Pioneering new frontiers in circadian medicine chronotherapies for cardiovascular health.

Cardiovascular disease (CVD) is a global health concern. Circadian medicine improves cardiovascular care by aligning treatments with our body's daily rhythms and their underlying cellular circadian mechanisms. Time-based therapies, or chronotherapies, show special promise in clinical cardiology. They optimize treatment schedules for better outcomes with fewer side effects by recognizing the profound influence of rhythmic body cycles. In this review, we focus on three chronotherapy areas (medication, light, and meal timing) with potential to enhance cardiovascular care. We also highlight pioneering research in the new field of rest, the gut microbiome, novel chronotherapies for hypertension, pain management, and small molecules that targeting the circadian mechanism.

Circadian Influences on Myocardial Ischemia-Reperfusion Injury and Heart Failure.

The impact of circadian rhythms on cardiovascular function and disease development is well established, with numerous studies in genetically modified animals emphasizing the circadian molecular clock's significance in the pathogenesis and pathophysiology of myocardial ischemia and heart failure progression. However, translational preclinical studies targeting the heart's circadian biology are just now emerging and are leading to the development of a novel field of medicine termed circadian medicine. In this review, we explore circadian molecular mechanisms and novel therapies, including (1) intense light, (2) small molecules modulating the circadian mechanism, and (3) chronotherapies such as cardiovascular drugs and meal timings. These promise significant clinical translation in circadian medicine for cardiovascular disease. (4) Additionally, we address the differential functioning of the circadian mechanism in males versus females, emphasizing the consideration of biological sex, gender, and aging in circadian therapies for cardiovascular disease.

Nrf2 activation rescues stress-induced depression-like behaviour and inflammatory responses in male but not female rats.

BACKGROUND: Major depressive disorder (MDD) is a recurring affective disorder that is two times more prevalent in females than males. Evidence supports immune system dysfunction as a major contributing factor to MDD, notably in a sexually dimorphic manner. Nuclear factor erythroid 2-related factor 2 (Nrf2), a regulator of antioxidant signalling during inflammation, is dysregulated in many chronic inflammatory disorders; however, its role in depression and the associated sex differences have yet to be explored. Here, we investigated the sex-specific antidepressant and immunomodulatory effects of the potent Nrf2 activator dimethyl fumarate (DMF), as well as the associated gene expression profiles. METHODS: Male and female rats were treated with vehicle or DMF (25 mg/kg) whilst subjected to 8 weeks of chronic unpredictable stress. The effect of DMF treatment on stress-induced depression- and anxiety-like behaviours, as well as deficits in recognition and spatial learning and memory were then assessed. Sex differences in hippocampal (HIP) microglial activation and gene expression response were also evaluated. RESULTS: DMF treatment during stress exposure had antidepressant effects in male but not female rats, with no anxiolytic effects in either sex. Recognition learning and memory and spatial learning and memory were impaired in chronically stressed males and females, respectively, and DMF treatment rescued these deficits. DMF treatment also prevented stress-induced HIP microglial activation in males. Conversely, females displayed no HIP microglial activation associated with stress exposure. Last, chronic stress elicited sex-specific alterations in HIP gene expression, many of which were normalized in animals treated with DMF. Of note, most of the differentially expressed genes in males normalized by DMF were related to antioxidant, inflammatory or immune responses. CONCLUSIONS: Collectively, these findings support a greater role of immune processes in males than females in a rodent model of depression. This suggests that pharmacotherapies that target Nrf2 have the potential to be an effective sex-specific treatment for depression.

