Quarky calcium release in the heart

Abstract

Rationale: In cardiac myocytes, "Ca(2+) sparks" represent the stereotyped elemental unit of Ca(2+) release arising from activation of large arrays of ryanodine receptors (RyRs), whereas "Ca(2+) blinks" represent the reciprocal Ca(2+) depletion signal produced in the terminal cisterns of the junctional sarcoplasmic reticulum. Emerging evidence, however, suggests possible substructures in local Ca(2+) release events.

Objective: With improved detection ability and sensitivity provided by simultaneous spark-blink pair measurements, we investigated possible release events that are smaller than sparks and their interplay with regular sparks.

Methods and results: We directly visualized small solitary release events amid noise: spontaneous Ca(2+) quark-like or "quarky" Ca(2+) release (QCR) events in rabbit ventricular myocytes. Because the frequency of QCR events in paced myocytes is much higher than the frequency of Ca(2+) sparks, the total Ca(2+) leak attributable to the small QCR events is approximately equal to that of the spontaneous Ca(2+) sparks. Furthermore, the Ca(2+) release underlying a spark consists of an initial high-flux stereotypical release component and a low-flux highly variable QCR component. The QCR part of the spark, but not the initial release, is sensitive to cytosolic Ca(2+) buffering by EGTA, suggesting that the QCR component is attributable to a Ca(2+)-induced Ca(2+) release mechanism. Experimental evidence, together with modeling, suggests that QCR events may depend on the opening of rogue RyR2s (or small cluster of RyR2s).

Conclusions: QCR events play an important role in shaping elemental Ca(2+) release characteristics and the nonspark QCR events contribute to "invisible" Ca(2+) leak in health and disease.

Figure 1. Spark-blink pairs

A, Linescan images of simultaneous measurement of a Ca²⁺ spark (rhod-2) (left) and its companion Ca²⁺ blink (fluo-5N) (middle) in an intact rabbit ventricular myocyte are shown after background subtraction. Unsubtracted fluo-5N image (right) shows the enrichment for the fluo-5N dye in the jSR at the Z-disk. The spark-blink pair is centered on the jSR band which is labeled as “J”. B, Spatial profiles of the spark-blink pair. Arrows mark jSR locations (J, J−1, J−2, J+1 and J+2). C, Time courses of the spark-blink pair.

Figure 2. Quarky Ca²⁺ Release (QCR)

A, Succession of Ca²⁺ sparks and QCR events on the same jSR (top), the corresponding Ca²⁺ blinks and quarky SR Ca²⁺ depletion (QCD) events (middle) and their time courses (bottom). Ticks to the left of the bottom image mark locations of the Z-disks. Arrows denote QCR and QCD on the images and time course plots. B, Enlarged view of the last succession of QCR and spark (top left) and the corresponding QCD and blink (top right) from panel A, the automated detection of QCD/blink (middle right) and the corresponding time courses (bottom). Arrows denote QCR and QCD on the images and time course plot. C&D, Kinetics (C) and amplitude (D) of QCR and QCD. * P<0.05. E, Histogram distribution of amplitude of Ca²⁺ release events (QCR and spark).

Figure 3. QCR in electrically paced cardiomyocytes

A, Simultaneous measurement of diastolic QCR (rhod-2) (top) and QCD (fluo-5N) (middle) events in a paced (0.5 Hz) cardiomyocyte. Ticks to the left of the bottom image mark locations of the Z-disks. Traces on the bottom show time courses of 2 QCR-QCD events identified during diastole along with the transient. The traces of the QCR-QCD events have been enlarged in the inserts. B, Frequency of sparks and QCR events (* P<0.05). C, Averaged spark and QCR signal mass (* P<0.05). D, Summed Ca²⁺ leak of sparks or QCR events.

Figure 4. Spark-blink characteristics

A, Histogram distributions of spark decay time and blink recovery time (t₆₇). B, Scatter plot of spark and blink t₆₇ along with their regression line. C, Top, Average traces of sparks and blinks for t₆₇ (spark) <50 ms (24 events), 5<t₆₇<70 ms (19 events) and t₆₇>70 ms (7 events). Bottom, the same traces after normalization by the amplitude. D, Bar graphs of t_peak and t_nadir for sparks and blinks, respectively, for the same t₆₇ groups as in panel C. E, Peak amplitudes of sparks and blinks for the same t₆₇ groups as in panel C. F, Relationship between spark kinetics and amplitudes. Scatter plot of spark t₆₇ and amplitudes along with their regression line.

Figure 5. Effect of EGTA on kinetics and amplitudes of spark-blink pairs

A, Linescan images of a spark (left) and its conjugate blink (center left) obtained in 0.5 mmol/L EGTA after background subtraction along with the blink automated detection (center right). The time courses of the spark and blink are also shown (right). The black lines between the images mark the positions of jSR located at the Z-disks. B, Same as A, except in a solution containing 2 mmol/L EGTA. C, Bar graph of spark and blink t_peak/t_nadir and t₆₇ in 0.5 and 2 mmol/L EGTA, * P<0.05. D, Scatter plot of spark and blink t₆₇ values in 0.5 and 2 mmol/L EGTA along with their regression lines. E, Spark and blink amplitudes in 0.5 and 2 mmol/L EGTA. * P<0.05.

Figure 6. QCR-QCD events during long spark-blink pairs

A through C, Three examples of long sparks (left) and long blinks (middle), with the corresponding time courses on the right. Ticks next to the images denote the position of the jSR at the Z-disks.

Figure 7. Schematic model of QCR activation

Ca²⁺ spark arising from a CRU composed of 2 clusters of RyR2s and several rogue RyR2s (left). This Ca²⁺ spark may contain QCR events. B. QCR arising from rogue RyR2s on the same CRU as in A.