L8: Circadian Rhythms and Hypertension

Introduction and Learning Objectives

• This lecture focuses on the intersection of circadian rhythms and hypertension, which means how our body’s internal clock relates to high blood pressure. It’s divided into two distinct parts to make the information easier to understand.

• Learning Objectives: We aim to:

  • Discuss the importance of circadian rhythms—these are physical, mental, and behavioral changes that follow a daily cycle—and how they relate to blood pressure, with a focus on why this matters in real life (Part 1).

  • Describe how the molecular circadian clock works and what role it plays in controlling blood pressure (Part 2).

  • Provide examples from research studies that show how specific proteins involved in our body’s internal clock can influence blood pressure regulation (Part 2).

• Structural Overview: In Part 1, we will start with an introductory case study titled "A Tale of Two Postdocs," which sets the stage for understanding rhythms in humans. We will discuss how these rhythms are clinically relevant and how we measure them. Part 2 will dive deeper into the molecular mechanisms—essentially the biology—and provide evidence on how clock proteins are important for regulating blood pressure.

The Discovery of Circadian Variation in Proteins: A Tale of Two Postdocs

• Historical Context: This story begins in the laboratory of Dr. Uli Schibler, a scientist at the University of Geneva, back in the early 1990s. His team made a pivotal discovery related to how our body clock works.

• The Initial Finding (1990): A groundbreaking paper was published in the journal Cell, which explained the discovery of a new transcription factor named DBP. A transcription factor is a protein that helps turn specific genes on or off. They found that DBP’s expression—or how much of the protein was available—happened specifically in the liver and that this expression was controlled after the messenger RNA (mRNA) was formed and not while the gene was being copied.

  • The first postdoc who worked on this research was successful in identifying and characterizing this protein, meaning they helped prove what the protein was and what it did.

• The Conflict: A second postdoc joined the lab, aiming to build upon the first’s work. They performed experiments using a method called Western blots to detect DBP in the samples collected but couldn’t find the protein signal.

• The Resolution via Time Course Study: The second postdoc did something smart—they performed a time series study, which means they collected mouse liver tissue every 4 hours throughout the day, such as at $8:00 AM$, Noon, $4:00 PM$, $8:00 PM$, Midnight, and $4:00 AM$. This method would help them see how the expression of DBP changes over time.

  • Protein Staining (Coomassie): This initial step showed that overall protein levels seemed to stay pretty constant throughout the day and night.

  • Western Blot (Anti-DBP Antibody): However, when they specifically looked for DBP using antibodies (which are like tiny flags that help us identify proteins), they found that DBP expression was actually circadian (meaning it followed a regular cycle). They only detected the protein at certain times, particularly at $4:00 PM$ and $8:00 PM$.

  • Immunofluorescence: They examined liver cross-sections and noticed strong staining for DBP at $8:00 PM$ but no signal at $9:00 AM$, which meant that if they didn’t collect samples at the right time, they wouldn’t find the protein.

• The Explanation: The first postdoc was someone who worked like a “night owl,” starting their day at noon and collecting samples in the late afternoon, when DBP was easily identifiable. The second postdoc, on the other hand, was an “early bird” who came in at 7:00 AM and collected samples at 8:00 AM—a time when the protein simply wasn’t present. This conflict highlighted how biological timing can influence scientific research outcomes.

• Impact: This investigation expanded Dr. Schibler’s lab into circadian rhythm research, showcasing how the timing of biological processes can affect the reliability and reproducibility of scientific studies, meaning that understanding time can help ensure accurate results.

The Nobel Prize and the Molecular Foundation of Circadian Rhythms

• The 2017 Nobel Prize in Physiology or Medicine: This prestigious award recognized the work of three scientists—Jeffrey Hall, Michael Rosbash, and Michael Young—for their research on circadian rhythms.

• Model Organism: Their studies were carried out using Drosophila, commonly known as fruit flies, which are used in scientific research because they have a short lifecycle and their genetics are easier to manipulate.

• Primary Discovery: They were the first to identify a gene that influences circadian rhythms, naming it period. This gene plays a critical role in maintaining the body’s daily cycle.

The Human 24-Hour Physiological Clock

• Our bodies have a natural 24-hour clock that affects many functions, peaking at different times each day:

  • Early Morning: This is when cortisol, a stress hormone, is released, contributing to the highest increase in blood pressure during what is known as the morning surge.

  • Late Morning: During this time, people experience their peak alertness, which affects their ability to think and perform tasks.

