Ultrastructure Of Myofibrils Research Articles

EFFECTS OF CHRONIC MATERNAL ALCOHOLIZATION ON THE CARDIOMYOCYTE CONTRACTILITY IN RAT OFFSPRINGS

The effects of intrauterine alcohol exposure on heart development, specifically the contractile apparatus and myofibril distribution within cardiomyocytes, remain controversial. Aim: This study investigates the postnatal dynamics of myofibril ultrastructure in rat ventricular cardiomyocytes following chronic maternal alcohol exposure. Methods: We analyzed cardiac tissue from offspring of chronically alcoholized rats at various postnatal stages using transmission electron microscopy. Quantitative parameters of cardiomyocyte myofibrils in different ventricular myocardial zones were assessed. Results: Chronic maternal alcohol exposure appeared to inhibit sarcomerogenesis and reduced overall myofibril content in the hearts of newborn offspring. Furthermore, we observed increased myofibril surface area and disrupted spatial orientation in subendocardial left ventricular cardiomyocytes and intramural zones of both ventricles. Even in mature offspring, the myocardial contractile apparatus remained irreversibly damaged, exhibiting reduced myofibril density, increased surface area, and disrupted orientation.

Ultrastructural Assessment and Proteomic Analysis in Myofibrillogenesis in the Heart Primordium After Heartbeat Initiation in Rats.

Myofibrillogenesis is an essential process for cardiogenesis and is closely related to excitation-contraction coupling and the maintenance of heartbeat. It remains unclear whether the formation of myofibrils and sarcomeres is associated with heartbeat initiation in the early embryonic heart development. Here, we investigated the association between the ultrastructure of myofibrils assessed by transmission electron microscopy and their proteomic profiling assessed by data-independent acquisition mass spectrometry (DIA-MS) in the rat heart primordia before and after heartbeat initiation at embryonic day 10.0, when heartbeat begins in rats, and in the primitive heart tube at embryonic day 11.0. Bundles of myofilaments were scattered in a few cells of the heart primordium after heartbeat initiation, whereas there were no typical sarcomeres in the heart primordia both before and after heartbeat initiation. Sarcomeres with Z-lines were identified in cells of the primitive heart tube, though myofilaments were not aligned. DIA-MS proteome analysis revealed that only 43 proteins were significantly upregulated by more than 2.0 fold among a total of 7,762 detected proteins in the heart primordium after heartbeat initiation compared with that before heartbeat initiation. Indeed, of those upregulated proteins, 12 (27.9%) were constituent proteins of myofibrils and 10 (23.3%) were proteins that were accessories and regulators for myofibrillogenesis, suggesting that upregulated proteins that are associated with heartbeat initiation were enriched in myofibrillogenesis. Collectively, our results suggest that the establishment of heartbeat is induced by development of bundles of myofilaments with upregulated proteins associated with myofibrillogensis, whereas sarcomeres are not required for the initial heartbeat.

The effect of sodium chloride levels on the taste and texture of dry-cured ham

In order to investigate the effect of NaCl levels (1.5%, 3.5%, 5.5% and 13%) on the taste and texture of dry-cured ham, TPA, myofibril ultrastructure, proteolysis index and free amino acids were evaluated. The proteolysis index (30.76% ± 2.05–11.88% ± 1.90), cohesiveness (0.74 ± 0.04–0.60 ± 0.01), springiness (0.83 ± 0.03–0.71 ± 0.04), and the total free amino acids (9885.3 mg/100 g ± 677.98–3505.15 mg/100 g ± 378.20) decreased with the increase of salt addition, while the hardness (8701.99 g ± 513.37–12057.53 g ± 783.89) and adhesiveness (− 15.73 ± 2.53 to − 32.01 ± 4.95) increased with the increase of salt addition. The myofibril ultrastructure indicated that hams with higher proteolysis index (> 20%) showed more obvious degradation of M-line and Z-disk, and more disordered fiber structure, which was closely related to the degradation of myofibrillar proteins (actin, desmin and troponin-T). Among four groups, the dry-cured ham with 5.5% salt had the highest levels of moisture (45.73% ± 1.13) and umami amino acids (1422.77 mg/100 g ± 119.87), as well as the lowest chewiness (3346.89 ± 121.19). The ham with 5.5% salt also had higher sensory scores of texture (4.83 ± 0.1), taste (5.50 ± 0.2) and overall acceptance (5.51 ± 0.09) as compared to the ham added with other salt levels. These results indicated that 5.5% NaCl treatment showed positive effects on the texture and taste of dry-cured ham, which meets the consumer's demand for dry-cured meat products.

