Quo Vadis Oncological Hyperthermia (2020)?

Heating as a medical intervention in cancer treatment is an ancient approach, but effective deep heating techniques are lacking in modern practice. The use of electromagnetic interactions has enabled the development of more reliable local-regional hyperthermia (LRHT) techniques whole-body hyperthermia (WBH) techniques. Contrary to the relatively simple physical-physiological concepts behind hyperthermia, its development was not steady, and it has gone through periods of failures and renewals with mixed views on the benefits of heating seen in the medical community over the decades. In this review we study in detail the various techniques currently available and describe challenges and trends of oncological hyperthermia from a new perspective. Our aim is to describe what we believe to be a new and effective approach to oncologic hyperthermia, and a change in the paradigm of dosing. Physiological limits restrict the application of WBH which has moved toward the mild temperature range, targeting immune support. LRHT does not have a temperature limit in the tumor (which can be burned out in extreme conditions) but a trend has started toward milder temperatures with immune-oriented goals, developing toward immune modulation, and especially toward tumor-specific immune reactions by which LRHT seeks to target the malignancy systemically. The emerging research of bystander and abscopal effects, in both laboratory investigations and clinical applications, has been intensified. Our present review summarizes the methods and results, and discusses the trends of hyperthermia in oncology.

Review of the Clinical Evidences of Modulated Electro-Hyperthermia (mEHT) Method: An Update for the Practicing Oncologist.

Background: Modulated electro-hyperthermia (mEHT) is a variation of the conventional hyperthermia which selectively targets the malignant cell membranes in order to heat the malignant tissue and sensitize the tissue to oncology treatments. Although widely applied, the formulation of guidelines for the use thereof is still in progress for many tumors. Aim: In this paper we review the literature on the effects of mEHT in cancer patients on local disease control and survival. Methodology: Our review on data presents the collected experience with capacitive hyperthermia treatments with the EHY-2000+ device (OncoTherm Ltd., Germany). A literature search was conducted in Pubmed and articles were grouped and discussed according to: trial type, animal studies, in vitro studies, and reviews. Search results from Conference Abstracts; Trial Registries; Thesis and Dissertations and the Oncothermia Journal were included in the discussions. Results: Modulated electro-hyperthermia is a safe form of hyperthermia which has shown to effectively sensitizes deep tumors, regardless of the thickness of the adipose layers. The technology has demonstrated equal benefits compared to other forms of hyperthermia for a variety of tumors. Given the effective heating ability to moderate temperatures, the improved tumor perfusion, and ability to increase drug absorption, mEHT is a safe and effective heating technology which can be easily applied to sensitize tumors which have demonstrated benefits with the addition of hyperthermia. Modulated electro-hyperthermia also appears to improve local control and survival rates and appears to induce an abscopal (systemic) response to ionizing radiation. Conclusion: Based on clinical studies, the method mEHT is a feasible hyperthermia technology for oncological applications. Concomitant utilization of mEHT is supported by the preclinical and clinical data.

Oncological hyperthermia: The correct dosing in clinical applications.

The problem with the application of conventional hyperthermia in oncology is firmly connected to the dose definition, which conventionally uses the concept of the homogeneous (isothermal) temperature of the target. Its imprecise control and complex evaluation is the primary barrier to the extensive clinical applications. The aim of this study was to show the basis of the problems of the misleading dose concept. A clear clarification of the proper dose concept must begin with the description of the limitations of the present doses in conventional hyperthermia applications. The surmounting of the limits the dose of oncologic hyperthermia has to be based on the applicability of the Eyring transition state theory on thermal effects. In order to avoid the countereffects of thermal homeostasis, the use of precise heating on the nanoscale with highly efficient energy delivery is recommended. The nano­scale heating allows for an energy­based dose to control the process. The main aspects of the method are the following: i) It is not isothermal (no homogeneous heating); ii) malignant cells are heated selectively; and iii) it employs high heating efficacy, with less energy loss. The applied rigorous thermodynamical considerations show the proper terminology and dose concept of hyperthermia, which is based on the energy­absorption (such as in the case of ionizing radiation) instead of the temperature­based ideas. On the whole, according to the present study, the appropriate dose in oncological hyperthermia must use an energy­based concept, as it is well­known in all the ionizing radiation therapies. We propose the use of Gy (J/kg) in cases of non­ionizing radiation (hyperthermia) as well.