  • Late Afternoon: Coordination is at its best, meaning people can respond to tasks more quickly.

  • Evening: Body temperature and blood pressure peak during late afternoon to early evening, showing that energy levels are higher at this time.

  • Night: Blood pressure drops during sleep, and the pineal gland begins to secrete melatonin, which helps regulate sleep patterns and is often referred to as the "dark hormone."

• Melatonin: This hormone is a key marker in studies of circadian phases and shows how our body knows when to sleep.

Predictive Homeostasis and the Evolutionary Advantage

• The Concept: This term was coined by researcher Martin Moore-Ede in the 1980s. He introduced it to explain how our internal clock helps us adapt to environmental changes.

• Definition: It suggests that our body’s clock evolved to give organisms a better chance of survival by predicting daily changes in light and darkness, which can affect lifestyle and behavior.

• Evidence in Renal Physiology: Moore-Ede showed that kidney functions such as urine production and the excretion of important minerals follow strong circadian rhythms. This means our bodies are tuned to operate efficiently.

• The Gut Clock Example: If a person plans to wake up at $6:00 AM$ and have breakfast at $7:00 AM$, their gastrointestinal (GI) system starts preparing early. It increases the production of enzymes and transporters that help absorb nutrients, meaning our bodies are ready to digest food more effectively when we eat.

Circadian Disruption and Clinical Pathologies

• Modern Disruption: In today’s world, things like shift work, where people work at different hours than what is typical, and exposure to bright artificial light at night can disturb these circadian rhythms.

• Risk Factors: Not following normal circadian rhythms can increase the risk for:

  • Chronic Kidney Disease (CKD).

  • Hypertension (high blood pressure).

  • Cardiovascular Disease (CVD).

  • Metabolic Syndrome, which involves a cluster of conditions that raise the risk of heart disease and diabetes.

• Bidirectional Relationship: It’s important to note that while a disrupted circadian rhythm can lead to health issues, existing health problems can also worsen these rhythms. This creates a “vicious cycle” where one condition exacerbates the other, making it harder to escape the negative health effects.

Measuring and Categorizing Blood Pressure Rhythms

• Normal Dipping Pattern: Clinically, this is defined as a drop in blood pressure during sleep that is at least $10\%$ lower than the blood pressure during daytime. This is considered healthy.

• Ambulatory Blood Pressure Monitoring (ABPM): A method used in clinical settings involving a wearable cuff that measures blood pressure automatically at regular intervals—every 15 minutes when awake and every 30 minutes at night for 24 hours.

• Dipping Classifications and Survival Risks:

  • Dippers: Those who achieve a normal $10\\%$ dip in blood pressure at night are associated with the lowest risk for cardiovascular events, meaning they have the best heart health.

  • Extreme Dippers: They experience a larger $20\\%$ difference in blood pressure between night and day.

  • Non-dippers: Fail to achieve the necessary $10\\%$ dip. This failure is linked to a greatly increased risk of heart failure and higher cardiovascular mortality rates.

  • Reverse Dippers (Risers): These individuals actually have higher blood pressure at night than during the day, placing them at the highest risk for severe cardiovascular events like strokes or heart attacks.

Key Clinical Studies on Circadian Blood Pressure

• JAMA (2006): A significant study published in the journal JAMA showed that elderly men who were non-dippers had much lower chances of surviving without heart failure starting about four years into the study.

• Hemodialysis Patients: Another study found that patients receiving treatment for end-stage renal failure who were classified as non-dippers had a noticeable separation in heart health outcomes after 2.5 years, with non-dippers showing significantly worse results.

• Resistant Hypertension: Patients taking multiple antihypertensive medications who exhibited a non-dipping pattern experienced a significant decline in heart health compared to those who were dippers.

• Italian Cohort Study: In a study that followed over 3,000 patients with untreated essential hypertension for 20 years, researchers confirmed that reverse dippers had the highest rate of adverse health events, with non-dippers being next, followed by dippers and extreme dippers who faced the lowest risks.

Technological Advances: Wearables and AI

• The Rise of Wearables: Devices like smartwatches and fitness trackers can now monitor not just heart rate, but also heart rhythm, variability, and even breathing rates. This technology represents a significant advancement in personal health monitoring.

• Clinical Applications: They are being used for telemonitoring (keeping track of someone's health remotely), diagnosing diseases, and guiding people on necessary physical activities to maintain optimal health.

