneous Ca²⁺ sparks in permeabilized myocytes A, representative line scan images of Ca²⁺ sparks in cells infected with adenoviral constructs. B, C, D and E, bar graphs of pooled values of spark frequency (B), amplitude of 5% of brightest events (C), rise time (D) and spark duration at half-amplitude (E) for the same groups of cells. *Significantly different at P < 0.05; one-way ANOVA (n of Ca²⁺ sparks was 388 and 565; n of cells was 73 and 76 for Ad-Control and Ad-TrdPt-infected myocytes, respectively).

Figure 5

Effects of expression of TrdPt on properties of cytoplasmic and intra-SR Ca²⁺ waves in permeabilized myocytes A, line-scan images along with time-dependent profiles of Rhod-2 and Fluo-5N fluorescence. B, C, D and F, pooled data on wave frequency (B), cytosolic wave amplitude measured with Rhod-2 (C), changes in the baseline [Ca²⁺]_SR and intra-SR [Ca²⁺] wave nadir measured with SR-entrapped Fluo-5N (D) and amplitude of cytosolic caffeine-induced Ca²⁺ transients (F). E, caffeine-evoked Ca²⁺ transients (upper panel) and depletion signals (lower panel) recorded in myocytes infected with Ad-Control and Ad-TrdPt vectors. *Significantly different at P < 0.05; one-way ANOVA (n from 8 to 17).

Figure 6

SERCA2a-mediated SR Ca²⁺ uptake is not significantly altered in TrdPt expressing myocytes A, time course of SR Ca²⁺ uptake, measured with SR-entrapped Fluo-5N in permeabilized control and TrdPt-expressing myocytes in the presence of 10 μm ruthenium red. Intra-SR [Ca²⁺] was depleted with 10 mm caffeine; caffeine was washed out with a Ca²⁺-free solution; then ruthenium red was applied and SR Ca²⁺ uptake initiated by raising cytosolic [Ca²⁺] to 100 nm[Ca²⁺]. B, pooled data for the time constants from single exponential fits of SR Ca²⁺ uptake. n was 11 and 12 for control and TrdPt expressing cells, respectively.

Figure 7

A synthetic decoy peptide corresponding to CASQ2 binding domain of Trd (residues 200–224) interferes with CASQ2 inhibitory action on RyR2 channel complex Trd 200–224 does not modulate activity of purified RyR2 channels. A, representative single RyR2 channel traces. B, pooled data on open probability of purified RyR2s before and after addition to the trans chamber 5–50 μm of the Trd decoy peptide (n = 8). C, representative single-channel traces illustrating the irreversibility of the effects of 5 mm luminal Ca²⁺ and restoration of initial low activity by CASQ2 added to the trans chamber in native RyR2s (upper panel). Trd 200–224 interferes with the ability of CASQ2 to inhibit native RyR2s (lower panel). The P_o values were 0.06 ± 0.01 for low trans[Ca²⁺] (20 μm); 0.29 ± 0.07 for high trans[Ca²⁺] (5 mm); 0.41 ± 0.12 reverting to low trans[Ca²⁺] (20 μm); and 0.04 ± 0.01 (n = 6) versus 0.48 ± 0.08 (n = 4)* after application of 5 μg ml⁻¹ of CASQ2 alone or with 50 μg ml⁻¹ of Trd 200–224. The data are presented as means ±s.e.m.*Significantly different versus CASQ2 alone at P < 0.05 (one-way ANOVA).