Is a taurine supplement necessary in fishmeal-based feeds for juvenile European sea bass (Dicentrarchus labrax)?

The effect of taurine supplementation of fishmeal-based feeds for juvenile European sea bass (Dicentrarchus labrax) was investigated. Fish (initial weight ca. 5 g) were reared for 90 days in 12 concrete tanks (1 m3), with 50 fish in each tank. Four feeds (44% crude protein (CP) and 17% lipids (L)) containing 0 (control (CTRL)), 0.4 (T4), 0.7 (T7) and 1.0 (T10) % taurine were prepared and were fed to apparent visual satiation to triplicate groups of fish. The effects on fish growth, feed utilization, proximate chemical composition, haematology, immune biomarkers and muscle morphometry were examined. An increase in the dietary taurine incorporation resulted in progressive increases in the growth metrics, with the highest values being observed for fish receiving the T10 feed (weight gain 22 vs 18 g/fish in CTRL; protein productive value 31 vs 28% in CTRL). Taurine addition at all levels significantly increased the fish protein percentage, but the percentage lipid was reduced significantly in T4 fish only (7.8% compared with 9.4% in CTRL fish). Taurine supplementation resulted in elevated blood lymphocyte and monocyte counts and increased serum phagocytic and lysozyme activities. Taurine addition at 1% (T10 feed), the suggested level to boost growth, altered the dorsal muscle cellularity and myofibril ultrastructure, suggesting enhanced muscle function and firmness in comparison with that of the fish given the CTRL feed.

The Effect of µ/m-Calpain on Protein Degradation of Chicken Breast Meat.

This study was designed to determine the effects ofµ/m-calpain on the degradation of cytoskeletal proteins in pectoralis major. Four chickens were slaughtered and the breasts were removed and stored for 12hr at 4 °C. Each sample was divided into three groups and respectively immersed in control reagent, calpain inhibitor, and caspase inhibitor at 4°C. The samples were used to evaluate troponin-T and desmin degradation, calpain activity, and myofibril ultrastructure at 12hr, day 1, day 3, and day 7. Casein zymography revealed thatµ-calpain could not be detected in all samples after 12hr postmortem. The calpain inhibitor inhibitedµ/m-calpain activity and reduced troponin-T and desmin degradation during 7day postmortem. The caspase inhibitor inhibitedµ/m-calpain activity and, troponin-T and desmin degradation before day 3 postmortem. The findings suggest that,µ/m-calpain had an effect on cytoskeletal protein degradation after 12hr postmortem.

A net-shaped multicellular formation facilitates the maturation of hPSC-derived cardiomyocytes through mechanical and electrophysiological stimuli.

The use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is limited in drug discovery and cardiac disease mechanism studies due to cell immaturity. Although many approaches have been reported to improve the maturation of hiPSC-CMs, the elucidation of the process of maturation is crucial. We applied a small-molecule-based differentiation method to generate cardiomyocytes (CMs) with multiple aggregation forms. The motion analysis revealed significant physical differences in the differently shaped CMs, and the net-shaped CMs had larger motion amplitudes and faster velocities than the sheet-shaped CMs. The net-shaped CMs displayed accelerated maturation at the transcriptional level and were more similar to CMs with a prolonged culture time (30 days) than to sheet-d15. Ion channel genes and gap junction proteins were up-regulated in net-shaped CMs, indicating that robust contraction was coupled with enhanced ion channel and connexin expression. The net-shaped CMs also displayed improved myofibril ultrastructure under transmission electron microscopy. In conclusion, different multicellular hPSC-CM structures, such as the net-shaped pattern, are formed using the conditioned induction method, providing a useful tool to improve cardiac maturation.

Effect of fast pH decline during the early postmortem period on calpain activity and cytoskeletal protein degradation of broiler M. pectoralis major

The objective of this study was to determine the effects of fast pH decline during the early postmortem period on calpain activity and the degradation of cytoskeletal proteins in broilers. Eighty broilers were randomly categorized into two groups: physical restraint (PR) and free struggle (FS). M. pectoralis major (PM) was used for determination of calpain activity, shear value, ultrastructure of myofibrils, and the degradation of desmin, titin, nebulin, and troponin-T. The pH (6.05) of FS group is significantly low than PR group (6.38) at 0.3 h postmortem. Fast pH decline during the early postmortem period led to a decrease of μ/m-calpain activities at 0.3 and 3 h postmortem (P < 0.05), but did not affect the ultimate μ/m-calpain activity. An initial fast decrease in pH increased the degradation of desmin, titin, nebulin, and increased the 30 kDa degradation fragments of troponin-T. Therefore, the fast pH decline during the early postmortem period decreased the μ/m-calpain activity and increased the degradation of cytoskeletal proteins in broiler muscle. It is possible that the fast pH decline experienced an earlier activation of calpains that resulted in earlier protein degradation and ultimately lower shear force.

