Agonist-stimulated oscillations and cycling of intracellular free calcium in individual cultured muscle cells

Receptor regulation of [Ca2+]i was monitored in individual BC3H-1 muscle cells with intracellularly trapped fura-2 using digital imaging analysis techniques. Activation of alpha 1-adrenergic or H1-histaminergic receptors resulted in multiple bursts, or oscillations, of elevated [Ca2+]i with an average interval frequency of approximately 1.8 min-1. The duration of oscillatory behavior was generally more prolonged in response to phenylephrine than in response to histamine. Additionally, a larger fraction of the cells responded with [Ca2+]i oscillations to phenylephrine (approximately 90%) than to histamine (approximately 60%), although the majority of cells produced oscillations in response to both agonists. In most cells, the receptor-mediated [Ca2+]i oscillations continued for several minutes in the absence of extracellular Ca2+, although the amplitude of the individual peaks gradually decreased. The activation of [Ca2+]i oscillations by H1-receptors was more dependent upon extracellular Ca2+ than those elicited by alpha 1-receptors, reflecting the greater dependency of the histaminergic response on Ca2+ influx. Readdition of Ca2+ to the incubation buffer resulted in the resumption of the [Ca2+]i oscillations. These results indicate that considerable cycling of Ca2+ between the cytoplasm and the endoplasmic reticulum must occur. Receptor-mediated [Ca2+]i oscillations were much more prevalent in subconfluent cells than in confluent cells, possibly due to increased coupling of the cells at higher densities. The cells were capable of responding independently of one another, since sister cells displayed unique temporal responses immediately following cell division. Thus, the linkage of receptor occupancy to [Ca2+]i elevation is a functionally unique property for each individual cell and can be influenced by epigenetic factors.     

Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channels

The human umbilical cord is a biological sample that can be easily obtained just after birth. A methodology was developed to perform cultures of human umbilical artery smooth muscle cells (HUASMC) expressing contractile proteins and functional ionic channels. To avoid fibroblast and endothelial cell contamination, we mechanically separated the tunica media, which only contains HUASMC and matrix proteins. To isolate the cells, collagenase V and elastase were used as hydrolyzing enzymes. The isolated cells were plated in collagen-coated dishes to obtain cultures of HUASMC. The cells obtained after different passages (1 to 6) exhibit the characteristic vascular smooth cell morphology and express smooth muscle alpha-2 actin, myosin heavy chain SM1, and alpha subunits of L- and T-type calcium channels (Cav 1.2, Cav 1.2, and Cav 3.2). Electrophysiology recordings for L- and T-type calcium channels were made, indicating that these channels are functional in the cultured cells. In conclusion, the procedure developed allows obtaining cultures of HUASMC expressing contractile proteins and also functional ionic channels. These cells could be used to study cellular and molecular aspects about the regulation of the vascular function.     

Genetic variation in histidine rich proteins among Indian Plasmodium falciparum population: possible cause of variable sensitivity malaria of rapid diagnostic tests

ABSTRACT: BACKGROUND: Rapid diagnostic tests (RDTs) have revolutionized the diagnosis of malaria. Among the various factors affecting RDTs sensitivity is genetic variation of the antigen used. The genetic variation in PfHRP2 and PfHRP3 proteins was studied among the Indian Plasmodium falciparum isolates. METHODS: One hundred and forty isolates of P. falciparum were collected from six geographical regions of India. Target genes encoding PfHRP2 and PfHRP3 antigens were sequenced to study genetic polymorphism. Minimum detection limit giving a positive rapid diagnostic test was also determined. RESULTS: Extensive variations were observed in amino acid repeat types of PfHRP2 and PfHRP3. PfHRP2 exhibited more polymorphism than PfHRP3. Significant relation was observed between type 2 and type 7 repeats and RDT detection rate as higher number of these repeats showed better sensitivity with RDTs. CONCLUSION: The results provide insights into the genetic diversity of Pfhrp2 and Pfhrp3 genes among Indian P. falciparum population and its relation to RDT sensitivity.     

