The vascular endothelium is a monolayer of cells that lines the inner surface of blood vessels and, through production of various bioactive factors, closely regulates vessel tone, blood flow, and microvascular permeability. Endothelial nitric oxide synthase (eNOS)-mediated production of NO is one of the most important systems regulating the microvasculature, controlling both vessel diameter and permeability. With regard to the latter, in healthy microvasculature eNOS actively maintains endothelial barrier integrity and inhibition of NOS rapidly increases permeability [1]. We hypothesized that native (non-contrast-enhanced) T1 mapping of the heart during NOS inhibition would detect elevated myocardial T1 corresponding to increased interstitial water content resulting from increased microvascular permeability, providing a novel means to noninvasively probe eNOS regulation of the coronary microvasculature. The ability to noninvasively image eNOS regulation of coronary microvascular permeability may potentially be useful as a means of detecting coronary endothelial dysfunction, as this finely controlled system may be impaired under conditions such as inflammation and oxidative stress that lead to vascular disease. Wild type male C57Bl/6 mice underwent MRI studies using a 7T system (Clinscan, Bruker). During MRI, mice were anesthetized with 1.25% isoflurane and maintained at 36 ± 0.5°C using circulating warm water. After localizer imaging, T1 mapping was performed in a mid-ventricular short-axis slice at baseline and at 5 and 20 minutes after intravenous injection of LNAME, a NOS inhibitor that has been shown to rapidly increase coronary microvascular permeability in rodents [1]. The T1-mapping method used fuzzy-clustering of spiral k-space data to ensure heart rate independence as previously described [2], and was accelerated using sparse sampling and compressed-sensing to reduce the scan time to 7 minutes. Two doses of LNAME, 2 mg/kg (n = 8) and 4 mg/kg (n = 6), were administered in different imaging sessions. The average heart rates at baseline, 5 minutes, and 20 minutes after administering 2mg/kg LNAME were 486 ± 37, 418 ± 17 and 423 ± 25 respectively, and 482 ± 17, 419 ± 13 and 419 ± 13 for 4mg/kg. Figure 1 shows example R1 (1/T1) maps of the heart acquired before and 5 minutes after injection of 4mg/kg LNAME, demonstrating the decrease in R1 observed in response to NOS inhibition. Figure 2 shows example myocardial T1 relaxation curves immediately prior to and 5 minutes post LNAME injection (4mg/kg). The estimated T1 values were 1337 ms and 1542 ms for pre- and post-LNAME, respectively, in the example shown. Figure 3 summarizes data from all the mice and shows that T1 mapping after the injection of LNAME detected a transient increase in T1, as T1 was elevated at 5 minutes post injection and was returning toward its baseline value at 20 minutes post injection. These data suggest a dose dependent effect, as the increase in T1 at 5 minutes after LNAME was greater for the higher dose (ΔT1 = 113 ± 15 ms at 4mg/kg, and ΔT1 = 73 ± 20 ms at 2mg/kg), however only ΔT1 at 4mg/kg LNAME vs. baseline reached statistical significance (ANOVA on ranks, p<0.05). We showed, for the first time, that native T1 mapping detects increased myocardial T1 after administration of LNAME, a NOS inhibitor. As LNAME is known to increase coronary microvascular permeability [1], the increased T1 is likely due to an increase in myocardial water content. NOS inhibition leads to vasoconstriction and decreased coronary blood flow [3], thus it is unlikely that the increase in T1 is due to an increase in blood volume. These methods are likely probing eNOS regulation of coronary microvascular permeability, which may represent a novel means of noninvasively assessing the health of the coronary endothelium. Future studies in eNOS knockout mice and mouse models of diabetes will elucidate the mechanisms underlying these observations and will test whether these methods can detect coronary microvascular disease. These methods can potentially be translated for use in humans, as NOS inhibition with LNAME has been performed previously at 4mg/kg in human subjects [4].