Danon disease is a lethal X-linked genetic syndrome resulting from radical mutations in the LAMP2 gene. LAMP2 protein deficiency results in defective lysosomal function, autophagy arrest and a multisystem disorder primarily involving the heart, skeletal muscle and the central nervous system. Cardiomyopathy is the main cause of morbidity and mortality. To investigate the mechanisms of and develop therapies for cardiac Danon disease we engineered a mouse model carrying an exon 6 deletion human mutation in LAMP2, which recapitulates the human cardiac disease phenotype. Mice develop cardiac hypertrophy followed by left ventricular dilatation and systolic dysfunction, in association with progressive fibrosis, oxidative stress, accumulation of autophagosomes and activation of proteasome. Stimulation of autophagy in Danon mice (by exercise training, caloric restriction, and rapamycin) aggravate the disease phenotype, promoting dilated cardiomyopathy. Inhibiting autophagy (by high fat diet or hydroxychloroquine) is better tolerated by Danon mice compared to wild type but is not curative. Inhibiting proteasome by Velcade was found to be highly toxic to Danon mice, suggesting that proteasome is activated to compensate for defective autophagy. In conclusion, activation of autophagy should be avoided in Danon patients. Since Danon's is a lifelong disease, we suggest that lifestyle interventions to decrease cardiac stress may be useful to slow progression of Danon's cardiomyopathy. While Danon mice better tolerate high fat diet and sedentary lifestyle, the benefit regarding cardiomyopathy in humans needs to be balanced against other health consequences of such interventions.

In response to the need for reliable cellular models that reflect complex tumor microenvironmental properties, and enable more precise testing of anti-cancer therapeutics effects on humans, a co-culture platform for in-vitro model that enhances the physiology of breast cancer (BC) microenvironment is presented. A six well imaging plate wherein each macro-well contains several separate compartments was designed. Three-dimensional (3D) cancer spheroids are generated and cultured in the inner compartment which is embossed with an array of nano-liter micro-chambers made of hydrogel. Stromal cells are cultured in the outer chambers. The two cell types are cultured side-by-side, sharing a common space, thus enabling extra-cellular communication via secreted molecules. As proof of concept, a model of BC tumor microenvironment was recapitulated by co-cultivating 3D MCF7 spheroids in the presence of tumor-associated macrophages (TAMs). The presence of TAMs induced an aggressive phenotype by promoting spheroid growth, enhancing survivin expression levels and enabling invasive behavior. Moreover, TAMs influenced the response of BC spheroids to cytotoxic treatment as well as hormonal drug therapy, and enhanced the effects of nitric oxide donor. The platform enables time-lapse imaging and treatment without losing spatial location of the measured spheroids, thereby allowing measurements and analysis at individual-object resolution in an easy and efficient manner.

It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and nature. A native of Israel, David grew up in the historic years that preceded the birth of the State of Israel. He was a member of the group that established Kibbutz Revivim in the Negev desert, and in 1948 participated in Israel’s War of Independence. David and Ruth eventually joined Kibbutz Givat Brenner by Rehovot, permitting David to be both a kibbutz member and a life-long researcher at the Weizmann Institute of Science, where David received his PhD in 1959. David returned to the Institute after his postdoc at Stanford. Here, after several years of researching a number of tissues as models for studying the process of differentiation, David entered the myogenesis field and stayed with it to his last day. With his dedication to the field of myogenesis and his commitment to furthering the understanding of the People and the Land of Israel throughout the international scientific community, David organized the first ever myogenesis meeting that took place in Shoresh, Israel in 1975. This was followed by the 1980 myogenesis meeting at the same place and many more outstanding meetings, all of which brought together myogenesis, nature and scenery. Herein, through the preparation and publication of this current manuscript, we are meeting once again at a “David Yaffe myogenesis meeting". Some of us have been members of the Yaffe lab, some of us have known David as his national and international colleagues in the myology field. One of our contributors has also known (and communicates here) about David Yaffe’s earlier years as a kibbutznick in the Negev. Our collective reflections are a tribute to Professor David Yaffe. We are fortunate that the European Journal of Translational Myology has provided us with tremendous input and a platform for holding this 2020 distance meeting "Farwell to Professor David Yaffe - A Pillar of the Myogenesis Field".

