Dynamic metabolism of endothelial triglycerides protects against atherosclerosis in mice.

Blood vessels are continually exposed to circulating lipids, and elevation of ApoB-containing lipoproteins causes atherosclerosis. Lipoprotein metabolism is highly regulated by lipolysis, largely at the level of the capillary endothelium lining metabolically active tissues. How large blood vessels, the site of atherosclerotic vascular disease, regulate the flux of fatty acids (FAs) into triglyceride-rich (TG-rich) lipid droplets (LDs) is not known. In this study, we showed that deletion of the enzyme adipose TG lipase (ATGL) in the endothelium led to neutral lipid accumulation in vessels and impaired endothelial-dependent vascular tone and nitric oxide synthesis to promote endothelial dysfunction. Mechanistically, the loss of ATGL led to endoplasmic reticulum stress-induced inflammation in the endothelium. Consistent with this mechanism, deletion of endothelial ATGL markedly increased lesion size in a model of atherosclerosis. Together, these data demonstrate that the dynamics of FA flux through LD affects endothelial cell homeostasis and consequently large vessel function during normal physiology and in a chronic disease state.

TNFα increases the degradation of pyruvate dehydrogenase kinase 4 by the Lon protease to support proinflammatory genes.

The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and 13C-glucose and 13C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation.

SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.

Multiaxial pressure-strain analysis of regional myocardial work in the setting of graded coronary stenoses and dobutamine stress.

We present a detailed analysis of regional myocardial blood flow and work to better understand the effects of coronary stenoses and low-dose dobutamine stress. Our analysis is based on a unique open-chest model in anesthetized canines that features invasive hemodynamic monitoring, microsphere-based blood flow analysis, and an extensive three-dimensional (3-D) sonomicrometer array that provides multiaxial deformational assessments in the ischemic, border, and remote vascular territories. We use this model to construct regional pressure-strain loops for each territory and quantify the loop subcomponent areas that reflect myocardial work contributing to the ejection of blood and wasted work that does not. We demonstrate that reductions in coronary blood flow markedly alter the shapes and temporal relationships of pressure-strain loops, as well as the magnitudes of their total and subcomponent areas. Specifically, we show that moderate stenoses in the mid-left anterior descending coronary artery decrease regional midventricle myocardial work indices and substantially increase indices of wasted work. In the midventricle, these effects are most pronounced along the radial and longitudinal axes, with more modest effects along the circumferential axis. We further demonstrate that low-dose dobutamine can help to restore or even improve function, but often at the cost of increased wasted work. This detailed, multiaxial analysis provides unique insight into the physiology and mechanics of the heart in the presence of ischemia and low-dose dobutamine, with potential implications in many areas, including the detection and characterization of ischemic heart disease and the use of inotropic support for low cardiac output.NEW & NOTEWORTHY Our unique experimental model assesses cardiac pressure-strain relationships along multiple axes in multiple regions. We demonstrate that moderate coronary stenoses decrease regional myocardial work and increase wasted work and that low-dose dobutamine can help to restore myocardial function, but often with further increases in wasted work. Our findings highlight the significant directional variation of cardiac mechanics and demonstrate potential advantages of pressure-strain analyses over traditional, purely deformational measures, especially in characterizing physiological changes related to dobutamine.

Inflammatory stress signaling via NF-kB alters accessible cholesterol to upregulate SREBP2 transcriptional activity in endothelial cells.

There is a growing appreciation that a tight relationship exists between cholesterol homeostasis and immunity in leukocytes; however, this relationship has not been deeply explored in the vascular endothelium. Endothelial cells (ECs) rapidly respond to extrinsic signals, such as tissue damage or microbial infection, by upregulating factors to activate and recruit circulating leukocytes to the site of injury and aberrant activation of ECs leads to inflammatory based diseases, such as multiple sclerosis and atherosclerosis. Here, we studied the role of cholesterol and a key transcription regulator of cholesterol homeostasis, SREBP2, in the EC responses to inflammatory stress. Treatment of primary human ECs with pro-inflammatory cytokines upregulated SREBP2 cleavage and cholesterol biosynthetic gene expression within the late phase of the acute inflammatory response. Furthermore, SREBP2 activation was dependent on NF-κB DNA binding and canonical SCAP-SREBP2 processing. Mechanistically, inflammatory activation of SREBP was mediated by a reduction in accessible cholesterol, leading to heightened sterol sensing and downstream SREBP2 cleavage. Detailed analysis of NF-κB inducible genes that may impact sterol sensing resulted in the identification of a novel RELA-inducible target, STARD10, that mediates accessible cholesterol homeostasis in ECs. Thus, this study provides an in-depth characterization of the relationship between cholesterol homeostasis and the acute inflammatory response in EC.

