Salt Responsive Gut Microbiota Induces Sex Specific Blood Pressure Changes.

BACKGROUND: Tryptophan metabolism is important in blood pressure regulation. The tryptophan-indole pathway is exclusively mediated by the gut microbiota. ACE2 (angiotensin-converting enzyme 2) participates in tryptophan absorption, and a lack of ACE2 leads to changes in the gut microbiota. The gut microbiota has been recognized as a regulator of blood pressure. Furthermore, there is ample evidence for sex differences in the gut microbiota. However, it is unclear whether such sex differences impact blood pressure differentially through the tryptophan-indole pathway. METHODS: To study the sex-specific mechanisms of gut microbiota-mediated tryptophan-indole pathway in hypertension, we generated a novel rat model with Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9)-targeted deletion of Ace2 in the Dahl salt-sensitive rat. Cecal microbiota transfers from donors of both sexes to female S recipients were performed. Also, Dahl salt-sensitive rats of both sexes were orally gavaged with indole to investigate blood pressure response. RESULTS: The female gut microbiota and its tryptophan-indole pathway exhibited greater buffering capacity when exposed to tryptophan, due to Ace2 deficiency, and salt. In contrast, the male gut microbiota and its tryptophan-indole pathway were more vulnerable. Female rats with male cecal microbiota responded to salt with a higher blood pressure increase. Indole, a tryptophan-derived metabolite produced by gut bacteria, increased blood pressure in male but not in female rats. Moreover, salt altered host-mediated tryptophan metabolism, characterized by reduced serum serotonin of both sexes and higher levels of kynurenine derivatives in the females. CONCLUSIONS: We uncovered a novel sex-specific mechanism in the gut microbiota-mediated tryptophan-indole pathway in blood pressure regulation. Salt tipped the tryptophan metabolism between the host and gut microbiota in a sex-dependent manner. Our study provides evidence for a novel concept that gut microbiota and its metabolism play sex-specific roles in the development of salt-sensitive hypertension.

Gut microbiota-derived Metabolite, Shikimic Acid, inhibits vascular smooth muscle cell proliferation and migration.

Gut microbiota dysbiosis is linked to vascular wall disease, but the mechanisms by which gut microbiota cross-talk with the host vascular cells remain largely unknown. Shikimic acid (SA) is a biochemical intermediate synthesized in plants and microorganisms, but not mammals. Surprisingly, recent metabolomic profiling data demonstrate that SA is detectable in human and murine blood. In this study, analyzing data from germ-free rats, we provide evidence in support of SA as a bona fide gut microbiota-derived metabolite, emphasizing its biological relevance. Since vascular cells are the first cells exposed to circulating metabolites, in this study, we examined, for the first time, the effects and potential underlying molecular mechanisms of SA on vascular smooth muscle cell (VSMC) proliferation and migration, which play a key role in occlusive vascular diseases, such as post-angioplasty restenosis and atherosclerosis. We found that SA inhibits the proliferation and migration of human coronary artery SMCs. At the molecular level, unexpectedly, we found that SA activates, rather than inhibits, multiple pro-mitogenic signaling pathways in VSMCs, such as ERK1/2, AKT, and mTOR/p70S6K. Conversely, we found that SA activates the anti-proliferative AMP-activated protein kinase (AMPK) in VSMCs, a key cellular energy sensor and regulator. However, loss-of-function experiments demonstrate that AMPK does not mediate the inhibitory effects of SA on VSMC proliferation. In conclusion, these studies demonstrate that a microbiota-derived metabolite, SA, inhibits VSMC proliferation and migration in vitro and prompt further evaluation of the possible underlying molecular mechanisms and the potential protective role in VSMC-related vascular wall disease in vivo.

Spontaneous vascular dysfunction in Dahl salt-sensitive male rats raised without a high-salt diet.

