Bmal1-deficient mouse fibroblast cells do not provide premature cellular senescence in vitro

Bmal1 is a core circadian clock gene. Bmal1^?/? mice show disruption of the clock and premature aging phenotypes with a short lifespan. However, little is known whether disruption of Bmal1 leads to premature aging at cellular level. Here, we established primary mouse embryonic fibroblast (MEF) cells derived from Bmal1^?/? mice and investigated its effects on cellular senescence. Unexpectedly, Bmal1^?/? primary MEFs that showed disrupted circadian oscillation underwent neither premature replicative nor stress-induced cellular senescence. Our results therefore uncover that Bmal1 is not required for in vitro cellular senescence, suggesting that circadian clock does not control in vitro cellular senescence.     

New titin (connectin) isoforms and their functional role in striated muscles of mammals: Facts and suppositions

This review summarizes results of our studies on titin isoform composition in vertebrate striated muscles under normal conditions, during hibernation, real and simulated microgravity, and under pathological conditions (stiff-person syndrome, post-apoplectic spasticity, dilated cardiomyopathy, cardiac hypertrophy). Experimental evidence for the existence in mammalian striated muscles of higher molecular weight isoforms of titin (NT-isoforms) in addition to the known N2A-, N2BA-, and N2B-titin isoforms was obtained. Comparative studies of changes in titin isoform composition and structure-functional properties of human and animal striated muscles during adaptive and pathological processes led to a conclusion about the key role of NT-isoforms of titin in maintenance of sarcomere structure and contractile function of these muscles.     

Passive tension of rat skeletal soleus muscle fibers: effects of unloading conditions.

In this work we studied changes in passive elastic properties of rat soleus muscle fibers subjected to 14 days of hindlimb unloading (HU). For this purpose, we investigated the titin isoform expression in soleus muscles, passive tension-fiber strain relationships of single fibers, and the effects of the thick filament depolymerization on passive tension development. The myosin heavy chain composition was also measured for all fibers studied. Despite a slow-to-fast transformation of the soleus muscles on the basis of their myosin heavy chain content, no modification in the titin isoform expression was detected after 14 days of HU. However, the passive tension-fiber strain relationships revealed that passive tension of both slow and fast HU soleus fibers increased less steeply with sarcomere length than that of control fibers. Gel analysis suggested that this result could be explained by a decrease in the amount of titin in soleus muscle after HU. Furthermore, the thick filament depolymerization was found to similarly decrease passive tension in control and HU soleus fibers. Taken together, these results suggested that HU did not change titin isoform expression in the soleus muscle, but rather modified muscle stiffness by decreasing the amount of titin.     

Sex and dosing-time dependencies in irinotecan-induced circadian disruption

Circadian disruption accelerates malignant growth; thus, it should be avoided in anticancer therapy. The circadian disruptive effects of irinotecan, a topoisomerase I inhibitor, was investigated according to dosing time and sex. In previous work, irinotecan achieved best tolerability following dosing at zeitgeber time (ZT) 11 in male and ZT15 in female mice, whereas worst toxicity corresponded to treatment at ZT23 and ZT3 in male and female mice, respectively. Here, irinotecan (50 mg/kg intravenous [i.v.]) was delivered at the sex-specific optimal or worst circadian timing in male and female B6D2F1 mice. Circadian disruption was assessed with rest-activity, body temperature, plasma corticosterone, and liver mRNA expressions of clock genes Rev-erbα, Per2, and Bmal1. Baseline circadian rhythms in rest-activity, body temperature, and plasma corticosterone were more prominent in females as compared to males. Severe circadian disruption was documented for all physiology and molecular clock endpoints in female mice treated at the ZT of worst tolerability. Conversely, irinotecan administration at the ZT of best tolerability induced slight alteration of circadian physiology and clock-gene expression patterns in female mice. In male mice, irinotecan produced moderate alterations of circadian physiology and clock-gene expression patterns, irrespective of treatment ZT. However, the average expression of Rev-erbα, Per2, and Bmal1 were down-regulated 2- to 10-fold with irinotecan at the worst ZT, while being minimally or unaffected at the best ZT, irrespective of sex. Corticosterone secretion increased acutely within 2?h with a sex-specific response pattern, resulting in a ZT-dependent phase-advance or -delay in both sex. The mRNA expressions of irinotecan clock-controlled metabolism genes Ce2, Ugt1a1, and Top1 were unchanged or down-regulated according to irinotecan timing and sex. This study shows that the circadian timing system represents an important toxicity target of irinotecan in female mice, where circadian disruption persists after wrongly timed treatment. As a result, the mechanisms underling cancer chronotherapeutics are expectedly more susceptible to disruption in females as compared to males. Thus, the optimal circadian timing of chemotherapy requires precise determination according to sex, and should involve the noninvasive monitoring of circadian biomarkers.     

