Uncovering a Sex-Specific cAMP Landscape in Vascular Smooth Muscle

The second messenger, cyclic adenosine monophosphate (cAMP), plays a crucial role in regulating cellular function, including in vascular smooth muscle (VSM). While extensive research has focused on the compartmentalization of cAMP signaling in various cell types, the influence of biological sex on distinct cAMP pools and their functional implications in the VSM remains largely unexplored. To address this knowledge gap, we employed a multiscale experimental approach spanning targeted cAMP biosensors, wire myography, live-cell calcium imaging, proximity ligation assays, and in vivo ultrasound imaging. Our findings reveal a sex-specific distribution of cAMP signaling domains, with the cAMP pool selectively present in the SR of females but not males in the VSM. Ovariectomy abolished this sexual dimorphism, with SR cAMP pools disappearing in ovx females, mirroring the pattern observed in male cells. Males and ovx female VSM exhibited a significantly stronger RyR-PDE3/PDE4 association than sham females, suggesting that ovarian hormone-dependent sequestration of phosphodiesterases away from the SR licenses cAMP signaling in this compartment in female cells. Functionally, this sexually dimorphic cAMP compartmentalization correlates with smaller Ca ²⁺ spark amplitude, reduced isoproterenol-induced relaxation, and higher pulse wave velocity in male and ovx compared to female VSM/vessels/mice. Together, these findings establish biological sex as a fundamental determinant of subcellular cAMP organization in VSM, with implications for understanding sex differences in vascular function and cardiovascular disease.

Sex-dependent compartmentation of cAMP signaling in vascular smooth muscle. In females, β-adrenergic stimulation generates robust plasma membrane, cytosolic, and sarcoplasmic reticulum-localized cAMP signals that support RyR-dependent Ca ²⁺ sparks, vasodilation, and lower pulse wave velocity. In males and ovariectomized females, PDE3/PDE4 activity constraints sarcoplasmic reticulum cAMP pool, which correlates with reduced Ca ²⁺ spark activity, reduced vasodilation, and increased pulse wave velocity.

Using compartment-targeted cAMP biosensors, we found that β-adrenergic stimulation raises cAMP at the plasma membrane and cytosol in vascular smooth muscle of both sexes but generates a sarcoplasmic reticulum cAMP pool only in female cells.Ovariectomy abolishes the female-specific pool, indicating that the sarcoplasmic reticulum response depends on ovarian hormones.Phosphodiesterase (PDE) transcript levels are similar across groups, but PDE3 and PDE4 sit closer to the ryanodine receptor in males and ovariectomized females, and inhibiting either PDE unmasks the sarcoplasmic reticulum cAMP pool. Absence of the sarcoplasmic reticulum cAMP pool in male and ovariectomized female VSM is accompanied by smaller Ca ²⁺ spark amplitude, weaker isoproterenol-induced dilatation, and higher pulse wave velocity. Results suggest subcellular cAMP organization is a sex-dependent determinant of cellular function, vascular reactivity and arterial stiffness.