Structures from intact myofibrils reveal mechanism of thin filament regulation through nebulin.

In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.

Simple implementation of muscle tissue into routine workflow of blood analysis in forensic cases - A validated method for quantification of 29 drugs in postmortem blood and muscle samples by UHPLC-MS/MS.

Whole blood is most often the matrix of choice for postmortem analysis but it is not always available. In these cases, muscle tissue can be used as an alternative matrix. Therefore, an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the quantification of 29 drugs and metabolites of toxicological interest in postmortem muscle tissue was developed and validated. Additionally, a validation of whole blood was carried out to compare the results from the two matrices. Solid-phase extraction was performed by an automated robotic system to minimize manual labour and risk of human errors, and increase robustness, sample throughput and sample traceability. The method was validated in terms of selectivity, matrix effect, extraction recovery, process efficiency, measuring range, lower limit of quantification, carry-over, stability, precision and accuracy. To correct for any inter-individual variability in matrix effects on analyte accuracy and precision, deuterated analogues of each analyte were used as internal standards. The lower limit of quantification in both blood and muscle homogenate ranged between 0.002 and 0.005 mg/kg, while the upper limit of quantification spanned from 0.20 to 1.0 mg/kg. Corrected with the 4-fold dilution factor, the corresponding concentrations in muscle tissue were 0.008-0.02 mg/kg at the lower limit of quantification and 0.80-4.0 mg/kg at the upper limit of quantification. The method showed acceptable precision and accuracy, with precision below 12% and accuracies ranging from 87% to 115% at up to 6 levels for all analytes in both matrices. In addition, comparison between calibration standards in spiked muscle homogenate and spiked blood showed that analyte concentrations in muscle samples could be quantified by using spiked blood samples as calibration standards with acceptable precision and accuracy when using deuterated analogues as internal standards. The investigation of matrix effects showed no great difference between blood and homogenates of non-decomposed and decomposed muscle tissue for most analytes. In the samples where high ion suppression or enhancement was observed, the results were corrected by the internal standards. Statistical comparison of quality control samples in blood and muscle tissue showed no obvious differences, and therefore muscle tissue was included in the routine method for analysis of blood samples and used in autopsy cases where no blood was available. By adding a semi-automated homogenization step before the remaining automated sample preparation, muscle tissue samples were easily incorporated into the workflow of the existing routine method. The present method has been successfully implemented in routine analysis of blood and muscle tissue since 2019.

Integrated lipidomics and targeted metabolomics analyses reveal changes in flavor precursors in psoas major muscle of castrated lambs.

Castration may decrease off-odors and improve meat flavor. Meat flavor is generated through complex chemical reactions that involve hydrophilic and hydrophobic flavor precursors. In this study, we investigated the flavor precursors in psoas major muscles of castrated and intact sheep using lipidomics and targeted metabolomics. Castration decreased testosterone levels and increased intramuscular fat content. Six hundred fourteen lipid molecules confirmed showed a separation between castrated and intact sheep based on principal component analysis. Fourteen lipid species and 224 lipid molecules increased in castrated sheep. Targeted metabolomics analysis showed that 18 hydrophilic metabolites were affected by castration; however, only hypoxanthine significantly increased in the castration group. Among 45 volatiles identified, 1-octen-3-ol and hexanal were significantly higher in castrated sheep. These results revealed that lipids, hydrophilic metabolites, and volatile compounds in lamb were affected by castration, which might be beneficial in lamb quality.

The influence of habitat and sex on feral fallow deer meat lipid fraction.

BACKGROUND: The study tested the hypothesis that habitat and sex influence the composition of meat lipid fraction. Therefore, this study aims to characterize the effects of habitat and sex on feral fallow deer (Dama dama) tenderloin (psoas major muscle) intramuscular fat composition. RESULTS: Fallow deer meat from the Atlantic forest presented higher total cholesterol and total monounsaturated fatty acids contents, while fallow deer meat from Montado presented significantly higher proportion of total branched chain fatty acids, n-6 polyunsaturated fatty acids and dimethyl acetals. Regarding the sex influence on meat's fatty acid composition, it was observed that female fallow deer displayed higher contents of total saturated fatty acids than their male counterparts. CONCLUSION: Independently of the habitat the feral fallow deer's meat can be classified as extra lean. However, the high peroxidability index and the low total vitamin E content suggests that it is prone to lipid peroxidation. © 2020 Society of Chemical Industry.