Leptin is an adipokine of pleiotropic effects linked to energy metabolism, satiety, the immune response, and cardioprotection. We have recently shown that leptin causally conferred resistance to myocardial infarction-induced damage in transgenic αMUPA mice overexpressing leptin compared to their wild type (WT) ancestral mice FVB/N. Prompted by these findings, we have investigated here if leptin can counteract the inflammatory response triggered after LPS administration in tissues in vivo and in cardiomyocytes in culture. The results have shown that LPS upregulated in vivo and in vitro all genes examined here, both pro-inflammatory and antioxidant, as well as the leptin gene. Pretreating mice with leptin neutralizing antibodies further upregulated the expression of TNFα and IL-1β in the adipose tissue of both mouse types, and in the αMUPA heart. The antibodies also increased the levels of serum markers for cell toxicity in both mouse types. These results indicate that under LPS, leptin actually reduced the levels of these inflammatory-related parameters. In addition, pretreatment with leptin antibodies reduced the levels of HIF-1α and VEGF mRNAs in the heart, indicating that under LPS leptin increased the levels of these mRNAs. In cardiomyocytes, pretreatment with exogenous leptin prior to LPS reduced the expression of both pro-inflammatory genes, enhanced the expression of the antioxidant genes HO-1, SOD2 and HIF-1α, and lowered ROS staining. In addition, results obtained with leptin antibodies and the SMLA leptin antagonist indicated that endogenous and exogenous leptin can inhibit leptin gene expression. Together, these findings have indicated that under LPS, leptin concomitantly downregulated pro-inflammatory genes, upregulated antioxidant genes, and lowered ROS levels. These results suggest that leptin can counteract inflammation in the heart and adipose tissue by modulating gene expression.

Background Diabetes mellitus type 2 (DM2) is a risk factor for developing heart failure but there is no specific therapy for diabetic heart disease. Sodium glucose transporter 2 inhibitors (SGLT2I) are recently developed diabetic drugs that primarily work on the kidney. Clinical data describing the cardiovascular benefits of SGLT2Is highlight the potential therapeutic benefit of these drugs in the prevention of cardiovascular events and heart failure. However, the underlying mechanism of protection remains unclear. We investigated the effect of Dapagliflozin—SGLT2I, on diabetic cardiomyopathy in a mouse model of DM2. Methods Cardiomyopathy was induced in diabetic mice (db/db) by subcutaneous infusion of angiotensin II (ATII) for 30 days using an osmotic pump. Dapagliflozin (1.5 mg/kg/day) was administered concomitantly in drinking water. Male homozygous, 12–14 weeks old WT or db/db mice (n = 4–8/group), were used for the experiments. Isolated cardiomyocytes were exposed to glucose (17.5–33 mM) and treated with Dapagliflozin in vitro. Intracellular calcium transients were measured using a fluorescent indicator indo-1. Results Angiotensin II infusion induced cardiomyopathy in db/db mice, manifested by cardiac hypertrophy, myocardial fibrosis and inflammation (TNFα, TLR4). Dapagliflozin decreased blood glucose (874 ± 111 to 556 ± 57 mg/dl, p < 0.05). In addition it attenuated fibrosis and inflammation and increased the left ventricular fractional shortening in ATII treated db/db mice. In isolated cardiomyocytes Dapagliflozin decreased intracellular calcium transients, inflammation and ROS production. Finally, voltage-dependent L-type calcium channel (CACNA1C), the sodium–calcium exchanger (NCX) and the sodium–hydrogen exchanger 1 (NHE) membrane transporters expression was reduced following Dapagliflozin treatment. Conclusion Dapagliflozin was cardioprotective in ATII-stressed diabetic mice. It reduced oxygen radicals, as well the activity of membrane channels related to calcium transport. The cardioprotective effect manifested by decreased fibrosis, reduced inflammation and improved systolic function. The clinical implication of our results suggest a novel pharmacologic approach for the treatment of diabetic cardiomyopathy through modulation of ion homeostasis.

