The Clock Gene,brain And Muscle Arnt-like 1,regulates Autophagy In High-glucose-induced Cardiomyocyte Injury

Diabetic cardiomyopathy(DCM)refers to a disease process that affects the myocardium in the diabetic heart,thereby causing a variety of structural abnormalities and ultimately leading to left ventricular hypertrophy and dysfunction.Hyperglycemia is an independent risk factor for diabetic cardiac injury,the development and progression of DCM is closely associated with the duration and severity of high glucose.However,current treatments cannot effectively reduce DCM in diabetic patients,indicating that other mechanisms remain to be discovered.Autophagy is a dynamic process in which damaged or aged intracellular organelles and long-lived proteins are sequestered into double-membraned vesicles termed autophagosomes.Autophagosomes then fuse with lysosomes for the degradation and recycling of their contents.Autophagy has long been described as a protective mechanism that lets the cell to maintain homeostasis and viability under stress conditions and starvation.However,low level of autophagy is protective to cardiomyocytes,excessive autophagy may aggravate cell injury even causes cell death.Autophagy is a double-edged sword that could be either beneficial or harmful depending on the cellular milieu,the intensity of the stimulus and the levels of autophagy.It has been reported that high-glucose inhibits autophagy in cardiomyocytes,while autophagy suppression is protective against high-glucose-induced cardiomyocyte injury.These results indicate an association between altered autophagy and diabetic cardiac injury.Numerous studies in both human subjects and animal models have confirmed that perturbations of the internal clock system constitute risk factors for such disorders as obesity,type 2 diabetes,and cardiovascular disease.The circadian clock is an evolutionarily conserved fundamental mechanism in organisms,a biological response to Earth's diurnal cycles.In mammals,circadian rhythms are driven by a group of clock genes that include Bmal1,Clock,Cry,and Per.The clocks are present even when cells are in culture,indicating that the basic mechanism is intrinsic and self-sustained.As a core clock gene,Bmal1 plays a key role in the generation and maintenance of circadian rhythms.Bmal1-knockout mice display a complete loss of circadian rhythms.In addition,alterations of circadian clock activity are present in the Streptozotocin-induced diabetic heart.Genetic disruption of Bmal1 induces an abnormal metabolic phenotype characterized by impaired gluconeogenesis,hyperleptinemia,glucose intolerance,and dyslipidemia.Given this evidence,we hypothesized that the circadian gene Bmal1 might participate in the pathogenesis of DCM and that such a connection would furnish new opportunities for mechanism-based DCM therapeutics.In this study,we use neonatal rat cardiomyocytes to produce an injury model of hyperglycemia,we sought to address the impact of altered circadian clock gene Bmal1 expression on cardiomyocyte viability in response to high glucose and the molecular mechanism underlying it.Our study includes the following parts: Part 1:Effects of high glucose on cardiomyocyte viability and autophagyObjectives:1 To build up neonatal rat cardiomyocyte injury model in response to high glucose.2 To explore the effects of high glucose on cardiomyocyte viability.3 To explore the effects of high glucose on cardiomyocyte autophagic activity.Methods:Cardiomyocytes were derived from 2 to 3-day old neonatal SD rats.The cells were divided into 2 groups:(1)Normal glucose(5.5mM)group,(2)High glucose(25mM)group.Cardiomyocyte viability was calculated with the Live/Dead Viability/Cytotoxicity.To detect cardiomyocyte apoptosis,a terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)assay was performed,along with analysis of caspase 3 and PARP cleavage.Beclin-1(BECN1),microtubule-associated protein light chain 3(LC3),ATG12-5,and p62 were used as autophagy-related markers.The levels of BECN1,LC3-II/LC-I and ATG12–5 conjugate are proportional to the number of autophagosomes,while the level of p62 has a negative correlation with autophagic activity.The levels of autophagy-related markers were measured by Western blot.As a more accurate assessment of autophagy,autophagic flux reflects the number of autophagosomes that are delivered to and degraded in the lysosome and can be detected by the difference between LC3-II protein levels in the absence or presence of lysosomal inhibitors(e.g.,bafilomycin A1).Results:1 Neonatal rat cardiomyocyte