Dahl salt-sensitive (SS) rats fed a high-salt diet, but not low-salt, exhibit vascular dysfunction. Several substrains of SS rats exist that differ in their blood pressure phenotypes and salt sensitivity. The goal of this study was to investigate whether the John-Rapp-derived SS rat (SS/Jr), which exhibits spontaneous hypertension on a low-salt diet, presents with hallmarks of vascular dysfunction observed in another experimental model of hypertension independent of dietary salt, the spontaneously hypertensive rat (SHR). Endothelium-intact aortic rings and mesenteric resistance arteries were isolated from low-salt fed adult male SS/Jr rats and SHRs, or their respective controls, for isometric wire myography. Vessels were challenged with cumulative concentrations of various vasoactive substances, in the absence or presence of nitric oxide synthase or cyclooxygenase inhibitors. Despite showing some differences in their responses to various vasoactive substances, both SS/Jr rats and SHRs exhibited key features of vascular dysfunction, including endothelial dysfunction and hyperresponsiveness to vasocontractile agonists. In conclusion, this study provides evidence to support the utility of the SS/Jr rat strain maintained on a low-salt diet as a valid experimental model for vascular dysfunction, a key feature of human hypertension.

Hypertension Increases Susceptibility to Experimental Malaria in Mice.

Global prevalence of hypertension is on the rise, burdening healthcare, especially in developing countries where infectious diseases, such as malaria, are also rampant. Whether hypertension could predispose or increase susceptibility to malaria, however, has not been extensively explored. Previously, we reported that hypertension is associated with abnormal red blood cell (RBC) physiology and anemia. Since RBC are target host cells for malarial parasite, Plasmodium, we hypothesized that hypertensive patients with abnormal RBC physiology are at greater risk or susceptibility to Plasmodium infection. To test this hypothesis, normotensive (BPN/3J) and hypertensive (BPH/2J) mice were characterized for their RBC physiology and subsequently infected with Plasmodium yoelii (P. yoelii), a murine-specific non-lethal strain. When compared to BPN mice, BPH mice displayed microcytic anemia with RBC highly resistant to osmotic hemolysis. Further, BPH RBC exhibited greater membrane rigidity and an altered lipid composition, as evidenced by higher levels of phospholipids and saturated fatty acid, such as stearate (C18:0), along with lower levels of polyunsaturated fatty acid like arachidonate (C20:4). Moreover, BPH mice had significantly greater circulating Ter119+ CD71+ reticulocytes, or immature RBC, prone to P. yoelii infection. Upon infection with P. yoelii, BPH mice experienced significant body weight loss accompanied by sustained parasitemia, indices of anemia, and substantial increase in systemic pro-inflammatory mediators, compared to BPN mice, indicating that BPH mice were incompetent to clear P. yoelii infection. Collectively, these data demonstrate that aberrant RBC physiology observed in hypertensive BPH mice contributes to an increased susceptibility to P. yoelii infection and malaria-associated pathology.

Sex Dimorphic Effects of Bile Acid Metabolism in Liver Cancer in Mice.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a male-dominant disease, but targeted sex hormone therapies have not been successful. Bile acids are a potential liver carcinogen and are biomolecules with hormone-like effects. A few studies highlight their potential sex dimorphism in physiology and disease. We hypothesized that bile acids could be a potential molecular signature that explains sex disparity in HCC. METHODS & RESULTS: We used the farnesoid X receptor knockout (FxrKO) mouse model to study bile acid-dependent HCC. Temporal tracking of circulating bile acids determined more than 80% of FxrKO females developed spontaneous cholemia (ie, serum total bile acids ≥40 µmol/L) as early as 8 weeks old. Opposingly, FxrKO males were highly resistant to cholemia, with ∼23% incidence even when 26 weeks old. However, FxrKO males demonstrated higher levels of deoxycholate than females. Compared with males, FxrKO females had more severe cholestatic liver injury and further aberrancies in bile acid metabolism. Yet, FxrKO females expressed more detoxification transcripts and had greater renal excretion of bile acids. Intervention with CYP7A1 (rate limiting enzyme for bile acid biosynthesis) deficiency or taurine supplementation either completely or partially normalized bile acid levels and liver injury in FxrKO females. Despite higher cholemia prevalence in FxrKO females, their tumor burden was less compared with FxrKO males. An exception to this sex-dimorphic pattern was found in a subset of male and female FxrKO mice born with congenital cholemia due to portosystemic shunt, where both sexes had comparable robust HCC. CONCLUSIONS: Our study highlights bile acids as sex-dimorphic metabolites in HCC except in the case of portosystemic shunt.