An allometric approach of tumor-angiogenesis.

Angiogenesis is one of the main supporting factors of tumor-progression. It is a complex set of interactions together with hypoxia and inflammation, regulating tumor growth. The objective of this study is to examine the effect of angiogenesis with an allometric approach applied to angiogenesis and the regulating factors. The results show that allometry has the potential to describe this aspect, including the sigmoid-like transport function. There are particular conditions under which the complex control maximizes the relative tumor mass. Linear growth of malignancy diameter with an allometric approach was proven.

Nicotine interferes with purinergic signaling in smooth muscle cells isolated from urinary bladders of patients with lower urinary tract symptoms.

In patients with outlet obstruction, the contraction of the base is reduced compared to that of healthy individuals, while the contraction of the dome is not affected. Here, we investigated the cellular mechanisms that might be responsible for cholinergic effects blocking non-adrenergic non-cholinergic contractions in the base of the urinary bladder. Smooth muscle cells either from the base or from the dome of human urinary bladders were cultured to determine the contribution of cholinergic and purinergic mechanisms to their Ca2+ homeostasis. While ATP evoked Ca2+ transients in all the cells, nicotine and carbachol induced Ca2+ transients only in 56% and 44% of the cells, respectively. When ATP was administered together with nicotine or carbachol, the amplitudes of the Ca2+ transients recorded from cells prepared from the base of bladders were significantly smaller (42 ± 6% with nicotine and 56 ± 9% with carbachol) than those evoked by ATP alone. This inhibition was much less apparent in the dome of bladders. The inhibition between the cholinergic and purinergic signaling pathways reported in this work may decrease the strength of the contraction of the base of the urinary bladder in patients with outlet obstruction during voiding.

Hyperthermia versus Oncothermia: Cellular Effects in Complementary Cancer Therapy.

Hyperthermia means overheating of the living object completely or partly. Hyperthermia, the procedure of raising the temperature of a part of or the whole body above normal for a defined period of time, is applied alone or as an adjunctive with various established cancer treatment modalities such as radiotherapy and chemotherapy. However, hyperthermia is not generally accepted as conventional therapy. The problem is its controversial performance. The controversy is originated from the complications of the deep heating and the focusing of the heat effect. The idea of oncothermia solves the selective deep action on nearly cellular resolution. We would like to demonstrate the force and perspectives of oncothermia, as a highly specialized hyperthermia in clinical oncology. Our aim is to prove the ability of oncothermia to be a candidate to become a widely accepted modality of the standard cancer care. We would like to show the proofs and the challenges of the hyperthermia and oncothermia applications to provide the presently available data and summarize the knowledge in the topic. Like many early stage therapies, oncothermia lacks adequate treatment experience and long-range, comprehensive statistics that can help us optimize its use for all indications.

Rehabilitation of stroke patients using Yamamoto New Scalp Acupuncture: a pilot study.

OBJECTIVES: The aim of this study was to investigate whether acupuncture, especially Yamamoto's New Scalp Acupuncture (YNSA), is of value in additional to standard poststroke motor rehabilitation. DESIGN: A prospective, assessor-blinded randomized control trial was carried out in an inpatient stroke rehabilitation unit with day hospital service. After inclusion, patients were stratified into control group and acupuncture group, randomly. OUTCOME MEASURES: The Barthel Index, the Rivermead Scale Index, and the Visual Analogue Scale were used to follow the efficacy of treatment. RESULTS: In the acupuncture group, all the sensory, motor, and functional scores improved significantly during the examination period until 2 years after injury. The Barthel Index is increased from 4±2 to 95±4 in the acupuncture group. This index also increased in the control group (from 4±2 to 75±4), but the changes were significantly less than in the acupuncture group. A significant spontaneous recovery during the 2-year follow-up was found, but the YNSA treatment facilitated the functional recovery. Improved moving function and more flexible joints and ligaments were observed in comparison to the patients' condition prior to treatment. CONCLUSIONS: The data suggest that the YNSA is a useful method to treat stroke patients and enhance their quality of life.

A possible role of the cholinergic and purinergic receptor interaction in the regulation of the rat urinary bladder function.