Major depressive disorder is two times more prevalent in females than males. Further, immune system dysfunction has been shown to contribute to the development of depression, with previous studies consistently reporting chronic low-grade inflammation in depressed individuals. Not surprisingly, the immune system dysfunction associated with depression appears to be sex specific. As such, whilst anti-inflammatory drugs have shown antidepressant effects in preclinical studies, the sex differences in these effects are seldomly investigated. Thus, this study sought to determine the sex-specific antidepressant and cognitive effects of dimethyl fumarate (DMF) treatment. DMF is a drug that activates the protein nuclear factor erythroid 2-related factor 2 to initiate anti-inflammatory processes. Here, male and female rats were exposed to 8 weeks of chronic stress whilst receiving daily DMF treatment. Subsequently, their expression of depression- and anxiety-like behaviours, as well as learning and memory deficits were assessed. Alterations in gene expression were also evaluated. DMF treatment had antidepressant effects in male rats only but did not have anti-anxiety effects in either sex. The learning and memory deficits in both sexes were rescued with DMF treatment. Notably, DMF normalized several of the sex-specific gene alterations induced by chronic stress, with many of the male-specific genes relating to inflammatory processes. These data suggest that DMF may be an effective antidepressant treatment in males.

One Health: Circadian Medicine Benefits Both Non-human Animals and Humans Alike.

Circadian biology's impact on human physical health and its role in disease development and progression is widely recognized. The forefront of circadian rhythm research now focuses on translational applications to clinical medicine, aiming to enhance disease diagnosis, prognosis, and treatment responses. However, the field of circadian medicine has predominantly concentrated on human healthcare, neglecting its potential for transformative applications in veterinary medicine, thereby overlooking opportunities to improve non-human animal health and welfare. This review consists of three main sections. The first section focuses on the translational potential of circadian medicine into current industry practices of agricultural animals, with a particular emphasis on horses, broiler chickens, and laying hens. The second section delves into the potential applications of circadian medicine in small animal veterinary care, primarily focusing on our companion animals, namely dogs and cats. The final section explores emerging frontiers in circadian medicine, encompassing aquaculture, veterinary hospital care, and non-human animal welfare and concludes with the integration of One Health principles. In summary, circadian medicine represents a highly promising field of medicine that holds the potential to significantly enhance the clinical care and overall health of all animals, extending its impact beyond human healthcare.

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.

Disrupting circadian control of peripheral myogenic reactivity mitigates cardiac injury following myocardial infarction.

AIMS: Circadian rhythms orchestrate important functions in the cardiovascular system: the contribution of microvascular rhythms to cardiovascular disease progression/severity is unknown. This study hypothesized that (i) myogenic reactivity in skeletal muscle resistance arteries is rhythmic and (ii) disrupting this rhythmicity would alter cardiac injury post-myocardial infarction (MI). METHODS AND RESULTS: Cremaster skeletal muscle resistance arteries were isolated and assessed using standard pressure myography. Circadian rhythmicity was globally disrupted with the ClockΔ19/Δ19 mutation or discretely through smooth muscle cell-specific Bmal1 deletion (Sm-Bmal1 KO). Cardiac structure and function were determined by echocardiographic, hemodynamic and histological assessments. Myogenic reactivity in cremaster muscle resistance arteries is rhythmic. This rhythm is putatively mediated by the circadian modulation of a mechanosensitive signalosome incorporating tumour necrosis factor and casein kinase 1. Following left anterior descending coronary artery ligation, myogenic responsiveness is locked at the circadian maximum, although circadian molecular clock gene expression cycles normally. Disrupting the molecular clock abolishes myogenic rhythmicity: myogenic tone is suspended at the circadian minimum and is no longer augmented by MI. The reduced myogenic tone in ClockΔ19/Δ19 mice and Sm-Bmal1 KO mice associates with reduced total peripheral resistance (TPR), improved cardiac function and reduced infarct expansion post-MI. CONCLUSIONS: Augmented microvascular constriction aggravates cardiac injury post-MI. Following MI, skeletal muscle resistance artery myogenic reactivity increases specifically within the rest phase, when TPR would normally decline. Disrupting the circadian clock interrupts the MI-induced augmentation in myogenic reactivity: therapeutics targeting the molecular clock, therefore, may be useful for improving MI outcomes.

A brief morning rest period benefits cardiac repair in pressure overload hypertrophy and postmyocardial infarction.