Isolation and Mechanical Measurements of Myofibrils from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

SummaryTension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G). These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations.

Changes in the Anatomic and Microscopic Structure and the Expression of HIF-1α and VEGF of the Yak Heart with Aging and Hypoxia.

The study aimed to identify the changes of anatomic and microscopic structure and the expression and localization of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in the myocardium and coronary artery of the yak heart adapted to chronic hypoxia with aging. Thirty-two yaks (1 day, 6 months, 1 year, 2 years, and 5 year old) were included, and immunoelectronmicroscopy, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used. Right ventricular hypertrophy was not present in yaks with aging. There was no intima thickening phenomenon in the coronary artery. The ultrastructure of myofibrils, mitochondria, and collagen fibers and the diameter and quantity of collagen changed significantly with aging. The enzymatic activity of complexes I, II, and V increased with age. Immunogold labeling showed the localization of HIF-1α protein in the cytoplasm and nuclei of endothelial cells and cytoplasm of cardiac muscle cells, and VEGF protein in the nuclei and perinuclei areas of smooth muscle cells of coronary artery, and in the cytoplasm and nuclei of endothelial cells. ELISA results showed that HIF-1α secretion significantly increased in the myocardium and coronary artery from an age of 1 day to 2 years of yaks and decreased in old yaks. However, VEGF protein always increased with aging. The findings of this study suggest that 6 months is a key age of yak before which there are some adaptive changes to deal with low-oxygen environment, and there is a maturation of the yak heart from the age of 6 months to 2 years.

High pressure processing of dry-cured ham: Ultrastructural and molecular changes affecting sodium and water dynamics

Abstract High pressure processing (HPP) has become a popular method to reduce bacterial load on dry-cured ham. However, HPP processed hams tend to present higher saltiness than non-processed ones. In this study was evaluated the effect of processing biceps femoris samples of 9 dry-cured hams aged for 15 months at a pressure of 0.1, 300, 600 and 900 MPa. 23Na-Nuclear Magnetic Resonance (NMR) relaxation values were used to determine the mobility of sodium. Ultrastructure changes at 600 MPa were evaluated by transmission electron microscopy (TEM) analysis. Double quantum NMR experiments demonstrated that pressure promoted tighter binding of bound sodium to proteins. However, a small proportion of sodium was liberated. The modification of myofibril ultrastructure at 600 MPa observed by TEM and the denaturation observed between 300 and 600 MPa facilitate the liberation of a proportion of sodium. That together with the decrease of water holding capacity between unpressurised and 900 MPa samples makes sodium more noticeable for saltiness perception, which could be of interest for sodium reduced dry-cured meat products. Industrial relevance High pressure processing of cured ham is a non-thermal pasteurization actually used in Spain. Even if the technology answer the microbiological goals to control microorganisms, an increase of saltiness perception have been detected. The understanding of this phenomenon from an ultrastructural and molecular perspective could help to present high pressure processing treatments as an alternative solution for salt reduction in dry-cured meat products in order to obtain healthier food products and good consumer acceptance.

Absence of SOCS3 in the Cardiomyocyte Increases Mortality in a gp130-Dependent Manner Accompanied by Contractile Dysfunction and Ventricular Arrhythmias

