Abstract 577: S100A1 Decreases Calcium Spark Frequency and Alters their Spatial Characteristics in Ventricular Cardiomyocytes
Background: S100A1, a Ca2+-sensor protein of the EF-hand type, exerts positive inotropic effects in the heart via enhanced cardiac ryanodine receptor (RyR2) activity. Although we have shown that S100A1 enhances excitation-contraction coupling gain in cardiomyocytes, the effect of S100A1 on diastolic RyR2 activity and SR Ca2+-leak has not been investigated yet.
Methods and Results: Confocal imaging and coimmunoprecipitation studies show that S100A1 protein (0.1μM) interacts with the RyR2 in resting permeabilized cardiomyocytes at free Ca2+-levels comparable to diastolic Ca2+-concentrations (~150nM). Preincubation with the S100A1 C-terminus (S100A1-ct, aa 75–94, 10μM) inhibited both S100A1 protein colocalization and coimmunoprecipitation with the RyR2. Alterations of RyR2 function due to S100A1 binding was assessed via analysis of Ca2+-spark characteristics. Ca2+-spark frequency, amplitude and duration were all significantly reduced upon perfusion with 0.1μM S100A1 protein (n=350 sparks, n=12 myocytes) by 38%, 14% and 18%, respectively, compared to control (n=336 sparks, n=10 myocytes, p<0.05). Most likely, these effects were conveyed through the S100A1 C-terminus (S100A1-ct; amino acids 75–94) as the corresponding S100A1-ct peptide (0.1μM, n=374 sparks, n=12 myocytes) that inhibited S100A1 protein binding to the RyR2 similarly attenuated frequency, amplitude and duration of Ca2+-sparks by 52%, 8% and 26%, respectively, compared to control (n=368 sparks, n=10 myocytes, p<0.05). Accordingly, S100A1 (n=15) significantly increased sarcoplasmic reticulum Ca2+-content (+15%, p<0.05) but did not alter the stoichiometry of other accessory RyR2 modulators such as sorcin or FKBP12.6 as assessed by Western blotting.
Conclusion: Hence, we propose S100A1 as a novel inhibitory modulator of RyR2 function at diastolic Ca2+-concentrations in cardiomyocytes that might contribute to prevention of diastolic SR calcium leak through inhibition of RyR2 activity at rest but enhances the calcium-activated state of the channel in systole.