Cyanide concentrations in blood and tissues of fire victims.

Cyanide poisoning has been regarded to contribute the fatal outcome in fire victims. The toxicity of inhaled hydrogen cyanide (HCN) at the cellular level was evaluated considering the impact of methemoglobin (MetHb) produced by fire gases. Cyanide (CN) concentrations and total hemoglobin contents were measured in right heart blood (RHB) and seven organs/tissues (basal ganglia, brain stem, heart, lung, liver, kidney and psoas muscle) collected from 20 fire fatalities. MetHb and carboxyhemoglobin saturations were also measured in RHB. The amount of CN probably bound to the cytochrome c oxidase of the tissue cells (CCO-CN) was extrapolated from CN and hemoglobin contents in RHB and organs/tissues, MetHb saturation in RHB and binding capacity of MetHb for CN. CN concentrations in RHB showed a wide range with the highest concentration of 8.927 µg/mL. The lung contained the largest CN content among organs/tissues with the mean concentration of 2.219 µg/g, then the heart (0.259 µg/g) and it was lower than 0.100 µg/g in others. Exceedingly large amount of CN in the lung could be explained by high hemoglobin content, being the port of entry of HCN and postmortem diffusion of fire gases. CCO-CN was theoretically present in about 20% of organ/tissue samples, most commonly in the basal ganglia (10 samples, with the mean of 0.059 µg/g) followed by heart (eight samples, with the mean of 0.109 µg/g). No CCO-CN was found in liver and kidney. HCN might have the effect on brain and heart.

Organ distribution of diclazepam, pyrazolam and 3-fluorophenmetrazine.

The organ distribution of 3-fluorophenmetrazine (3-FPM), pyrazolam, diclazepam as well as its main metabolites delorazepam, lormetazepam and lorazepam, was investigated. A solid phase extraction (SPE) and a QuEChERS (acronym for quick, easy, cheap, effective, rugged and safe) - approach were used for the extraction of the analytes from human tissues, body fluids and stomach contents. The detection was performed on a liquid chromatography-tandem mass spectrometry system (LCMS/MS). The analytes of interest were detected in all body fluids and tissues. Results showed femoral blood concentrations of 10 µg/L for 3-FPM, 28 µg/L for pyrazolam, 1 µg/L for diclazepam, 100 µg/L for delorazepam, 6 µg/L for lormetazepam, and 22 µg/L for lorazepam. Tissues (muscle, kidney and liver) and bile exhibited higher concentrations of the mentioned analytes than in blood. Additional positive findings in femoral blood were for 2-fluoroamphetamine (2-FA, approx. 89 µg/L), 2-flourometamphetamine (2-FMA, hint), methiopropamine (approx. 2.2 µg/L), amphetamine (approx. 21 µg/L) and caffeine (positive). Delorazepam showed the highest ratio of heart (C) and femoral blood (P) concentration (C/P ratio = 2.5), supported by the concentrations detected in psoas muscle (430 µg/kg) and stomach content (approx. 210 µg/L, absolute 84 µg). The C/P ratio indicates that delorazepam displays susceptibility for post-mortem redistribution (PMR), supported by the findings in muscle tissue. 3-FPM, pyrazolam, diclazepam, lorazepam and lormetazepam did apparently not exhibit any PMR. The cause of death, in conjunction with autopsy findings was concluded as a positional asphyxia promoted by poly-drug intoxication by arising from designer benzodiazepines and the presence of synthetic stimulants.

Use of the Randox Evidence Investigator immunoassay system for near-body drug screening during post-mortem examination in 261 forensic cases.

