Circadian Variations of Vasoconstriction and Blood Pressure in Physiology and Diabetes

Abstract

The intrinsic vascular smooth muscle contraction and vasoconstriction show time-of-day variations, contributing to the blood pressure circadian rhythm, which is essential for cardiovascular health. This brief review provides an overview of our current understanding of the mechanisms underlying the time-of-day variations of vascular smooth muscle contraction. We discuss the potential contribution of the time-of-day variations of vasoconstriction to the physiological blood pressure circadian rhythm. Finally, we survey the data obtained in the type 2 diabetic db/db mouse model that demonstrate the alterations of the time-of-day variations of vasoconstriction and the non-dipping blood pressure in diabetes.

Vasculature serves as the conduit that delivers blood to every cell and tissue in the body. The vessel’s radius, which is regulated by the vascular smooth muscle contractile state, is a dominant factor that controls blood flow, peripheral resistance, and blood pressure. The blood pressure exhibits circadian rhythm, with a higher level during the day and a lower value during the night. Disruption of blood pressure circadian rhythm is associated with a significantly higher risk of cardiovascular disease events [1]. While the circadian rhythms in neural and humoral systems are critical in regulating the physiological circadian rhythm of blood pressure, accumulating evidence indicates that the intrinsic responses of vasoconstriction and dilatation to the same stimuli also exhibit time-of-day variations. Such time-of-day variations in vasoconstriction likely significantly contribute to the physiological circadian rhythm of blood pressure. On the other hand, disturbance in vasoconstriction time-of-day variations likely contributes to the disrupted blood pressure circadian rhythm and increased adverse cardiovascular events. This brief review will summarize the current understanding of the mechanisms underlying and the role of molecular clocks in the time-of-day variations in vasoconstriction, its potential contribution to physiological blood pressure circadian rhythm, and their disruptions in diabetes.

Circadian rhythm is defined as a 24-hour oscillation that persists under constant environmental conditions, i.e., constant dark. Diurnal or nocturnal rhythms refer to 24-hour oscillation in the presence of natural synchronizer input, i.e., light. Since blood pressure and many other diurnal rhythms are found also to be circadian[2], we used the term circadian rhythm for blood pressure, regardless of external input. For the vasoconstriction, since a 24-hour oscillation was not verified due to the limited time points and time period of sampling, we used the term “time-of-day variation”.

The time-of-day variations of vasoconstrictions

Extensive evidence indicates the intrinsic vasoconstriction response varies throughout a 24-hour day. Numerous studies have demonstrated that when the aorta, mesenteric arteries, renal arteries are isolated from the animal at a different time during the day, the amplitude of constriction in response to the same stimuli is different. These include the responses to G-protein coupled receptor agonists phenylephrine (α1-adrenergic receptor agonist), 5-hydroxytryptamine, angiotensin II [3–8], and to high potassium depolarization-induced contraction [4, 7]. The difference in high potassium induced vasoconstriction suggests the involvement of a voltage-sensitive mechanism in the process. Notably, the time-of-day variations of vasoconstriction are also observed in vivo. The immediate increases in blood pressure in response to bolus vasoconstrictor injection exhibit time-of-day variations in humans [9, 10] and animals [7, 11]. Endothelium significantly contributes to the time-of-day variations in vasoconstriction. For example, eNOS phosphorylation and endothelium-dependent relaxation exhibit time-of-day variations [12, 13], which are impaired in diseases [14]. Inhibition of eNOS activity by L-NAME abolished the time-of-day contractile difference in isolated rat mesenteric arteries rings [6]. The important role of endothelium has been extensively studied and summarized in excellent reviews [15–17]. We have not identified any report that studied the specific contribution of the adventitial fibroblasts in the circadian rhythms of vasoconstriction and blood pressure. Accordingly, the current review will briefly present the mechanisms underlying the time-of-day variations in vascular smooth muscle contraction.