Microbiota-associated mechanisms underlying sexual dimorphism in hypertension.

Consistent research over the last 20 years has shown that there are clear sex differences in the pathogenesis of hypertension, the leading risk factor for the development of cardiovascular diseases. More recently, there is evidence in both humans and experimental animal models that causally implicates the gut microbiota in hypertension. It therefore follows that sex differences in the gut microbiota may mediate the extent of disease between sexes. This new field is rapidly changing and advancing, and the purpose of this review is to cover the most up-to-date evidence regarding the sexual dimorphism of the gut microbiota and its potential influence on the differential manifestation of hypertension in males versus females. Emphasis will be placed on the mechanisms thought to contribute to these sex differences in both the gut microbiota and hypertension, including sex steroid hormones, gut-derived metabolites, the immune system, and pregnancy.

Sex hormones, sex chromosomes, and microbiota: Identification of Akkermansia muciniphila as an estrogen-responsive microbiota.

Microbiota composition is known to be linked to sex. However, separating sex hormones and sex chromosome roles in gut microbial diversity is yet to be determined. To investigate the sex chromosome role independent of sex hormones, we used the four-core genotype mouse model. In this mouse model, males with testes and females with ovaries have XX or XY sex chromosome complement. In gonadectomized four-core genotype mice, we observed a significant decrease in the levels of estradiol (P<0.001) and progesterone (P<0.03) in female and testosterone (P<0.0001) in male mice plasma samples. Independent of sex chromosome complement, microbial α diversity was increased in gonadectomized female but not male mice compared to sex-matched gonad-intact controls. ß diversity analysis showed separation between male (P<0.05) but not female XX and XY mice. Importantly, Akkermansia muciniphila was less abundant in gonadectomized compared to gonadal intact female mice (P<0.0001). In the presence of ß-estradiol, Akkermansia muciniphila growth exponentially increased, providing evidence for the identification of a female sex hormone-responsive bacterium (P<0.001).

Genetically engineered Lactobacillus paracasei rescues colonic angiotensin converting enzyme 2 (ACE2) and attenuates hypertension in female Ace2 knock out rats.

Engineered gut microbiota represents a new frontier in medicine, in part serving as a vehicle for the delivery of therapeutic biologics to treat a range of host conditions. The gut microbiota plays a significant role in blood pressure regulation; thus, manipulation of gut microbiota is a promising avenue for hypertension treatment. In this study, we tested the potential of Lactobacillus paracasei, genetically engineered to produce and deliver human angiotensin converting enzyme 2 (Lacto-hACE2), to regulate blood pressure in a rat model of hypertension with genetic ablation of endogenous Ace2 (Ace2-/- and Ace2-/y). Our findings reveal a sex-specific reduction in blood pressure in female (Ace2-/-) but not male (Ace2-/y) rats following colonization with the Lacto-hACE2. This beneficial effect of lowering blood pressure was aligned with a specific reduction in colonic angiotensin II, but not renal angiotensin II, suggesting the importance of colonic Ace2 in the regulation of blood pressure. We conclude that this approach of targeting the colon with engineered bacteria for delivery of ACE2 represents a promising new paradigm in the development of antihypertensive therapeutics.

Urine-based Detection of Congenital Portosystemic Shunt in C57BL/6 Mice.

