Seasonal changes in the composition of titin isoforms in muscles of hibernating ground squirrels

An electophoretic study of changes in the content of intact titin isoforms, N2B-, N2BA-, N2A-titins and T2 in skeletal and cardiac muscles of ground squirrel (Spermophillus undulatus) is made in different periods: summer activity, autumnal activity, hibernation, arousal, and winter activity. In atria and ventricles of ground squirrels in the period of autumnal activity an increase (by ～1.5 times) in the N2BA to N2B ratio was observed, in comparison with that in cardiac muscle in summer activity. During hibernation, the decrease in the relative content of N2B-, N2BA-titins and T2 in cardiac muscle as well as of N2A-titin and T2 in skeletal muscles was determined against the background of preservation of the relative amount of intact titin isoforms. At waking of ground squirrels and in a short period of winter activity, a rapid restoration of the content of N2B-, N2BA-, N2A-titisns and T2 in muscles was observed. In the myocardium of hibernating, waking ground squirrels and of those during winter activity the increased N2BA to N2B ratio was retained. The changes in the titin content are discussed in the aspect of adaptation of ground squirrels to hibernation.     

Adult cardiac muscle cells in long-term serum-free culture: Myofibrillar organization and expression of myosin heavy chain isoforms

Summary A culture system for adult rat cardiac muscle cells has been established without exposure of cells to serum at any step of the procedure. The methodology has been standardized and optimized to obtain better quality and high yield of cells and culture. Subsequent to enzyme perfusion, the release of myocytes from enzyme-perfused tissues was carried out in enzyme-free Joklik's medium instead of exposing cells to proteolytic enzyme(s) as done previously. Approximately 5 million cylindrical muscle cells per ventricle were obtained. The culture medium contained Eagle's minimum essential medium with Earle's salts, basic fibroblast growth factor, epidermal growth factor, insulin, transferrin, selenium, norepinephrine, triiodothyronine (T₃), bovine serum albumin, nonessential amino acids, and ascorbic acid. The plating efficiency of the experimental cultures was comparable to that of the control cultures grown in the presence of serum. The cells in the serum-free medium contained myofibrillar and myosin isoforms characteristics of the adult myocytes. The cells underwent cellular reorganization comparable to that of the controls. The initial phase of reorganization involved the breakdown of myofibrils and extrusion of mitochondria, degraded myofibrils, and other cellular organelles. The latter phase of reorganization included myofibrillogenesis and organellogenesis resulting in the development of myofibrillar apparatus with cellular organelles. Myocytes were contractile throughout the culture period. Cardiac myocytes grown, in serum-free medium expressed the predominant myosin isoform V1 similar to their counterparts in vivo. T₃ is essential for the expression of isomyosin V1. This study demonstrates that adult cardiac muscle cells can be maintained in long-term serum-free culture from seeding to termination. The cells in serum-free conditions maintain at least two differentiated characteristics of adult myocytes investigated, namely, abundant organized myofibrils and predominant myosin isoform V1. This work is supported by grant DCB-8709594 from the National Science Foundation, Washington, DC     

Circadian rhythm of intracellular protein synthesis signaling in rat cardiac and skeletal muscles

Intracellular signaling exhibits circadian variation in the suprachiasmatic nucleus and liver. However, it is unclear whether circadian regulation also extends to intracellular signaling pathways in the cardiac and skeletal muscles. Here, we examined circadian variation in the intracellular mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase 1 (p70S6K) and extracellular signal-regulated kinase (ERK) pathways, which regulate protein synthesis in rat cardiac and skeletal muscles. Seven-week-old male Wistar rats were assigned to six groups: Zeitgeber time (ZT) 2, ZT6, ZT10, ZT14, ZT18, and ZT22 (ZT0, lights on; ZT12, lights off). The cardiac, plantaris, and soleus muscles were removed after a 12-h fasting period, and signal transducers involved in protein synthesis (mTOR, p70S6K, and ERK) were analyzed by western blotting. Circadian rhythms of signal transducers were observed in both cardiac (mTOR, p70S6K, and ERK) and plantaris (p70S6K and ERK) muscles (p<0.05), but not in the soleus muscle. In the cardiac muscle, the phosphorylation rate of mTOR was significantly higher at ZT6 (peak) than at ZT18 (bottom), and the phosphorylation rate of p70S6K was significantly higher at ZT2 (peak) than at ZT18 (bottom). In contrast, in the plantaris muscle, the phosphorylation rate of ERK was significantly lower at ZT2 (bottom) than at ZT18 (peak). Our data suggested that protein synthesis via mTOR/p70S6K and ERK signaling molecules exhibits circadian variation in rat cardiac and fast-type plantaris muscles.     