Mechanisms mediating the time-of-day variations of vasoconstriction

Smooth muscle contraction is primarily regulated by the reversible phosphorylation of the 20 kDa myosin light chain (MLC₂₀) and thin-filament mediated mechanisms [18, 19]. Until recently, it was unclear whether either of the two mechanisms was involved in the time-of-day variations in vasoconstriction. Recent evidence indicates an important role of MLC₂₀ phosphorylation. In cultured smooth muscle cells, the Thr-18 and Ser19-double MLC₂₀ phosphorylation exhibit time-of-day variations in response to thrombin [20]. In isolated mouse aorta, MLC₂₀ phosphorylation in response to thromboxane A2 agonist U46619 and non-hydrolyzable GTP analog GTPγS is higher at ZT0 (Zeitgeber Time, 0 is the light on time) than at ZT12 (light off time) [20]. In isolated mouse mesenteric arteries, MLC₂₀ phosphorylation increase is larger at ZT5 than at ZT17 in response to phenylephrine plus 5-hydroxytryptamine [8]. The MLC₂₀ phosphorylation and contraction in response to agonists stimulation may be mediated by enhanced cytoplasmic free Ca²⁺ or by an increase in the sensitivity of the contractile filaments to Ca²⁺, the so-called “calcium sensitization.” There is data suggesting the involvement of both pathways in regulating the time-of-day variations in the vasoconstriction.

Accumulating evidence indicates an important role of the rhoA-ROCK mediated calcium sensitization pathway in the time-of-day variations of vasoconstriction and MLC₂₀ phosphorylation. Saito et al. demonstrated that, in permeabilized vascular preparations, the Ca²⁺-induced contraction shows no time-of-day difference. However, GTPγS- as well as U46619-induced Ca²⁺-sensitization mediated contractions exhibits time-of-day differences [20]. This indicates the signaling pathway upstream of cytoplasmic free Ca²⁺ underlying the time-of-day variations in G-protein activation-induced contractions. The rhoA-ROCK-inhibition of myosin phosphatase is a critical pathway mediating agonist-induced calcium sensitization of contraction and MLC₂₀ phosphorylation in smooth muscle [18, 19], thus potentially involved in the G-protein activation-induced time-of-day variations of contraction. Indeed, ROCK2 mRNA, protein, and activity (based on the myosin phosphatase phosphorylation at Thr853) show time-of-day differences [8, 20]. Inhibition of ROCK with Y27632 in isolated mesenteric arteries abolishes the time-of-day differences in the contractile response to 5-HT [8].

The observation that high potassium depolarization-induced vasoconstriction exhibited time-of-day variations suggests voltage-dependent cytoplasmic free calcium fluctuations likely also contribute to the time-of-day variations of vasoconstriction. Voltage-dependent L-type calcium channel Cav1.2, a main Ca²⁺ influx pathway in vascular smooth muscle, plays a vital role in regulating vascular smooth muscle contraction. A recent study demonstrated that the activity and protein, but not mRNA, of Cav1.2, exhibits circadian variation, which mediates the time-of-day variations in cerebrovascular contractility [21].

Taken together, the time-of-day dependent vascular smooth muscle contraction is mediated by MLC₂₀ phosphorylation via the Ca²⁺-sensitization rho-ROCK pathway and the Cav1.2 mediated cytoplasmic free Ca²⁺ increase. These findings lead to the question: what is the upstream mechanism(s) that caused such time-of-day variations in the rho-ROCK pathway an d the calcium channel Cav1.2 level/activity. Recent evidence indicates the molecular clocks Bmal1 play a pivotal role, which will be discussed in the next section.