Sporadic occurrence of congenital portosystemic shunt (PSS) at a rate of ∼1 out of 10 among C57BL/6 J mice, which are widely used in biomedical research, results in aberrancies in serologic, metabolic, and physiologic parameters. Therefore, mice with PSS should be identified as outliers in research. Accordingly, we sought methods to, reliably and efficiently, identify PSS mice. Serum total bile acids ≥ 40 µm is a bona fide biomarker of PSS in mice but utility of this biomarker is limited by its cost and invasiveness, particularly if large numbers of mice are to be screened. This led us to investigate if assay of urine might serve as a simple, inexpensive, noninvasive means of PSS diagnosis. Metabolome profiling uncovered that Krebs cycle intermediates, that is, citrate, α-ketoglutarate, and fumarate, were strikingly and distinctly elevated in the urine of PSS mice. We leveraged the iron-chelating and pH-lowering properties of such metabolites as the basis for 3 urine-based PSS screening tests: urinary iron-chelation assay, pH strip test, and phenol red assay. Our findings demonstrate the feasibility of using these colorimetric assays, whereby their readout can be assessed by direct observation, to diagnose PSS in an inexpensive, rapid, and noninvasive manner. Application of our urinary PSS screening protocols can aid biomedical research by enabling stratification of PSS mice, which, at present, likely confound numerous ongoing studies.

Epigenetic histone modification by butyrate downregulates KIT and attenuates mast cell function.

Short-chain fatty acid butyrate is produced from the bacterial fermentation of indigestible fiber in the intestinal lumen, and it has been shown to attenuate lung inflammation in murine asthma models. Mast cells (MCs) are initiators of inflammatory response to allergens, and they play an important role in asthma. MC survival and proliferation is regulated by its growth factor stem cell factor (SCF), which acts through the receptor, KIT. It has previously been shown that butyrate attenuates the activation of MCs by allergen stimulation. However, how butyrate mechanistically influences SCF signalling to impact MC function remains unknown. Here, we report that butyrate treatment triggered the modification of MC histones via butyrylation and acetylation, and inhibition of histone deacetylase (HDAC) activity. Further, butyrate treatment caused downregulation of SCF receptor KIT and associated phosphorylation, leading to significant attenuation of SCF-mediated MC proliferation, and pro-inflammatory cytokine secretion. Mechanistically, butyrate inhibited MC function by suppressing KIT and downstream p38 and Erk phosphorylation, and it mediated these effects via modification of histones, acting as an HDAC inhibitor and not via its traditional GPR41 (FFAR3) or GPR43 (FFAR2) butyrate receptors. In agreement, the pharmacological inhibition of Class I HDAC (HDAC1/3) mirrored butyrate's effects, suggesting that butyrate impacts MC function by HDAC1/3 inhibition. Taken together, butyrate epigenetically modifies histones and downregulates the SCF/KIT/p38/Erk signalling axis, leading to the attenuation of MC function, validating its ability to suppress MC-mediated inflammation. Therefore, butyrate supplementations could offer a potential treatment strategy for allergy and asthma via epigenetic alterations in MCs.

Loss of toll-like receptor 5 potentiates spontaneous hepatocarcinogenesis in farnesoid X receptor-deficient mice.