Selected contribution: mechanical strain increases force production and calcium sensitivity in cultured airway smooth muscle cells.

Cultured airway smooth muscle cells subjected to cyclic deformational strain have increased cell content of myosin light chain kinase (MLCK) and myosin and increased formation of actin filaments. To determine how these changes may increase cell contractility, we measured isometric force production with changes in cytosolic calcium in individual permeabilized cells. The pCa for 50% maximal force production was 6.6+/-0.4 in the strain cells compared with 5.9+/-0.3 in control cells, signifying increased calcium sensitivity in strain cells. Maximal force production was also greater in strain cells (8.6+/-2.9 vs. 5.7+/-3.1 microN). The increased maximal force production in strain cells persisted after irreversible thiophosphorylation of myosin light chain, signifying that increased force could not be explained by differences in myosin light chain phosphorylation. Cells strained for brief periods sufficient to increase cytoskeletal organization but insufficient to increase contractile protein content also produced more force, suggesting that strain-induced cytoskeletal reorganization also increases force production.     

Trypsin and forskolin decrease the sensitivity of L-type calcium current to inhibition by cytoplasmic free calcium in guinea pig heart muscle cells.

A key feature of trypsin action on ionic membrane currents including L-type Ca2+ current (ICa) is the removal of inactivation upon intracellular application. Here we report that trypsin also occludes the resting cytoplasmic free Ca2+ ([Ca2+]i)-induced inhibition of peak ICa in isolated guinea pig ventricular cardiomyocytes, using the whole-cell patch clamp in combination with the Fura-2 ratio-fluorescence technique. The effectiveness of trypsin to guard ICa against [Ca2+]i-induced inhibition was compared with that of forskolin, as cAMP-dependent phosphorylation had been suggested to confer protection against [Ca2+]i-induced inactivation. Intracellular dialysis of trypsin (1 mg/ml) augmented ICa by 7.2-fold, significantly larger than the threefold increase induced by forskolin (3 microM). Forskolin application after trypsin dialysis did not further enhance ICa. An increase in [Ca2+]i from resting levels (varied by 0.2, 10, and 40 mM EGTA dialysis) to submicromolar concentrations after replacement of external Na+ (Na(o)+) with tetraethylammonium (TEA+) resulted in monotonic inhibition of control ICa, elicited from a holding potential of -40 mV at 22 degrees C. AFter trypsin dialysis, however, ICa became less sensitive to submicromolar [Ca2+]i; the [Ca2+]i of half-maximal inhibition (K0.5, normally around 60 nM) increased by approximately 20-fold. Forskolin also increased the K0.5 by approximately threefold. These and accompanying kinetic data on ICa decay are compatible with a model in which it is assumed that Ca2+ channels can exist in two modes (a high open probability "willing" and a low open probability "reluctant" mode) that are in equilibrium with one another. An increase in [Ca2+]i places a larger fraction of channels in the reluctant mode. This interconversion is hindered by cAMP-dependent phosphorylation and becomes nearly impossible after tryptic digestion.     

Use of ryanodine for functional removal of the calcium store in smooth muscle cells of the guinea-pig

Calcium store of the skinned fibers of the guinea-pig portal vein, pulmonary artery and taenia caeci consisted of two classes: one with both Ca-induced Ca release (CICR) and inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanisms (S alpha) and the other only with IICR mechanisms (S beta). Ryanodine, applied during the CICR was activated, locked the CICR channels open, but the drug had practically no effect on the IICR mechanism. Thus, after the ryanodine treatment the Ca store with the CICR (S alpha) lost its capacity to hold Ca. Changes in the agonist-evoked contraction of intact muscle due to the ryanodine treatment suggest that agonists release Ca from S alpha which produces the initial phase of contractures.     