Molecular clocks control the time-of-day variations of vasoconstriction

The molecular mechanisms underlying the circadian rhythms are intrinsic clocks comprised of self-autonomous transcription-translational feedback loops. In mammals, Bmal1 (Aryl hydrocarbon receptor nuclear translocator-like protein-1) binds to Clock (Circadian Locomotor Output Cycles Kaput) or Npas2 (Neuronal PAS Domain Protein 2) to form a heterodimer that promotes the expression of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes. The Per and Cry proteins then form a heterodimer that translocates back into the nucleus and inhibits Bmal1 and Clock, thus, suppress their own transcription [22]. Another loop is composed of Bmal1, Rev-erbα (nuclear receptor subfamily 1, group D, member 1) and RORα (retinoic acid receptor-related orphan receptor alpha), in which Rev-erbα represses [23] while RORα stimulates Bmal1 [24].

The molecular clocks operate autonomously in every cell and tissue, including in vasculatures. The mRNA of the clock genes have been demonstrated to oscillate in the aorta, mesenteric arteries, cerebral arteries, and veins isolated at different times throughout a day [7, 25, 26]. Immunohistochemistry staining detected molecular clocks at the protein level in the endothelium, smooth muscle, and adventitia in the mouse vasculatures, with different temporal and cellular profiles depending on vasculature [27]. Using Per1 promoter-driven luciferase transgenic rats, Davidson et al. first demonstrated that the clock genes function in intact vascular tissues with a divergent phase of oscillation in different vasculatures [28]. Accumulating evidence suggests that the vascular clock genes control the time-of-day variations of the vasoconstriction.

How do the clock genes regulate the time-of-day variations of vasoconstriction? Studies reported clock gene Bmal1 works by modulating both the rho-ROCK Ca²⁺-sensitization and the Cav1.2 voltage-dependent calcium influx pathways. Bmal1 has been demonstrated to activate the ROCK2 promoter via binding to E-box motifs in a time-of-day-dependent manner in mouse mesenteric arteries [8]. The abolishment of the time-of-day variations of contraction by deleting smooth muscle Bmal1 is associated with eliminating the time-of-day variations in ROCK2 mRNA and activity [8]. A positive regulator of Bmal1, RORA, has also been demonstrated to regulate ROCK2 activity and Ca²⁺-sensitization-mediated contraction [20]. RORA mutant Stagger mice lose the time-of-day variations in Ca²⁺-sensitization mediated contraction and ROCK2 expression [20]. Besides, Bmal1 also regulates the Cav1.2 and Ca²⁺ influx pathway. Overexpression of Bmal1 in smooth muscle increases miR-103, which directly binds to 3’-UTR of the Cav1.2 α1C subunit, thus suppresses the Cav1.2 protein and activity [21]. Silencing Bmal1 in smooth muscle decreases miR-103 mRNA, increases Cav1.2 protein, and activity [21].

The circadian rhythm of the Bmal1 mRNA, protein, and activity controls, at least in part, the rho-ROCK and calcium pathways’ time-of-day variations. Bmal1 mRNA peaks during the later dark phase around ZT20 to ZT24 in the aorta, mesenteric artery, and cerebral artery [7, 21]. Bmal1 protein peaks early light phase around ZT0 to ZT4 [21]. The increased Bmal1 protein in the light phase correlates with the lower level of miR-103 and higher Cav1.2 protein and activity during the light phase [21]. Interestingly, the binding of Bmal1 to ROCK2 promoter is higher during dark phase ZT17 than during the light phase ZT5 [8], which is associated with higher ROCK2 mRNA and protein during the dark phase [7, 20]. Based on the current data with limited time resolution, Bmal1 protein level, and binding to ROCK peaks at a different time of the day. This suggests some additional modulatory mechanism that controls the Bmal1 binding activity in the vasculature. Indeed, numerous post-translational modification of Bmal1 has been reported [29].

Bmal1 also modulates vascular smooth muscle contraction indirectly via a paracrine mechanism. A recent study reported that selective deletion of Bmal1 in perivascular brown adipocyte suppresses angiotensinogen transcription and abolishes the PVAT (perivascular adipose tissue) extract’s ability to induce vasoconstriction [30]. This indicates clock Bmal1 plays a critical role in regulating the time-of-day variations of vasoconstriction via multiple direct and indirect mechanisms.