Type 2 diabetes mellitus (DM2) leads to cardiomyopathy characterized by cardiomyocyte hypertrophy, followed by mitochondrial dysfunction and interstitial fibrosis, all of which are exacerbated by angiotensin II (AT). SIRT1 and its transcriptional coactivator target PGC-1α (peroxisome proliferator-activated receptor-γ coactivator), and heme oxygenase-1 (HO-1) modulates mitochondrial biogenesis and antioxidant protection. We have previously shown the beneficial effect of caloric restriction (CR) on diabetic cardiomyopathy through intracellular signaling pathways involving the SIRT1–PGC-1α axis. In the current study, we examined the role of HO-1 in diabetic cardiomyopathy in mice subjected to CR. Methods: Cardiomyopathy was induced in obese diabetic (db/db) mice by AT infusion. Mice were either fed ad libitum or subjected to CR. In an in vitro study, the reactive oxygen species (ROS) level was determined in cardiomyocytes exposed to different glucose levels (7.5–33 mM). We examined the effects of Sn(tin)-mesoporphyrin (SnMP), which is an inhibitor of HO activity, the HO-1 inducer cobalt protoporphyrin (CoPP), and the SIRT1 inhibitor (EX-527) on diabetic cardiomyopathy. Results: Diabetic mice had low levels of HO-1 and elevated levels of the oxidative marker malondialdehyde (MDA). CR attenuated left ventricular hypertrophy (LVH), increased HO-1 levels, and decreased MDA levels. SnMP abolished the protective effects of CR and caused pronounced LVH and cardiac metabolic dysfunction represented by suppressed levels of adiponectin, SIRT1, PPARγ, PGC-1α, and increased MDA. High glucose (33 mM) increased ROS in cultured cardiomyocytes, while SnMP reduced SIRT1, PGC-1α levels, and HO activity. Similarly, SIRT1 inhibition led to a reduction in PGC-1α and HO-1 levels. CoPP increased HO-1 protein levels and activity, SIRT1, and PGC-1α levels, and decreased ROS production, suggesting a positive feedback between SIRT1 and HO-1. Conclusion: These results establish a link between SIRT1, PGC-1α, and HO-1 signaling that leads to the attenuation of ROS production and diabetic cardiomyopathy. CoPP mimicked the beneficial effect of CR, while SnMP increased oxidative stress, aggravating cardiac hypertrophy. The data suggest that increasing HO-1 levels constitutes a novel therapeutic approach to protect the diabetic heart. Brief Summary: CR attenuates cardiomyopathy, and increases HO-1, SIRT activity, and PGC-1α protein levels in diabetic mice. High glucose reduces adiponectin, SIRT1, PGC1-1α, and HO-1 levels in cardiomyocytes, resulting in oxidative stress. The pharmacological activation of HO-1 activity mimics the effect of CR, while SnMP increased oxidative stress and cardiac hypertrophy. These data suggest the critical role of HO-1 in protecting the diabetic heart.

The Schumann Resonances (ScR) are Extremely Low Frequency (ELF) electromagnetic resonances in the Earth-ionosphere cavity excited by global lightning discharges. This natural electromagnetic noise has likely existed on the Earth ever since the Earth had an atmosphere and an ionosphere, hence surrounding us throughout our evolutionary history. The purpose of this study was to examine the influence of extremely weak magnetic fields in the ScR first mode frequency range on the spontaneous contractions, calcium transients and Creatine Kinase (CK) release of rat cardiac cell cultures. We show that applying 7.8 Hz, 90 nT magnetic fields (MF) causes a gradual decrease in the spontaneous calcium transients’ amplitude, reaching 28% of the initial amplitude after 40 minutes of MF application, and accompanied with a gradual decrease in the calcium transients’ rise time. The mechanical spontaneous contractions cease after the ScR fields have been applied for more than 30 minutes, when the calcium transient’s amplitude reached ~60% of its initial value. The influence of the ScR MF was reversible, independent of the field magnitude in the range 20 pT-100 nT, and independent of the external DC magnetic field. However, the effect is frequency dependent; the described changes occurred only in the 7.6–8 Hz range. In addition, applying 7.8 Hz, 90 nT MF for 1.5 hours, reduced the amount of CK released to the buffer, during normal conditions, hypoxic conditions and oxidative stress induced by 80 μM H 2 O 2 . We show that the ScR field induced reduction in CK release is associated with a stress response process and has a protective character.

TLR4, a member of the toll-like receptors (TLRs) family, serves as a pattern recognition receptor in the innate immune response to different microbial pathogens. [...]

Activation of the PKA catalytic unit by small peptide (SE1). Development of peptidomimetics.

