Extended Data Figure 3: Calcium transient effects of miR-25 compared with that of siRNAs against SERCA2a and IP3R isoforms.

Cardiomyocyte-like HL-1 cells were transfected with miR-25, siRNA to SERCA2a (siSerca2a) (left panels) or siRNAs to IP3R1 or IP3R2 (siIP3R1, siIP3R2) (right panels), and analysed 72 h later by kinetic imaging cytometry. Kinetic parameters are CaTD₅₀, CaTD_75–25 and V_max upstroke. Data are represented as whisker plots, with the box denoting the 25th and 75th percentiles, the whiskers the 5th and 95th percentiles, the middle bar the median, and outliers indicated as individual dots. Note that siSerca2a and miR-25 elicited comparable effects, both markedly delaying the Ca²⁺ uptake phase parameters CaTD₅₀ and CaTD_75–25 without appreciably altering the V_max upstroke kinetics (n > 550 cells per group). Box defines interquartile range; whiskers indicate ± 5th and 95th percentile; dots indicate outliers. Also note that siIP3R1 only minimally affected the Ca²⁺ kinetic parameters. siIP3R2 slowed V_max upstroke and broadened the distribution of uptake phase kinetic parameters CaTD₅₀ and CaTD_75–25. miR-25 in contrast slowed the uptake phase parameters but did not appreciably affect V_max upstroke (n > 550 cells per group). Box defines interquartile range; whiskers indicate ± 5th and 95th percentile; dots indicate outliers. Although IP3R1 might be a direct target of miR-25, several lines of evidence suggest that it is unlikely to mediate the effect of miR-25 in heart failure. IP3Rs are intracellular ligand-gated Ca²⁺ release channels⁴¹ that in the sarcoplasmic reticulum are associated with excitation–contraction coupling or spontaneous Ca²⁺ release and enhanced Ca²⁺ transients, whereas in the nuclear envelope they promote nuclear Ca²⁺ signalling^42,43,44, but a specific role for IP3R1 in heart failure has not been identified. Nonetheless, the control of IP3R1 by miR-25 might have a critical role under conditions that sensitize cardiomyocytes to inositol-1,4,5-trisphosphate, such as in response to endothelin 1, angiotensin and phenylephrine²³ or in local Ca²⁺ control²⁴. Experiments were replicated two times.