Suppressor of cytokine signaling-3 (SOCS3) is a key negative-feedback regulator of the gp130 receptor that provides crucial signaling for cardiac hypertrophy and survival; however, an in vivo role of SOCS3 regulation on cardiac gp130 signaling remains obscure. We generated cardiac-specific SOCS3 knockout (SOCS3 cKO) mice. These mice showed increased activation of gp130 downstream signaling targets (STAT3, ERK1/2, AKT, and p38) from 15 weeks of age and developed cardiac dysfunction from approximately 25 weeks of age with signs of heart failure. Surprisingly, SOCS3 cKO failing hearts had minimal histological abnormalities with intact myofibril ultrastructure. In addition, Ca(2+) transients were significantly increased in SOCS3 cKO failing hearts compared with wild-type hearts. We also found that Ser23/24 residues of troponin I were hypophosphorylated in SOCS3 cKO hearts before the manifestation of cardiac dysfunction. These data suggested the presence of abnormalities in myofilament Ca(2+) sensitivity in SOCS3 cKO mice. In addition to the contractile dysfunction, we found various ventricular arrhythmias in SOCS3 cKO nonfailing hearts accompanied by a sarcoplasmic reticulum Ca(2+) overload. To determine the contribution of gp130 signaling to the cardiac phenotype that occurs with SOCS3 deficiency, we generated cardiac-specific gp130 and SOCS3 double KO mice. Double KO mice lived significantly longer and had different histological abnormalities when compared with SOCS3 cKO mice, thus demonstrating the importance of gp130 signaling in the SOCS3 cKO cardiac phenotype. Our results demonstrate an important role of SOCS3 regulation on cardiac gp130 signaling in the pathogenesis of contractile dysfunction and ventricular arrhythmias.

Preliminary study on the effect of caspase-6 and calpain inhibitors on postmortem proteolysis of myofibrillar proteins in chicken breast muscle

The objective was to determine the effect of three different protease inhibitors, caspase-6 specific inhibitor VEID-CHO (N-Acetyl-Val-Glu-Ile-Asp-al), calpain inhibitor leupeptin or calpain inhibitor EGTA on protein degradation, ultrastructure of myofibrils and calpain activity during postmortem (PM) aging of chicken muscle. Results showed that proteolysis of nebulin, troponin-T and desmin during 14-days postmortem storage were inhibited significantly by leupeptin. Inhibitive effects of VEID-CHO and EGTA on these protein degradations were significant only during 1-day postmortem storage. The activities of calpains were inhibited noticeably by leupeptin and EGTA, but not by VEID-CHO. Samples treated with VEID-CHO, leupeptin and EGTA retarded structural disruption of chicken muscle fibers. These results demonstrate that calpain is a major contributor to PM tenderization; while caspase-6 plays, if any, a minimal role in the conversion of chicken muscle to meat.

Interactive effects of corticosteroid and mechanical ventilation on diaphragm muscle function

Information on the interactive effects of methylprednisolone, controlled mechanical ventilation (CMV), and assisted mechanical ventilation (AMV) on diaphragm function is sparse. Sedated rabbits received 2 days of CMV, AMV, and spontaneous breathing (SB), with either methylprednisolone (MP; 60 mg/kg/day intravenously) or saline. There was also a control group. In vitro diaphragm force, myofibril ultrastructure, αII-spectrin proteins, insulin-like growth factor-1 (IGF-1), and muscle atrophy F-box (MAF-box) mRNA were measured. Maximal tetanic tension (P(o)) decreased significantly with CMV. Combined MP plus CMV did not decrease P(o) further. With AMV, P(o) was similar to SB and controls. Combined MP plus AMV or MP plus SB decreased P(o) substantially. Combined MP plus CMV, MP plus AMV, or MP plus SB induced myofibrillar disruption that correlated with the reduced P(o). αII-spectrin increased, IGF-1 decreased, and MAF-box mRNA increased in both the CMV group and MP plus CMV group. Short-term, high-dose MP had no additive effects on CMV-induced diaphragm dysfunction. Combined MP plus AMV impaired diaphragm function, but AMV alone did not. We found that acute, high-dose MP produces diaphragm dysfunction depending on the mode of mechanical ventilation.

Close cardiac surveillance of patients treated with anthracyclines, also necessary after chemotherapy?