Structural and functional mutations of the perlecan gene cause Schwartz-Jampel syndrome, with myotonic myopathy and chondrodysplasia

Perlecan, a large heparan sulfate proteoglycan, is a component of the basement membrane and other extracellular matrices and has been implicated in multiple biological functions. Mutations in the perlecan gene (HSPG2) cause two classes of skeletal disorders: the relatively mild Schwartz-Jampel syndrome (SJS) and severe neonatal lethal dyssegmental dysplasia, Silverman-Handmaker type (DDSH). SJS is an autosomal recessive skeletal dysplasia characterized by varying degrees of myotonia and chondrodysplasia, and patients with SJS survive. The molecular mechanism underlying the chondrodystrophic myotonia phenotype of SJS is unknown. In the present report, we identify five different mutations that resulted in various forms of perlecan in three unrelated patients with SJS. Heterozygous mutations in two patients with SJS either produced truncated perlecan that lacked domain V or significantly reduced levels of wild-type perlecan. The third patient had a homozygous 7-kb deletion that resulted in reduced amounts of nearly full-length perlecan. Unlike DDSH, the SJS mutations result in different forms of perlecan in reduced levels that are secreted to the extracellular matrix and are likely partially functional. These findings suggest that perlecan has an important role in neuromuscular function and cartilage formation, and they define the molecular basis involved in the difference in the phenotypic severity between DDSH and SJS.     

Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor

Calcium serves as a second messenger in glucose-triggered insulin secretion of pancreatic cells. Less is known about sugar signaling in non-excitable cells. Here, the high sensitivity FRET calcium sensor TN-XXL was used to characterize glucose-induced calcium responses in non-excitable human embryonic kidney HEK293T cells. HEK293T cells responded to perfusion with glucose with a sustained and concentration-dependent increase in cytosolic calcium levels. Sucrose and mannitol triggered comparable calcium responses, suggesting that the increase of the calcium concentration was caused by osmotic effects. HEK293T cells are characterized by low endogenous glucose uptake capacity as shown with a high sensitivity glucose sensor. Consistently, when glucose influx was artificially increased by co-expression of GLUT glucose transporters, the glucose-induced calcium increase was significantly reduced. Neither calcium depletion, nor gadolinium or thapsigargin were able to inhibit the calcium accumulation. Taken together, membrane impermeable osmolytes such as sucrose and mannitol lead to an increase in calcium levels, while the effect of glucose depends on the cell's glucose uptake capacity and will thus vary between cell types in the body that differ in their glucose uptake capacity.     

Platelet-derived growth factor and angiotensin II cause increases in cytosolic free calcium by different mechanisms in vascular smooth muscle cells