• Artificial Intelligence (AI) and Machine Learning: As these devices collect large amounts of data, AI and machine learning become crucial for effectively managing and interpreting all this information. An example is a study from 2025 in Biosensors, where a research group from China used circadian heart rate variability (HRV) data combined with machine learning techniques to estimate important heart function levels in patients with high blood pressure.

• Circadian Medicine: This is an emerging field focused on integrating data from these wearables into healthcare systems to improve how health data is managed and set clinical guidelines, allowing for better patient care.

Factors Influencing the Circadian Rhythm of Blood Pressure

• Demographics: Factors such as age and body mass index (BMI) influence how blood pressure rhythms behave in different individuals.

• Physiology: Overall blood pressure levels, how sensitive one is to changes, activity from the sympathetic nervous system (which governs body responses to stress), and the levels of hormones like Angiotensin II and Aldosterone, are all part of this dynamic and follow their own cycles. Additionally, the presence of Atrial Natriuretic Peptide (ANP) affects how our blood pressure changes throughout the day.

• Behavior: Individual behaviors, including physical activity, mental stress, and even how we position our bodies during sleep or waking hours, directly impact these rhythms.

• Environment: External factors such as a diet high in salt or smoking habits also play a significant role in how our circadian rhythms function.

• Comorbidities: Conditions like diabetes and Chronic Kidney Disease (CKD) can markedly raise the risk of experiencing a non-dipping blood pressure pattern, indicating worsening overall health and clock function.

Consequences of Rhythm Loss

• Exaggerated Morning Surge: This refers to a drastic increase in blood pressure when waking up. This surge is often linked to a higher frequency of heart attacks and strokes occurring in the morning hours, which can lead to severe health outcomes.

• Blood Pressure Variability: This means that blood pressure can fluctuate greatly throughout the day, which is not ideal for heart health.

• Mechanisms Contributing to Loss of Rhythm:

  • Increased sodium retention in the body can affect blood pressure levels negatively.

  • Over-activation of the sympathetic nervous system can lead to higher blood pressure.

  • Over-activation of the Renin-Angiotensin-Aldosterone System (RAAS) contributes to stress and pressure on the heart and blood vessels.

  • Additional causes include over-activation of a hormone called Endothelin, arterial stiffness, inflammation, oxidative stress, and dysfunctions in the cells lining the blood vessels.

• Target Organ Damage: When there’s a loss of the nighttime dip in blood pressure, it reduces the body’s recovery phase, hence increasing damage to various organs and accelerating diseases related to the heart and kidneys.

• The circadian system is organized hierarchically throughout the body, as depicted in a review article published by the speaker's group in 2022. This means there are different levels or layers of biological clocks that interact with each other.

• The Central Clock: Located in the suprachiasmatic nucleus ($SCN$), which lies within the hypothalamus of the brain. The SCN is like the master clock of the body, controlling the timing of various biological processes by sending signals to other parts of the brain and body.

• Entrainment Mechanisms: The central clock is directly entrained by light cues transmitted through the retinal hypothalamic tract directly to the $SCN$. This means that the SCN uses light from the environment to adjust its timing. For example, daylight helps the body know it's time to be awake and alert.

  • Non-photic cues include food intake and exercise. Other factors, like when we eat or how much we move, can also influence our internal clock.

• Signal Transmission: In response to these cues, the central clock sends out signals via hormonal and neuronal signaling to synchronize peripheral clocks. These signals help different parts of the body stay in sync with the central clock, ensuring that all systems are working together efficiently.

• Peripheral Clocks: Nearly every cell type in the body contains a molecular clock. Specific tissues mentioned include:

  • Liver

  • Kidney

  • Adrenal glands

  • Heart

  • Lungs
    These peripheral clocks help regulate various functions in specific tissues according to the time of day, so our body can respond appropriately depending on whether it’s daytime or nighttime.

• Output Rhythms: These cell-autonomous clocks generate circadian rhythms in physiological functions across the entire organism. This means each cell's clock helps maintain the timing of important processes, like metabolism and hormone release, which can vary based on the time of day.

Aldosterone Synthesis and Rhythmic Patterns

• Aldosterone is a hormone secreted by the adrenal gland that exhibits a clear circadian rhythm in humans. This means that the levels of aldosterone fluctuate during the day, rising and falling in a predictable pattern.

• Healthy Human Pattern: Humans are diurnal, meaning they are more active when lights are on. Being diurnal means we are designed to function best during the daytime, with sleep at night.