Type 2 diabetes mellitus (DM2) follows impaired glucose tolerance in obesity and is frequently associated with hypertension, causing adverse myocardial remodelling and leading to heart failure. The DNA bound protein PARP (poly ADP ribose) polymerase catalyses a post translational modification (polymerization of negatively charged ADP-ribose chains) of nuclear proteins. PARP-1 activation is NAD(+) dependent and takes part in DNA repair and in chromatin remodelling and has a function in transcriptional regulation, intracellular trafficking and energy metabolism. PARP-1 is activated in diabetic cardiomyopathy. We hypothesized that PARP-1 inhibition in diabetic mice may protect cardiomyocytes from inflammation and ROS production. Methods: Obese Leptin resistant (db/db) mice suffering from DM2, were treated with angiotensin II (AT) for 4 weeks to enhance the development of cardiomyopathy. Mice were concomitantly treated with the PARP-1 inhibitor INO1001. Neonatal cardiomyocytes exposed to high levels of glucose (33 mM) with or without AT were treated with INO1001. or with SIRT inhibitor (EX-527) in the presence of INO1001 were tested in-vitro. Results: The in-vivo tests show that hearts from AT treated DM2 mice exhibited cardiac hypertrophy, fibrosis and an increase in the inflammatory marker TNF alpha. DM2 mice had an increased oxidative stress, concomitant with elevated PARP-1 activity and reduced Sirtuin-1 (SIRT1) expression. PARP-1 inhibition led to increased SIRT1 and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 alpha) levels, attenuating oxidative stress, inflammation and fibrosis. In-vitro experiments demonstrated that inhibition of PARP-1 in cardiomyocytes exposed to high levels of glucose and AT led to a significant reduction in ROS (P < 0.01), which was abolished in the presence of the SIRT1 inhibitor together with increased protein expression of SIRT1 and PGC-1 alpha. Conclusion: PARP1 inhibitor INO1001 attenuated cardiomyopathic features in diabetic mice through the activation of SIRT1 and its downstream antioxidant defence mechanisms. The results of this study suggest a pivotal role of PARP-1 inhibition in treating diabetic and AT-induced cardiomyopathy.

Calcium-sensitive fluorescent indicators fall broadly into two categories, ratiometric (dual-wavelength) or single-wavelength indicators based on their response to a calcium elevation. Ratiometric indicators shift either their excitation or their emission wavelengths in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. Fura-2 is one of the most common ratiometric fluorescent Ca2+ indicators, which has an emission maximum at 510 nm whereas its excitation maximum changes from 380 to 340 nm in response to calcium binding. Historically, a combination of arc lamp and monochromators has been used as the light source for Fura-2 ratiomatric fluorescence microscopy. In recent years, different combinations of LEDs such as 350/380 nm or 360/380 nm have been used to excite Fura-2. To precisely match the Fura-2 excitation, in this study, we built a new fast switchable 340/385-nm LED excitation light source (Prizmatix Ltd., Israel). The newly constructed light source has been exploited in Fura-2 ratiometric calcium imaging of skeletal muscle cells. The spontaneously elicited Ca2+ transients in the cells were recorded with high temporal resolution. The light source utilized for the demonstrated instrumentation in this report optimally matches the excitation wavelengths of either calcium-free or bound states of Fura-2. The high-intensity stability and fast switching of the 340/385-nm LED illuminators indicate their potential as a preferred light source for Fura-2 ratiometric calcium imaging over the existing light sources.

TLR4, a member of the Toll-like receptor (TLR) family, serves as a pattern recognition receptor in the innate immune response to microbial pathogens. TLR4 also regulates the inflammatory reaction to ischemic injury in the heart. The TRIF-related adaptor molecule (TRAM) is an adapter that recruits the Toll/interleukin 1 receptor (TIR) domain, which contains adapter-inducing IFN-β (TRIF), to activate TLR4, following TRIF-dependent cytokine gene transcription. On the basis of a known TRAM-derived decoy peptide, 10 of its peptidomimetics were synthesized. One of them, 1-benzyl-5-methyl-4-( n-octylamino)pyrimidin-2(1 H)-one (21), exhibited high potency and efficacy in vitro. In vitro results and in silico analysis provided evidence for the possible direct interaction of 21 with the TLR4 complex. Administered in mice, 21 was able to block the pathophysiological manifestation of MI, restoring the concomitant tissue damage, with a 100% survival rate. Thus, inhibition of TLR4-mediated inflammation in postischemic myocardium could be used as an approach for developing cardioprotective drugs.