Application of in vitro motility assay to studying the calcium-mechanical relationship in skeletal and cardiac muscles

In a set of experiments on regulated contractile systems (i.e., in vitro motility assay with a reconstructed thin filament), the velocity of a thin filament on the surface coated with rabbit skeletal or rat cardiac myosin was estimated at various calcium ion concentrations in solution (pCa 4–8). The velocity versus pCa curve proved to be sigmoid. The velocity of a regulated thin filament at a saturating calcium concentration (pCa 4) exceeded that of a nonregulated thin filament by 65 and 87% for skeletal and cardiac myosin, respectively. The Hill coefficient was 1.95 and 2.5 for skeletal and cardiac muscles, respectively; this difference was discussed in terms of the different contributions of cooperativity mechanisms of contractile and regulatory proteins to the regulation of contraction in these types of muscle.     

Assessment of fertility and infertility in boron-exposed Turkish subpopulations: 3. Evaluation of fertility among sibs and in "borate families"

As a part of a work to reveal the health effects of boron and its compounds, fertility and infertility states of sibs of probands, contacted and interviewed in the field, and of their spouses were given. The purposes were to prevent duplications seemingly inevitable in a relatively small community with prevailing consanguinity while analyzing marriages over respective generations and to reveal if there occurred an aggregation of infertile couples. Any family without offspring after about the second year of marriage was considered primary infertile as adopted throughout the study and such families were ascertained through the individual pedigree charts set up according to the instructions of the proband, he (she) himself (herself) being excluded. The rates of childless families of this type were 0.0–3.4% among male and 0.9–3.8% among female sibs of the participant, and 2.3–10.0% among male and 0.0–5.6% among female sibs of his (her) spouse with averages of 2.3% of 1589, 2.6% of 1589, 4.0% of 1314, and 3.3% of 1436 instances, respectively. The differences were insignificant and the rates were not different from those concerning probands themselves and that of a comparable segment of the Turkish population. “Borate families/kindreds” with two or more members engaged in the borate industry were also assessed in order to detect if there was a significant clustering of infertiles within the kindred. Although it was difficult to compare with a matched group, few couples were examples of familial concentration of infertility. These results provided further support that boron exposure does not affect human reproduction primarily and most probably secondarily. This work was partly presented to the 10th International Symposium on Trace Elements in Man and in Animal (TEMA-10), Evian, France, May 2–7, 1999.     

Comparison of the effects of the membraneassociated Ca2+/calmodulin-dependent protein kinase on Ca2+-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca²⁺-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca²⁺-ATPase. Studies comparing, the effects of PKA and CaM kinase on cardiac Ca²⁺-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca²⁺-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca²⁺-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 M) directly to the Ca²⁺ transport assay medium results in minimal ( 112–130% of control) stimulation of Ca²⁺ uptake activity when the Ca²⁺ uptake reaction is initiated by the addition of either ATP or Ca²⁺/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca²⁺ transport assay medium results in stimulation of Ca²⁺ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca²⁺ uptake markedly (215% of control) when the Ca²⁺ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca²⁺/EGTA but minimally (130% of control) when the Ca²⁺ uptake reaction is initiated by the addition of ATP. These findings imply that (a) phospholamban is coupled to the Ca²⁺-ATPase in slow-twitch skeletal muscle SR (as in cardiac SR), and (b) the amount of Ca²⁺ uptake stimulation seen upon the addition of calmodulin or PKA depends strongly on the assay conditions employed. Our observations help to explain the wide range of effects of calmodulin or PKA addition reported in previous studies. It should be noted that, since CaM kinase is now known to phosphorylate the Ca²⁺-ATPase in addition to phospholamban, further studies are required to determine the relative contributions of phospholambanversus Ca²⁺-ATPase phosphorylation in the stimulation of Ca²⁺-ATPase function by CaM kinase. Also, earlier studies attributing all of the effects of CaM kinase stimulation of Ca²⁺ uptake and Ca²⁺-ATPase activity to phospholamban phosphorylation need to be re-examined.     