injury model in response to high glucose was successfully built up.2 Compared with normal glucose group,high glucose led to an increase in cardiomyocyte death and apoptosis.The exacerbated apoptosis was confirmed by examining the abundance of cleaved caspase 3 and PARP.3 The levels of LC3-II/LC-I,BECN1,and ATG12–5 conjugates in cardiomyocytes exposed to high glucose were markedly lower than those in cells cultured under normal glucose concentrations,while the p62 protein levels were higher in 25 mM glucose than in 5.5 mM glucose.The autophagic flux clearly showed that high glucose inhibited autophagic activity in cardiomyocytes.Conclusions:1 High glucose induces neonatal rat cardiomyocyte injury.2 High glucose inhibited autophagic activity in cardiomyocytes.Part 2:Effects of altered Bmal1 expression on cardiomyocyte viability in response to high glucoseObjectives:To investigate whether and how altered Bmal1 expression affect cardiomyocyte viability in response to high glucose.Methods:Lentivirus was used to deliver a short hairpin RNA(shRNA)against Bmal1 mRNA that would knock down(KD)the expression of the Bmal1 gene in cultured neonatal rat cardiomyocytes.Bmal1 cDNA was deliver into cultured cardiomyocytes to overexpression of the Bmal1 gene.Scrambled control(SC)shRNA and p cDNA were used as control groups.We then exposed the cells to normal and high concentrations of glucose to study the effects of circadian disruption on cardiomyocyte survival in response to high glucose.The cells were assigned into the following groups:(1)Bmal1 cDNA + high glucose treatment,(2)p cDNA + high glucose treatment,(3)Bmal1 cDNA + normal glucose treatment,(4)p cDNA + normal glucose treatment,(5)Bmal1 shRNA + high glucose treatment,(6)SC shRNA + high glucose treatment,(7)Bmal1 sh RNA + normal glucose treatment,(8)SC shRNA + normal glucose treatment.Cardiomyocyte viability was calculated with the Live/Dead Viability/Cytotoxicity.To detect cardiomyocyte apoptosis,TUNEL assay was performed,along with analysis of caspase 3 and PARP cleavage.Results:1 Compared with the SC shRNA group,KD of Bmal1 led to an increase in cardiomyocyte death under high-glucose conditions.The proportion of apoptotic cells in the Bmal1 silencing group were dramatically higher compared with SC shRNA control group.The exacerbated apoptosis was confirmed by examining the abundance of cleaved caspase 3 and PARP.2 In addition,overexpression of Bmal1(cDNA)protected cardiomyocytes against high-glucose injury,as shown by attenuated cell death,decreased TUNEL-positive cells,and less cleaved caspase 3 and PARP,as shown by Western blot analysis.However,in cardiomyocytes under normal glucose conditions,altering the expression of Bmal1 did not obviously impact cardiomyocyte survival or death.Conclusions:Alterations in Bmal1 levels had an effect only under hyperglycemic conditions,as disruption of Bmal1 aggravated hyperglycemic toxicity as evaluated by both cardiomyocyte death and apoptosis.Part 3 : Altered expression of Bmal1 affects high-glucose-induced cardiomyocyte injury by mediating autophagyObjectives:To investigate whether Bmal1 can influence cardiomyocyte survival in hyperglycemic conditions by mediating autophagic activity.Methods:Lentivirus was used to deliver a short hairpin RNA(shRNA)against Bmal1 mRNA that would knock down(KD)the expression of the Bmal1 gene in cultured neonatal rat cardiomyocytes.Bmal1 cDNA was deliver into cultured cardiomyocytes to overexpression of the Bmal1 gene.Scrambled control(SC)shRNA and p cDNA were used as control groups.We then exposed the cells to normal and high concentrations of glucose to study the effects of circadian disruption on cardiomyocyte survival in response to high glucose.The cells were assigned into the following groups:(1)Bmal1 cDNA + high glucose treatment,(2)p cDNA + high glucose treatment,(3)Bmal1 cDNA + normal glucose treatment,(4)p cDNA + normal glucose treatment,(5)Bmal1 shRNA + high glucose treatment,(6)SC shRNA + high glucose treatment,(7)Bmal1 sh RNA + normal glucose treatment,(8)SC shRNA + normal glucose treatment.BECN1,LC3,ATG12-5,and p62 were used as autophagy-related markers.The levels of BECN1,LC3-II/LC-I and ATG12–5 conjugate are proportional to the number of autophagosomes,while the level of p62 has a negative correlation with autophagic activity.The levels of autophagy-related markers were measured by Western blot.As a more accurate assessment of autophagy,autophagic flux reflects the number of autophagosomes that are delivered to and degraded in the lysosome and can be detected