Platelet-derived growth factor (PDGF) and angiotensin II (AII) are thought to mediate their biological effects in vascular smooth muscle cells (VSMCs) by causing alterations in cytosolic free calcium ([ Ca2+]i). In this study we examine the pathways by which PDGF and AII alter [Ca2+]i in VSMCs. Addition of PDGF resulted in a rapid, transient, concentration-dependent increase in [Ca2+]i; this rise in [Ca2+]i was blocked completely by preincubation of cells with ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) or CoCl2, by the voltage-sensitive Ca2+-channel antagonists verapamil or nifedipine, by 12-O-tetradecanoylphorbol-13-acetate (TPA), or by pertussis toxin. AII also caused an increase in [Ca2+]i; however, AII-stimulated alterations in [Ca2+]i displayed different kinetics compared with those caused by PDGF. Pretreatment of cells with 8-(diethylamine)-octyl-3,4,5-trimethyoxybenzoate hydrochloride (TMB-8), almost totally inhibited AII-induced increases in [Ca2+]i. EGTA or CoCl2 only slightly diminished AII-stimulated increases in [Ca2+]i. Nifedipine, verapamil, TPA, and pertussis toxin pretreatment were without effect on AII-induced increases in [Ca2+]i. PDGF and AII both stimulated increases in total inositol phosphate accumulation, although the one-half maximal concentration (ED50) for alterations in [Ca2+]i and phosphoinisitide hydrolysis differed by a factor of 10 for PDGF (3 X 10(-10) M for Ca2+ vs. 2.5 X 10(-9) M for phosphoinositide hydrolysis), but they were essentially identical for AII (7.5 X 10(-9) M for Ca2+ vs. 5.0 X 10(-9) M for phosphoinositide hydrolysis). PDGF stimulated mitogenesis (as measured by [3H]-thymidine incorporation into DNA) in VSMCs with an ED50 similar to that for PDGF-induced alterations in phosphoinositide hydrolysis. PDGF-stimulated mitogenesis was blocked by pretreatment of cells with voltage-sensitive Ca2+ channel blockers, TPA, or pertussis toxin. These results suggest that PDGF and AII cause alterations in [Ca2+]i in VSMCs by at least quantitatively distinct mechanisms. PDGF binding activates a pertussis-toxin-sensitive Ca2+ influx into cells via voltage-sensitive Ca2+ channels (blocked by EGTA, verapamil, and nifedipine), as well as stimulating phosphoinositide hydrolysis leading to release of Ca2+ from intracellular stores. AII-induced alterations in [Ca2+]i are mainly the result of phosphoinositide hydrolysis and consequent entry of Ca2+ into the cytoplasm from intracellular stores. Our data also suggest that changes in [Ca2+]i caused by PDGF are required for PDGF-stimulated mitogenesis.     

Mechanisms of calcium signaling in smooth muscle cells explored with fluorescence confocal imaging

A rise in the intracellular concentration of ionized calcium ([Ca²⁺]_i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca²⁺ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca²⁺ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca²⁺]_i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP₃Rs) is an important determinant of the [Ca²⁺]_i dynamics and recruits neighboring Ca²⁺-release sites to generate [Ca²⁺]_i waves; (v) [Ca²⁺]_i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca²⁺-dependent K⁺ and Cl^- channels sense the local changes in [Ca²⁺]_i during a Ca²⁺ spark and thereby may couple changes in [Ca²⁺]_i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mI _cat) does not mirror changes in [Ca²⁺]_i, thus reflecting the complexity of [Ca²⁺]_i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca²⁺ release, either spontaneous or caffeine-induced, does not augment mI _cat; (ix) intracellular flash release of Ca²⁺ is less effective in augmentation of mI _cat than flash release of IP₃, suggesting that IP₃ may sensitize muscarinic cationic channels to Ca²⁺; (x) intracellular flash release of IP₃ fails to augment mI _cat in SMCs, in which [Ca²⁺]_i was strongly buffered, suggesting that IP₃ exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca²⁺]_i rather than depletion of the IP₃-dependent Ca²⁺ store; and (xi) predominant expression of IP₃R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP₃R-mediated Ca²⁺ release and muscarinic cationic channel opening.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 455–465, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole cell oscillations in smooth muscle cells

In vitro, alpha-adrenoreceptor stimulation of rat mesenteric small arteries often leads to a rhythmic change in wall tension, i.e., vasomotion. Within the individual smooth muscle cells of the vascular wall, vasomotion is often preceded by a period of asynchronous calcium waves. Abruptly, these low-frequency waves may transform into high-frequency whole cell calcium oscillations. Simultaneously, multiple cells synchronize, leading to rhythmic generation of tension. We present a mathematical model of vascular smooth muscle cells that aims at characterizing this sudden transition. Simulations show calcium waves sweeping through the cytoplasm when the sarcoplasmic reticulum (SR) is stimulated to release calcium. A rise in cGMP leads to the experimentally observed transition from waves to whole cell calcium oscillations. At the same time, membrane potential starts to oscillate and the frequency approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes a uniform opening of L-type calcium channels on the cell surface, stimulating a synchronized release of SR calcium and inducing the shift from waves to whole cell oscillations. The effect of the channel is therefore to couple the processes of the SR with those of the membrane. We hypothesize that the shift in oscillatory mode and the associated onset of oscillations in membrane potential within the individual cell may underlie sudden intercellular synchronization and the appearance of vasomotion.     

Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection

Laser-capture microdissection was coupled with PCR to define the mitochondrial genotype of aged muscle fibers exhibiting mitochondrial enzymatic abnormalities. These electron transport system (ETS) abnormalities accumulate with age, are localized segmentally along muscle fibers, are associated with fiber atrophy and may contribute to age-related fiber loss. DNA extracted from single, 10 µm thick, ETS abnormal muscle fibers, as well as sections from normal fibers, served as templates for PCR-based deletion analysis. Large mitochondrial (mt) DNA deletion mutations (4.4–9.7 kb) were detected in all 29 ETS abnormal fibers analyzed. Deleted mtDNA genomes were detected only in the regions of the fibers with ETS abnormalities; adjacent phenotypically normal portions of the same fiber contained wild-type mtDNA. In addition, identical mtDNA deletion mutations were found within different sections of the same abnormal region. These findings demonstrate that large deletion mutations are associated with ETS abnormalities in aged rat muscle and that, within a fiber, deletion mutations are clonal. The displacement of wild-type mtDNAs with mutant mtDNAs results in concomitant mitochondrial enzymatic abnormalities, fiber atrophy and fiber breakage.     

Agonist-induced calcium transients in cultured smooth muscle cells: measurements with fura-2 loaded monolayers

Elevation of cytosolic Ca2+ in response to depolarization and various receptor agonists was measured in several types of cultured smooth muscle cells (DDT1, A10, rabbit aorta) loaded with the either quin-2 or fura-2, and assayed either in suspension or in monolayer cultures attached to plastic cover slips. Agonists (norepinephrine, vasopressin) induced both the release of intracellular Ca2+ and the influx of extracellular Ca2+. Agonist-induced Ca2+ influx was not blocked by dihydropyridines, and depolarization did not induce Ca2+ influx. However, in fura-2 loaded monolayers of PC12 cells, depolarization did induce dihydropyridine-sensitive Ca2+ influx. Thus cultured smooth muscle cells appear to express receptor-operated Ca2+ channels, but not functional voltage-operated Ca2+ channels.     

Familial hypertrophic cardiomyopathy mutations from different functional regions of troponin T result in different effects on the pH and Ca2+ sensitivity of cardiac muscle contraction

To understand the molecular function of troponin T (TnT) in the Ca(2+) regulation of muscle contraction as well as the molecular pathogenesis of familial hypertrophic cardiomyopathy (FHC), eight FHC-linked TnT mutations, which are located in different functional regions of human cardiac TnT (HCTnT), were produced, and their structural and functional properties were examined. Circular dichroism spectroscopy demonstrated different secondary structures of these TnT mutants. Each of the recombinant HCTnTs was incorporated into porcine skinned fibers along with human cardiac troponin I (HCTnI) and troponin C (HCTnC), and the Ca(2+) dependent isometric force development of these troponin-replaced fibers was determined at pH 7.0 and 6.5. All eight mutants altered the contractile properties of skinned cardiac fibers. E244D potentiated the maximum force development without changing Ca(2+) sensitivity. In contrast, the other seven mutants increased the Ca(2+) sensitivity of force development but not the maximal force. R92L, R92W, and R94L also decreased the change in Ca(2+) sensitivity of force development observed on lowering the pH from 7 to 6.5, when compared with wild type TnT. The examination of additional mutants, H91Q and a double mutant H91Q/R92W, suggests that mutations in a region including residues 91-94 in HCTnT can perturb the proper response of cardiac contraction to changes in pH. These results suggest that different regions of TnT may contribute to the pathogenesis of TnT-linked FHC through different mechanisms.     