BACKGROUND: This paper describes the performance of four Randox drug arrays, designed for whole blood, for the near-body analysis of drugs in a range of post-mortem body specimens. METHODS: Liver, psoas muscle, femoral blood, vitreous humor and urine from 261 post-mortem cases were screened in the mortuary and results were obtained within the time taken to complete a post-mortem. Specimens were screened for the presence of amfetamine, barbiturates, benzodiazepines, benzoylecgonine, buprenorphine, cannabinoids, dextropropoxyphene, fentanyl, ketamine, lysergide, methadone, metamfetamine, methaqualone, 3,4-methylenedioxymetamfetamine, opioids, paracetamol, phencyclidine, salicylate, salicylic acid, zaleplon, zopiclone and zolpidem using the DOA I, DOA I+, DOA II and Custom arrays. RESULTS: Liver and muscle specimens were obtained from each of the 261 post-mortem cases; femoral blood, vitreous humor and urine were available in 98%, 92% and 72% of the cases, respectively. As such, the equivalent of 12,978 individual drug-specific, or drug-group, immunoassay tests were undertaken. Overall >98% of the 12,978 screening tests undertaken agreed with laboratory confirmatory tests performed on femoral blood. CONCLUSIONS: There is growing interest in the development of non-invasive procedures for determining the cause of death using MRI and CT scanning however these procedures are, in most cases, unable to determine whether death may have been associated with drug use. The Randox arrays can provide qualitative and semi-quantitative results in a mortuary environment enabling pathologists to decide whether to remove specimens from the body and submit them for laboratory analysis. Analysis can be undertaken on a range of autopsy specimens which is particularly useful when conventional specimens such as blood are unavailable.

Post-mortem distribution of the synthetic cannabinoid MDMB-CHMICA and its metabolites in a case of combined drug intoxication.

This case report centres on the post-mortem distribution of the synthetic cannabinoid MDMB-CHMICA and its metabolites in the case of a 27-year-old man found dead after falling from the 24th floor of a high-rise building. Toxicological analysis of post-mortem samples confirmed, besides consumption of the synthetic cannabinoids MDMB-CHMICA (1.7 ng/mL femoral blood) and EG-018, the abuse of THC (9.3 ng/mL femoral blood), amphetamine (1050 ng/mL femoral blood), MDMA (275 ng/mL femoral blood), and cocaine. Regarding EG-018 and cocaine, only traces were detected in heart blood as well as in the brain (EG-018) and urine (cocaine), respectively, which is why no quantification was conducted in the femoral blood sample. It was concluded from femoral blood analysis that, at the time of death, the man was under the influence of the synthetic cannabinoid MDMB-CHMICA, THC, amphetamine and MDMA. Comprehensive screenings of all post-mortem specimens were conducted to elucidate the post-mortem distribution of MDMB-CHMICA and its metabolites. The MDMB-CHMICA concentrations ranged between 0.01 ng/mL (urine) and 5.5 ng/g (brain). Comparably low concentrations were detected in cardiac and femoral blood (2.1 ng/mL and 1.7 ng/mL, respectively) as well as in the psoas major muscle (1.2 ng/g). Higher concentrations were found in the lung (2.6 ng/g), liver (2.6 ng/g), and kidney (3.8 ng/g). Gastric content yielded a MDMB-CHMICA concentration of 2.4 ng/g (1.1 µg absolute). Screening for MDMB-CHMICA metabolites resulted in the detection of mainly monohydroxylated metabolites in the blood, kidney, and liver specimens. Results indicated that monohydroxylated metabolites of MDMB-CHMICA are appropriate target analytes for detecting MDMB-CHMICA intake.

Metabolites of Heroin in Several Different Post-mortem Matrices.

In some forensic autopsies blood is not available, and other matrices are sampled for toxicological analysis. The aims of the present study were to examine whether heroin metabolites can be detected in different post-mortem matrices, and investigate whether analyses in other matrices can give useful information about concentrations in peripheral blood. Effects of ethanol on the metabolism and distribution of heroin metabolites were also investigated. We included 45 forensic autopsies where morphine was detected in peripheral blood, concomitantly with 6-acetylmorphine (6-AM) detected in any matrix. Samples were collected from peripheral blood, cardiac blood, pericardial fluid, psoas muscle, lateral vastus muscle, vitreous humor and urine. Opioid analysis included 6-AM, morphine, codeine, and morphine glucuronides. The 6-AM was most often detected in urine (n = 39) and vitreous humor (n = 38). The median morphine concentration ratio relative to peripheral blood was 1.3 (range 0-3.6) for cardiac blood, 1.4 (range 0.07-5.3) for pericardial fluid, 1.2 (range 0-19.2) for psoas muscle, 1.1 (range 0-1.7) for lateral vastus muscle and 0.4 (range 0.2-3.2) for vitreous humor. The number of 6-AM positive cases was significantly higher (P = 0.03) in the ethanol positive group (n = 6; 86%) compared to the ethanol negative group (n = 14; 37%) in peripheral blood. The distribution of heroin metabolites to the different matrices was not significantly different between the ethanol positive and the ethanol negative group. This study shows that toxicological analyses of several matrices could be useful in heroin-related deaths. Urine and vitreous humor are superior for detection of 6-AM, while concentrations of morphine could be assessed from peripheral or cardiac blood, pericardial fluid, psoas muscle and lateral vastus muscle.