It is worth pointing out that additional molecular clocks are also involved in the regulation of the time-of-day variations of vasoconstriction. Clocks can regulate cellular processes as a transcription factor independent of their clock function. The prominent role of the molecular clock Bmal1 in the regulation of vascular contractility raises the possibility that Bmal1 may work as a specific transcription factor in this process. However, clock molecules Cry1/Cry2 also affects the time-of-day vasoconstriction, as the higher pressor response to phenylephrine injection during the light-phase is abolished in Cry1/Cry2 double knockout mice [11], suggesting the involvement of clock function.

Time-of-day variations of vasoconstrictions in physiological blood pressure circadian rhythm

Blood pressure rises during the early morning, maintains at a higher level throughout the day, followed by a decrease of about 10%–20% at night rest. An essential role of clocks in the normal blood pressure circadian rhythm is well established. Constitutively or conditionally global Bmal1 deletion [31, 32], clock D19 mutation [33], or global Cry1/Cry2 double deletion [11, 34] abolish or significantly diminish the blood pressure circadian rhythm (reviewed in [35, 36]). The loss of blood pressure circadian rhythm in the global Cry1/Cry2 double knockout mouse [11, 34] is associated with a loss of the time-of-day variations in vasoconstriction [11], suggesting an involvement of the time-of-day variations of vasoconstriction in the blood pressure circadian rhythm. However, these studies do not identify the specific contribution of the vascular clock and time-of-day variations of vasocontraction in blood pressure circadian rhythm. Our group developed a smooth muscle selective Bmal1-KO mouse model to address this specific issue. We found that the deletion of Bmal1 selectively from smooth muscle abolishes the time-of-day variations of vasoconstriction, which is associated with significant alteration in blood pressure circadian rhythm [8]. Smooth muscle Bmal1 deletion dampens the amplitude and advances the acrophase without affecting the period length of the blood pressure circadian rhythm [8]. While the pulse pressure circadian oscillation is also significantly dampened, the level of pulse pressure is significantly increased in the smooth muscle Bmal1 knockout mice due to the larger decrease in diastolic than in systolic pressure [8]. Of note, the heart rate circadian rhythm is unaltered, and the changes in blood pressure circadian rhythms in response to constant dark or constant light conditions are unaltered in the smooth muscle Bmal1 knockout mice [8]. Interestingly, endothelial selective Bmal1 deficiency (by ≈40% reduction in Bmal1 mRNA expression in endothelial cells) caused a moderate decrease in blood pressure at distinct time points within the active phase without affecting the 24-hour harmonic in blood pressure [37]. Brown adipose tissue-selective Bmal1 deletion decreased the blood pressure selectively during the light phase, resulted in a “superdipper” phenotype [30], which is associated with a loss of the perivascular adipose tissue extract-induced vasoconstriction. Together these results indicate that vascular clock and the time-of-day variations in vasoconstriction are essential for physiological blood pressure circadian rhythm.

When assessing the vasocontractility during the 12-hour resting and 12-hour active phase, higher vasoconstriction is usually observed during the resting phase in vivo [7, 11] and isolated vasculature [5–7]. Hemodynamic measurements in humans [38] and rat [39] also show that higher blood pressure during the active phase is associated with a decreased total peripheral resistance. This is surprising since the resting phase is a time of the day when the blood pressure is at a lower level. These findings suggest the relationship between the time-of-day variations of vasoconstriction and blood pressure circadian rhythm is complex rather than linear. This is not entirely unexpected since vasoconstriction-induced blood pressure change will trigger autonomic, renal, humoral and paracrine responses. For example, the spontaneous baroreflex sensitivity show time-of-day variations [8, 9, 11]. Functionally, the higher vasocontractility during the resting phase may serve to prevent blood pressure drop too low to ensure the appropriate tissue blood supply. The lower vasocontractility during the active phase may serve to prevent blood pressure rising too high thus causing adverse cardiovascular events.