The contractile activation of the upper (dome) and lower (base) parts of the urinary bladder show some differences. Cellular mechanisms that might be responsible for cholinergic effects blocking non-adrenergic non-cholinergic contractions in the base of the rat urinary bladder were investigated. Smooth muscle cells were thus freshly isolated or cultured both from the dome and the base of the rat urinary bladder and the contribution from cholinergic and purinergic pathways to their Ca(2+) homeostasis was examined. The expression of nicotinic acetylcholine (nAChR) and P2X2 purinergic receptors on the cultured cells and on tissue sections was investigated. The ATP-evoked Ca(2+) transients in rat smooth muscle cells did not show any desensitization. However, when ATP was administered together with carbamylcholine (CCh), the latter essentially prevented ATP from evoking Ca(2+) transients in smooth muscle cells from the base (suppression to 12 ± 2.5% of control, n = 57; p < 0.01), but not from the dome (99 ± 5% of control, n = 52; p > 0.05) of the rat urinary bladder. While atropine was unable to modify (6 ± 3% of control, n = 14; p < 0.05), α-bungarotoxin (118 ± 12% of control, n = 20; p > 0.05) blocked the inhibitory effects of CCh. Additionally, α7 subunits of nAChR and P2X2 purinergic receptors were identified using immunocytochemistry, immunohistochemistry, and Western blot in cultured urinary bladder smooth muscle cells, in urinary bladder sections, and in urinary bladder muscle strips, respectively, suggesting that the activation of nAChR modifies the action of ATP.

[Microvolt T-wave alternant: pathomechanism and evaluation of a new marker of arrhythmia risk]. / A fokozott arrhythmiarizikó új markere: a mikrovolt T-hullám-alternáns patomechanizmusa és vizsgálati módszerei.

Microvolt T-wave alternant (microV-TWA) is a beat-to-beat fluctuation in the amplitude of T-wave at a microvolt level. The amount of variation is small, on the order of microvolts, so sensitive digital signal processing techniques are required to detect microV-TWA. The appearance of microV-TWA has been suggested as a predictor of susceptibility to lethal ventricular tachyarrhythmias, and sudden cardiac death in different patients' populations. During the last decade, theoretical, experimental and clinical research efforts have focused primarily on microV-TWA, examining its mechanisms and predictive value using time-invariant cutoff values. The cellular mechanisms involved are not well-defined and are the subject of this investigation. This review discusses the bench-to-bedside literature that, over decades, has linked alternans of repolarization in cellular, whole-heart, and human studies with spatial dispersion of repolarization, alternans of cellular action potential, and fluctuations in ionic currents, intracellular calcium regulation, role of the beta adrenergic receptors and connexins that may lead to ventricular arrhythmias. This review then provides a contemporary framework for the use of microV-TWA methods to enhance risk stratification for sudden cardiac death, identifying populations for whom microV-TWA is best established.

Calcium entry is regulated by Zn2+ in relation to extracellular ionic environment in human airway epithelial cells.

The extracellular pH, sodium and divalent cation concentrations influence the ATP-induced changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)). This elevation of [Ca(2+)](i) and activation of Ca(2+)-dependent Cl(-) channels represent a possible therapeutic approach in cystic fibrosis (CF). We investigated the changes of [Ca(2+)](i) in different external ionic environment, and P2X purinergic receptors (P2XRs) expression in the control and CF airway epithelial cells. The parallel removal of Na(+) and alkalinization of the extracellular solution increased the amplitude of sustained ATP-induced Ca(2+) signals independent of wild-type or mutant CFTR expression. The ATP-induced Ca(2+) entry was either inhibited or stimulated by Zn(2+) depending on the extracellular Na(+) concentration. In Na(+)-free environment, Zn(2+) and other divalent cations elicited a biphasic Ca(2+) signal. Immunohistochemical data suggest that, multiple subtypes of P2XRs are expressed in these airway epithelial cells. In conclusion, Ca(2+) entry is finely regulated by external ionic environment. Therefore, we speculate that properly compiled aerosols could influence efficacy of zinc-based therapy in CF.

Altered expression of triadin 95 causes parallel changes in localized Ca2+ release events and global Ca2+ signals in skeletal muscle cells in culture.

