The ryanodine receptor stabilizer S107 ameliorates contractility of adult Rbm20 knockout rat cardiomyocytes

Figures

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  FIGURE 1. Titin size switching and resting stiffness with in vitro and in vivo measurements. (a) Detection of titin size switching in WT and KO rat heart with ageing using gel electrophoresis; (b) resting tension measurements with in vitro isolated individual myofibrils in WT group (three biological repeats C, B, and E) and in KO group (three biological repeats A, D, and F); (c) diastolic stiffness measured with PV‐loop in 3 months old WT and KO rats; (d) representative example of PV loops of an inferior vena cava (IVC) occlusion experiment; the end‐diastolic PV relationship (EDPVR) is shown as a blue line. KO, RBM20 knockout; M, month; MyHC, myosin heavy chain; N2B, smaller titin isoform; N2BA‐A1 and G, larger titin isoforms; T2, degraded titin; WT, wild type; Mean ± SEM (n = 3 and 6), *p < .05, **p < .01

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  FIGURE 2. In vivo assessment of cardiac function. (a) and (c) Increase in left ventricle (LV) internal dimension in both diastole and systole in KO at the age of 3 (n = 8 and 9), 6 (n = 7 and 12), 9 (n = 7 and 10), and 12 months (n = 6 and 6); (b) and (d) decrease in LV posterior wall dimension in both diastole and systole in KO rat at the age of 3, 6, 9, and 12 months; (e) fractional shortening value decreases in KO rat at the age of 3, 6, 9, and 12 months; (f) cardiac output decreases in KO at 6 and 9 months determined by PV‐Loop analysis. CO, cardiac output; FS, fractional shortening; KO, RBM20 knockout; LVIDd, left ventricular end‐diastolic internal dimension; LVIDs, left ventricular end systolic internal dimension; LVPWd, left ventricular end‐diastolic posterior wall dimension; LVPWs, left ventricular end systolic posterior wall dimension; WT, wild type. Mean ± SEM, *p < .05, **p < .01, ***p < .001

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  FIGURE 3. Ryr2 variant and cardiomyocyte contractility measurements in WT and KO cardiomyocytes treated with and without S107. (a) RyR2 expression, phosphorylation and splicing variant; (b) resting sarcomere length treated with S107; (c) peak shortening treated with S107; (d) maximum velocity of shortening treated with S107; (e) maximum velocity of relengthening treated with S107; (f) time to 50% peak shortening treated with S107; (g) time to 50% relengthening treated with S107; KO, knockout; sTPS50, time to 50% peak shortening; sTR50, time to 50% relengthening; WT, wild type. Mean ± SEM (n = 100 cells from three biological replicates), *p < .05, **p < .01

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  FIGURE 4. Intracellular calcium transient measurements. (a) Resting fura‐2 fluorescence intensity (FFI) with S107 treatment; (b) electrically stimulated rise in fura‐2 fluorescence intensity (∆FFI) with S107 treatment; (c) intracellular calcium decay rate (single exponential) with S107 treatment. KO, knockout; WT, wild type. Mean ± SEM (n = 100 cells from three biological replicates), *p < .05, ***p < .001

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  FIGURE 5. In vivo assessment of cardiac function with S107 treatment. (a) and (b) Left ventricle (LV) internal dimension in both diastole and systole in KO rats with/without S107 treatment; (c) and (d) LV posterior wall dimension in both diastole and systole in KO rats treated with/without S107; (e) ejection fraction in KO rats treated with/without S107. EF, ejection fraction; KO, RBM20 knockout; LVIDd, left ventricular end‐diastolic internal dimension; LVIDs, left ventricular end systolic internal dimension; LVPWd, left ventricular end‐diastolic posterior wall dimension; LVPWs, left ventricular end systolic posterior wall dimension. Mean ± SEM (n = 9 and 6), ns, not significant.