In women, breast cancer is the second most common form of cancer and the leading cause of death caused by malignancy. Anthracyclines are well known chemotherapeutics for treatment of these tumors, other solid tumors and hematologic malignancies. As adjuvant treatment in breast cancer it improves survival and reduces recurrence [1]. Unfortunately, their clinical use is currently limited by the development of a progressive dose-dependent cardiomyopathy that irreversibly evolves toward congestive heart failure, which is usually refractory to conventional therapy [2]. Studies have reported that 10–26% of patients administered cumulative anthracycline doses above those, develop congestive heart failure, and that more than 50% of patients administered these doses will experience measurable functional impairment months to years after the end of therapy [3]. The pathophysiology of this cardiomyopathy is incompletely understood. Anthracyclines decrease b-andrenoceptor density in the cell membrane, increase the permeability of myocytes and alter the ultrastructure of myofibrils and actin networks [4]. Risk factors for anthracycline-induced cardiotoxicity include high cumulative anthracycline doses, high anthracycline dose intensity, and radiotherapy. Radiotherapy in patients with cancer treated with anthracyclines can exacerbate cardiac tissue damage. Cardiovascular complications of treatment may be observed during administration of the drugs, days to months following administration, or years to decades following exposure. Several strategies have been developed to decrease the risk of cardiotoxicity while maintaining efficacy. These strategies include altered schedules of drug administration and adjunctive treatment with dexrazoxane. Unfortunately, none of these approaches have been successful, and patients remain at risk for the development of anthracycline cardiomyopathy, particularly with higher cumulative doses. Therefore careful serial monitoring is required for optimal medical management. Cardiomyopathy disease progression can be delayed in adults by using carvedilol or angiotensin-converting enzyme inhibitors [5, 6]. The permanent risk of cardiomyopathy by anthracyclines has made monitoring for the earliest evidence of cardiotoxicity necessary. In clinical practice monitoring is mostly performed using echocardiography or radionuclide angiography. The study of Appel et al. in the current issue of the International Journal of Cardiac Imaging shows that development of anthracycline toxicity is difficult to predict [7]. Although all patient in this study had a normal left ventricular function before receiving chemotherapy, 33% of the patients developed heart failure within a time period Editorial comment on the article of Appel et al. (doi:10.1007/ s10554-009-9518-2).

Melatonin is as Effective as Testosterone in the Prevention of Soleus Muscle Atrophy Induced by Castration in Rats

The purpose of this experiment was to compare the weight, insulin-like growth factor-I (IGF-I) expression, and ultrastructure of the soleus muscle in growing castrated rats treated with testosterone or melatonin. In this study, adult male Wistar albino rats were used. The groups were arranged as sham, castrated, and testosterone- or melatonin-injected groups after castration. The soleus muscle samples were fixed in Bouin's solution for immunohistochemistry, and in 2.5% gluteraldehyde in 0.1 M phosphate buffer (pH 7.4). Whereas castration reduced the soleus weight and fiber diameter, testosterone and melatonin administration increased them. IGF-I immunostaining observed in the satellite cells and periphery of the myofibers was least intense in the castrated group. Strong staining of IGF-I was observed in the testosterone- and melatonin-administered groups. The ultrastructure of the soleus muscle in castrated animals showed the important ultrastructural modifications related to degeneration. In these groups, degenerative mitochondria, glycogen clusters under the sarcolemma, irregular Z lines, and loss of lamina externa were observed. The ultrastructure of myofibrils in the testosterone- and melatonin-injected groups was similar to that in sham groups in view of structure. In conclusion, we suggest that melatonin is as effective as testosterone in the prevention of atrophy induced by castration through the IGF-I axis.

Postmortem Calcium Chloride Injection Alters Ultrastructure and Improves Tenderness of Mature Chinese Yellow Cattle Longissimus Muscle

ABSTRACT: This study was conducted to test the hypothesis that postmortem calcium injection could activate the calpain system in mature Chinese Yellow Cattle muscle, thereby promoting meat tenderization through disruption of the myofibril structure during aging. A 10% (w/w) injection of CaCl2 (300 mM) solution lowered the Warner-Bratzler shear values of longissimus muscle by more than 30% (P < 0.05), even with only 24 h postmortem storage when compared with noninjected or water-injected controls. The accelerated meat tenderization by the Ca2+ treatment paralleled the changes in myofibril fragmentation index and fracture of the myofibril ultrastructure throughout the sarcomere but most notably around the I-bands and the Z-disks. Injection of ZnCl2 (50 mM) largely inhibited these proteolytic changes. The colorimetric L* and a* values were not affected by CaCl2 nor by ZnCl2 injection. The results suggest that postmortem CaCl2 injection can be used to help resolve the toughness problem of mature Chinese Yellow Cattle meat and shorten the aging time required to achieve adequate tenderness.

Decline in titin content in rat skeletal muscle after denervation

Titin, an elastic and giant myofibrillar protein, is responsible for generating passive tension and maintaining sarcomere structure in striated muscles. Several studies have reported attenuation of passive tension and disorganization of sarcomere in atrophic muscles, but the changes of titin have not been investigated after denervation. For this purpose, we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunofluorescent staining to examine titin in innervated and denervated tibialis anterior (TA) muscles of the rat. With increasing denervation time, we found a greater loss of titin than myosin heavy chain (MHC) and actin contents in atrophic TA muscle. The ratios of titin/MHC and titin/actin gradually decreased following denervation. In contrast, ratios of MHC/actin in the denervated groups showed no significant differences with the controls even at 56 days postdenervation. The ultrastructure of myofibrils also showed disturbed arrangements of myofilaments and a disorganized contractile apparatus in denervated muscle. Immunofluorescent staining displayed translocation of the titin epitope from the Z-line to the I-band, suggesting that the apparent cleavage of titin occurred near the Z-line region during the atrophying process. Our study provides evidence that titin is more sensitive to degradation than MHC and actin after denervation. Moreover, the titin decline results in the loss of titin-based sarcomeric integrity in atrophic muscle.