BACKGROUND: HCC is the most common primary liver cancer and a leading cause of cancer-related mortality. Gut microbiota is a large collection of microbes, predominately bacteria, that harbor the gastrointestinal tract. Changes in gut microbiota that deviate from the native composition, that is, "dysbiosis," is proposed as a probable diagnostic biomarker and a risk factor for HCC. However, whether gut microbiota dysbiosis is a cause or a consequence of HCC is unknown. METHODS: To better understand the role of gut microbiota in HCC, mice deficient of toll-like receptor 5 (TLR5, a receptor for bacterial flagellin) as a model of spontaneous gut microbiota dysbiosis were crossed with farnesoid X receptor knockout mice (FxrKO), a genetic model for spontaneous HCC. Male FxrKO/Tlr5KO double knockout (DKO), FxrKO, Tlr5KO, and wild-type (WT) mice were aged to the 16-month HCC time point. RESULTS: Compared with FxrKO mice, DKO mice had more severe hepatooncogenesis at the gross, histological, and transcript levels and this was associated with pronounced cholestatic liver injury. The bile acid dysmetabolism in FxrKO mice became more aberrant in the absence of TLR5 due in part to suppression of bile acid secretion and enhanced cholestasis. Out of the 14 enriched taxon signatures seen in the DKO gut microbiota, 50% were dominated by the Proteobacteria phylum with expansion of the gut pathobiont γ-Proteobacteria that is implicated in HCC. CONCLUSIONS: Collectively, introducing gut microbiota dysbiosis by TLR5 deletion exacerbated hepatocarcinogenesis in the FxrKO mouse model.

Artificial Intelligence in Medicine: Microbiome-Based Machine Learning for Phenotypic Classification.

Advanced computational approaches in artificial intelligence, such as machine learning, have been increasingly applied in life sciences and healthcare to analyze large-scale complex biological data, such as microbiome data. In this chapter, we describe the experimental procedures for using microbiome-based machine learning models for phenotypic classification.

Conjugated bile acids are nutritionally re-programmable antihypertensive metabolites.

BACKGROUND: Hypertension is the largest risk factor affecting global mortality. Despite available medications, uncontrolled hypertension is on the rise, whereby there is an urgent need to develop novel and sustainable therapeutics. Because gut microbiota is now recognized as an important entity in blood pressure regulation, one such new avenue is to target the gut-liver axis wherein metabolites are transacted via host-microbiota interactions. Knowledge on which metabolites within the gut-liver axis regulate blood pressure is largely unknown. METHOD: To address this, we analyzed bile acid profiles of human, hypertensive and germ-free rat models and report that conjugated bile acids are inversely correlated with blood pressure in humans and rats. RESULTS: Notably intervening with taurine or tauro-cholic acid rescued bile acid conjugation and reduced blood pressure in hypertensive rats. Subsequently, untargeted metabolomics uncovered altered energy metabolism following conjugation of bile acids as a mechanism alleviating high blood pressure. CONCLUSION: Together this work reveals conjugated bile acids as nutritionally re-programmable anti-hypertensive metabolites.

Survey and Evaluation of Hypertension Machine Learning Research.

Background Machine learning (ML) is pervasive in all fields of research, from automating tasks to complex decision-making. However, applications in different specialities are variable and generally limited. Like other conditions, the number of studies employing ML in hypertension research is growing rapidly. In this study, we aimed to survey hypertension research using ML, evaluate the reporting quality, and identify barriers to ML's potential to transform hypertension care. Methods and Results The Harmonious Understanding of Machine Learning Analytics Network survey questionnaire was applied to 63 hypertension-related ML research articles published between January 2019 and September 2021. The most common research topics were blood pressure prediction (38%), hypertension (22%), cardiovascular outcomes (6%), blood pressure variability (5%), treatment response (5%), and real-time blood pressure estimation (5%). The reporting quality of the articles was variable. Only 46% of articles described the study population or derivation cohort. Most articles (81%) reported at least 1 performance measure, but only 40% presented any measures of calibration. Compliance with ethics, patient privacy, and data security regulations were mentioned in 30 (48%) of the articles. Only 14% used geographically or temporally distinct validation data sets. Algorithmic bias was not addressed in any of the articles, with only 6 of them acknowledging risk of bias. Conclusions Recent ML research on hypertension is limited to exploratory research and has significant shortcomings in reporting quality, model validation, and algorithmic bias. Our analysis identifies areas for improvement that will help pave the way for the realization of the potential of ML in hypertension and facilitate its adoption.

Bacterial flagellin is a dominant, stable innate immune activator in the gastrointestinal contents of mice and rats.