Targeting the vasculature in cardiometabolic disease.

Obesity has reached epidemic proportions and is a major contributor to insulin resistance (IR) and type 2 diabetes (T2D). Importantly, IR and T2D substantially increase the risk of cardiovascular (CV) disease. Although there are successful approaches to maintain glycemic control, there continue to be increased CV morbidity and mortality associated with metabolic disease. Therefore, there is an urgent need to understand the cellular and molecular processes that underlie cardiometabolic changes that occur during obesity so that optimal medical therapies can be designed to attenuate or prevent the sequelae of this disease. The vascular endothelium is in constant contact with the circulating milieu; thus, it is not surprising that obesity-driven elevations in lipids, glucose, and proinflammatory mediators induce endothelial dysfunction, vascular inflammation, and vascular remodeling in all segments of the vasculature. As cardiometabolic disease progresses, so do pathological changes in the entire vascular network, which can feed forward to exacerbate disease progression. Recent cellular and molecular data have implicated the vasculature as an initiating and instigating factor in the development of several cardiometabolic diseases. This Review discusses these findings in the context of atherosclerosis, IR and T2D, and heart failure with preserved ejection fraction. In addition, novel strategies to therapeutically target the vasculature to lessen cardiometabolic disease burden are introduced.

Prototype device for endoventricular beta-emitting radiotracer detection and molecularly-guided intervention.

BACKGROUND: We have set out to develop a catheter-based theranostic system that: (a) identifies diseased and at-risk myocardium via endocardial detection of systemically delivered ß-emitting radiotracers and (b) utilizes molecular signals to guide delivery of therapeutics to appropriate tissue via direct intramyocardial injection. METHODS: Our prototype device consists of a miniature ß-radiation detector contained within the tip of a flexible intravascular catheter. The catheter can be adapted to incorporate an injection port and retractable needle for therapeutic delivery. The performance of the ß-detection catheter was assessed in vitro with various ß-emitting radionuclides and ex vivo in hearts of pigs following systemic injection of 18F-fluorodeoxyglucose (18F-FDG) at 1-week post-myocardial infarction. Regional catheter-based endocardial measurements of 18F activity were compared to regional tissue activity from PET/CT images and gamma counting. RESULTS: The ß-detection catheter demonstrated sensitive in vitro detection of ß-radiation from 22Na (ß+), 18F (ß+), and 204Tl (ß-), with minimal sensitivity to γ-radiation. For 18F, the catheter demonstrated a sensitivity of 4067 counts/s/µCi in contact and a spatial resolution of 1.1 mm FWHM. Ex vivo measurements of endocardial 18F activity with the ß-detection catheter in the chronic pig infarct model demonstrated good qualitative and quantitative correlation with regional tissue activity from PET/CT images and gamma counting. CONCLUSION: The prototype ß-detection catheter demonstrates sensitive and selective detection of ß- and ß+ emissions over a wide range of energies and enables high-fidelity ex vivo characterization of endocardial activity from systemically delivered 18F-FDG.

Feasibility study of PET dynamic imaging of [18F]DHMT for quantification of reactive oxygen species in the myocardium of large animals.

OBJECTIVES: We aimed to develop a dynamic imaging technique for a novel PET superoxide tracer, [18F]DHMT, to allow for absolute quantification of myocardial reactive oxygen species (ROS) production in a large animal model. METHODS: Six beagle dogs underwent a single baseline dynamic [18F]DHMT PET study, whereas one animal underwent three serial dynamic studies over the course of chronic doxorubicin administration (1 mg·kg-1·week-1 for 15 weeks). During the scans, sequential arterial blood samples were obtained for plasma metabolite correction. The optimal compartment model and graphical analysis method were identified for kinetic modeling. Values for the left ventricular (LV) net influx rate, Ki, were reported for all the studies and compared with the LV standard uptake values (SUVs) and the LV-to-blood pool SUV ratios from the 60 to 90 minute static images. Parametric images were also generated. RESULTS: [18F]DHMT followed irreversible kinetics once oxidized within the myocardium in the presence of superoxide, as evidenced by the fitting generated by the irreversible two-tissue (2Ti) compartment model and the linearity of Patlak analysis. Myocardial Ki values showed a weak correlation with LV SUV (R2 = 0.27), but a strong correlation with LV-to-blood pool SUV ratio (R2 = 0.92). Generation of high-quality parametric images showed superior myocardial to blood contrast compared to static images. CONCLUSIONS: A dynamic PET imaging technique for [18F]DHMT was developed with full and simplified kinetic modeling for absolute quantification of myocardial superoxide production in a large animal model.

