Coordinated regulation of murine cardiomyocyte contractility by nanomolar (-)-epigallocatechin-3-gallate, the major green tea catechin.

Green tea polyphenolic catechins exhibit biological activity in a wide variety of cell types. Although reports in the lay and scientific literature suggest therapeutic potential for improving cardiovascular health, the underlying molecular mechanisms of action remain unclear. Previous studies have implicated a wide range of molecular targets in cardiac muscle for the major green tea catechin, (-)-epigallocatechin-3-gallate (EGCG), but effects were observed only at micromolar concentrations of unclear clinical relevance. Here, we report that nanomolar concentrations of EGCG significantly enhance contractility of intact murine myocytes by increasing electrically evoked Ca(2+) transients, sarcoplasmic reticulum (SR) Ca(2+) content, and ryanodine receptor type 2 (RyR2) channel open probability. Voltage-clamp experiments demonstrate that 10 nM EGCG significantly inhibits the Na(+)-Ca(2+) exchanger. Of importance, other Na(+) and Ca(2+) handling proteins such as Ca(2+)-ATPase, Na(+)-H(+) exchanger, and Na(+)-K(+)-ATPase were not affected by EGCG ≤ 1 µM. Thus, nanomolar EGCG increases contractility in intact myocytes by coordinately modulating SR Ca(2+) loading, RyR2-mediated Ca(2+) release, and Na(+)-Ca(2+) exchange. Inhibition of Na(+)-K(+)-ATPase activity probably contributes to the positive inotropic effects observed at EGCG concentrations >1 µM. These newly recognized actions of nanomolar and micromolar EGCG should be considered when the therapeutic and toxicological potential of green tea supplementation is evaluated and may provide a novel therapeutic strategy for improving contractile function in heart failure.

Functional and biochemical properties of ryanodine receptor type 1 channels from heterozygous R163C malignant hyperthermia-susceptible mice.

Mutations in ryanodine receptor type 1 (RyR1) confer malignant hyperthermia susceptibility. How inherent impairments in Ca(2+) channel regulation affect skeletal muscle function in myotubes and adult fibers under basal (nontriggering) conditions are not understood. Myotubes, adult flexor digitorum brevis (FDB) fibers, and sarcoplasmic reticulum skeletal membranes were isolated from heterozygous knockin R163C and wild-type (WT) mice. Compared with WT myotubules, R163C myotubes have reduced Ca(2+) transient amplitudes in response to electrical field pulses; however, R163C FDB fibers do not differ in their responses to electrical stimuli, despite heightened cellular cytoplasmic resting Ca(2+) ([Ca(2+)](rest)) and sensitivity to halothane. Immunoblotting of membranes from each genotype shows similar expression of RyR1, FK506 binding protein 12 kDa, and Ca(2+)-ATPase, but RyR1 (2844)Ser phosphorylation in R163C muscle is 31% higher than that of WT muscle (p < 0.001). RyR1 channels reconstituted in planar lipid bilayers reveal ∼65% of R163C channels exhibit ≥2-fold greater open probability (P(o)) than WT, with prolonged mean open dwell times and shortened closed dwell times. [(3)H]Ryanodine (Ry) binding and single-channel analyses show that R163C-RyR1 has altered regulation compared with WT: 1) 3-fold higher sensitivity to Ca(2+) activation; 2) 2-fold greater [(3)H]Ry receptor occupancy; 3) comparatively higher channel activity, even in reducing glutathione buffer; 4) enhanced RyR1 activity both at 25 and 37°C; and 5) elevated cytoplasmic [Ca(2+)](rest). R163C channels are inherently more active than WT channels, a functional impairment that cannot be reversed by dephosphorylation with protein phosphatase. Dysregulated R163C channels produce a more overt phenotype in myotubes than in adult fibers in the absence of triggering agents, suggesting tighter negative regulation of R163C-RyR1 within the Ca(2+) release unit of adult fibers.

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+.

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.

Green tea catechins are potent sensitizers of ryanodine receptor type 1 (RyR1).

Catechins, polyphenols extracted from green tea leaves, have a broad range of biological activities although the specific molecular mechanisms responsible are not known. At the high experimental concentrations typically used polyphenols bind to membrane phospholipid and also are easily auto-oxidized to generate superoxide anion and semiquinones, and can adduct to protein thiols. We report that the type 1 ryanodine receptor (RyR1) is a molecular target that responds to nanomolar (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG). Single channel analyses demonstrate EGCG (5-10nM) increases channel open probability (Po) twofold, by lengthening open dwell time. The degree of channel activation is concentration-dependent and is rapidly and fully reversible. Four related catechins, EGCG, ECG, EGC ((-)-epigallocatechin) and EC ((-)-epicatechin) showed a rank order of activity toward RyR1 (EGCG>ECG>>EGC>>>EC). EGCG and ECG enhance the sensitivity of RyR1 to activation by < or =100microM cytoplasmic Ca(2+) without altering inhibitory potency by >100microM Ca(2+). EGCG as high as 10microM in the extracellular medium potentiated Ca(2+) transient amplitudes evoked by electrical stimuli applied to intact myotubes and adult FDB fibers, without eliciting spontaneous Ca(2+) release or slowing Ca(2+) transient recovery. The results identify RyR1 as a sensitive target for the major tea catechins EGCG and ECG, and this interaction is likely to contribute to their observed biological activities.