• Temporal Dynamics of Aldosterone:
  

  • Levels are higher in the morning (around $08:00$). This is when our body starts to wake up and get active.

  • Levels decline throughout the day. As the day progresses and we begin to tire, levels of aldosterone decrease.

  • Levels peak as the individual is about to wake up (near the end of the sleep cycle/midnight to early morning phase). Just before waking, our body ramps up certain hormones like aldosterone, preparing us for the day ahead.

The Molecular Clock Mechanism and Transcription-Translation Feedback Loop

• The primary function of the molecular clock is the regulation of gene expression. This means that our internal clocks control when and how genes are turned on or off, which ultimately influences the production of proteins within our cells.

• The Positive Arm:

  • Clock-controlled genes contain sequence-specific DNA elements called E-box response elements. These are specific sequences of DNA where clock proteins can bind to influence gene activity.

  • Two clock proteins, $BMAL1$ and $CLOCK$, heterodimerize (bind together). These proteins must work together to regulate gene activity effectively.

  • This heterodimer interacts with the E-box response elements to drive the transcription of clock-controlled genes. When bound to these elements, they enhance the production of certain genes that help maintain our circadian rhythms.

• The Negative Arm:

  • Two of the genes transcribed by the positive arm are Period ($PER$) and Cryptochrome ($CRY$). These genes play an essential role in the feedback loop that keeps our clock accurate.

  • These are translated into proteins in the cytosol. Once the genes are expressed, their messenger RNA is translated into proteins in the cell's cytoplasm.

  • $PER$ and $CRY$ proteins interact with each other and translocate back into the nucleus. After functioning in the cytoplasm, these proteins return to the nucleus to take part in regulating the transcription process again.

  • Inside the nucleus, they inhibit the actions of $BMAL1$ and $CLOCK$, thereby inhibiting their own transcription. By doing this, $PER$ and $CRY$ provide a self-regulating mechanism that maintains a consistent pace of the circadian cycle.

• Cycle Duration: This feedback loop repeats every $24\, hours$. This regularity ensures our biological processes occur in sync with the day-night cycle.

Clock-Controlled Genes (CCGs) and Hypertension Targets

• The function of the clock is tissue-specific. Some genes regulated by the molecular clock are called clock-controlled genes (CCGs), and they play various roles depending on the tissue. Types of CCGs include ion channels, transporters, enzymes, receptors, transcription factors, and signaling molecules.

• Examples Relevant to Hypertension Treatment:
  

  • $NCC$ (Thiazide-sensitive sodium chloride cotransporter): An ion transporter in the distal convoluted tubule of the kidney. This transporter helps control sodium levels in the body and is a target for thiazide diuretics, which are used to treat high blood pressure.

  • $ACE$ (Angiotensin-converting enzyme): Located in the lungs; it is the target for ACE inhibitors. ACE plays a significant role in regulating blood pressure, and blocking it can help lower hypertension.

  • Mineralocorticoid Receptor: Regulated by the clock mechanism, specifically shown in human vascular tissues (blood vessels). This receptor is crucial for managing how the body handles salt and water, which directly affects blood pressure.

Species Differences: Diurnal Humans vs. Nocturnal Rodents

• Humans (Diurnal): Awake and active when lights are on. Our bodies tend to follow a schedule where we have our most energy during the day. Blood pressure peaks during the active phase (noon) and dips during the rest phase (midnight). This means our blood pressure is typically highest when we are active in the daytime and low when we are at rest during the night.

• Mice and Rats (Nocturnal): Awake and active when lights are off. Their physiological rhythms are inverted compared to humans. For mice, the active period is at night ($midnight$ on a clock diagram), and the rest period is during the day ($noon$). Because of this, their biological functions, including blood pressure, are also opposite to those of humans.

• Impact on Research: It is critical to account for this inversion when translating preclinical data from rodents to humans. Research findings in rodents may not apply directly to humans because of these differences in activity patterns and resultant biological rhythms.

Methodology in Blood Pressure Monitoring

• Ambulatory Blood Pressure Monitoring (Humans): - Humans wear a blood pressure cuff that measures pressure throughout the day and night during regular activity. This method helps track how blood pressure changes with daily activities.

  • This is used to identify non-dipping hypertension, which increases cardiovascular risk. Non-dipping means blood pressure does not fall as expected during nighttime rest, which is a risk factor for heart disease.