Morphological and functional changes in Lewis lung carcinoma cells after treatment with A23187 calcium ionophore

Cultured Lewis Lung carcinoma cells (3LL) treated with A23187 calcium ionophore were studied by transmission electron microscopy and HPLC analysis. Results showed that A23187 calcium ionophore induces on osmiophilic inclusion bodies of 3LL cells similar process of lamellar bodies formation to those reported in the development of the osmiophilic inclusions bodies within the granular pneumocyte of normal lungs. HPLC analysis shows that calcium ionophore generates quantitative changes in the 3LL cytoplasmic protein content expressed by an increase of 18-22 kDa and 4-9 kDa proteins.     

The calcium homeostasis and the membrane potential of cultured muscle cells from patients with myotonic dystrophy

Using the fluorescence indicator, quin2, we compared the cytoplasmic Ca2+ concentration ([Ca2+]i) of cultured myotubes obtained from control subjects and myotonic dystrophy (MyD) patients. In Ca2(+)-free buffer the [Ca2+]i of the cultured MyD muscle cells was not significantly different from that of the control cells. In the presence of 1 mM external Ca2+ the cultured MyD muscle cells showed a significantly higher [Ca2+]i, which was due to the influx of Ca2+ through voltage-operated nifedipine-sensitive Ca2+ channels. In the presence of external Ca2+, MyD myotubes did not respond to acetylcholine, whereas control myotubes showed a transient increase in [Ca2+]i after addition of acetylcholine. This increase was inhibited by the addition of nifedipine. The differences in Ca2(+)-homeostasis between cultured MyD muscle cells and control cells were not due to differences in the resting membrane potential or the inability of the MyD cells to depolarize as a response to acetylcholine. Therefore, cultured MyD muscle cells exhibit altered nifedipine-sensitive voltage-operated channels which are active under conditions in which they are normally present in the inactive state, and which are unable to respond to depolarization caused by acetylcholine.     

Decoupling of heart muscle cells: Correlation with increased cytoplasmic calcium activity and with changes of nexus ultrastructure

Summary Purkinje fibers of the sheep heart were exposed to (a) 0.1mm dihydro-ouabain (DHO), followed by (b) 0.1mm DHO in Na-free solution or to (c) 1mm dinitrophenol (DNP). The degree of electrical decoupling was characterized in terms of the inside longitudinal resistancer _i as measured with a 3-microelectrode voltage-clamp technique. Procedurea increasedr _i by a factor of 3.7±1.1 (mean±sd),b by a factor of 9.8±2.2, whereas inc incomplete voltage control indicated nearly complete uncoupling. Intracellular calcium activity (aCa _i ) was monitored with a microelectrode system. At control conditionsaCa _i was below 0.1 m. The procedures listed above increasedaCa _i to (a) 4±1.5 m, (b) 8±2 m, and (c) 36±12 m. The increase ofaCa _i was in good correlation with the changes in core resistance. Effects on nexus ultrastructure, investigated with freeze-fracture techniques, are shown in histograms. At control conditions, the particle diameter distributed around a single peak (8.3±0.5 nm). Proceduresb andc induced a second population at 10.8 nm; increased decoupling reduced the control population in favor of the 10.8 nm population. Decoupling enlarged the width of the nexus gap by a factor of 1.6; again, the control population decreased in favor of a new population. In the decoupled state the height of the particle was smaller. Pits on the E-face displayed a more regular array and a nearly unchanged center-to-center spacing. Separation into several peaks was not possible due to scatter of the data.We interpret the findings to mean that elevatedaCa _i induces a conformational change of the nexus subunits which corresponds to a transition from an open to a closed state. The conformational change can be formally described by a particle contraction which disrupts the continuity with the particle of the adjacent membrane. Purkinje fibers exposed to DNP for 1 hr showed thinned (7.7±0.5 mm) and elongated particles. We suggest that this is a secondary event and not a precursor of functional uncoupling.