Epigallocatechin-3-Gallate Accelerates Relaxation and Ca2+ Transient Decay and Desensitizes Myofilaments in Healthy and Mybpc3-Targeted Knock-in Cardiomyopathic Mice

Figures

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  Figure 1. EGCg effect on Mybpc3 WT cardiac myocyte contractility. (A) After isolation of ventricular myocytes from adult Mybpc3 WT mice, paired (before/after EGCg) concentration-response curves were performed. Graph depicts % of fractional sarcomere shortening as readout with increasing EGCg concentrations (10−8 = 100 nM, 10−7 = 10 nM, 10−6 = 1 μM, 10−5.74 = 1.8 μM, 10−5.52 = 3 μM, 10−5 = 10 μM, 10−4.52 = 30 μM, 10−4 = 100 μM; n = 3–9/concentration. Concentrations above ≥3 μM EGCg caused a significant increase in sarcomere shortening. (B,C) Representative contractions of a cardiac myocyte at baseline conditions (left) and after 5 min of exposure to 1.8 (B, right) or 30 μM EGCg (C, right).

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  Figure 2. Contractile parameters of Mybpc3 WT and KI cardiomyocytes before and after treatment with EGCg. After isolation of ventricular myocytes from adult Mybpc3 WT and KI mice, paired (before/after EGCg) measurements of contractile function were exerted. (A) Averaged sarcomere shortening traces of Mybpc3 WT (black) and KI (red) cells in baseline and with EGCg. (B) Fractional sarcomere shortening, (C) contraction time (time from stimulation to peak of contraction), (D) diastolic sarcomere length, and (E) relaxation time (time from peak of contraction to 50% relaxation) were analyzed. (F) Delta of relaxation time (before/after EGCg). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. WT in the same condition, unpaired Student's t-test; #P < 0.05 vs. baseline, paired Student's t-test, n = 17–22, N = 5.

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  Figure 3. Ca2+ transients and kinetics of Mybpc3 WT and KI cardiomyocytes before and after treatment with EGCg. After isolation of cardiac myocytes from adult Mybpc3 WT and KI mice, paired (before/after EGCg) measurements of Ca2+ transients were performed in Fura-2 loaded cells. (A) Averaged Ca2+ transients of Mybpc3 WT and KI cells in baseline and with EGCg. (B) Ca2+ peak height, (C) time to peak Ca2+ (from stimulation to peak of 340/380 ratio), (D) diastolic Ca2+, and (E) time to 50% Ca2+ decay (from peak of 340/380 ratio to 50% decay) were analyzed. (F) Sarcomere length of Mybpc3 WT cells plotted against the Fura-2 signal ratio F340/380 indicating the Ca2+ transient in the absence (black loop) or presence (dotted black loop) of 1.8 μM EGCg, respectively. Loops proceed in a counter-clockwise direction. **P < 0.01 vs. WT in the same condition, unpaired Student's t-test; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. baseline, paired Student's t-test, n = 20–26, N = 5. For loops: n = 9.

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  Figure 4. Force-Ca2+ relationship of permeabilized cardiac muscle strips of WT and KI mice before and after treatment with 30 μM EGCg. Force-Ca2+ concentration in (A) WT strips, (B) KI strips. (C) The pCa50 represents the measure of myofilament Ca2+-sensitivity. (D) Delta of pCa50before and after EGCg. (E) nHill coefficient±EGCg. **P < 0.01 and ***P < 0.001 vs. WT in the same condition, unpaired Student's t-test; ##P < 0.001 and ###P < 0.001 vs. baseline, paired Student's t-test, concentration response curves were fitted to the data points and curve comparison was done by using extra sum-of-squares F-test; n = 10; N = 6 mice/genotype.