Some limitations should be borne in mind with these interpretations. Firstly, the very limited time resolution of assessing vasocontractility during a day prevents evaluation of the precise temporal relationship between blood pressure and vasocontractility. It takes hours to measure vasocontractility, and freshly isolated tissues are required. Therefore, most studies determined the vasocontractility in two, sometimes four-time points within a 24 day. Secondly, the phase of the clock gene oscillation and likely the time-of-day variations in vasoconstriction are vascular bed specific. In summary, the mechanisms via which the vascular clock and time-of-day variations of vasoconstriction modulate blood pressure circadian rhythm are likely to be complex and remain to be established. Importantly, additional human studies are needed to verify further the significance of the time-of-day vascular reactivity variations and clock gene mutations in the non-dipping blood pressure and adverse cardiovascular outcomes.

The time-of-day vasoconstrictions and non-dipping blood pressure in diabetes

Cardiovascular complications remain a leading cause of the increased morbidity and mortality among people with diabetes mellitus [40]. Among the diabetic vascular abnormalities are endothelial dysfunction and enhanced vasocontractility [41]. Due to the brief nature of this review, we will summarize information obtained using the diabetic db/db mouse model on the clock gene, time-of-day variations in vasoconstriction, and non-dipping blood pressure.

The db/db mouse is an extensively used monogenic type 2 diabetes model in which an inactivation mutation in the leptin receptor leads to obesity, insulin resistance, and marked hyperglycemia [42]. In addition to the well-recognized endothelial dysfunction [43], it is well documented that vasoconstrictions are enhanced in the aorta and mesenteric arteries isolated from db/db mice compared with non-diabetic controls [44–47]. Potential mechanisms underlying the enhanced vasocontractility include an increase in cyclooxygenase dependent production of vaso-constrictive prostaglandins [44, 48, 49] and impaired perivascular adipocyte-derived anti-contractile adipokines [50, 51]. Of note, accumulating data implicates a contribution of the ROCK up-regulation and activation. ROCK mRNA, protein, and activity are increased in the mesenteric artery, coronary artery, and cerebral vasculature from the db/db mice compared to controls [50–53]. Inhibition of ROCK by fasudil or Y-27632 or H-1152 ameliorated the enhanced vasocontractility [51–53]. Interestingly, while the intracellular calcium release was enhanced in vascular smooth muscle from db/db mice, such enhanced calcium release does not contribute to the enhanced vasocontractility in the aorta or mesenteric arteries [54]. However, these studies did not pay attention to the time-of-day. It is unclear whether the animals were takedown at the same time of the day, and if so, what time of the day. Our group demonstrated that the normal time-of-day variations in vasoconstriction to phenylephrine, angiotensin II and high potassium depolarization are lost in aorta isolated from db/db mice [7]. Moreover, the time-of-day variations of the in vivo immediate pressor responses to phenylephrine and angiotensin II are also lost in db/db mice [7]. Such loss of the time-of-day variations in vasocontractility in the db/db mice is associated with alterations in clock gene expression level or their oscillation phases in various tissues [7, 55–60] including vasculature [7, 58, 60]. Consistent with the important role of ROCK2 in mediating clock gene regulation of the time-of-day variations in smooth muscle contraction, ROCK2 mRNA oscillations were abolished in the aorta and mesenteric arteries isolated from db/db mice [7]. This is associated with the loss of mRNA oscillation of several additional contractile regulatory proteins, including CPI-17 and SM22α in db/db mice [7].