Rest has long been considered beneficial to patient healing; however, remarkably, there are no evidence-based experimental models determining how it benefits disease outcomes. Here, we created an experimental rest model in mice that briefly extends the morning rest period. We found in 2 major cardiovascular disease conditions (cardiac hypertrophy, myocardial infarction) that imposing a short, extended period of morning rest each day limited cardiac remodeling compared with controls. Mechanistically, rest mitigates autonomic-mediated hemodynamic stress on the cardiovascular system, relaxes myofilament contractility, and attenuates cardiac remodeling genes, consistent with the benefits on cardiac structure and function. These same rest-responsive gene pathways underlie the pathophysiology of many major human cardiovascular conditions, as demonstrated by interrogating open-source transcriptomic data; thus, patients with other conditions may also benefit from a morning rest period in a similar manner. Our findings implicate rest as a key driver of physiology, creating a potentially new field - as broad and important as diet, sleep, or exercise - and provide a strong rationale for investigation of rest-based therapy for major clinical diseases.

The Impact of Sex, Circadian Disruption, and the ClockΔ19/Δ19 Genotype on Alcohol Drinking in Mice.

Shift work is associated with increased alcohol drinking, more so in males than females, and is thought to be a coping mechanism for disrupted sleep cycles. However, little is presently known about the causal influence of circadian rhythm disruptions on sex differences in alcohol consumption. In this study, we disrupted circadian rhythms in female and male mice using both environmental (i.e., shifting diurnal cycles) and genetic (i.e., ClockΔ19/Δ19 mutation) manipulations, and measured changes in alcohol consumption and preference using a two-bottle choice paradigm. Alcohol consumption and preference, as well as food and water consumption, total caloric intake, and weight were assessed in adult female and male ClockΔ19/Δ19 mutant mice or wild-type (WT) litter-mates, housed under a 12-hour:12-hour light:dark (L:D) cycle or a shortened 10-hour:10-hour L:D cycle. Female WT mice (under both light cycles) increased their alcohol consumption and preference over time, a pattern not observed in male WT mice. Compared to WT mice, ClockΔ19/Δ19 mice displayed increased alcohol consumption and preference. Sex differences were not apparent in ClockΔ19/Δ19 mice, with or without shifting diurnal cycles. In conclusion, sex differences in alcohol consumption patterns are evident and increase with prolonged access to alcohol. Disrupting circadian rhythms by mutating the Clock gene greatly increases alcohol consumption and abolishes sex differences present in WT animals.

Mitochondrial autophagy and cell survival is regulated by the circadian Clock gene in cardiac myocytes during ischemic stress.