Comparison of myosin heavy chain content determined by label-free quantification between porcine longissimus thoracis, psoas major and semimembranosus muscles.

The present study was conducted to quantify MHCs in porcine longissimus thoracis (LT), psoas major (PM) and semimembranosus (SM) muscles through the label-free quantification (LFQ). To estimate the accuracy of LFQ, quantitative RT-PCR (qRT-PCR), immunohistochemistry (IHC), and Western-blotting (WB) were performed. MHCs 2x, 2a, 2b and slow were identified by LC-MS/MS analysis and 279 ion spectra were selected for LFQ analysis. The content of MHC 2b was higher in LT and in SM than in PM (p<0.05), while the content of MHC slow was highest in PM among the muscles (p<0.01) regardless of LFQ types. Positive correlation coefficients of MHC 2b and MHC slow between LFQ and IHC (relative area composition) and qRT-PCR results partially supported the LFQ results. Though low-abundant peptides should be considered to estimate MHC contents via the spectral count method, LFQ enables the determination of MHC contents at protein level regardless of LFQ types.

High-pressure effects on the molecular aggregation and physicochemical properties of myosin in relation to heat gelation.

Myosin was extracted from the M. psoas muscle of rabbits, and dissolved in 0.6M KCl buffer (pH6.5). Effects of high-pressure (HP, 100 to 300MPa, 9min, 25°C) treatment on myosin solubility, molecular traits (molecular weight and morphology), flow behavior and strength of heat-induced myosin gels were studied and compared with the untreated controls. Myosin subjected to 200MPa HP treatment had lower solubility than samples treated at other pressures (P<0.05). Molecular dimerization and morphological swelling of myosin was observed using gel-permeation chromatography and atomic-force microscopy. Additionally, the shear-thinning behavior of myosin solutions (10mg/mL) was improved by HP treatment (≥200MPa), and a positive trend in gel-strength enhancement was inferred. It is postulated that significant morphological changes in myosin accounted for changes in its functional properties, by the influence of HP treatment on protein-protein and/or protein-water interactions. There is a relationship between molecular morphology and the coalescing behavior of myosin, since significant changes of both attributes were observed at pressures ≥200MPa.

Assessment of STR Typing Success Rate in Soft Tissues from Putrefied Bodies Based on a Quantitative Grading System for Putrefaction.

To date, there is no systematic investigation of the association of short tandem repeat (STR) typing success rate in soft tissues with different signs of putrefaction. Herein, putrefaction was rated using a newly developed 19-parameter system in soft tissues from a collective of 68 decaying bodies, and DNA yield was determined in 408 samples. DNA integrity was rated using a self-devised pentaplex PCR generating an "integrity score" (Si). STR typing success rate was then assessed for selected cases. DNA yield and Si differed significantly between tissues with kidney on average exhibiting the highest Si values. Statistical analysis revealed that nine parameters were significantly and positively correlated with Si . The observed values for each of these nine parameters were summed up to generate a putrefaction score (Sp) for each sample. Our results show that STR typing success rate can be predicted based on Sp before expensive multiplex STR profiling is performed.

How reliable are parenchymal tissues for the evaluation of carbon monoxide poisoning? A pilot study.

Dealing with burnt bodies, the forensic pathologist must first of all answer the question whether the victim was alive at the moment of the fire. This study aims at clarifying whether some human solid tissues may be reliably used for the forensic diagnosis of Co poisoning on burnt bodies providing no collectable blood during the autopsy. From 34 selected cases, both cardiac blood and parenchymal samples were collected to perform CO-oxymeter, spectrophotometry, and gas chromatography tests: blood CO estimations (blood COHb% and blood[CO]) and parenchymal[CO] values have been compared with special focus on R values. The solid tissues having the best correlations with blood CO amount turned out to be the lung (R 0.84), the liver (R 0.83), the kidney (R 0.79), and the spleen (R 0.92).

