In the heart, Ca2+ is released from the intracellular Ca2+ store called the sarcoplasmic reticulum (SR). Release is gated by ryanodine receptors (RyRs), which are grouped in clusters of about a 100. Because a cluster of RyRs acts in concert to release Ca2+, the cluster is called a Ca2+ release unit or CRU. When the cardiomyocyte is loaded with a fluorescent Ca2+ indicator, we can see the Ca2+ that is released from a CRU as brief points of light, which were imaginatively christened Ca2+ sparks by their discoverers Heping Cheng, Jon Lederer, and Mark Cannell.Click on the image on the right to see a movie of Ca2+ sparks obtained with our high-speed 2D scanning confocal microscope.
RyRs have the remarkable property that the probability that they will open (and release Ca2+) increases as the Ca2+ concentration around it increases. What this means that when Ca2+ is released from a CRU it raises the Ca2+ concentration around a neighboring CRU, which makes it more likely that the neighbor will also open or “fire”. CRUs are analogous to piles of gunpowder. One can well imagine that if enough energy is released from one gunpowder pile, if the gunpowder is sufficiently unstable, and if the piles are close enough to one another then lighting one or a few will start a wave of burning. CRUs can interact strongly enough with each other and produce waves of Ca2+ release as seen in this movie.
What makes the Ca2+ control system unstable?
A Ca2+ wave is a hallmark of an unstable Ca2+ control system. Our interest in when the Ca2+ control system becomes unstable stems from the fact that spontaneously generated Ca2+ waves can trigger an action potential. If Ca2+ waves occur in enough heart cells then arrhythmias and sudden cardiac death may occur. Therefore, we are interested in determining what factors underlie the stability of the Ca2+ control system.In our gunpowder analogy, a wave of burning would occur if (a) the gunpowder was unstable enough, (b) each pile gave off enough energy, and (c) the piles were close enough to each other. The stability of the Ca2+ control system is controlled by analogous factors: (a’) sensitivity of the CRUs to the Ca2+ concentration, (b’) the amount of Ca2+ released by a CRU, and (c’) the distances between CRUs. These factors determine how one CRU influences another. When the influence is high, Ca2+ waves can spontaneously form.
Eavesdropping on the social lives of Ca2+ sparks
We developed a mathematical method to quantitatively measure the influence of one CRU on another, effectively allowing us to eavesdrop on how CRUs talk to each other. The idea behind the analysis is simple and can be understood in terms of raindrops on a pond. Imagine being able to see only the ripples caused by raindrops but not the raindrops themselves. The figure below shows the two ways of generating two sets of ripples. In A, the two sets are generated by two raindrops falling independently; in B, the two sets are generated by one raindrop and a splash. Likewise, two Ca2+ sparks could be independently generated or one spark could have been triggered by another. Now a raindrop is unlikely to generate a ripple very far away and similarly one spark is unlikely to trigger a CRU very far away. From the spatial distribution of Ca2+ sparks (figure at right) we can determine how the firing of one spark will increase the probability of that a CRU at a distance r will fire.
How the spatial distribution of CRUs affects stability
One would intuitively guess that if the CRUs were very close to each other then one spark will trigger the next, generate a Ca2+ waves, and the heart would never function. Of course, the heart works fine most of the time because the CRUs are not packed like sardines. On the other hand, the CRUs cannot be too far apart because the Ca2+ concentration would not rise high enough to activate contraction of the myofibrils. Therefore, the CRU spacing represents a compromise between the need for stability (large spacing) and the need for myofibrillar activation (small spacing).
The bright spots in the figure at the right are CRUs that were fluorescently tagged with antibodies to the RyRs. We measured the CRU spacing of the CRUs in ventricular and atrial cells of normal rats,and used the spacing values to construct a mathematical model of the Ca2+ control system. We found that the stability of the Ca2+ control system was very sensitive to the CRU spacing. The three figures below are movies of three computer simulations where all that is changed is the spacing of the z-lines (big blue circles) where the CRUs are located. In the leftmost simulation video the z-line spacing is 2 µm. The triggered sparks (cyan-colored exploding ellipses) at the start of the simulation activate a few other sparks but otherwise nothing happens. In the middle simulation the z-line is shortened 10% to 1.8 µm. Now the triggered sparks activate a Ca2+ wave. Shortening the z-lines by another 0.2 µm makes the Ca2+ control system even more unstable and the Ca2+ wave starts even more quickly. These simulations show how sensitive the Ca2+ control system is to even subtle changes in the CRU spatial distribution.
Understanding arrhythmias in FHC
The sensitivity of the Ca2+ control system to changes in the spatial distribution of CRUs could provide an explanation for why people with some forms of familial hypertrophic cardiomyopathy (FHC) are highly susceptible to arrhythmia and sudden cardiac death. FHC involves mutations of the sarcomeric proteins involved in muscle contraction. There is a long-standing question of how a mutation of a contractile protein leads to an increase in electrical arrhythmias. We’re hypothesizing that these mutations are causing the CRUs to be closer to each other and thereby decreasing the stability of the Ca2+ control system.
We are beginning to test this hypothesis in collaboration with Jil Tardiff at Albert Einstein in New York. Stay tuned.
