| Background and ObjectiveThe temporal organization of physiology is critical for human healthy,and the environmental light–dark cycles allowed intrinsic circadian rhythms to adapt the light and dark changes.Since the wide adoption of electronic lighting equipment,the exposure to nighttime lighting is extremely common,such as night shift work appears,light pollution,even polar day and night in the north and south poles and so forth.These light exposure environments disturb the the boundaries of day and night,making it difficult to synchronize biological processes.It is bound to affect human health and cause a variety of diseases.Many systems are under circadian control,including sleep-wake,hormone secretion,cellular function and gene expression.The effects of circadian disruption induced by exposure to nighttime light on human health attract more and more researchers'attention,which is associated with the increasing incidence of metabolic dysfunction and mood disorders,cardiovascular events?myocardial infarction,stroke,sudden cardiac arrest?,endocrine disorder,even cancer.Research suggested that circadian disruption induced by light exposure may be associated with the abnormal regulation of peripheral rhythm system,and exposure to light inhibits the secretion of melatonin to cause a disorder in the clock gene in central and peripheral areas.The research of the circadian system regulating glucocorticoid indicated that light exposure interacts with the hypothalamic–pituitary–adrenal axis and stress responsibility.Much of the evidence from rodent species experiments showed that light exposure stimulated the ganglion cells of the cone and rod to produce a neural circuit through SCN directly or indirectly projecting to other brain areas,and affects neuroplasticity and neurotransmission.Meanwhile,constant light exposure could cause complex cardiovascular effects,leading to the tissue and organs dysfunction.Recently,researchers have found that lighting has multiple effects on cardiovascular function.It is important for autonomic regulation to cardiovascular function.It has been documented that several brain areas such as hypothalamus and medulla oblongata are involved in autonomic processing.The sympathetic activity is mainly regulated by the rostral ventrolateral medulla?RVLM?in central nervous system.The C1neurons in the RVLM not only receive afferent fiber projection from paraventricular hypothalamic nucleus?PVN?and caudal ventrolateral medulla?CVLM?and other neural nuclei of cardiovascular regulation,but the efferent fibers in the RVLM also occur single synapse connection with the presympathetic ganglion neurons to control the sympathetic activity and cardiovascular function in peripheral region.The light exposure activated PVN.Moreover,there is direct evidence that suprachiasmatic nucleus?SCN?participates in the light-induced sympatho-excitation,while the roles of light exposure on the RVLM are not clarified.Thus,to maximally imitate the regular circumstances,this study utilized LED light?250-300 lux?to explore the effects of light exposure on sympathetic outflow in the RVLM and cardiovascular function and the possible mechanisms.There are an important role and profound significance for exploring neuronal circuit linked the circadian rhythm system with cardiovascular system.Heart Failure?HF?is induced by multifactorial cardiovascular syndrome,and the myocardial ischemia,even infarction is the critical factor for rapid progression to heart failure,and it becomes an important reason for cardiovascular diseases patients to sudden death.Circadian disruption induced by constant light exposure increased the risk of cardiovascular diseases,such as myocardial infarction and sudden cardiac arrest,suggesting that circadian system is critical for maintaining normal cardiovascular function and prevention of diseases.Thus,this study will comprehensively explore the impact of constant light exposure on cardiac function in normal and heart failure rats to provide evidence for the prevention of cardiovascular diseases.As is well known,myocardial ischemia quickly activates the sympathetic nervous system,resulting in an increased release of catecholamines.In acute phase,excessive catecholamines activate?