Intestinal contents comprise the largest repository of immunogenic ligands of microbial origin. We undertook this study to assess the predominant microbe-associated molecular patterns (MAMPs) present therein and the receptors) that mediate the innate immune responses to them. Here, we demonstrated that intestinal contents from conventional, but not germ-free, mice and rats triggered robust innate immune responses in vitro and in vivo. Such immune responses were abrogated in the absence of either myeloid differentiation factor 88 (MyD88) or Toll-like receptor (TLR) 5, but not TLR4, suggesting that the stimuli was flagellin (i.e., protein subunit of flagella that drives bacterial motility). Accordingly, pre-treating intestinal extracts with proteinase, thereby degrading flagellin, was sufficient to block their ability to activate innate immune responses. Taken together, this work serves to underscore flagellin as a major, heat-stable and bioactive MAMP in the intestinal content that confers this milieu strong potential to trigger innate immune responses.

Toward Precision Medicine: Circadian Rhythm of Blood Pressure and Chronotherapy for Hypertension - 2021 NHLBI Workshop Report.

Healthy individuals exhibit blood pressure variation over a 24-hour period with higher blood pressure during wakefulness and lower blood pressure during sleep. Loss or disruption of the blood pressure circadian rhythm has been linked to adverse health outcomes, for example, cardiovascular disease, dementia, and chronic kidney disease. However, the current diagnostic and therapeutic approaches lack sufficient attention to the circadian rhythmicity of blood pressure. Sleep patterns, hormone release, eating habits, digestion, body temperature, renal and cardiovascular function, and other important host functions as well as gut microbiota exhibit circadian rhythms, and influence circadian rhythms of blood pressure. Potential benefits of nonpharmacologic interventions such as meal timing, and pharmacologic chronotherapeutic interventions, such as the bedtime administration of antihypertensive medications, have recently been suggested in some studies. However, the mechanisms underlying circadian rhythm-mediated blood pressure regulation and the efficacy of chronotherapy in hypertension remain unclear. This review summarizes the results of the National Heart, Lung, and Blood Institute workshop convened on October 27 to 29, 2021 to assess knowledge gaps and research opportunities in the study of circadian rhythm of blood pressure and chronotherapy for hypertension.

Combating hypertension beyond genome-wide association studies: Microbiome and artificial intelligence as opportunities for precision medicine.

The single largest contributor to human mortality is cardiovascular disease, the top risk factor for which is hypertension (HTN). The last two decades have placed much emphasis on the identification of genetic factors contributing to HTN. As a result, over 1,500 genetic alleles have been associated with human HTN. Mapping studies using genetic models of HTN have yielded hundreds of blood pressure (BP) loci but their individual effects on BP are minor, which limits opportunities to target them in the clinic. The value of collecting genome-wide association data is evident in ongoing research, which is beginning to utilize these data at individual-level genetic disparities combined with artificial intelligence (AI) strategies to develop a polygenic risk score (PRS) for the prediction of HTN. However, PRS alone may or may not be sufficient to account for the incidence and progression of HTN because genetics is responsible for <30% of the risk factors influencing the etiology of HTN pathogenesis. Therefore, integrating data from other nongenetic factors influencing BP regulation will be important to enhance the power of PRS. One such factor is the composition of gut microbiota, which constitute a more recently discovered important contributor to HTN. Studies to-date have clearly demonstrated that the transition from normal BP homeostasis to a state of elevated BP is linked to compositional changes in gut microbiota and its interaction with the host. Here, we first document evidence from studies on gut dysbiosis in animal models and patients with HTN followed by a discussion on the prospects of using microbiota data to develop a metagenomic risk score (MRS) for HTN to be combined with PRS and a clinical risk score (CRS). Finally, we propose that integrating AI to learn from the combined PRS, MRS and CRS may further enhance predictive power for the susceptibility and progression of HTN.

Repurposing a Drug Targeting Inflammatory Bowel Disease for Lowering Hypertension.