Estrogen-induced apoptosis has become a successful treatment for postmenopausal metastatic, estrogen receptor-positive breast cancer. Nitric oxide involvement in the response to this endocrine treatment and its influence upon estrogen receptor-positive breast cancer progression is still unclear. Nitric oxide impact on the MCF7 breast cancer line, before and after estrogen-induced apoptosis, was investigated in 3D culture systems using unique live-cell imaging methodologies. Spheroids were established from MCF7 cells vulnerable to estrogen-induced apoptosis, before and after exposure to estrogen. Spheroids derived from estrogen-treated cells exhibited extensive apoptosis levels with downregulation of estrogen receptor expression, low proliferation rate and reduced metabolic activity, unlike spheroids derived from non-treated cells. In addition to basic phenotypic differences, these two cell cluster types are diverse in their reactions to exogenous nitric oxide. A dual effect of nitric oxide was observed in the breast cancer phenotype sensitive to estrogen-induced apoptosis. Nitric oxide, at the nanomolar level, induced cell proliferation, high metabolic activity, downregulation of estrogen receptor and enhanced collective invasion, contributing to a more aggressive phenotype. Following hormone supplementation, breast cancer 3D clusters were rescued from estrogen-induced apoptosis by these low nitric oxide-donor concentrations, since nitric oxide attenuates cell death levels, upregulates survivin expression and increases metabolic activity. Higher nitric oxide concentrations (100nM) inhibited cell growth, metabolism and promoted apoptosis. These results suggest that nitric oxide, in nanomolar concentrations, may inhibit estrogen-induced apoptosis, playing a major role in hormonal therapy. Inhibiting nitric oxide activity may benefit breast cancer patients and ultimately reduce tumor recurrence.

Background This study was designed to assess the changes of acetylcholine receptor (AChR) and creatine kinase (CK) levels, which are important biochemical markers for muscle viability in cases of long-term muscle denervation. Scientists and peripheral nerve surgeons may find these data important regarding maximal range of muscle viability applicable for timing of effective peripheral nerve reconstructive surgery. Methods The study was conducted on 48 rats (96 gastrocnemius muscles), whose right legs were denervated by removing a 10-mm segment of sciatic nerve, while their left legs remained intact. Under general anesthesia, the rats were euthanized at seven points in time, on days 7, 14, 21, 30, 60, 120, and 210. In both legs, AChR was quantified by 125I-α-bungarotoxin, whereas CK activity was measured using a spectrophotometric method. Results CK levels in the denervated limb reached a minimal level of 34% on day 30 in comparison to the intact limb and remained at this level up to 210 days after operation. AChR levels in the denervated limb reached a minimal level of 38% on day 120 in comparison to the intact limb and remained at this level up to 210 days after operation. Conclusion The present study shows that AChR and CK levels in rat denervated muscles remain constant at about third of its intact condition for a period of at least a third of rat's lifetime postinjury.

BACKGROUND The recessive form of catecholaminergic polymorphic ventricular tachycardia 2 (CPVT2) is caused by mutations in cardiac calsequestrin (CASQ2), leading to protein deficiency. OBJECTIVES The aims of this study were to develop a viral-delivered gene therapy for CPVT2 and to determine the relationship between CASQ2 expression and antiarrhythmic efficacy in a murine model.METHODS We used a murine model of CPVT2 caused by the D307H human mutation (CASQ2(D307H)) or CASQ2 knockout (CASQ2(Delta/Delta)). Adeno-associated virus (AAV) particles containing the CASQ2 gene (AAV(CASQ2)) were injected into the heart or intraperitoneally to 12-week-old mice. A telemetry device was implanted, and mice underwent provocation testing 7-8 weeks after gene therapy.RESULTS CASQ2(Delta/Delta) mice injected intracardiacally with AAVCASQ2 expressed 40% +/- 25% of the normal CASQ2 protein level, which was increased compared to untreated CASQ2(Delta/Delta) mice (n 5 10; P,.05). Intraperitoneal therapy led to a significantly elevated expression of the CASQ2 protein, which was comparable in CASQ2(D307H) (n = 12) and CASQ2(Delta/Delta) (n = 4) mice. All control mice with CPVT2 had nonsustained ventricular tachycardia (VT) and 8 of 13 had sustained VT on provocation. Expressing >= 33% of the normal CASQ2 level was needed to protect from nonsustained VT as well as stress-induced premature ventricular contractions. Lower levels of expression prevented sustained VT in AAVCASQ2-treated mice (0 of 26; P,. 001 vs controls).CONCLUSION AAV(CASQ2) displays a long-lasting capacity to attenuate and potentially cure CPVT2. Systemic delivery is feasible and convenient, reproducibly providing adequate levels of transgene expression. Antiarrhythmic efficacy depends on the CASQ2 level: >= 33% of the normal CASQ2 level is needed to prevent arrhythmia. However, even lower levels of protein protect from sustained VT, thereby potentially reducing the risk of sudden death.

