Nitric oxide (NO) relaxes vascular smooth muscle cells (SMCs) and dilates blood vessels by increasing intracellular levels of cyclic guanosine monophosphate (cGMP), which stimulates the activity of cGMP-dependent protein kinase (PKG). However, the vasodilator mechanisms downstream of PKG remain incompletely understood. Here, we found that transient receptor potential melastatin 4 (TRPM4) cation channels, which are activated by Ca2+ released from the sarcoplasmic reticulum (SR) through inositol triphosphate receptors (IP3Rs) under native conditions, are essential for SMC membrane depolarization and vasoconstriction. We hypothesized that signaling via the NO/cGMP/PKG pathway causes vasodilation by inhibiting TRPM4. We found that TRPM4 currents activated by stretching the plasma membrane or directly activating IP3Rs were suppressed by exogenous NO or a membrane-permeable cGMP analog, the latter of which also impaired IP3R-mediated release of Ca2+ from the SR. The effects of NO on TRPM4 activity were blocked by inhibition of soluble guanylyl cyclase or PKG. Notably, upon phosphorylation by PKG, IRAG (IP3R-associated PKG substrate) inhibited IP3R-mediated Ca2+ release, and knockdown of IRAG expression diminished NO-mediated inhibition of TRPM4 activity and vasodilation. Using superresolution microscopy, we found that IRAG, PKG, and IP3Rs form a nanoscale signaling complex on the SR of SMCs. We conclude that NO/cGMP/PKG signaling through IRAG inhibits IP3R-dependent activation of TRPM4 channels in SMCs to dilate arteries. Significance Statement: Nitric oxide (NO) is a gaseous vasodilator produced by endothelial cells that is essential for cardiovascular function. Although NO-mediated signaling pathways have been intensively studied, the mechanisms by which they relax smooth muscle cells (SMCs) to dilate blood vessels remain incompletely understood. In this study, we show that NO causes vasodilation by inhibiting the activity of Ca2+-dependent TRPM4 (transient receptor potential melastatin 4) cation channels. Probing further, we found that NO does not act directly on TRPM4 but instead initiates a signaling cascade that inhibits its activation by blocking the release of Ca2+ from the sarcoplasmic reticulum. Thus, our findings reveal the essential molecular pathways of NO-induced vasodilation—a fundamental unresolved concept in cardiovascular physiology.

Gould syndrome is a rare multisystem disorder resulting from autosomal dominant mutations in the collagen-encoding genes COL4A1 and COL4A2. Human patients and Col4a1 mutant mice display brain pathology that typifies cerebral small vessel diseases (cSVDs), including white matter hyperintensities, dilated perivascular spaces, lacunar infarcts, microbleeds, and spontaneous intracerebral hemorrhage. The underlying pathogenic mechanisms are unknown. Using the Col4a1 +/G394V mouse model, we found that vasoconstriction in response to internal pressure—the vascular myogenic response—is blunted in cerebral arteries from middle-aged (12 mo old) but not young adult (3 mo old) animals, revealing age-dependent cerebral vascular dysfunction. The defect in the myogenic response was associated with a significant decrease in depolarizing cation currents conducted by TRPM4 (transient receptor potential melastatin 4) channels in native cerebral artery smooth muscle cells (SMCs) isolated from mutant mice. The minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP 2 ) is necessary for TRPM4 activity. Dialyzing SMCs with PIP 2 and selective blockade of phosphoinositide 3-kinase (PI3K), an enzyme that converts PIP 2 to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP 3 ), restored TRPM4 currents. Acute inhibition of PI3K activity and blockade of transforming growth factor-beta (TGF-β) receptors also rescued the myogenic response, suggesting that hyperactivity of TGF-β signaling pathways stimulates PI3K to deplete PIP 2 and impair TRPM4 channels. We conclude that age-related cerebral vascular dysfunction in Col4a1 +/G394V mice is caused by the loss of depolarizing TRPM4 currents due to PIP 2 depletion, revealing an age-dependent mechanism of cSVD.

Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) in brain capillary endothelial cells, leading to the loss of inwardly rectifier K+ (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP2 by converting it to phosphatidylinositol (3,4,5)-trisphosphate (PIP3), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD.

Humans and mice with mutations in COL4A1 and COL4A2 manifest radiological and clinical hallmarks of cerebral small vessel disease (cSVD), but the pathogenic mechanisms are unknown. Here we report that mice with a missense mutation in Col4a1 at amino acid 1344 (Col4a1+/G1344D) exhibit age-dependent intracerebral hemorrhage (ICH), brain lesions detected by magnetic resonance imaging, and memory deficits. This pathology was associated with the loss of myogenic vasoconstriction, a vascular response essential for autoregulation of cerebral blood flow. Electrophysiological analyses showed that the loss of myogenic constriction resulted from blunted pressure-induced smooth muscle cell (SMC) membrane depolarization. Further, we found that dysregulation of membrane potential was associated with impaired Ca2+-dependent activation of transient receptor potential melastatin 4 (TRPM4) cation channels and disruptions in sarcoplasmic reticulum (SR) Ca2+ signaling. Treating Col4a1+/G1344D mice with 4-phenylbutyrate, a compound that promotes the trafficking of misfolded proteins and alleviates SR stress, averted the loss of myogenic tone, prevented ICH, and reduced memory defects. We conclude that alterations in SR Ca2+ handling that impair TRPM4 channel activity results in dysregulation of SMC membrane potential and loss of myogenic tone which contributes to age-related cSVD in Col4a1+/G1344D mice.