• Radio Telemetry (Mice/Rats): - The gold standard for measuring blood pressure in preclinical models. This method allows for continuous blood pressure monitoring in small animals.

  • A radio telemetry device is surgically implanted; a probe is threaded through the carotid artery so the tip rests in the free-flowing blood of the aorta. This allows for accurate reading of blood pressure in real-time.

  • Capabilities: Continuous recording of heart rate, blood pressure, pulse pressure, and activity levels. This detailed monitoring provides more comprehensive data on the animal's cardiovascular health.

  • Cost: Extremely expensive; individual transmitters cost well over $\$3,000$ each. A study with $10$ to $20$ mice is a significant financial venture. This high cost can limit the number of studies that can be conducted.

Primary Literature: Clock Gene Knockouts and Phenotypes

• Every Clock-gene knockout mouse studied exhibits a blood pressure phenotype. This means that researchers have found distinct blood pressure characteristics when specific clock genes are disabled in these mice.

$BMAL1$ Knockout

• Phenotype: Lower blood pressure compared to wild-type controls. This suggests that when the $BMAL1$ gene is lacking, blood pressure tends to decrease.

• Circadian Rhythms: Exhibited as a "non-riser" pattern (blood pressure stays low during the night instead of rising during the active phase). A typical pattern would see blood pressure increase when awake, but this pattern is disrupted in these mice.

• Additional Traits:
  

  • Lower aldosterone levels during the active period (night). This hormonal drop may contribute to the observed low blood pressure.

  • Renal sodium handling defect (sodium wasting/increased excretion in urine). This means their kidneys might not be processing sodium correctly, impacting hydration and blood pressure.

  • Vascular defects (Curtis et al., 2007). This refers to issues with blood vessel structure or function that may affect how blood flows through the body.

$CLOCK$ Knockout

• Phenotype: Lower blood pressure. Similar to the $BMAL1$ knockout, this gene's absence is linked to reduced blood pressure.

• Additional Traits:
  

  • Lower aldosterone levels. Again, this hormonal decrease may connect to their blood pressure problems.

  • Wasting of sodium in the urine. This suggests improper regulation of sodium, leading to further complications in blood pressure.

  • Circadian defect: Not apparent in initial studies. While early studies didn't reveal clear rhythms, further investigation may uncover more.

  • Vascular defect: To be determined ($TBD$). Research is ongoing to fully understand how this knockout affects blood vessels.

$CRY1$ and $CRY2$ Double Knockout

• Phenotype: Higher blood pressure that is salt-sensitive. This indicates their blood pressure rises unusually in response to salt intake, a troubling sign for cardiovascular health.

• Additional Traits:
  

  • Higher levels of aldosterone. Elevated aldosterone may contribute to their blood pressure issues.

  • Disrupted circadian rhythm. Their internal clock is not functioning as it should, leading to fluctuations in blood pressure across the day.

  • No vascular or kidney defects described yet. More research is needed to determine if there are any issues in these organs.

Period 1 ($PER1$) Knockout

• Phenotype: Higher blood pressure in response to salt and an aldosterone analog. This suggests a strong link between this gene’s function and how the body controls blood pressure with dietary salt.

• Renal Handling: Altered night-to-day ratio of urine sodium excretion. These mice might handle sodium very differently based on the time of day.

• Vascular Phenotype: Vascular stiffening observed in male mice (unpublished data from the speaker's lab). Changes in blood vessel structure can affect how well blood flows.

• Circadian Defect: Non-dipping hypertension. Again, this refers to their blood pressure failing to drop as it should during the night, which can lead to health problems over time.

Time of Day as a Key Biological Variable

• In a study of global $BMAL1$ knockout mice, blood pressure was measured using zeitgeber time ($ZT$). Zeitgeber time is a way to measure time based on environmental cues, like light.

• $ZT\,0$: Lights on ($6\,AM$). This marks the beginning of the active day for these mice.

• $ZT\,12$: Lights off ($6\,PM$). This indicates when the lights go out and the mice expect to rest.

• Misinterpretation Risk: If researchers only measured blood pressure at one time point, such as $4\,PM$ ($ZT\,10$), they would see no genotype difference between wild-type and knockout mice. Only measuring at one time can lead to misleading conclusions about the effects of gene knockout.

• Conclusion: Continuous $24\, hour$ monitoring is essential to avoid erroneously concluding that a genotype has no effect on blood pressure. To really understand how these genes impact blood pressure, it's important to observe them over a full day.