-adrenergic receptors??-AR?to maintain cardiac output and systemic blood pressure by compensation,and the persistent sympathetic excitation accelerates the disease development.Researchers believed that activation of?1-AR has damage to heart through myocardial fibrosis remodeling and myocardial cell death.Therefore,the main emphasis for this study is to explore the roles of sympathetic nerve activity in cardiovascular dysfunction induced by light exposure.The RVLM is a critical area for controlling sympathetic outflow in heart failure,and the increasing of sympathetic outflow is the main reason of complications and death for paitents.Thus,it is of great importance to explore the effect of the RVLM on sympathetic nerve activity on cardiovascular regulation induced by light exposure.Recent studies suggested that constant light exposure caused oxidative stress to increase in tissue and organs,while the underlying mechanisms of effects of light exposure on oxidative stress in the RVLM have not been clarified.Reactive oxygen species?ROS?resulted in the increase of oxidative stress,and it is the important mechanism for sympathetic hyperactivity.Meanwhile,administration of the ROS inhibitor Tempol?SOD mimic?educes sympathetic hyperactivity,thereby improves cardiovascular function.In that way,whether light exposure resulted in sympathetic hyperactivity through increasing oxidative stress in the RVLM?It is reported that the generation of reactive oxygen was regulated by nuclear transcription factor Nrf2,and there is an important role for Nrf2 in antioxidant defence.Nrf2 phosphorylation facilitates anti-oxidative stress protein transcription and inhibits the level of oxidative stress.Nrf2 knockout mice with Angiotensin II?Ang II?perfusing,showed the increased cardiac hypertrophy and oxidative stress.Overexpression of Nrf2 inhibited the worsening of chronic obstructive pulmonary disease.However,the effects of constant light exposure on oxidative stress and the specific mechanisms in the RVLM in normal and heart failure rats have not been elucidated.Studies indicated that the angiotensin-converting enzyme 2?ACE2?catalyzes Ang II and generate Ang1-7.Up-regulated ACE2/Ang1-7/Mas R functional axis reduces sympathetic excitation to produce cardiovascular protective effect in the pathological condition of heart failure and hypertension.Overexpression of ACE2 in the RVLM decreases sympathetic nerve activity and blood pressure in hypertensive rats via reducing excitability of synaptic transmission.Meanwhile,up-regulated ACE2/Ang1-7/Mas R functional axis inhibits oxidative stress and protects neuronal functions.However,whether up-regulated ACE2/Ang1-7/Mas R functional axis may improve oxidative stress due to exposure to light is unclear.Number researches on circadian disruption induced by exposure to light suggested that the effects of light exposure on sympathetic outflow might be associated with the circadian rhythm disorder in the RVLM.Studies have indicated that constant light exposure affects normal biological rhythms and increases sleep disturbances,resulting in behavioral change.The balance of circadian rhythms plays an important role in maintaining a stable of biological rhythms,and the regulated center of circadian rhythm locates in the SCN,mainly affected by light.At the molecular level,circadian rhythm is regulated by clock gene.There is also a variety of clock genes expression in the RVLM.Circadian rhythm disorders are closely associated with cardiovascular diseases,such as heart failure,hypertension,myocardial infarction and arrhythmia.Interestingly,studies reported that the SCN receives light stimulation through the retina to connect directly or indirectly with multiple brain areas,such as PVN,Medial preoptic area?MPOA?,paraventricular nucleus of the thalamus?PVT?,lateral habenula?LHb?,ventral tegmental area?VTA?,dorsal raphe?DR?and so forth,producing complex neural network regulation.It is not clear whether there has a direct or indirect anatomical link between SCN