Unexpectedly, a post-translational modification of DNA-binding proteins, initiating the cell response to single-strand DNA damage, was also required for long-term memory acquisition in a variety of learning paradigms. Our findings disclose a molecular mechanism based on PARP1-Erk synergism, which may underlie this phenomenon. A stimulation induced PARP1 binding to phosphorylated Erk2 in the chromatin of cerebral neurons caused Erk-induced PARP1 activation, rendering transcription factors and promoters of immediate early genes (IEG) accessible to PARP1-bound phosphorylated Erk2. Thus, Erk-induced PARP1 activation mediated IEG expression implicated in long-term memory. PARP1 inhibition, silencing, or genetic deletion abrogated stimulation-induced Erk-recruitment to IEG promoters, gene expression and LTP generation in hippocampal CA3-CA1-connections. Moreover, a predominant binding of PARP1 to single-strand DNA breaks, occluding its Erk binding sites, suppressed IEG expression and prevented the generation of LTP. These findings outline a PARP1-dependent mechanism required for LTP generation, which may be implicated in long-term memory acquisition and in its deterioration in senescence.

Toll-like receptor 4 (TLR4), the receptor for lipopolysaccharide (LPS) of gram-negative pathogens expressed in the heart, is activated by several endogenous ligands associated with tissue injury in response to myocardial infarction (MI). The aim of this study was to investigate the involvement of TLR4 signaling in cardiomyocytes dysfunction following hypoxia (90min) using multiple methodologies such as knocking down TLR4 and small interfering RNA (siTLR4). Cardiomyocytes of C57Bl/6 mice (WT) subjected to hypoxic stress showed increased cardiac release of LDH, HMGB1, IκB, TNF-α and myocardial apoptotic and necrotic markers (BAX, PI) compared to TLR4 knock out mice (TLR4KO). Treating these cardiomyocytes with siRNA against TLR4 decreased the damage markers (LDH, IκB, TNF-α). TLR4 silencing during hypoxic stress resulted in the activation of the p-AKT and p-GSK3β (by ∼25%). The latter is an indicator that there is a reduction of mitochondrial permeability transition pore (mPTP) opening following hypoxic myocardial induced injury leading to preserved mitochondrial membrane potential. Silencing TLR4 in cardiomyocytes improved cell survival following hypoxic injury through activation of the AKT/GSK3β pathway, reduced inflammatory and apoptotic signals. These findings suggest that TLR4 may serve as a potential target in the treatment of ischemic myocardial injury. Moreover, RNA interfering targeting TLR4 expression represents a therapeutic strategy.

Weak electromagnetic fields (WEF) enhance Ca(2+) entry into cells via voltage-gated Ca(2+) channels and affect various aspects of metabolism, structure, and function. However, little information is available on the effect of WEF on skeletal muscle, which depends primarily on intracellular Ca(2+) stores for function and metabolism. Here, we examine the effects of 30 min exposure of rat primary myotube cultures to WEF (1.75 μT, 16 Hz) on Ca(2+) handling and creatine kinase (CK) release. Free myoplasmic Ca(2+) concentration ([Ca(2+) i]) was measured with the ratiometric dye indo-1. WEF did not affect basal [Ca(2+)]i but decreased the twitch [Ca(2+)]i transient in a time-dependent manner, and the twitch amplitude was decreased to ∼30 % after 30 min. WEF completely abolished the increase in [Ca(2+)]i induced by potassium chloride (∼60 mM) but had no effect on the increase induced by caffeine (∼6 mM). Hypoxia (2 h exposure to 100 % argon) resulted in a marked loss of CK into the medium (400 % of normoxic value), as well as a rapid (within 20 min) and sustained increase in basal [Ca(2+)]i (∼20 % above baseline). However, during exposure to WEF, basal [Ca(2+)]i remained constant during the initial 60 min of hypoxia and, thereafter, increased to levels similar to those observed in the absence of WEF. Finally, WEF blocked about 80 % of hypoxia-mediated CK release (P < 0.05). These data demonstrate that WEF inhibits increases in [Ca(2+)]i by interfering with muscle excitation and protects against muscle damage induced by hypoxia. Thus, WEF may have therapeutic/protective effects on skeletal muscle.