Role of the fetal and alpha/beta exons in the function of fast skeletal troponin T isoforms: correlation with altered Ca2+ regulation associated with development

In mammalian fast skeletal muscle, constitutive and alternative splicing from a single troponin T (TnT) gene produce multiple developmentally regulated and tissue specific TnT isoforms. Two exons, alpha (exon 16) and beta (exon 17), located near the 3' end of the gene and coding for two different 14 amino acid residue peptides are spliced in a mutually exclusive manner giving rise to the adult TnTalpha and the fetal TnTbeta isoforms. In addition, an acidic peptide coded by a fetal (f) exon located between exons 8 and 9 near the 5' end of the gene, is specifically present in TnTbeta and absent in the adult isoforms. To define the functional role of the f and alpha/beta exons, we constructed combinations of TnT cDNAs from a single human fetal fast skeletal TnTbeta cDNA clone in order to circumvent the problem of N-terminal sequence heterogeneity present in wild-type TnT isoforms, irrespective of the stage of development. Nucleotide sequences of these constructs, viz. TnTalpha, TnTalpha + f, TnTbeta - f and TnTbeta are identical, except for the presence or absence of the alpha or beta and f exons. Our results, using the recombinant TnT isoforms in different functional in vitro assays, show that the presence of the f peptide in the N-terminal T1 region of TnT, has a strong inhibitory effect on binary interactions between TnT and other thin filament proteins, TnI, TnC and Tm. The presence of the f peptide led to reduced Ca2+-dependent ATPase activity in a reconstituted thin filament, whereas the contribution of the alpha and beta peptides in the biological activity of TnT was primarily modulatory. These results indicate that the f peptide confers an inhibitory effect on the biological function of fast skeletal TnT and this can be correlated with changes in the Ca2+ regulation associated with development in fast skeletal muscle.     

Distinct lipidomic profiles in models of physiological and pathological cardiac remodeling,and potential therapeutic strategies

Cardiac myocyte membranes contain lipids which remodel dramatically in response to heart growth and remodeling. Lipid species have both structural and functional roles. Physiological and pathological cardiac remodeling have very distinct phenotypes, and the identification of molecular differences represent avenues for therapeutic interventions. Whether the abundance of specific lipid classes is different in physiological and pathological models was largely unknown. The aim of this study was to determine whether distinct lipids are regulated in settings of physiological and pathological remodeling, and if so, whether modulation of differentially regulated lipids could modulate heart size and function. Lipidomic profiling was performed on cardiac-specific transgenic mice with 1) physiological cardiac hypertrophy due to increased Insulin-like Growth Factor 1 (IGF1) receptor or Phosphoinositide 3-Kinase (PI3K) signaling, 2) small hearts due to depressed PI3K signaling (dnPI3K), and 3) failing hearts due to dilated cardiomyopathy (DCM). In hearts of dnPI3K and DCM mice, several phospholipids (plasmalogens) were decreased and sphingolipids increased compared to mice with physiological hypertrophy. To assess whether restoration of plasmalogens could restore heart size or cardiac function, dnPI3K and DCM mice were administered batyl alcohol (BA; precursor to plasmalogen biosynthesis) in the diet for 16 weeks. BA supplementation increased a major plasmalogen species (p18:0) in the heart but had no effect on heart size or function. This may be due to the concurrent reduction in other plasmalogen species (p16:0 and p18:1) with BA. Here we show that lipid species are differentially regulated in settings of physiological and pathological remodeling. Restoration of lipid species in the failing heart warrants further examination.     

The dual face of connexin-based astroglial Ca communication: A key player in brain physiology and a prime target in pathology

For decades, studies have been focusing on the neuronal abnormalities that accompany neurodegenerative disorders. Yet, glial cells are emerging as important players in numerous neurological diseases. Astrocytes, the main type of glia in the central nervous system , form extensive networks that physically and functionally connect neuronal synapses with cerebral blood vessels. Normal brain functioning strictly depends on highly specialized cellular cross-talk between these different partners to which Ca^2 +, as a signaling ion, largely contributes. Altered intracellular Ca^2 + levels are associated with neurodegenerative disorders and play a crucial role in the glial responses to injury. Intracellular Ca^2 + increases in single astrocytes can be propagated toward neighboring cells as intercellular Ca^2 + waves, thereby recruiting a larger group of cells. Intercellular Ca²⁺ wave propagation depends on two, parallel, connexin (Cx) channel-based mechanisms: i) the diffusion of inositol 1,4,5-trisphosphate through gap junction channels that directly connect the cytoplasm of neighboring cells, and ii) the release of paracrine messengers such as glutamate and ATP through hemichannels (‘half of a gap junction channel’). This review gives an overview of the current knowledge on Cx-mediated Ca^2 + communication among astrocytes as well as between astrocytes and other brain cell types in physiology and pathology, with a focus on the processes of neurodegeneration and reactive gliosis. Research on Cx-mediated astroglial Ca^2 + communication may ultimately shed light on the development of targeted therapies for neurodegenerative disorders in which astrocytes participate. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.