and the sympathetic center RVLM?Based on it,we will further explore the constant light exposure on sympathetic nerve activity from the perspective of neuroscience for providing a new perspective to elucidate the functional link between the circadian system and cardiovascular system.Thus,the main objective of this study was to define the effects of constant light exposure on sympathetic outflow in the RVLM and cardiac function in normal and heart failure rats,and further explore whether the effects of constant light exposure on oxidative stress is associated with Nrf2 pathway and the down-regulation of ACE2/Ang1-7/Mas functional axis.We also explore the effects of constant light exposure on the circadian rhythm.This study will clarify the effects of light exposure on circadian rhythm at the level of RVLM and observe whether there is a direct or indirect neuronal projection between the SCN and RVLM.Based on the background above,this study is to clarify:1)The effects of constant light exposure on cardiac function in normal and heart failure rats,and whether sympathetic nerve activity is involved in the effects of light exposure on cardiac function;2)Whether constant light exposure causes an increase of oxidative stress in the RVLM to affect the cardiac function;3)Whether Nrf2 pathway is involved in the effects of ACE2overexpresstion in the RVLM on improving cardiac function caused by constant light exposure;4)Whether constant light exposure affects the normal circadian rhythm in the RVLM,and whether there is a direct or indirect connection between SCN and RVLM.Methods1.All SD rats were placed in the time-controlled lighting chambers?Circadian Time?CT?07:00 Light on,ZT 0;Circadian Time?CT?19:00 Light off,ZT 12?in a 12 h Light/12h Dark?LD?alternating environment concentrated feeding,at least which were adapted for two weeks before the experiment.Then,certain animals were fed in 24-hour continuous light environment?Constant Light,CL?.The cardiac functional differences were observed and compared between LD and CL.2.Continuous dynamic monitoring was utilized to determine the level of blood pressure?SBP,DBP?and heart rate?HR?by tail-cuff measurement for one week in ZT 12in LD and CL groups.Then plasma norepinephrine?NE?,epinephrine?E?,angiotensin II?Ang II?,glucocorticoids?GC?,atrial natriuretic peptide?ANP?and brain natriuretic peptide?BNP?concentrations were measured by orbital vein blood collection for 5 weeks in ZT 12in LD and CL environment.The changes of cardiac function were examined by echocardiography,cardiac catheterization of Millar and Masson staining in light exposure after four or eight weeks.Renal sympathetic nerve activity was used to detect the baseline renal sympathetic nerve activity?RSNA?in light exposure environment for four or eight weeks,and further determine the minimum time based on the change of cardiac function.3.Establishment of Heart Failure model.Heart Failure?HF?model was induced by coronary artery ligation and the sham-operated?Sham?animal was without the ligation operation.The rats in two groups were fed in LD.After four weeks,the cardiac function was measured by echocardiography and cardiac catheterization of Millar,and the heart failure model was further confirmed again through H&E staining.Then the normal and HF rats were grouped into LD group,CL group,HF-LD group and HF-CL group before experiments.4.After the end of light exposure,the cardiac function was measured in LD,CL,HF-LD and HF-CL groups by echocardiography and cardiac catheterization.The ejection fraction was recorded in HF-LD and HF-CL groups for two weeks by echocardiography.Then the heart tissue was perfused with paraformaldehyde,and Masson staining of heart slices were done.The cardiac fibrosis area?