• Implications: Limiting preclinical studies to "typical working hours" (when mice are resting) can miss critical phenotypes that only appear during the active phase. This highlights the need for thorough data collection throughout the day and night.

Smooth Muscle Specific $BMAL1$ Knockout (Gong et al., 2015)

• Researchers used Cre-Lox technology to generate a smooth muscle-specific knockout of $BMAL1$. This advanced technique allows scientists to study the function of genes in a specific tissue type.

• Findings:

  • Blood pressure is lower in knockouts compared to wild-type. This finding underscores the importance of $BMAL1$ in regulating blood pressure.

  • The magnitude of the "dip" (night-day difference) is significantly decreased in the knockout mice. Their usual drop in blood pressure at night is not as pronounced, indicating a disruption in their natural rhythm.

• Significance: This study proved that $BMAL1$ functions specifically within smooth muscle cells to regulate the overall level and the circadian rhythm of blood pressure. Understanding where and how BMAL1 works can help researchers find new treatments for blood pressure issues.

Cryptochrome Double Knockout Study (Okamura et al., 2010)

• Study Design:

  • Used $CRY1$/$CRY2$ double knockout mice. This combination of knockouts allows for a clearer understanding of their impact on health.

  • Evaluated blood pressure on normal salt and high salt diets. This helps see how dietary changes affect blood pressure in these mice.

  • Applied the mineralocorticoid receptor inhibitor Eplerenone. This is a drug that blocks the action of aldosterone to see how it interacts with the lack of cry genes.

• Results:

  • High salt diet caused significantly elevated blood pressure in knockouts, but not in wild-type mice (who are salt-resistant). This indicates that knockouts react poorly to salt, highlighting the importance of the cry genes in regulating blood pressure.

  • Eplerenone brought knockout blood pressure back toward normal, suggesting an aldosterone-driven mechanism. This result reinforces the idea that aldosterone plays a critical role in controlling blood pressure, especially when clock genes are disrupted.

• Adrenal Transcriptomics:

  • A gene called $HSD3B1$ in mice ($HSD3B6$ in humans) was dramatically upregulated in the adrenal glands of the knockouts. This indicates that without the cry genes, other processes in the adrenal glands go awry.

  • Other enzymes like $STAR$ or $CYP11B2$ (aldosterone synthase) were not significantly changed. This suggests that the pathways regulating aldosterone production may become overactive in the absence of clock genes.

• Conclusion: Absence of $CRY$ proteins results in the over-expression of a critical enzyme for aldosterone production, leading to salt-sensitive hypertension. This finding helps establish a link between circadian biology and blood pressure regulation, showing how timing affects our health.

Period 1 ($PER1$) Knockout and Sex Differences

• Data from the speaker's lab utilized $C57BL/6$ mice with global $PER1$ knockout. This specific strain of mice is commonly used in research because of its well-understood genetic background.

• Experimental Treatments: High salt diet plus injection of DOCP (desoxycorticosteroid), a long-acting aldosterone analog. This combined treatment helps identify how the lack of $PER1$ affects blood pressure regulation under stress.

• Male Mice Findings:

  • On a normal diet, blood pressure is relatively normal. This suggests that male mice can handle a regular diet without issues.

  • On high salt + $DOCP$, male knockouts exhibit non-dipping hypertension. This means their blood pressure does not drop at night as it should.

  • The calculated dip in male knockouts was only $5.7\%$, compared to over $11\%$ in wild-type males ($10\%$ is the clinical threshold for a normal dip). This demonstrates how critically the $PER1$ gene works to maintain healthy blood pressure patterns.

• Female Mice Findings:

  • Female wild-type and knockout mice both exhibited healthy dipping patterns ($12\%$ to $13\%$ dip). This means that the female mice maintained a normal blood pressure rhythm regardless of the gene status.

  • Female mice remained resistant to the non-dipping phenotype caused by high salt and $DOCP$. This shows potential differences between the sexes in blood pressure regulation related to their circadian clock.

• Clinical Connection: A $2009$ study in the Canadian Journal of Cardiology found that human women are $16$ times more likely to develop non-dipping hypertension post-menopause than pre-menopause. This highlights the importance of studying sex differences in hypertension and how age-related hormonal changes can influence risk.

• Take-home Point: Age and sex are critical biological variables. Preclinical studies must control for age and cycling status to accurately model human circadian defects in blood pressure. Understanding these variables can lead to better treatments and insights for both men and women experiencing high blood pressure.