by the difference between LC3-II protein levels in the absence or presence of lysosomal inhibitors(e.g.,bafilomycin A1).And then the autophagy-inducing agent rapamycin(Rap)and the autophagy inhibitor 3-methyladenine(3-MA)were furtherly used to manipulate autophagic activity.Cardiomyocyte viability was calculated with the Live/Dead Viability/Cytotoxicity.To detect cardiomyocyte apoptosis,TUNEL assay was performed,along with analysis of caspase 3 and PARP cleavage.Results:1 Neither overexpression nor silencing of Bmal1 had a significant effect on the autophagic activity of cardiomyocytes cultured in normal glucose concentrations.However,overexpression of Baml1 strikingly increased the abundance of LC3-II,BECN1,and ATG12-5 in cardiomyocytes exposed to high levels of glucose.Meanwhile,autophagic flux increased to 2-fold that of p cDNA cardiomyocytes,as evidenced by ?LC3-II levels.Conversely,KD of Bmal1 further reduced autophagic flux to 16% of the SC control in cardiomyocytes cultured in 25 mM glucose,indicating that high glucose and shBmal1 synergistically inhibited autophagy.2 Cardiomyocyte survival caused by overexpression of Bmal1 was reduced by 3-MA,as demonstrated by increased proportions of dead cells in live/dead staining and TUNEL-positive cells and by enhanced cleavage of caspase 3 and PARP.Meanwhile,Rap attenuated the Bmal1KD-induced enhancement of hyperglycemic cardiotoxicity,as shown by reduced fractions of dead cells in live/dead staining and TUNEL-positive cells and by lower levels of cleaved caspase 3 and PARP.Conclusions:These results suggested that although overexpression of Bmal1 under hyperglycemic conditions did not further enhance autophagic flux beyond basal levels,it reversed the autophagy inhibition that was triggered by high levels of glucose.Altered expression of Bmal1 affects high-glucose-induced cardiomyocyte injury by mediating autophagy.Bmal1 attenuates high-glucose-induced cardiomyocyte injury by inducing autophagic activity.Part 4:The clock gene Bmal1 induces autophagy by activating the mTORC1 signaling pathwayObjectives:To investigate the molecular mechanisms of Bmal1 regulating cardiomyocyte autophagy.To explore whether mTOR signaling pathway is involved in Bmal1-induced autophagic activity.Methods:Lentivirus was used to deliver a short hairpin RNA(shRNA)against Bmal1 mRNA that would knock down(KD)the expression of the Bmal1 gene in cultured neonatal rat cardiomyocytes.Bmal1 cDNA was deliver into cultured cardiomyocytes to overexpression of the Bmal1 gene.Scrambled control(SC)shRNA and p cDNA were used as control groups.We then exposed the cells to normal and high concentrations of glucose to study the effects of circadian disruption on cardiomyocyte survival in response to high glucose.The cells were assigned into the following groups:(1)Bmal1 cDNA + high glucose treatment,(2)p cDNA + high glucose treatment,(3)Bmal1 cDNA + normal glucose treatment,(4)p cDNA + normal glucose treatment,(5)Bmal1 shRNA + high glucose treatment,(6)SC shRNA + high glucose treatment,(7)Bmal1 sh RNA + normal glucose treatment,(8)SC shRNA + normal glucose treatment.mTORC1 downstream effectors such as p70 ribosomal S6 subunit kinase(p70S6K),ribosomal S6 protein(S6),and elongation factor 4E binding protein(4EBP)were measured by Western blot.To further verify the role of mTORC1 in the regulation of autophagic activity in response to altered Bmal1 expression,Bmal1-overexpressing cardiomyocytes were infected with a lentivirus expressing mTOR and then exposed to high glucose.Bmal1 KD cardiomyocytes were subjected to mTORC1 inhibitor Rap,according to assigned groups:(1)Bmal1 cDNA +mTOR + high glucose treatment,(2)Bmal1 cDNA + high glucose treatment,(3)p cDNA +mTOR + high glucose treatment,(4)p cDNA + high glucose treatment,(5)Bmal1 shRNA +Rap + high glucose treatment,(6)Bmal1 shRNA + high glucose treatment,(7)SC shRNA + Rap + high glucose treatment,(8)SC shRNA + high glucose treatment.Results:Overexpression of Bmal1 severely inhibited the mTOR signaling pathway,as shown by the attenuated phosphorylation of such mTOR downstream effectors as p70S6 K,S6,and 4EBP.Conversely,mTOR signaling was greatly enhanced by Bmal1 KD.Overexpression of mTOR decreased autophagic flux.Indeed,the mTOR inhibitor Rap antagonized the downregulation of autophagic activity triggered by Bmal1 KD,as indicated by ?LC3-II levels.Together,these results demonstrated thatConclusions:mTOR signaling pathway is involved in Bmal1-induced autophagic activity.The autophagy induction triggered by Bmal1 is mediated by inhibition of mTOR.