%?were observed in LD,CL,HF-LD and HF-CL groups.5.The renal sympathetic nerve was separated,and the baseline renal sympathetic nerve activity?RSNA?was compared among LD,CL,HF-LD and HF-CL groups.The plasma NE concentration was tested by ELISA in four groups.Finally,the rats were perfused,and the brain slices including the RVLM were stained by DAB immunohistochemistry,and then the percentage of c-fos positive neurons was compared among LD,CL,HF-LD and HF-CL groups.Furthermore,the RVLM c-fos protein expression was detected by Western Blot in four groups.6.Detection of the expressions of ACE2,Mas and Nrf2 pathway proteins and the ROS levels in the RVLM by Western Blot and DHE immunohistochemistry in LD,CL,HF-LD and HF-CL groups.The Nrf2 phosphorylation levels were detected in the RVLM in LD and CL groups by Western Blot.The overexpresstion of ACE2 was performed through microinjection of lentivirus particle in the bilateral RVLM.The ROS levels in the RVLM were tested by DHE in LD+Lenti-GFP,LD+Lenti-ACE2,CL+Lenti-GFP and CL+Lenti-ACE2 groups,and then the cardiac function was measured in four groups by echocardiography and catheterization.At last,the expression of Nrf2/HO-1/NQO-1pathway protein were detected in the RVLM by Western Blot.7.The continuous monitoring of spontaneous activities were recorded?ZT 0-12 and ZT 12-24?in LD and CL groups by wireless telemetry.Comparing the average activity counts in 24 hours between LD and CL groups was performed.The circadian secretion of melatonin and cortisol were tested by ELISA in ZT 0-12 and ZT 12-24 in LD and CL groups;the core clock gene Bmal1 and Clock oscillatory expression for 24 hours were tested by RT-PCR in the two groups.At last,the retrogradely labeled AAV-virus(AAVretro-cre-h Syn-EGFP)was injected into the RVLM in ROSA26-EGFP mice.The GFP expression of neurons was observed in the whole brain by whole brain sections continuously,which was subjected to confirm whether there is a primary fiber connection between RVLM and SCN.Results1.Constant light exposure elevated blood pressure and sympathetic nerve activity in normal ratsLight exposure resulted in the increasing of blood pressure?SBP,DBP?in rats with conscious state for one week?P<0.05 vs.LD?,and the NE,Ang II,GC,BNP and ANP were significantly increased in light exposure for four weeks.Light exposure for four weeks?CL4W?resulted in the increase of left ventricular systolic pressure?LVESP?,left ventricular diastolic pressure?LVEDP?and the cardiac fibrosis area,and the decrease of±d P/d T?P<0.05 vs.LD?.The LVESP and cardiac±d P/d T were decreased,and cardiac fibrosis area?%?was increased in light exposure for eight weeks?CL 8W??P<0.01 vs.LD?.Compared with CL 4W,the cardiac±d P/d T was further decreased and the cardiac fibrosis area was further increased in CL 8W?P<0.05 vs.CL 4W?.Meanwhile,the sympathetic activity was significantly increased in CL 4W?8.64±0.48vs.20.02±1.24%Max,P<0.001 vs.LD?,and there was no significant difference between CL 4W and LD?8.64±0.48vs.10.16±0.38%Max,P>0.05 vs.LD?.In summary,constant light exposure for four weeks led to the decrease in cardiac function to and caused sympathetic activation.2.Constant light exposure exerted detrimental effects on cardiac function in normal and heart failure ratsLight exposure increased LVIDs in normal rats,whereas decreased LVEF and LVFS?79.42±2.91%vs.93.64±2.02%;43.14±3.03%vs.63.24±3.91%,P<0.001 vs.LD?,and reduced+d P/d T?5751.89±69.01 vs.7477.08±604.2 mm Hg/s,P<0.01 vs.LD?.Light exposure resulted in further decrease in the+d P/d T in HF rats?2414.57±138.5vs.3211.90±202 mm Hg/s,P<0.05vs.HF-LD?,and further increase in cardiac fibrosis?P<0.0001vs.HF-LD?.The other cardiac function parameters showed no significant difference.The dynamic monitoring results of ejection fraction in HF-LD and HF-CL rats showed that light exposure resulted in accelerated decline in EF?P<0.001vs.HF-LD?.3.Constant light exposure resulted in the increase of sympathetic outflow and c-fos expression in the RVLM in normal and heart failure ratsLight exposure caused a significant increase in baseline RSNA?