Dr. Alessandra Stangherlin

Most living organisms have an endogenous clock system to align with the external time, which is dictated by the day/night cycle. As a result, many aspects of mammalian physiology and behavior are organized into “circadian” rhythms (from the Latin circa dies = about a day) to anticipate changes in environmental conditions. The sleep-wake cycle is probably the most noticeable circadian rhythm, but body temperature, alertness, physical performance, and the memory process also change during the course of the day. Misalignment with the external time, due for example to night-shift work or jet lag, is associated with disease states such as cancer, obesity, and neurodegeneration. How aging perturbs the function of internal clocks remains an open question, as well as their contribution to age-associated diseases.

Our research: We recently described a new homeostatic control mechanism that allows cells to preserve cell volume and osmotic homeostasis upon acute and circadian changes in their macromolecular content. Cells import and export Na⁺, K⁺, and Cl^- ions with a 24 h rhythm to osmotically compensate for mTORC1-mediated daily changes in soluble protein abundance. Whether ion rhythms feed back to regulate protein abundance is still an open question. Our laboratory investigates how changes in ion abundance and composition modulate mTORC-mediated protein synthesis and how this impacts the circadian proteome and the associated cellular functions. The intracellular ionic composition changes with age but how this affects ion rhythms has not been investigated. We aim to monitor ion rhythms in aging mice, examine the underlying mechanisms, and study how they impact cellular homeostasis and physiological outputs.

Our goals:

• Investigate circadian ion dynamics at higher spatial resolution and how they modulate cellular functions
• Understand how cellular clocks are affected by ageing and how they contribute to age-related diseases
• Design interventions to restore cellular circadian rhythms to delay the onset of age-related diseases.

Our successes:

A buffering mechanism for daily changes in soluble protein abundance

Between 5-20% of the cellular proteome show circadian variation in abundance due to rhythms in transcription, mTORC activity, protein synthesis and degradation. We observed that the abundance of soluble cytosolic protein also has a circadian rhythm. If not counterbalanced, such a change in macromolecule content could trigger a compensatory movement of water, affecting cell volume, protein stability, and ultimately cell viability. To compensate for changes in cytosolic protein, we found that

mammalian cells import and export Na⁺, K⁺, and Cl^- with a 24 h rhythm. Mechanistically, we have identified the SLC12A family of cotransporters as important players in this process, with WNK and OXRS1 kinases being the signaling node where ion and protein rhythms converge. Besides revealing that the intracellular concentration of ions is not constant during the day, as generally assumed, this new homeostatic control mechanism suggests a link between osmoregulation and protein homeostasis.

An endogenous clock within cardiomyocytes contributes to circadian rhythms in HR

In healthy individuals, heart rate (HR) increases in the morning and decreases during the night. This has been mainly attributed to the circadian regulation of sympathetic and parasympathetic systems via the systemic release of neurotransmitters. In our recent work, we have shown that the circadian regulation of HR is mediated by a cell-autonomous clock within cardiomyocytes, which works independently of the central nervous system. Compensatory ion rhythms in Na⁺, K⁺, and Cl^- also occur in cardiomyocytes and significantly affect their electrical functions. They modulate the depolarization phase of the action potential, leading to an increased firing rate towards the end of the night and a decreased firing rate at the end of the 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 impairment of the buffering mechanism we have described could render the heart more vulnerable to stress in the morning when a change in HR is required.

Our methods/techniques: Besides traditional tissue culture and biochemical techniques, our laboratory performs live-cell bioluminescence assays to monitor the cellular circadian clock across several days in standard culture conditions. We have also established an ionomics platform based on microwave plasma atomic emission spectroscopy, which allows us to determine the elemental composition of cells and tissues. In the future, we plan to complement this technique with fluorescence-based ion sensors to increase our spatial resolution and investigate ion dynamics at the subcellular level.