Background The gut and gut microbiota, which were previously neglected in blood pressure regulation, are becoming increasingly recognized as factors contributing to hypertension. Diseases affecting the gut such as inflammatory bowel disease (IBD) present with aberrant energy metabolism of colonic epithelium and gut dysbiosis, both of which are also mechanisms contributing to hypertension. We reasoned that current measures to remedy deficits in colonic energy metabolism and dysbiosis in IBD could also ameliorate hypertension. Among them, 5-aminosalicylic acid (5-ASA; mesalamine) is a PPARγ (peroxisome proliferator-activated receptor gamma) agonist. It attenuates IBD by a dual mechanism of selectively enhancing colonic epithelial cell energy metabolism and ameliorating gut dysbiosis. Methods and Results A total of 2 groups of 11- to 12-week-old male, hypertensive, Dahl salt-sensitive (S) rats were gavaged with (n=10) or without (n=10) 5-aminosalicylic acid (150 mg/kg) for 4 weeks. Rats receiving 5-aminosalicylic acid treatment had a lower mean blood pressure than controls (145±3 mm Hg versus 153±4 mm Hg; P<0.0001). This reduction in blood pressure was accompanied by increased activity of PPARγ, increased expression of energy metabolism-related genes, and lowering of the Firmicutes/Bacteroidetes ratio in the colon, the reduction of which is a marker for the correction of gut dysbiosis. Furthermore, these data were consistent with the American Gut Project wherein the Firmicutes/Bacteroidetes ratio of non-IBD (n=611) patients was significantly lower than patients with IBD (n=631). Conclusions 5-Aminosalicylic acid could be repurposed for hypertension by specifically enhancing the gut energy metabolism and correction of microbiota dysbiosis.

Perceptions of US Medical Students on Artificial Intelligence in Medicine: Mixed Methods Survey Study.

BACKGROUND: Given the rapidity with which artificial intelligence is gaining momentum in clinical medicine, current physician leaders have called for more incorporation of artificial intelligence topics into undergraduate medical education. This is to prepare future physicians to better work together with artificial intelligence technology. However, the first step in curriculum development is to survey the needs of end users. There has not been a study to determine which media and which topics are most preferred by US medical students to learn about the topic of artificial intelligence in medicine. OBJECTIVE: We aimed to survey US medical students on the need to incorporate artificial intelligence in undergraduate medical education and their preferred means to do so to assist with future education initiatives. METHODS: A mixed methods survey comprising both specific questions and a write-in response section was sent through Qualtrics to US medical students in May 2021. Likert scale questions were used to first assess various perceptions of artificial intelligence in medicine. Specific questions were posed regarding learning format and topics in artificial intelligence. RESULTS: We surveyed 390 US medical students with an average age of 26 (SD 3) years from 17 different medical programs (the estimated response rate was 3.5%). A majority (355/388, 91.5%) of respondents agreed that training in artificial intelligence concepts during medical school would be useful for their future. While 79.4% (308/388) were excited to use artificial intelligence technologies, 91.2% (353/387) either reported that their medical schools did not offer resources or were unsure if they did so. Short lectures (264/378, 69.8%), formal electives (180/378, 47.6%), and Q and A panels (167/378, 44.2%) were identified as preferred formats, while fundamental concepts of artificial intelligence (247/379, 65.2%), when to use artificial intelligence in medicine (227/379, 59.9%), and pros and cons of using artificial intelligence (224/379, 59.1%) were the most preferred topics for enhancing their training. CONCLUSIONS: The results of this study indicate that current US medical students recognize the importance of artificial intelligence in medicine and acknowledge that current formal education and resources to study artificial intelligence-related topics are limited in most US medical schools. Respondents also indicated that a hybrid formal/flexible format would be most appropriate for incorporating artificial intelligence as a topic in US medical schools. Based on these data, we conclude that there is a definitive knowledge gap in artificial intelligence education within current medical education in the US. Further, the results suggest there is a disparity in opinions on the specific format and topics to be introduced.