Figure 2.
HCM mutations acutely decrease the apparent Kd of cytosolic Ca2+ buffering. Cardiomyocytes expressing WT or mutant cTnT/cTnI/α-TM were simultaneously assessed to measure [Ca2+]i by fura2 fluorescence and NCX current (to calculate [Ca2+]total) by voltage clamping upon the rapid application of 10 mm caffeine. A, plots of Δ[Ca2+]i versus Δ[Ca2+]total for cardiomyocytes containing WT cTnT (n = 20) versus R92Q cTnT (n = 23), WT cTnI (n = 9) versus R145G cTnI (n = 11), and WT α-TM (n = 12) versus D175N α-TM (n = 14) reveal increased buffering capacity in cells containing HCM mutants. The red lines show best fits to the following: [Ca2+]total = {Bmax*[Ca2+]i/(Kd + [Ca2+]i)} + Bmin. B, bar graph comparing mean Kd values for the fits calculated in A. C, bar graph showing the relative estimated changes in buffering capacity (calculated from relative Kd and Bmax values for individual cells undergoing the buffing protocol), where mutant calls have been compared with WT-infected cells set to 1. D, cytosolic buffering curves over a wide range of Ca2+ concentration drawn using the Kd and Bmax values calculated in A. Comparison of WT with HCM mutant: **, p < 0.01; *, p < 0.05.