In addition to the increased rate of hypertension, accumulating evidence demonstrated that the prevalence of blood pressure circadian rhythm disruption is much higher in diabetic subjects compared to non-diabetic subjects. Non-dipping blood pressure, defined as a less than 10% reduction in blood pressure from day to night, is the most common disruption of the blood pressure circadian rhythm. The prevalence of non-dipping blood pressure in type 2 diabetes is reported to be as high as 55% to 73% [61–64]. A large body of epidemiological evidence indicates that non-dipping blood pressure is associated with an increased risk of cardiovascular diseases and target organ damages in the heart, kidney, and brain [65]. We and others have found that db/db mice develop moderate hypertension and non-dipping blood pressure by 11–12 weeks of age using radiotelemetry [58, 66–69].

Multiple mechanisms likely contribute to the non-dipping blood pressure in diabetes. Disrupted clock function and circadian rhythm in one organ, for example, the loss of the time-of-day variations in vasoconstriction, could significantly contribute to the non-dipping blood pressure. In addition, recent evidence from a novel db/db-mPer2^luc mouse model suggests that the loss of synchrony among multiple blood pressure regulatory systems could also contribute to the non-dipping blood pressure in db/db mice [58]. The phase of the mPer2 daily oscillation was advance to different extents in the aorta, mesenteric artery, kidney, liver, and white adipose tissue explanted from the db/db-mPer2^luc mice [58]. Consistently, clock gene dysregulation is identified in multiple tissues, and altered circadian rhythms are reported in multiple physiological parameters, including heart rate, locomotor activity, food intake, and body temperature, sleep, and electroretinogram in db/db mice [69–72].

Potential contributions of disrupted circadian environmental cues to diabetes and non-dipping blood pressure

One point worth noting is that diabetes and clock gene disruption are bidirectional. While metabolic dysfunction in diabetes can disrupt the clock function, the clock gene mutation can lead to diabetes in humans [73–75] and rodents (reviewed in [76]). Light and food are two powerful environmental cues that entrain endogenous clocks. It is tempting to speculate that the higher rate of diabetes in modern society is related to increased light exposure during the night, a large number of shift workers, and frequent across-time zone travelers. Dark phase restricted feeding can correct liver Bmal1 oscillation [55], and a small molecule nobiletin targets the molecular oscillator was reported to enhance circadian rhythms and protects against metabolic syndrome in db/db mice [77]. It will be interesting to investigate whether these strategies would improve the functions of intrinsic clocks, vascular contractility, blood pressure circadian rhythm, and cardiovascular health in diabetes.

Summary and future directions

The intrinsic contractility of the vascular smooth muscle exhibits time-of-day variations. The circadian oscillating activity of the Bmal1 controls such time-of-day variation in smooth muscle contractility. Clock Bmal1 activates ROCK2 and Cav1.2, consequently results in MLC₂₀ phosphorylation and contraction. The vascular clock and the time-of-day variations in vasocontractility play an important role in physiological blood pressure circadian rhythm. The disruption of vascular clock oscillation and loss of the time-of-day variations in vasocontractility are associated with the non-dipping blood pressure in diabetic db/db mice.

In addition to further dissect the mechanisms underlying the time-of-day variations of vasocontractility and its contribution to the blood pressure circadian rhythm, many important questions remain to be addressed. How are the vascular clock and contractility entrained or adjusted in response to night-shift work or cross-time zone travel? In diabetes, what causes the dysregulation of the vascular clock and time-of-day variations of vasocontractility? Would modulating circadian rhythm improve the cardiovascular outcomes in individuals with diabetes? Furthermore, in addition to regulating vasoconstriction and dilatation, a body of literature demonstrated that vascular clocks play a critical role in vascular diseases, including atherosclerosis, restenosis, and aortic aneurysm, etc. With the increased appreciation of the fundamental significance of circadian rhythm in physiology and the availability of various genetic vascular clock animal models, we expect a large amount of new information about these topics will be forthcoming in the coming years, which could provide the knowledge that leads to novel strategies to improve cardiovascular health.