Age-associated disruption of molecular clock expression in skeletal muscle of the spontaneously hypertensive rat

It is well known that spontaneously hypertensive rats (SHR) develop muscle pathologies with hypertension and heart failure, though the mechanism remains poorly understood. Woon et al. (2007) linked the circadian clock gene Bmal1 to hypertension and metabolic dysfunction in the SHR. Building on these findings, we compared the expression pattern of several core-clock genes in the gastrocnemius muscle of aged SHR (80 weeks; overt heart failure) compared to aged-matched control WKY strain. Heart failure was associated with marked effects on the expression of Bmal1, Clock and Rora in addition to several non-circadian genes important in regulating skeletal muscle phenotype including Mck, Ttn and Mef2c. We next performed circadian time-course collections at a young age (8 weeks; pre-hypertensive) and adult age (22 weeks; hypertensive) to determine if clock gene expression was disrupted in gastrocnemius, heart and liver tissues prior to or after the rats became hypertensive. We found that hypertensive/hypertrophic SHR showed a dampening of peak Bmal1 and Rev-erb expression in the liver, and the clock-controlled gene Pgc1α in the gastrocnemius. In addition, the core-clock gene Clock and the muscle-specific, clock-controlled gene Myod1, no longer maintained a circadian pattern of expression in gastrocnemius from the hypertensive SHR. These findings provide a framework to suggest a mechanism whereby chronic heart failure leads to skeletal muscle pathologies; prolonged dysregulation of the molecular clock in skeletal muscle results in altered Clock, Pgc1α and Myod1 expression which in turn leads to the mis-regulation of target genes important for mechanical and metabolic function of skeletal muscle.     

Changes in structure and functional properties of cytoskeletal elastic protein titin in dilated cardiomyopathy

To elucidate the role of titin in the onset and development of dilated cardiomyopathy, the structure and functional properties of this protein from pathological myocardium (human left ventricle) were studied. By the use of SDS gel electrophoresis, a decrease in molecular weight of titin in dilated cardiomyopathy compared with norm (pig left ventricle) was revealed. The decrease correlated with the stage of the disease. A decrease in the length of molecules of pathological forms of titin was also found by electron microscopy, which confirms the results of electrophoresis tests. It was shown that, unlike titin from healthy muscle, pathological forms of titin do not activate but inhibit the main functional properties of control myosin: the actin-activated ATPase activity and its Ca2+ sensitivity. The direction of the changes in structure and functional properties of titin allows one to conclude about its contribution to the development of the pathology studied.     

Hypertrophic cardiomyopathy: two homozygous cases with "typical" hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy

About 10% of cases of hypertrophic cardiomyopathy (HCM) evolve into dilated cardiomyopathy (DCM) with unknown causes. We studied 11 unrelated patients (pts) with HCM who progressed to DCM (group A) and 11 who showed "typical" HCM (group B). Mutational analysis of the beta-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), and cardiac troponin T (TNNT2) genes demonstrated eight mutations affecting MYH7 or MYBPC3 gene, five of which were new mutations. In group A-pts, the first new mutation occurred in the myosin head-rod junction and the second occurred in the light chain-binding site. The third new mutation leads to a MYBPC3 lacking titin and myosin binding sites. In group B, two pts with severe HCM carried two homozygous MYBPC3 mutations and one with moderate hypertrophy was a compound heterozygous for MYBPC3 gene. We identified five unreported mutations, potentially "malignant" defects as for the associated phenotypes, but no specific mutations of HCM/DCM.     

BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis

Circadian timing is generated through a unique series of autoregulatory interactions termed the molecular clock. Behavioral rhythms subject to the molecular clock are well characterized. We demonstrate a role for Bmal1 and Clock in the regulation of glucose homeostasis. Inactivation of the known clock components Bmal1 (Mop3) and Clock suppress the diurnal variation in glucose and triglycerides. Gluconeogenesis is abolished by deletion of Bmal1 and is depressed in Clock mutants, but the counterregulatory response of corticosterone and glucagon to insulin-induced hypoglycaemia is retained. Furthermore, a high-fat diet modulates carbohydrate metabolism by amplifying circadian variation in glucose tolerance and insulin sensitivity, and mutation of Clock restores the chow-fed phenotype. Bmal1 and Clock, genes that function in the core molecular clock, exert profound control over recovery from insulin-induced hypoglycaemia. Furthermore, asynchronous dietary cues may modify glucose homeostasis via their interactions with peripheral molecular clocks.     