The 95 kDa triadin (Trisk 95), an integral protein of the sarcoplasmic reticular membrane in skeletal muscle, interacts with both the ryanodine receptor (RyR) and calsequestrin. While its role in the regulation of calcium homeostasis has been extensively studied, data are not available on whether the overexpression or the interference with the expression of Trisk 95 would affect calcium sparks the localized events of calcium release (LCRE). In the present study LCRE and calcium transients were studied using laser scanning confocal microscopy on C2C12 cells and on primary cultures of skeletal muscle. Liposome- or adenovirus-mediated Trisk 95 overexpression and shRNA interference with triadin translation were used to modify the level of the protein. Stable overexpression in C2C12 cells significantly decreased the amplitude and frequency of calcium sparks, and the frequency of embers. In line with these observations, depolarization-evoked calcium transients were also suppressed. Similarly, adenoviral transfection of Trisk 95 into cultured mouse skeletal muscle cells significantly decreased both the frequency and amplitude of spontaneous global calcium transients. Inhibition of endogenous triadin expression by RNA interference caused opposite effects. Primary cultures of rat skeletal muscle cells expressing endogenous Trisk 95 readily generated spontaneous calcium transients but rarely produced calcium sparks. Their transfection with specific shRNA sequence significantly reduced the triadin-specific immunoreactivity. Functional experiments on these cells revealed that while caffeine-evoked calcium transients were reduced, LCRE appeared with higher frequency. These results suggest that Trisk 95 negatively regulates RyR function by suppressing localized calcium release events and global calcium signals in cultured muscle cells.

[Atrial fibrillation and the autonomous nervous system]. / A pitvarfibrilláció és a vegetatív idegrendszer.

The autonomic nervous system has a crucial role in the genesis, maintenance and abruption of atrial fibrillation. The substrate and trigger mechanism of atrial fibrillation can be influenced by the changing autonomic tone. The authors summarize the current knowledge on the relationship between autonomic nervous system and atrial fibrillation. The special neuroanatomical status and the role of autonomic reflexes and baroreflex in the initiation, maintenance, and termination of arrhythmia are reviewed. Furthermore, the mechanism and consequences of autonomic effect of the curative radiofrequency catheter ablation of pulmonary vein with atrial vagal neuroablation are discussed. At the end we also summarize the pharmacologic therapy of atrial fibrillation. Classification of atrial fibrillation, as either vagal or adrenergic, has only limited impact on current management.

Activation of cholinergic receptors blocks non-adrenergic non-cholinergic contractions in the rat urinary bladder.

In the present study, the plasticity of the non-adrenergic non-cholinergic (NANC) response was investigated. Isolated rat bladder strips were electrically stimulated and the evoked contractions were isometrically recorded. The NANC part of the contractions were unmasked by applying 500 nM 4-DAMP, a potent muscarinic antagonist. Treatment of the bladder strips with 10 microM carbachol (a cholinergic agonist) increased the muscle tone but did not alter the neurally evoked contractions. However, carbachol decreased: (1) the NANC response from 74.6% to 33.3% of control and (2) the purinergic contractile response to alpha,beta-methylene ATP (alpha,beta-mATP) (10 microM) from 97.0% to 43.4% (p<0.05). Treatment with the cholinesterase inhibitor eserine (10 microM) also significantly decreased the NANC response to 21.1% (p<0.0001). The purinergic receptor antagonist suramin (100 microM) did not affect the neurally evoked contractions, however; subsequent addition of 4-DAMP decreased the contractions to 31%. Activation of the smooth muscle cholinergic receptors (with carbachol or eserine) and purinergic receptors (with alpha,beta-mATP) decreased the NANC contractions and the direct contractile response to alpha,beta-mATP. When the electrically evoked contractions were facilitated by the L-type Ca2+ channel activator, Bay-K 8644 the subsequent application of 4-DAMP did not unmask inhibited NANC contractions. We conclude that activation of muscarinic receptors by cholinergic agonist, carbachol or by endogenous acetylcholine (ACh) induce a cascade of events that leads to diminished purinergic response and consequently an inhibition of the bladder NANC response.

SEA0400 fails to alter the magnitude of intracellular Ca2+ transients and contractions in Langendorff-perfused guinea pig heart.