Cardiac function is highly reliant on mitochondrial oxidative metabolism and quality control. The circadian Clock gene is critically linked to vital physiological processes including mitochondrial fission, fusion and bioenergetics; however, little is known of how the Clock gene regulates these vital processes in the heart. Herein, we identified a putative circadian CLOCK-mitochondrial interactome that gates an adaptive survival response during myocardial ischemia. We show by transcriptome and gene ontology mapping in CLOCK Δ19/Δ19 mouse that Clock transcriptionally coordinates the efficient removal of damaged mitochondria during myocardial ischemia by directly controlling transcription of genes required for mitochondrial fission, fusion and macroautophagy/autophagy. Loss of Clock gene activity impaired mitochondrial turnover resulting in the accumulation of damaged reactive oxygen species (ROS)-producing mitochondria from impaired mitophagy. This coincided with ultrastructural defects to mitochondria and impaired cardiac function. Interestingly, wild type CLOCK but not mutations of CLOCK defective for E-Box binding or interaction with its cognate partner ARNTL/BMAL-1 suppressed mitochondrial damage and cell death during acute hypoxia. Interestingly, the autophagy defect and accumulation of damaged mitochondria in CLOCK-deficient cardiac myocytes were abrogated by restoring autophagy/mitophagy. Inhibition of autophagy by ATG7 knockdown abrogated the cytoprotective effects of CLOCK. Collectively, our results demonstrate that CLOCK regulates an adaptive stress response critical for cell survival by transcriptionally coordinating mitochondrial quality control mechanisms in cardiac myocytes. Interdictions that restore CLOCK activity may prove beneficial in reducing cardiac injury in individuals with disrupted circadian CLOCK.Abbreviations: ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATG14: autophagy related 14; ATG7: autophagy related 7; ATP: adenosine triphosphate; BCA: bovine serum albumin; BECN1: beclin 1, autophagy related; bHLH: basic helix- loop-helix; CLOCK: circadian locomotor output cycles kaput; CMV: cytomegalovirus; COQ5: coenzyme Q5 methyltransferase; CQ: chloroquine; CRY1: cryptochrome 1 (photolyase-like); DNM1L/DRP1: dynamin 1-like; EF: ejection fraction; EM: electron microscopy; FS: fractional shortening; GFP: green fluorescent protein; HPX: hypoxia; i.p.: intraperitoneal; I-R: ischemia-reperfusion; LAD: left anterior descending; LVIDd: left ventricular internal diameter diastolic; LVIDs: left ventricular internal diameter systolic; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN2: mitofusin 2; MI: myocardial infarction; mPTP: mitochondrial permeability transition pore; NDUFA4: Ndufa4, mitochondrial complex associated; NDUFA8: NADH: ubiquinone oxidoreductase subunit A8; NMX: normoxia; OCR: oxygen consumption rate; OPA1: OPA1, mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PBS: phosphate-buffered saline; PER1: period circadian clock 1; PPARGC1A/PGC-1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; qPCR: quantitative real-time PCR; RAB7A: RAB7, member RAS oncogene family; ROS: reactive oxygen species; RT: room temperature; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFAM: transcription factor A, mitochondrial; TFEB: transcription factor EB; TMRM: tetra-methylrhodamine methyl ester perchlorate; WT: wild -type; ZT: zeitgeber time.

Circadian influence on inflammatory response during cardiovascular disease.

Circadian rhythms follow a 24 h day and night cycle, regulate vital physiological processes, and are especially relevant to cardiovascular growth, renewal, repair, and remodeling. A recent flurry of clinical and experimental studies reveals a profound circadian influence on immune responses in cardiovascular disease. The first section of this review summarizes the importance of circadian rhythms for cardiovascular health and disease. The second section introduces the circadian nature of inflammatory responses. The third section combines these to elucidate a new role for the circadian system, influencing inflammation in heart disease, especially myocardial infarction. Particular focus is on circadian regulation of the NACHT, LRR, and PYD domains-containing protein 3 inflammasome, neutrophils, monocytes/macrophages, and T cells involved in cardiac repair. A role for biological sex is noted. The final section explores circadian influences on inflammation in other major cardiovascular conditions. Circadian regulation of inflammation has profound implications for benefitting the diagnosis, treatment, and prognosis of patients with cardiovascular disease.

Circadian mutant mice with obesity and metabolic syndrome are resilient to cardiovascular disease.