[Changes in titin and myosin heavy chain isoform composition in skeletal muscles of Mongolian gerbil (Meriones unguiculatus) after 12-day spaceflight].

Changes of titin and myosin heavy chain isoform composition in skeletal muscles (m. soleus, m. gastrocnemius, m. tibialis anterior, m. psoas major) in Mongolian Gerbil (Meriones unguiculatus ) were investigated after 12-day spaceflight on board of Russian space vehicle "Foton-M3". In m. psoas and m. soleus in the gerbils from "Flight" group the expected increase in the content of fast myosin heavy chain isoforms (IIxd and IIa, respectively) were observed. No significant differences were found in the content of IIxd and IIa isoforms of myosin heavy chain in m. tibialis anterior in the gerbils from control group as compared to that in "Flight" group. An unexpected increase in the content of slow myosin heavy chain I isoform and a decrease in the content of fast IIx/d isoform in m. gastrocnemius of the gerbils from "Flight" group were observed. In skeletal muscles of the gerbils from "Flight" group the relative content of titin N2A-isoform was reduced (by 1,2-1,7 times), although the content of its NT-isoform, which was revealed in striated muscles of mammals in our experiments earlier, remained the same. When the content of titin N2A-isoform was decreased, no predictable abnormalities in sarcomeric structure and contractile ability of skeletal muscles in the gerbils from "Flight" group were found. An assumption on the leading role of titin NT-isoform in maintenance of structural and functional properties of striated muscles of mammals was made.

Profiles and concentrations of heterocyclic aromatic amines formed in beef during various heat treatments depend on the time of ripening and muscle type.

Heterocyclic Aromatic Amine (HAA) profiles and concentrations depended on several factors. The largest changes in the HAA profile were observed in meat ripened (chill stored) for 5-10 days. Amines whos concentration varied most prominently included: Phe-P 1, harmane, AαC, IQ, IQx, PhIP, MeAαC, and MeIQx. HAA concentrations were strongly correlated with concentrations of the above compounds. Time of storage significantly affected the HAA profile and concentration. The profile changed dynamically for storage times up to 10 days. For longer times the profile stabilized, only the HAA content increased. A novel, highly precise and accurate HAA analytical method was developed for this study. Results may help to optimize meat processing technology from the point of view of reducing concentration of HAA formed during heat treatment, including the most carcinogenic; IQ, IQx, MeIQx and PhIP amines.

A preliminary investigation on the distribution of cannabinoids in man.

An LC/MS/MS procedure to determine THC along with its major metabolites 11-OH-THC, THC-COOH and its glucuronide as well as the cannabinoids CBD and CBN was applied to 5 post mortem cases to study their distribution into some less commonly studied matrices. Analytes were determined in fluids and tissue homogenates following protein precipitation and liquid-liquid extraction. Gall bladder fluid exhibited maximum concentrations of all analytes except THC, which was detectable in high concentrations in muscle tissue along with CBD. THC was also present in lung specimens, whereas its concentration in liver samples was low or not detectable at all. Liver und kidney specimens contained appreciable amounts of THC-COOglu. Findings from bile support extensive enterohepatic recirculation of the glucuronide. Muscle tissue seems an interesting specimen to detect multiple cannabis use, and brain may serve as an alternative specimen for blood; nevertheless, the present findings should be substantiated by further investigations.

Tropomyosin is in a reduced state in rabbit psoas muscle.

Tropomyosin (Tm) purified from skeletal and cardiac muscle often contains disulfide bonds due to oxidation of cysteine groups that are in close proximity in the coiled-coil structure. Are these disulfide crosslinks present in the muscle or produced by oxidation during preparation? To answer this question we reacted one part of freshly dissected rabbit psoas muscle fibers, which was permeabilized with Triton X-100, with N-ethyl maleimide (NEM) to block cysteine groups and another part with 5,5'-dithiobis(2-nitro benzoate) (DTNB) to facilitate disulfide bond formation by interchain sulfhydryl-disulfide exchange. We found, by high resolution gradient SDS polyacrylamide gels, that the NEM-treated muscle was only composed of uncrosslinked Tm and the DTNB treated muscle was composed of disulfide-crosslinked Tm. This work indicates that Tm exists in a reduced state in rabbit psoas muscle.

