How cellular clocks regulate cardiac physiology

In this interview, News-Medical talks to research assistant Dr. Alessandra Stangherlin on her latest research that provided new insight into the circadian rhythm of the heart.

Can you introduce yourself, tell us about your background in molecular biology, and what inspired your latest research into circadian rhythms?

My name is Alessandra Stangherlin and I have a long-standing interest in cellular circadian rhythms. I devoted the last five years of my career to studying how osmotic homeostasis is maintained during the circadian cycle.

Early observations showed that the amount of soluble cytosolic protein has a circadian rhythm with a change of 20%. If left unchecked, such a change in the intracellular amount of macromolecules can affect the osmotic potential of the cytosol, triggering the compensatory movement of water with negative consequences for cell viability.

I found that mammalian cells import and export Na, K and Cl at a 24 hour rate to compensate for the changes in cytosolic protein. This homeostatic control mechanism allows the cells to maintain a constant cell volume and maintain cell viability. In cardiomyocytes, the variation in ionic content provides an inherent daily rhythm of firing rate.

What is meant by the term ‘circadian rhythm’?

The adjective “circadian” is derived from the Latin words “circa” (around) and “door” (day). A circadian rhythm is a behavioral, physiological or cellular phenomenon that is repeated every 24 hours. Circadian rhythm provides an opportunity to synchronize an organism’s physiology with the outside world and the expectation of daily environmental changes in light, temperature and food availability.

Many aspects of human physiology have a circadian nature, such as sleep / wake cycle, release of more hormones and cellular metabolism. Very important, although they can be synchronized via external inputs, circadian rhythms are self-supporting and occur even in the absence of external timing signals.

circadian rhythmImage credit: VectorMine / Shuuterstock.com

Previously, how did we believe that the heart rhythm worked?

Pulse circadian rhythm has been known for decades. In healthy individuals, the heart rate rises in the morning and decreases during the night. Until recently, the circadian regulation of heart rate was mainly attributed to non-cell autonomic mechanisms controlled by the central clock located in the brain via sympathetic and parasympathetic modulation.

However, the contribution of cellular clocks has never been studied. We now show that a cell-autonomous clock in cardiomyocytes regulates the heart rate independently of the central nervous system and systemic signals. In fact, action potential of firing rate changes between day and night in isolated cardiomyocytes in culture. More importantly, we found that the daily variation in HR continues in vivo during autonomous blockade.

We also thought that the cellular ion concentrations involved in the circadian rhythm were fairly constant. Why has your research contradicted this theory, and how do these levels actually vary?

We have all learned from textbooks that the intracellular concentration of Na is about 10 mM, the concentration of K is about 140 mM, and we generally assumed that they remain fairly constant. We were surprised by our results, which showed that the intracellular concentration of many ions changes during the day by about 20-30%.

Our model suggests that the net imports and exports of Na, K and Cl change during the circadian cycle, leading to changes in the intracellular concentration of these ions. We have identified the SLC12A family of co-carriers as key players in this process, but we do not rule out the possibility that other carriers such as VRAC may also play a role.

Can you describe how you conducted your latest research into circadian rhythms?

We used various state-of-the-art techniques, from live cell microscopy and quantum dot detection to assess cytosolic crowding to microelectrode array technology to measure action potential firing rate. The key to detecting ion rhythms was to use an analytical technique called inductively coupled plasma mass spectrometry (ICP-MS). This technique can be used to determine the elemental composition of a sample with a high degree of sensitivity and specificity and overcomes the disadvantages of current colorimetric analyzes and electrophysiological methods.

How has this changed the way we think about heart-circadian rhythm?

This work has further confirmed the importance of cellular circadian rhythms in the regulation of basal cell functions and cardiac physiology. The 24 hour rhythm of Na, K and Cl abundance we have described has an important physiological implication as it affects the electrochemical gradient of these ions across the plasma membrane. In cardiomyocytes, this modulates the depolarization phase of the action potential, leading to increased firing rate when intracellular ions are high (end of night) and a decrease in the firing rate of action potential when ions are low (end of day).

Adverse cardiovascular events such as stroke, myocardial infarction, and sudden cardiac death occur with a higher incidence in the morning, but the underlying causes are unknown. Our data suggest that any deterioration of the buffer mechanism we have described may make the heart more vulnerable to stress in the morning when a change in demand is required.

How can you see that your research is influencing future treatment options for cardiovascular conditions?

Future treatments may involve pharmacological or behavioral therapies to maintain our circadian rhythm. For example, maintaining a healthy daily routine, which includes keeping the same sleep pattern, avoiding bright light before bedtime and avoiding eating at night, can help keep our body clocks in sync.

cardiovascular conditionsImage credit: BRO.vector / Shutterstock.com

Your research has also helped explain why shift workers are more vulnerable to heart problems. What preventative measures can shift workers take?

Our work and others suggest that shift workers become more vulnerable to heart problems due to a mismatch between the brain clock and the heart clocks. The brain clock is very sensitive to light from the surroundings, and therefore one must be very careful with light exposure.

There are various strategies that night shift workers could put in place to synchronize their body clocks to the new schedule. Most of these rely on adopting a regular sleep and light routine according to their chronotype (i.e., night owls may prefer to go to bed right after their shift). When you sleep during the day, it is important to get at least seven hours of sleep. Pay attention to the quality of sleep (use a comfortable bed, sleep in a dark and quiet room), and avoid consuming alcohol and caffeine for the few hours before going to bed, which can reduce the quality of sleep.

What are the implications of this research on the relationship between heart health and sleep?

Studies suggest that there is an interaction between circadian rhythm and sleep-wake-dependent processes on heart rate. In fact, the timed release of hormones such as cortisol and melatonin, regulated by the central clock in the brain and light exposure, modulates our sleep / wake cycle. Maintaining a regular sleep schedule and a proper light exposure pattern is thought to be essential to keeping our watches in sync and is very likely beneficial to heart health.

Collaboration has been a big part of your research. How important was this level of collaboration, and do you think that if more researchers collaborated, more scientific discoveries could be made?

Our work was supported by a long-term collaboration with many academic partners and AstraZeneca and was extremely interdisciplinary. We used a wide range of techniques, which has only been possible thanks to the unique expertise of our colleagues. Having a good network of colleagues and collaborators is fundamental to expanding with confidence and supporting the range of experiments one can perform.

What are the next steps for you and your research into circadian rhythms?

Next, I would like to examine ion rhythms at the subcellular solution and examine whether these rhythms deteriorate during aging.

Where can readers find more information?

About Dr. Alessandra Stangherli

I earned a master’s degree in Pharmaceutical Biotechnology and a Ph.D. in Cellular Biology from the University of Padova, Italy. Dr.  Alessandra StangherlinDuring my Ph.D. I focused on the regulation of cAMP and cGMP signaling and their role in cardiomyocyte contraction. In 2016, I joined Dr. John O’Neill’s Laboratory at the Laboratory of Molecular Biology (LMB) in Cambridge, UK, as a research fellow to study how cellular 24-hour clocks regulate cellular physiology. I am currently the lead researcher at CECAD, Cologne, where I will study the mechanisms of regulation of ion homeostasis during aging.

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