Obesity and metabolic syndrome commonly underlie cardiovascular disease. ClockΔ19/Δ19 mice fed a normal diet develop obesity and metabolic syndrome; however, it is not known whether they develop or are resilient to cardiovascular disease. We found that ClockΔ19/Δ19 mice do not develop cardiac dysfunction, despite their underlying conditions. Moreover, in contrast to wild-type controls fed a high-fat diet (HFD), ClockΔ19/Δ19 HFD mice still do not develop cardiovascular disease. Indeed, ClockΔ19/Δ19 HFD mice have preserved heart weight despite their obesity, no cardiomyocyte hypertrophy, and preserved heart structure and function, even after 24 wk of a HFD. To determine why ClockΔ19/Δ19 mice are resilient to cardiac dysfunction despite their underlying obesity and metabolic conditions, we examined global cardiac gene expression profiles by microarray and bioinformatics analyses, revealing that oxidative stress pathways were involved. We examined the pathways in further detail and found that 1) SIRT-dependent oxidative stress pathways were not directly involved in resilience; 2) 4-hydroxynonenal (4-HNE) increased in wild-type HFD but not ClockΔ19/Δ19 mice, suggesting less reactive oxygen species in ClockΔ19/Δ19 mice; 3) cardiac catalase (CAT) and glutathione peroxidase (GPx) increased, suggesting strong antioxidant defenses in the hearts of ClockΔ19/Δ19 mice; and 4) Pparγ was upregulated in the hearts of ClockΔ19/Δ19 mice; this circadian-regulated gene drives transcription of CAT and GPx, providing a molecular basis for resilience in the ClockΔ19/Δ19 mice. These findings shed new light on the circadian regulation of oxidative stress and demonstrate an important role for the circadian mechanism in resilience to cardiovascular disease.NEW & NOTEWORTHY We examined whether obesity and metabolic syndrome underlie the development of cardiac dysfunction in circadian mutant ClockΔ19/Δ19 mice. Surprisingly, we demonstrate that although ClockΔ19/Δ19 mice develop metabolic dysfunction, they are protected from cardiac hypertrophy, left ventricular remodeling, and diastolic dysfunction, in contrast to wild-type controls, even when challenged with a chronic high-fat diet. These findings shed new light on the circadian regulation of oxidative stress pathways, which can mediate resilience to cardiovascular disease.

Circadian influence on the microbiome improves heart failure outcomes.

Myocardial infarction (MI) leading to heart failure (HF) is a major cause of death worldwide. Previous studies revealed that the circadian system markedly impacts cardiac repair post-MI, and that light is an important environmental factor modulating the circadian influence over healing. Recent studies suggest that gut physiology also affects the circadian system, but how it contributes to cardiac repair post-MI and in HF is not well understood. To address this question, we first used a murine coronary artery ligation MI model to reveal that an intact gut microbiome is important for cardiac repair. Specifically, gut microbiome disruption impairs normal inflammatory responses in infarcted myocardium, elevates adverse cardiac gene biomarkers, and leads to worse HF outcomes. Conversely, reconstituting the microbiome post-MI in mice with prior gut microbiome disruption improves healing, consistent with the notion that normal gut physiology contributes to cardiac repair. To investigate a role for the circadian system, we initially utilized circadian mutant Clock∆19/∆19 mice, revealing that a functional circadian mechanism is necessary for gut microbiome benefits on post-MI cardiac repair and HF. Finally, we demonstrate that circadian-mediated gut responses that benefit cardiac repair can be conferred by time-restricted feeding, as wake time feeding of MI mice improves HF outcomes, but these benefits are not observed in MI mice fed during their sleep time. In summary, gut physiology is important for cardiac repair, and the circadian system influences the beneficial gut responses to improve post-MI and HF outcomes.

Differential effects of REV-ERBα/ß agonism on cardiac gene expression, metabolism, and contractile function in a mouse model of circadian disruption.

Cell-autonomous circadian clocks have emerged as temporal orchestrators of numerous biological processes. For example, the cardiomyocyte circadian clock modulates transcription, translation, posttranslational modifications, ion homeostasis, signaling cascades, metabolism, and contractility of the heart over the course of the day. Circadian clocks are composed of more than 10 interconnected transcriptional modulators, all of which have the potential to influence the cardiac transcriptome (and ultimately cardiac processes). These transcriptional modulators include BMAL1 and REV-ERBα/ß; BMAL1 induces REV-ERBα/ß, which in turn feeds back to inhibit BMAL1. Previous studies indicate that cardiomyocyte-specific BMAL1-knockout (CBK) mice exhibit a dysfunctional circadian clock (including decreased REV-ERBα/ß expression) in the heart associated with abnormalities in cardiac mitochondrial function, metabolism, signaling, and contractile function. Here, we hypothesized that decreased REV-ERBα/ß activity is responsible for distinct phenotypical alterations observed in CBK hearts. To test this hypothesis, CBK (and littermate control) mice were administered with the selective REV-ERBα/ß agonist SR-9009 (100 mg·kg-1·day-1 for 8 days). SR-9009 administration was sufficient to normalize cardiac glycogen synthesis rates, cardiomyocyte size, interstitial fibrosis, and contractility in CBK hearts (without influencing mitochondrial complex activities, nor normalizing substrate oxidation and Akt/mTOR/GSK3ß signaling). Collectively, these observations highlight a role for REV-ERBα/ß as a mediator of a subset of circadian clock-controlled processes in the heart.