%Max?in normal rats?8.64±0.48 vs.20.02±1.24%Max,P<0.001 vs.LD?,and resulted in the further increase in RSNA in HF rats?20.10±1.16vs.26.82±1.69%Max,P<0.05 vs.HF-LD?.With respect to mean arterial pressure?MAP?,there was no significant difference between LD and CL in anesthetized state?P>0.05vs.LD?,whereas the MAP of HF-LD was significantly decreased?P<0.05vs.LD?.Light exposure led to a significant increase in plasma norepinephrine?NE?in normal and HF rats?P<0.0001vs.LD;P<0.0001vs.HF-LD?.Meanwhile,light exposure increased the number of c-fos positive neuron in the RVLM in normal and HF rats?20.01±0.01%vs.13.02±0.10%,44.66±0.01%vs.13.02±0.10%,P<0.01 vs.LD;81.53±0.01%vs.44.66±0.01%,P<0.0001 vs.HF-LD?.And the expression of c-fos increased?P<0.01 vs.LD?,whereas there was no difference between the HF-LD and HF-CL.4.Constant light exposure resulted in an increase of ROS levels and the down-regulation of ACE2/Ang1-7/Mas R axis in the RVLM in normal and heart failure ratsThe DHE staining results showed that constant light exposure increased ROS levels in the RVLM?P<0.05 vs.LD?.Western Blot results showed that the Nrf2 phosphorylation and its downstream HO-1 and NQO-1 expressions were decreased?P<0.05 vs.LD?.Meanwhile,light exposure decreased ACE2 and Mas proteins expression in normal and HF rats?P<0.001 vs.LD?,and the RVLM ACE2 expression was further decreased in HF rats?P<0.0001 vs.HF-LD?.There was no difference of ROS levels,Nrf2 phosphorylation,and Mas expression between HF-LD and HF-CL.5.The overexpresstion of ACE2 in the RVLM decreased the light exposure-induced oxidative stress and improved the damage of cardiac function through enhancing Nrf2phosphorylationAfter the overexpression of ACE2 in the RVLM,compared with CL+Lenti-GFP,both LVEF,LVFS?89.10±0.79%vs.74.92±0.73%,P<0.001;53.91±1.18%vs.38.42±0.63%,P<0.05?and±d P/d T?5368.00±35.55 vs.4043.24±7.83 mm Hg/s;-4648.88±168 vs.-3376.37±144.2 mm Hg/s,P<0.05?were increased.The ROS levels was decreased in CL+Lenti-ACE2?P<0.05 vs.CL+Lenti-GFP?,and the Nrf2 phosphorylation was increased in CL+Lenti-ACE2?P<0.0001 vs.CL+Lenti-GFP?.There was no significant difference about ROS expression in the RVLM between LD+Lenti-ACE2 and LD+Lenti-GFP in RVLM.The downstream of HO-1 and NQO-1 expressions in Nrf2 pathway was increased in LD+Lenti-ACE2?P<0.01 vs.LD+Lenti-GFP?.And the expressions of HO-1 and NQO-1was significantly increased in CL+Lenti-ACE2?P<0.05 vs.CL+Lenti-GFP?.6.Constant light exposure resulted in circadian rhythm disorders in the RVLMConstant light exposure resulted in an increase in activity counts in normal rats,but the activity difference of ZT 0-12 and ZT 12-24 was disappeared?P>0.05?.Whereas in LD,the activity counts in ZT 12-24 was more than that in ZT 0-12,and there was significant difference in activity counts between ZT 0-12 and ZT 12-24?P<0.05?.The diurnal difference of melatonin and corticosterone secretion were appeared in LD?P<0.001?,while there was no difference about melatonin and corticosterone secretion between ZT0-12 and ZT 12-24 in CL.Moreover,the plasma melatonin of ZT 12-24 was reduced in CL compared with LD,and the melatonin decreased in ZT 0-12 compared with ZT 12-24 in LD.In addition,the plasma corticosterone of ZT 12-24 was increased in CL compared with ZT 12-24 in LD.Compared with ZT 0-12 in LD,the plasma corticosterone was also increased in ZT 0-12 in CL.Meanwhile,light exposure resulted in the abnormal oscillatory expression of Bmal1 and Clock in the RVLM in CL?P<0.01 vs.LD?.7.The RVLM receives an indirect neuronal projection from the SCNThe unilateral RVLM was injected with retrogradely labeled AAV-virus(AAVretro-cre-h Syn-EGFP)in ROSA26-EGFP mice.After four weeks,the whole brain mapping results indicated that there was no GFP expression in SCN,and the GFP expression was located in PVN,DM,LH LC and DR.In summary,constant light exposure resulted in circadian rhythm disorder,and the RVLM is associated with SCN through PVN,DM,LH,DR and LC to produce indirect neural network connection.ConclusionConstant light exposure resulted in an increase of sympathetic outflow in the RVLM and a decrease of cardiac function in normal and HF rats.The overexpression of ACE2 in the RVLM reduced light exposure-induced oxidative stress and the cardiac function damage by enhancing antioxidant effect of Nrf2 phosphorylation.Light exposure may cause circadian rhythm disorders in the RVLM,and the RVLM may be associated with SCN through PVN,DM,LH,DR and LC to produce indirect neural network connection. |
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