Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Dec 5:7:607.
doi: 10.3389/fphys.2016.00607. eCollection 2016.

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

Felix W Friedrich  1 , Frederik Flenner  1 , Mahtab Nasib  1 , Thomas Eschenhagen  1 , Lucie Carrier  1
Affiliations

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

Felix W Friedrich et al. Front Physiol. .

Abstract

Background: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac muscle disease with left ventricular hypertrophy, interstitial fibrosis and diastolic dysfunction. Increased myofilament Ca2+ sensitivity could be the underlying cause of diastolic dysfunction. Epigallocatechin-3-gallate (EGCg), a catechin found in green tea, has been reported to decrease myofilament Ca2+ sensitivity in HCM models with troponin mutations. However, whether this is also the case for HCM-associated thick filament mutations is not known. Therefore, we evaluated whether EGCg affects the behavior of cardiomyocytes and myofilaments of an HCM mouse model carrying a gene mutation in cardiac myosin-binding protein C and exhibiting both increased myofilament Ca2+ sensitivity and diastolic dysfunction. Methods and Results: Acute effects of EGCg were tested on fractional sarcomere shortening and Ca2+ transients in intact ventricular myocytes and on force-Ca2+ relationship of skinned ventricular muscle strips isolated from Mybpc3-targeted knock-in (KI) and wild-type (WT) mice. Fractional sarcomere shortening and Ca2+ transients were analyzed at 37°C under 1-Hz pacing in the absence or presence of EGCg (1.8 μM). At baseline and in the absence of Fura-2, KI cardiomyocytes displayed lower diastolic sarcomere length, higher fractional sarcomere shortening, longer time to peak shortening and time to 50% relengthening than WT cardiomyocytes. In WT and KI neither diastolic sarcomere length nor fractional sarcomere shortening were influenced by EGCg treatment, but relaxation time was reduced, to a greater extent in KI cells. EGCg shortened time to peak Ca2+ and Ca2+ transient decay in Fura-2-loaded WT and KI cardiomyocytes. EGCg did not influence phosphorylation of phospholamban. In skinned cardiac muscle strips, EGCg (30 μM) decreased Ca2+ sensitivity in both groups. Conclusion: EGCg hastened relaxation and Ca2+ transient decay to a larger extent in KI than in WT cardiomyocytes. This effect could be partially explained by myofilament Ca2+ desensitization.

Keywords: Ca2+ transient; Mybpc3; epigallocatechin-3-gallate; hypertrophic cardiomyopathy; myofilament Ca2+ sensitivity; relaxation.

PubMed Disclaimer

Figures

Figure 1
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).
Figure 2
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.
Figure 3
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.
Figure 4
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alvarez E., Campos-Toimil M., Justiniano-Basaran H., Lugnier C., Orallo F. (2006). Study of the mechanisms involved in the vasorelaxation induced by (-)-epigallocatechin-3-gallate in rat aorta. Br. J. Pharmacol. 147, 269–280. 10.1038/sj.bjp.0706507 - DOI - PMC - PubMed
    1. Alves M. L., Dias F. A., Gaffin R. D., Simon J. N., Montminy E. M., Biesiadecki B. J., et al. . (2014). Desensitization of myofilaments to Ca2+ as a therapeutic target for hypertrophic cardiomyopathy with mutations in thin filament proteins. Circ. Cardiovasc. Genet. 7, 132–143. 10.1161/CIRCGENETICS.113.000324 - DOI - PMC - PubMed
    1. Bao L., Lu F., Chen H., Min Q., Chen X., Song Y., et al. . (2015). High concentration of epigallocatechin-3-gallate increased the incidences of arrhythmia and diastolic dysfunction via beta2-adrenoceptor. J. Food Sci. 80, T659–T663. 10.1111/1750-3841.12803 - DOI - PubMed
    1. Barefield D., Kumar M., De Tombe P. P., Sadayappan S. (2014). Contractile dysfunction in a mouse model expressing a heterozygous MYBPC3 mutation associated with hypertrophic cardiomyopathy. Am. J. Physiol. Heart Circ. Physiol. 306, H807–H815. 10.1152/ajpheart.00913.2013 - DOI - PMC - PubMed
    1. Baudenbacher F., Schober T., Pinto J. R., Sidorov V. Y., Hilliard F., Solaro R. J., et al. . (2008). Myofilament Ca2+ sensitization causes susceptibility to cardiac arrhythmia in mice. J. Clin. Invest. 118, 3893–3903. 10.1172/jci36642 - DOI - PMC - PubMed

LinkOut - more resources