SEA0400 is a recently developed inhibitor of the Na+/Ca2+ exchanger (NCX) shown to suppress both forward and reverse mode operation of NCX. Present experiments were designed to study the effect of partial blockade of NCX on Ca handling and contractility in Langendorff-perfused guinea pig hearts loaded with the fluorescent Ca-sensitive dye fura-2. Left ventricular pressure and intracellular calcium concentration ([Ca2+]i) were synchronously recorded before and after cumulative superfusion with 0.3 and 1 muM SEA0400. SEA0400 caused no significant change in the systolic and diastolic values of left ventricular pressure and [Ca2+]i. Accordingly, pulse pressure and amplitude of the [Ca2+]i transient also remained unchanged in the presence of SEA0400. SEA0400 had no influence either on the time required to reach peak values of pressure and [Ca2+)]i or on half relaxation time. On the other hand, both 0.3 and 1 microM SEA0400 significantly increased the decay time constant of [Ca2+]i transients, obtained by fitting its descending limb between 30% and 90% of relaxation, from 127 +/- 7 to 165 +/- 7 and 177 +/- 14 ms, respectively (P < 0.05, n=6). In contrast to the guinea pig hearts, rat hearts responded to SEA0400 treatment with increased [Ca2+]i transients and contractility. These interspecies differences observed in the effect of SEA0400 can be explained by the known differences in calcium handling between the two species.

Alterations in the calcium homeostasis of skeletal muscle from postmyocardial infarcted rats.

In chronic heart failure, skeletal muscles develop a weakness that is not associated to an impaired circulatory function but rather to alterations in the skeletal muscle fibers themselves. To understand these changes, the steps in excitation-contraction coupling of rats that underwent a left anterior coronary artery occlusion were studied. About 24 weeks after the myocardial infarction, neither the total amount nor the voltage dependence of intramembrane charge were altered. In contrast, calcium release from the sarcoplasmic reticulum was considerably suppressed, and its voltage dependence shifted toward more positive voltages. Elementary calcium-release events showed altered morphology as the relative proportion of embers increased. Calcium sparks were smaller in amplitude and had larger time-to-peak values. Isolated ryanodine receptors (RyR) displayed an unusual rectification with increased single-channel conductance at positive (cis vs trans) voltages. In addition, the bell-shaped calcium dependence of channel activity was broader, with a slight shift of activation to lower and a larger shift in inactivation to higher calcium concentrations. These data indicate that the number of channels that open during a calcium-release event is decreased and that RyR function is altered; thus, calcium-release is suppressed after a myocardial infarction. These observations give an explanation for the impaired skeletal muscle function in these animals.

Functional genomics of calcium channels in human melanoma cells.

Ca(2+)-signaling of human melanoma is in the focus of intensive research since the identification of the role of WNT-signaling in melanomagenesis. Genomic and functional studies pointed to the important role of various Ca(2+) channels in melanoma, but these data were contradictory. In the present study we clearly demonstrate, in a number of different ways including microarray analysis, DNA sequencing and immunocytochemistry, that various human melanoma cell lines and melanoma tissues overexpress ryanodine receptor type 2 (RyR2) and express P2X(7) channel proteins as compared to melanocytes. These channels, although retain some of their usual characteristics and pharmacological properties, display unique features in melanoma cells, including a functional interaction between the two molecules. Unlike P2X(7), RyR2 does not function as a calcium channel. On the other hand, the P2X(7) receptor has an antiapoptotic function in melanoma cells, since ATP-activation suppresses induced apoptosis, while knock down of the gene expression significantly enhances that.

Hypotonic stress influence the membrane potential and alter the proliferation of keratinocytes in vitro.

Keratinocyte proliferation and differentiation is strongly influenced by mechanical forces. We investigated the effect of osmotic changes in the development of HaCaT cells in culture using intracellular calcium measurements, electrophysiological recordings and molecular biology techniques. The application of hypotonic stress (174 mOsmol/l) caused a sustained hyperpolarization of HaCaT cells from a resting potential of -27 +/- 4 to -51 +/- 9 mV. This change was partially reversible. The surface membrane channels involved in the hyperpolarization were identified as chloride channels due to the lack of response in the absence of the anion. Cells responded with an elevation of intracellular calcium concentration to hypotonic stress, which critically depended on external calcium. The presence of phorbol-12-myristate-13-acetate in the culture medium for 12 h augmented the subsequent response to hypotonic stress. A sudden switch from iso- to hypotonic solution increased cell proliferation and suppressed the production of involucrin, filaggrin and transglutaminase, markers of keratinocyte differentiation. It is concluded that sudden mechanical forces increase the proliferation of keratinocytes through alterations in their membrane potential and intracellular calcium concentration. These changes together with additional modifications in channel expression and intracellular signalling mechanisms could underlie the increased proliferation of keratinocytes in hyperproliferative skin diseases.