Resveratrol regulates circadian clock genes in Rat-1 fibroblast cells

Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 muM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.     

Daily torpor alters multiple gene expression in the suprachiasmatic nucleus and pineal gland of the Djungarian hamster (Phodopus sungorus)

Circadian rhythms are still expressed in animals that display daily torpor, implying a temperature compensation of the pacemaker. Nevertheless, it remains unclear how the clock works in hypothermic states and whether torpor itself, as a temperature pulse, affects the circadian system. To reveal changes in the clockwork during torpor, we compared clock gene and neuropeptide expression by in situ hybridization in the suprachiasmatic nucleus (SCN) and pineal gland of normothermic and torpid Djungarian hamsters (Phodopus sungorus). Animals from light-dark (LD) 8ratio16 were sacrificed at 8 time points throughout 24 h. To investigate the effect of a previous torpor episode on the clock, we sacrificed a group of normothermic hamsters 1 day after torpor. In normothermic animals, Per1 peaked at zeitgeber time (ZT)4; whereas, Bmal1 reached maximal expression between ZT16 and ZT19. AVP mRNA in the SCN showed highest levels at ZT7. On the day of torpor, the levels of all mRNAs investigated, except for AVP mRNA, were increased during the torpor bout. Moreover, the Bmal1 rhythm was advanced. On the day after the hypothermia, Bmal1 and AVP rhythms showed severely depressed amplitude. Those distinct amplitude changes of Bmal1 and AVP on the day after a torpor episode expression suggests that torpor affects the circadian system, probably by altered translational processes that might lead to a modified protein feedback on gene expression. In the pineal gland, an important clock output, Aanat expression, peaked between ZT16 and ZT22 in normothermic animals. Aanat levels were significantly advanced on the day of hypothermia, an effect which was still visible 1 day afterward. In summary, this study showed that daily torpor affects the phase and amplitude of rhythmic clock gene and clock-controlled gene expression in the SCN. Furthermore, the rhythmic gene expression in a peripheral oscillator, the pineal gland, is also affected.     

Facilitated physiological adaptation to prolonged circadian disruption through dietary supplementation with essence of chicken

Synchrony between circadian and metabolic processes is critical to the maintenance of energy homeostasis. Studies on essence of chicken (EC), a chicken meat extract rich in proteins, amino acids and peptides, showed its effectiveness in alleviating fatigue and promoting metabolism. A recent study revealed that it facilitated the re-entrainment of clock genes (Bmal1, Cry1, Dec1, Per1 and Per2) in the pineal gland and liver in a rat model of circadian disruption. Here, we investigated the role of EC-facilitated circadian synchrony in the maintenance of the energy homeostasis using a mouse model of prolonged circadian disruption. Prolonged circadian disruption (12 weeks) resulted in hepatic maladaptation, manifested by a mild but significant (p?<?0.05) hepatomegaly, accompanied by disturbed hepatic lipid metabolism and liver injury (indicated by increased circulating hepatic enzymes). Evidently, there was marked elevations of hepatic inflammatory mediators (interleukin-1beta and interleukin-6), suggesting an underlying inflammation leading to the hepatic injury and functional impairment. Importantly, the disruption paradigm caused the decoupling between key metabolic regulators (e.g. mTOR and AMPK) and hepatic clock genes (Per1, Cry1, Dec1, Bmal1). Further, we showed that the loss of circadian synchrony between the master and hepatic clock genes (Per1, Cry1, Dec1, Bmal1) could be the underlying cause of the maladaptation. When supplemented with EC, the functional impairment and inflammation were abolished. The protective effects could be linked to its effectiveness in maintaining the synchrony between the master and hepatic clocks, and the resultant improved coupling of the circadian oscillators (Per1, Cry1, Dec1, Bmal1) and metabolic regulators (mTOR, AMPK). Overall, EC supplementation promoted the physiological adaptation to the prolonged circadian disruption through facilitation of endogenous circadian synchrony and the coupling of circadian oscillators and metabolic regulators. This forms an important basis for further elucidation of the physiological benefits of EC-facilitated circadian synchrony.