Effects by doxorubicin on the myocardium are mediated by oxygen free radicals

We hypothesized that doxorubicin (DOX) induces cardiotoxicity of myocardium via oxygen radicals. The present study is aimed at examining the membrane alterations by oxygen radicals generated by DOX in adult rats and cultured neonatal myocytes. Our results showed that DOX 1) decreased β-adrenoceptor (BAR) density in the cell membrane, 2) increased the membrane permeability of cultured neonatal rat myocytes and 3) altered the ultrastructure of myofibrils and subplasmalemmal actin networks. These effects were reproducible by exogenous hydrogen peroxide. The antioxidant melatonin (MLT) inhibited enzyme leakage and peroxidation in a concentration-dependent manner. It is concluded that DOX induces cardiotoxicity through lipid peroxidation and melatonin is an effective antioxidant against the reactive oxygen intermediates generated by DOX.

Protein Extraction From Chicken Myofibrils Irrigated with Various Polyphosphate and NaCl Solutions

ABSTRACT:Physical changes in chicken gastrocnemius myofibrils incubated in 0.1 to 1.0 M NaCl solutions with or without 10 mM ortho‐(P), pyro‐(PP), tripoly‐(TPP) or hexameta‐ (HMP) phosphate at pH 6.0 were examined by phase‐contrast microscopy, electrophoresis, and solubility. PP and TPP performed similarly in promoting protein extraction, P had no apparent effect, and HMP exhibited an intermediate effect. PP, TPP, and HMP treatments markedly improved protein solubility in 0.3 and 0.4 M NaCl through the release of myosin, but the phosphate effect diminished in ≥ 0.6 M NaCl. Overall, phosphates influenced the ultrastructure of myofibrils and extraction of their constituents in the order: PP ∼ TPP &gt; HMP &gt; P ∼ nonphosphate control.

In vitro study of human skeletal muscle strips: Effect of nonesterified fatty acid supply on glucose storage

To examine the effect of increased nonesterified fatty acid concentration on glucose storage in human muscle, an in vitro method for study of glycogen synthesis in this tissue has been established. Muscle strips obtained from rectus abdominus during elective abdominal surgery were clamped at resting length, and adenosine triphosphate/total adenosine nucleotide ratios remained constant for 3 hours ex vivo. Leakage of enzyme markers of muscle damage was minimal, and electron microscopy showed preserved myofibril ultrastructure. Insulin stimulation brought about a dose-dependent increase in rates of glycogen synthesis with a half-maximal effect at 9 × 10 −10 mol/L insulin. In 15 consecutive studies, basal rates of glycogen synthesis were 4.1 ± 0.5, 3.2 ± 0.7, and 3.0 ± 0.3 nmol glucose/25 mg/h in the absence of palmitate, with 1.4 mmol/L and 2.8 mmol/L palmitate, respectively. Insulin-stimulated rates of glycogen synthesis were 8.6 ± 1.2, 6.0 ± 1.8, and 5.8 ± 0.8 nmol glucose/25 mg/h. Thus, increasing fatty acid concentrations decreased rates of glycogen synthesis both basally and with insulin stimulation. The insulin signal itself was not affected as the percentage stimulation over basal rates remained approximately constant in the presence or absence of fatty acid (2.1-, 1.9- and 1.9-fold, respectively). Insulin sensitivity in vivo is usually expressed as absolute rates of glucose uptake during euglycemic hyperinsulinemia, and if plasma fatty acid elevation were to be studied in vivo an erroneous conclusion may be reached of resistance to hormone action per se. These observations show that increased fatty acid availability brings about decreased rates of glucose storage in human muscle, not solely decreased rates of glucose oxidation as previously hypothesized. The precise mechanism underlying this metabolic interaction requires further study. Therapeutic manipulation of fatty acid metabolism in non-insulin-dependent diabetes mellitus is likely to modulate overall rates of glucose disposal.