Ryanodine receptor calcium channels and their partners as drug targets

Ryanodine receptors (RyRs) are high conductance intracellular cation channels that release calcium ions from stores such as the endoplasmic reticulum and sarcoplasmic reticulum. Although RyRs are expressed in many cell types, their roles have only been extensively characterised in tissues in which they are abundant: RyR1 is essential for excitation-contraction coupling in skeletal muscle; whereas RyR2 is required for the analogous signal transduction pathway in heart. Defects in RyR1 cause malignant hyperthermia and a spectrum of myopathies in skeletal muscle; whereas RyR2 dysregulation can result in fatal cardiac arrhythmias and is involved in heart failure. Altered RyR gating has been implicated in a range of other diseases, including epilepsy, neurodegeneration, pain and cancer. RyRs interact with a range of toxic substances, providing insights into their functional and structural properties. Consequently, these channel complexes represent potential therapeutic targets for treatment of numerous diseases. Furthermore, strategies for combating multicellular parasites and agricultural pests could exploit pharmacological differences between their RyRs and those of vertebrates. However, available pharmacological tools for manipulation of RyR gating are generally unsuitable for clinical, veterinary or agricultural use, owing to their lack of selectivity, inappropriate solubility in the aqueous or lipid environment, or generation of side-effects. The expression, subcellular distribution and gating of RyRs is modified by a wide variety of cellular proteins, some of which are expressed in a developmentally or tissue-restricted manner. This commentary examines the possibility of manipulating the expression and function of such proteins in order develop new drugs acting on RyR channel complexes.     

Age-related differences in excitation-contraction coupling in rat papillary muscle

Summary To investigate the possible role of an alteration in excitation-contraction coupling during development, aging and senescence we compared simultaneously recorded mechanical and electrical activity of left ventricular papillary muscles from 3, 6, 12, and 24-month-old male rats. In addition, the effects of calcium and verapamil on excitation-contraction coupling were evaluated.We recorded transmembrane action potentials during both isometric and isotonic contractions. At an external bath calcium=2.4 mM, action potential duration at 75% complete repolarization (APD₇₅) was significantly prolonged as a function of age (3 mo=28.2±2.7; 6 mo=29.5±2.6; 12 mo=49.5±5.6; 24 mo=121±8.5 msec) while peak developed tension (DT) was not significantly altered (3 mo=5.13±0.53; 6 mo=4.75±0.53; 12 mo=7.26±0.51; 24 mo=6.01±0.67 g/mm²). The correlation coefficient (r value) for APD₇₅ and DT was strong for 3-month-old animals (r=0.99) but weakened as a function of age (6 mo=0.93; 12 mo=0.81; 24 mo=0.57). Similar results were observed when APD₇₅ was correlated with time-to-peak tension (TPT) (3 mo=0.95; 6 mo=0.98; 12 mo=0.85; 24 mo=0.68), time-to-one-half relaxation (T1/2R) (3 mo=0.91; 6 mo=0.97; 12 mo=0.85; 24 mo=0.81) and time to peak shortening (TPS) (3 mo=0.89; 6 mo=0.81; 12 mo=0.82; 24 mo=0.51). Correlations between action potential duration and contractile parameters became weak in all age groups upon the addition of verapamil (V). The correlation between APD₇₅ and DT for 3-month-old animals decreased by 34% upon the addition of V while a 70% decrease was seen in 24-month-old animals. Similar results were seen when APD₇₅ was correlated with TPT, T1/2R and TPS when V was added to the perfusate. Our results indicate that excitation-contraction coupling, as evidence by alterations in not only the contractile apparatus but also in the surface membrane, may be altered in ventricular muscle as in function of age.Support in part by NIH Grants # HL 21933-04, # 07071-06 and a Herman Raucher Investigatorship Award of the New York Heart Association to Dr. Joseph M. Capasso. Animals used in this study were generously awarded to Dr. Capasso by the Animal Acquisitions Branch of the National Institute of Aging.     

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 (5-10 nM) 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 ≤100 μM cytoplasmic Ca²⁺ without altering inhibitory potency by >100 μM Ca²⁺. EGCG as high as 10 μM in the extracellular medium potentiated Ca²⁺ transient amplitudes evoked by electrical stimuli applied to intact myotubes and adult FDB fibers, without eliciting spontaneous Ca²⁺ release or slowing Ca²⁺ 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.     

Electrical and mechanical properties of skeletal muscle underlying increased fatigue in patients with amyotrophic lateral sclerosis

To investigate the mechanical efficiency of surviving motor units of anterior tibial muscle in patients with amyotrophic lateral sclerosis (ALS), we studied motor unit action potentials, muscle force, and muscle fatigability in patients with ALS and controls using 25 min of low to moderate intensity voluntary isometric exercise. During exercise, tetanic force (TF) and maximum voluntary contraction declined more in patients than in controls. The mean motor unit action potential duration, amplitude, and polyphasia were increased in patients compared to controls but did not change during 9 months of disease progression. The enlarged motor units in patients were negatively correlated to the muscle force and positively correlated to muscle fatigability. Furthermore, after a mean follow-up period of 9 months, the decline in force-generating capacity of the anterior tibial muscle in patients (twitch tension by 37.5 ± 11.2%, TF by 30.6 ± 7.4%) was greater than the decline in the amplitude of the compound muscle action potential (21.1 ± 8.8%, P < 0.05), suggesting a relative dissociation between electrical and mechanical properties. In conclusion, the enlarged motor units in patients with ALS are mechanically less efficient and fatigue relatively more than in healthy muscles, possibly due to an abnormality that is primarily distal to the muscle membrane. © 1996 John Wiley & Sons, Inc.     

Dystrophic skeletal muscle fibers display alterations at the level of calcium microdomains

The spatiotemporal properties of the Ca²⁺-release process in skeletal muscle fibers from normal and mdx fibers were determined using the confocal-spot detection technique. The Ca²⁺ indicator OGB-5N was used to record action potential-evoked fluorescence signals at consecutive locations separated by 200 nm along multiple sarcomeres of FDB fibers loaded with 10- and 30-mM EGTA. Three-dimensional reconstructions of fluorescence transients demonstrated the existence of microdomains of increased fluorescence around the Ca²⁺-release sites in both mouse strains. The Ca²⁺ microdomains in mdx fibers were regularly spaced along the fiber axis, displaying a distribution similar to that seen in normal fibers. Nevertheless, both preparations differed in that in 10-mM EGTA Ca²⁺ microdomains had smaller amplitudes and were wider in mdx fibers than in controls. In addition, Ca²⁺-dependent fluorescence transients recorded at selected locations within the sarcomere of mdx muscle fibers were not only smaller, but also slower than their counterparts in normal fibers. Notably, differences in the spatial features of the Ca²⁺ microdomains recorded in mdx and normal fibers, but not in the amplitude and kinetics of the Ca²⁺ transients, were eliminated in 30-mM EGTA. Our results consistently demonstrate that Ca²⁺-release flux calculated from release sites in mdx fibers is uniformly impaired with respect to those normal fibers. The Ca²⁺-release reduction is consistent with that previously measured using global detection techniques.