Stabilization of helical order in the thick filaments by blebbistatin: further evidence of coexisting multiple conformations of myosin.

The degree of helical order of the thick filament of mammalian skeletal muscle is highly dependent on temperature and the nature of the ligand. Previously, we showed that there was a close correlation between the conformation of the myosin heads on the surface of the thick filaments and the extent of their helical order. Helical order required the heads to be in the closed conformation. In addition, we showed that, with the same ligand bound at the active site, three conformations of myosin coexisted in equilibrium. Hitherto, however, there was no detectable helical order as measured by x-ray diffraction under the temperatures studied for myosin with MgADP and the nucleotide-free myosin, raising the possibility that the concept of multiple conformations has limited validity. In this study, blebbistatin was used to stabilize the closed conformation of myosin. The degree of helical order is substantially improved with MgATP at low temperature or with MgADP or in the absence of nucleotide. The thermodynamic parameters of the disorder<-->order transition and the characteristics of the ordered array were not significantly altered by binding blebbistatin. The simplest explanation is that the binding of blebbistatin increases the proportion of myosin in the closed conformation from being negligible to substantial. These results provide further evidence for the coexistence of multiple conformations of myosin under a wide range of conditions and for the closed conformation being directly coupled to helical order.

Characterization of water of hydration fractions in rabbit skeletal muscle with age and time of post-mortem by centrifugal dehydration force and rehydration methods.

Centrifugal dehydration force (CDF) and rehydration isotherm (RHI) methods were used to measure and characterize hydration fractions in rabbit psoas skeletal muscle. The CDF method assessed fluid flow rate from rabbit muscle and hydration capacity of the fractions. Bulk and multiple non-bulk water fractions were identified. The non-bulk water was divisible into the following fractions: two outer non-bulk fractions, a main chain proteins backbone or double water bridge fraction, and a single water bridge fraction. The total non-bulk water amounts to about 85% of the total water in the muscle. The sizes of the water fractions (in g water/g dry mass) agree with a recently proposed molecular stoichiometric hydration model (SHM) applicable to all proteins in and out of cells (Fullerton GD, Cameron IL. Water compartments in cells. Methods Enzymol, 2007; Cameron IL, Fullerton GD. Interfacial water compartments on tendon/collagen and in cells. In: Pollack GH, Chin WC, editors. Phase transitions in cells. Dordrecht, The Netherlands: Springer, 2008). Age of the rabbit significantly slowed the flow rate of the outer non-bulk water fraction by about 50%. Also, muscle of the older rabbit (26 weeks vs. 12 weeks old) had less bulk water and less outer non-bulk water but the same amount of main chain backbone water compared to muscle of the younger rabbit. Increase in time post-mortem from 30min to 4h resulted in rigor mortis and a significantly slower flow rate of water from the outer non-bulk water fraction, which is attributed to muscle contraction, increased packing of contractile elements and increased obstructions to flow of fluid from the muscle fibers.

Fluorescence lifetime imaging to detect actomyosin states in mammalian muscle sarcomeres.

We investigated the use of fluorescence lifetime imaging microscopy (FLIM) of a fluorescently labeled ATP analog (3'-O-{N-[3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP) to probe in permeabilized muscle fibers the changes in the environment of the nucleotide binding pocket caused by interaction with actin. Spatial averaging of FLIM data of muscle sarcomeres reduces photon noise, permitting detailed analysis of the fluorescence decay profiles. FLIM reveals that the lifetime of the nucleotide, in its ADP form because of the low concentration of nucleotide present, changes depending on whether the nucleotide is free in solution or bound to myosin, and on whether the myosin is bound to actin in an actomyosin complex. Characterization of the fluorescence decays by a multiexponential function allowed us to resolve the lifetimes and amplitudes of each of these populations, namely, the fluorophore bound to myosin, bound to actin, in an actomyosin complex, and free in the filament lattice. This novel application of FLIM to muscle fibers shows that with spatial averaging, detailed information about the nature of nucleotide complexes can be derived.