Differentiation-dependent alterations in the extracellular ATP-evoked calcium fluxes of cultured skeletal muscle cells from mice.

Although extracellular adenosine triphosphate (ATP) has been generally accepted as the regulator of cellular differentiation, the relative contribution of the various purinoreceptor subtypes to purinergic signalling at distinct stages of skeletal muscle differentiation is still poorly understood. Here we measured extracellular ATP-evoked changes in intracellular calcium concentration and surface membrane ionic currents (I (ATP)), calculated the calcium flux (FL) entering the myoplasmic space and compared these parameters at different stages of differentiation on cultured mouse myotubes. The ATP-evoked FL displayed an early peak and then declined to a steady level. With differentiation, the early peak became separated from the maintained component and was absent on mature myotubes. Repeated ATP applications caused desensitization of the response in both immature and differentiated myotubes, owing mainly to the reduction of the early peak of FL in the former and to a decline of both components in the latter group of cells. Depolarization of the cell or removal of external calcium suppressed the early peak. I (ATP) showed no inactivation, and its voltage dependence displayed strong inward rectification. The concentration dependence of I (ATP) can be fitted using a Hill equation, yielding an EC(50) of 56 microM. Results are consistent with the parallel activation of both P2X and P2Y receptors.

Contribution from P2X and P2Y purinoreceptors to ATP-evoked changes in intracellular calcium concentration on cultured myotubes.

Although the alteration of purinoreceptor pattern on skeletal muscle is known to accompany physiological muscle differentiation and the pathogenesis of muscle dystrophy, the exact identity of and the relative contribution from the individual receptor subtypes to the purinergic signal have been controversial. To identify these subtypes in cultured myotubes of 5-10 nuclei, changes in intracellular calcium concentration and surface membrane ionic currents were detected and calcium fluxes calculated after the application of the subtype-specific agonists 2'3'-O-(benzoyl-4-benzoyl)-ATP (BzATP), 2-methyltio-ADP and UTP. The effectiveness of these agonists together with positive immunocytochemical staining revealed the presence of P2X(4), P2X(5), P2X(7), P2Y(1) and P2Y(4) receptors. siRNA-reduced protein expression of P2X(5), P2X(7) and P2Y(1) receptors was accompanied by reduction in the ATP-evoked calcium transients. Furthermore, anti-P2X(7) siRNA caused a significant drop in the early peak and delayed steady component of the calculated calcium flux. The use of its antagonist, oxidized ATP, similarly to transfection with anti-P2X(7) siRNA caused significant reduction in the agonist-elicited ionic currents I (ATP) and I (BzATP), with a greater drop in the latter. Our results demonstrate that the activation of ionotropic P2X(4), P2X(5) and P2X(7) and metabotropic P2Y(1) and P2Y(4) purinoreceptors participates in forming the calcium transients of multinucleated myotubes.

Effect of TPEN on the calcium release of cultured C2C12 mouse myotubes.

N,N,N',N'-tetrakis(2-pyridylmethyl)-ethilenediamine (TPEN) is a membrane permeable heavy metal chelator that has been used to study intracellular calcium homeostasis but its exact mode of action is still unresolved. Here we examine the effects of TPEN on the Ca(2+) release from and the Ca(2+) uptake into the sarcoplasmic reticulum (SR) of cultured C2C12 skeletal muscle cells. Low concentrations (50 microM) of the drug evoked Ca(2+) transients in approximately 60% of C2C12 myotubes, while at high concentrations (500 microM) it significantly reduced the size of both depolarization-and caffeine-induced Ca(2+) transients, decreased the rate constant of decay and the calculated pump activity but failed to induce Ca(2+) transients. Experiments at low extracellular [Ca(2+)] revealed that it is the total rather than free TPEN concentration that is responsible for the observed effects. TPEN does not modify Ca(2+) release by Zn(2+) chelation, as evidenced by the unaltered effect seen after the removal of Zn(2+) from the extracellular space of the cells by chelating with EDPA. These findings provide experimental evidence that TPEN directly modifies both the release of Ca(2+) from the SR and its removal from the myoplasm.