SR9009 administered for one day after myocardial ischemia-reperfusion prevents heart failure in mice by targeting the cardiac inflammasome.

Reperfusion of patients after myocardial infarction (heart attack) triggers cardiac inflammation that leads to infarct expansion and heart failure (HF). We previously showed that the circadian mechanism is a critical regulator of reperfusion injury. However, whether pharmacological targeting using circadian medicine limits reperfusion injury and protects against HF is unknown. Here, we show that short-term targeting of the circadian driver REV-ERB with SR9009 benefits long-term cardiac repair post-myocardial ischemia reperfusion in mice. Gain and loss of function studies demonstrate specificity of targeting REV-ERB in mice. Treatment for just one day abates the cardiac NLRP3 inflammasome, decreasing immunocyte recruitment, and thereby allowing the vulnerable infarct to heal. Therapy is given in vivo, after reperfusion, and promotes efficient repair. This study presents downregulation of the cardiac inflammasome in fibroblasts as a cellular target of SR9009, inviting more targeted therapeutic investigations in the future.

The Clock Mechanism Influences Neurobiology and Adaptations to Heart Failure in Clock∆19/∆19 Mice With Implications for Circadian Medicine.

In this study we investigated the role of the circadian mechanism on cognition-relevant brain regions and neurobiological impairments associated with heart failure (HF), using murine models. We found that the circadian mechanism is an important regulator of healthy cognitive system neurobiology. Normal Clock∆19/∆19 mice had neurons with smaller apical dendrite trees in the medial prefrontal cortex (mPFC), and hippocampus, showed impaired visual-spatial memory, and exhibited lower cerebrovascular myogenic tone, versus wild types (WT). We then used the left anterior descending coronary artery ligation model to investigate adaptations in response to HF. Intriguingly, adaptations to neuron morphology, memory, and cerebrovascular tone occurred in differing magnitude and direction between Clock∆19/∆19 and WT mice, ultimately converging in HF. To investigate this dichotomous response, we performed microarrays and found genes crucial for growth and stress pathways that were altered in Clock∆19/∆19 mPFC and hippocampus. Thus these data demonstrate for the first time that (i) the circadian mechanism plays a role in neuron morphology and function; (ii) there are changes in neuron morphology and function in HF; (iii) CLOCK influences neurobiological gene adaptations to HF at a cellular level. These findings have clinical relevance as patients with HF often present with concurrent neurocognitive impairments. There is no cure for HF, and new understanding is needed to reduce morbidity and improve the quality of life for HF patients.

Circadian-Regulated Cell Death in Cardiovascular Diseases.

BACKGROUND: Over the past several years, a variety of human and animal studies have shown that circadian clocks regulate biological cardiovascular rhythms in both health and disease. For example, heart rate and blood pressure fluctuate over 24-hour daily periods, such that levels are higher in the morning and progressively decline in the evening. METHODS AND RESULTS: It is interesting to note that the timing of the administration of various cardiac treatments can also benefit some cardiovascular outcomes. Circadian rhythms have been implicated in the pathogenesis of a number of cardiovascular diseases, including myocardial infarction, ischemia-reperfusion injury after myocardial infarction, and heart failure. Cell death is a major component of ischemia-reperfusion injury and posited as the central underlying cause of ventricular remodeling and cardiac dysfunction following myocardial infarction. It is notable that the time of day profoundly influences cardiac tolerance and sensitivity to cardiac injury. CONCLUSIONS: Herein, we highlight the novel relationship between circadian rhythms and homeostatic processes that governs cell fate by apoptosis, necrosis, and autophagy. Understanding how these intricate processes interconnect at the cellular level is of paramount clinical importance for optimizing treatment strategies to achieve maximum cardiovascular outcome.