A shock, a threat, or even the pressure of a looming work deadline may cause our hearts to race. That stress response might help a mouse avoid a cat on the prowl or a person finish an important presentation, but it can also lead to a dangerous disruption of the heart’s rhythm, an arrhythmia. Surprisingly, some kinds of arrhythmias are more prevalent in men, while others appear more often in women (1).

“We know there are differences in males and females, even in humans, but no one really knows why or what’s causing it,” said Jessica Caldwell, a biologist at the University of California, Davis (UCD). Caldwell recently published a study that provides some answers (2).  

We know there are differences in males and females, even in humans, but no one really knows why or what’s causing it. 
- Jessica Caldwell, University of California, Davis

Caldwell’s team of biologists in Crystal Ripplinger’s group at UCD found that differences in molecular signaling between male and female mouse hearts lead to different electrical patterns in response to the stress hormone norepinephrine. They hope that this research will help scientists identify therapeutic targets for arrhythmias and other cardiac conditions.

In stressful situations, norepinephrine stimulates heart cells to release the signaling molecule cyclic adenosine monophosphate, or cyclic AMP. Cyclic AMP binds receptors on the surfaces of heart cells and causes the heart rate to speed up (3,4). 

To better understand cyclic AMP and electrical signaling in the heart, Caldwell’s team bred mice in which heart cells expressed a fluorescence resonance energy transfer (FRET) sensor on the cyclic AMP receptor protein. They then removed the mouse hearts but kept them artificially alive and beating via a system that pumps artificial blood through them. 

When the researchers shone blue light on excised mouse hearts, the hearts emitted both blue and yellow light. When cyclic AMP bound to its receptor, the researchers saw a drop in the yellow light signal and an increase in the blue light signal. As cyclic AMP disappeared, the yellow signal increased, and the blue light signal decreased. The researchers used these changing light emissions to monitor cyclic AMP binding over time as the hearts continued to beat. At the same time, they monitored the hearts’ electrical outputs with a voltage-sensitive red dye. 

When cyclic AMP is bound to heart cells, they appear yellow. In female hearts the signal disappears faster at the base of the heart, while in male hearts the signal disappears uniformly across the entire heart.

CREDIT: JESSICA CALDWELL/UCD

“We started seeing a real pattern of variation,” said Caldwell. In female mouse hearts, cyclic AMP disappeared from some parts of the heart faster than others. At the same time, the electrical response due to the ion flow changed at the same rate in the same locations. This meant that cells in different parts of the female hearts relaxed at different rates. In contrast, cells throughout male hearts relaxed at the same rate.

Caldwell said that although they have established a sex difference in signaling, it’s unclear if that difference hurts the heart or protects it. “It could be good,” she said. “We don’t actually know.” To find out, the researchers will need to perform this same analysis on diseased hearts predisposed to arrhythmia as well as other forms of heart malfunction.

This initial study combines mice with a heart-specific FRET sensor, whole heart imaging, and voltage monitoring all in one. It marks the first time anyone has tracked both the molecular and electrical response of whole mouse hearts. That combination is what allowed the researchers to connect molecular signaling to its effects on the heart. “We really need such studies,” said Viacheslav Nikolaev, a biologist and research director at the University Medical Center, Hamburg-Eppendorf who was not involved in the research. 

Nikolaev said that seeing such sex differences wasn’t totally unexpected because of the known differences in arrhythmias between men and women. But he said that this study represents the future of cardiac research. “Ten years ago, it was just basically the cyclic AMP imaging or voltage imaging on single cells,” he said. “Now it's more in combination and also in the intact heart, which gives a lot of new information, which really brings the field forward.”

Both Caldwell and Nikolaev cautioned that mouse and human hearts have a number of differences: their heart rates, electrical signaling patterns, and the enzymes that break down cyclic AMP. But the hearts of all mammals also have a lot in common in their molecular responses to stress, so both also agreed that the sex differences should translate to humans. 

Caldwell’s team next plans to expand this technology to track cyclic AMP’s effects on other cardiac pathways linked to both disease and sex differences, such as L-type calcium signaling (5).

“Having this technology is really important for studying many aspects of cardiac disease,” said Caldwell. She hopes that it will allow researchers to better understand all aspects of the heart and also offer a key tool for developing new therapies for arrhythmia and beyond. “There's a lot we can do out there,” she said.

References

1. Meng, L., Shivkumar, K., and Ajijola, O. Autonomic Regulation and Ventricular Arrhythmias.Curr Treat Options Cardio Med  20, 38 (2018). 
2. Caldwell, J.L. et al. Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics.Sci Adv  9, eadd5799 (2023).
3. Surdo, N.C. et al.FRET biosensor uncovers cAMP nano-domains at β-adrenergic targets that dictate precise tuning for cardiac contractility.Nat Comm  577, 695–700 (2020).
4. Wright, P.T. et al.  Cardiomyocyte Membrane Structure and cAMP Compartmentation Produce Anatomical Variation in β₂AR-cAMP Responsiveness in Murine Hearts.Cell Rep  23, 459–469 (2018).
5. Myles, R.C. et al.  Local β-adrenergic stimulation overcomes source-sink mismatch to generate focal arrhythmia.Circ Res  110, 1454–1464 (2012).