Association Between Impaired Myocardial Flow Reserve on 82Rubidium Positron Emission Tomography Imaging and Adverse Events in Patients With Autoimmune Rheumatic Disease.

BACKGROUND: Coronary microvascular dysfunction has been described in patients with autoimmune rheumatic disease (ARD). However, it is unknown whether positron emission tomography (PET)-derived myocardial flow reserve (MFR) can predict adverse events in this population. METHODS: Patients with ARD without coronary artery disease who underwent dynamic rest-stress 82Rubidium PET were retrospectively studied and compared with patients without ARD matched for age, sex, and comorbidities. The association between MFR and a composite end point of mortality or myocardial infarction or heart failure admission was evaluated with time to event and Cox-regression analyses. RESULTS: In 101 patients with ARD (88% female, age: 62±10 years), when compared with matched patients without ARD (n=101), global MFR was significantly reduced (median: 1.68 [interquartile range: 1.34-2.05] versus 1.86 [interquartile range: 1.58-2.28]) and reduced MFR (<1.5) was more frequent (40% versus 22%). MFR did not differ among subtypes of ARDs. In survival analysis, patients with ARD and low MFR (MFR<1.5) had decreased event-free survival for the combined end point, when compared with patients with and without ARD and normal MFR (MFR>1.5) and when compared with patients without ARD and low MFR, after adjustment for the nonlaboratory-based Framingham risk score, rest left ventricular ejection fraction, severe coronary calcification, and the presence of medium/large perfusion defects. In Cox-regression analysis, ARD diagnosis and reduced MFR were both independent predictors of adverse events along with congestive heart failure diagnosis and presence of medium/large stress perfusion defects on PET. Further analysis with inclusion of an interaction term between ARD and impaired MFR revealed no significant interaction effects between ARD and impaired MFR. CONCLUSIONS: In our retrospective cohort analysis, patients with ARD had significantly reduced PET MFR compared with age-, sex-, and comorbidity-matched patients without ARD. Reduced PET MFR and ARD diagnosis were both independent predictors of adverse outcomes.

Impaired Myocardial Flow Reserve on 82Rubidium Positron Emission Tomography/Computed Tomography in Patients With Systemic Sclerosis.

OBJECTIVE: To investigate the association between Raynaud phenomenon (RP) and coronary microvascular dysfunction, we measured myocardial flow reserve (MFR) using positron emission tomography/computed tomography (PET/CT) in patients with primary and secondary RP and controls. METHODS: Patients with RP, patient controls, and healthy participants who underwent dynamic rest-stress 82-rubidium PET/CT were studied. Differences in heart rate-blood pressure product-corrected MFR and clinical predictors of reduced MFR (< 2.0) were determined. RESULTS: Forty-nine patients with RP (80% female; aged 65 ± 11 yrs; 11 with primary RP, 18 with systemic sclerosis [SSc], and 20 with other autoimmune rheumatic diseases [AIRDs] including 6 with systemic lupus erythematosus, 6 with rheumatoid arthritis, 4 with overlap syndrome, 2 with Sjögren syndrome, and 2 with inflammatory arthritis), 49 matched patients without RP or AIRD (78% female; 64 ± 13 yrs), and 14 healthy participants (50% female; 35 ± 5 yrs) were studied. Patients with primary RP, matched patient controls, and healthy participants had comparable MFR. Patients with SSc-RP had significantly reduced MFR (1.62 ± 0.32) compared to matched patient controls (P = 0.03, 2.06 ± 0.61) and to healthy participants (P = 0.01, 2.22 ± 0.44). In multivariable logistic regression, SSc was an independent predictor of reduced MFR. We identified a correlation between time since AIRD diagnosis and MFR (r = -0.30, 95% CI -0.63 to -0.02, P = 0.04). CONCLUSION: Our findings suggest that only secondary, not primary, RP is associated with reduced MFR, and that patients with SSc-RP have reduced MFR compared to those with primary RP and patients with other AIRDs. Larger prospective studies are warranted to fully elucidate the prognostic value of MFR in patients with secondary RP.

Learning-Based Regularization for Cardiac Strain Analysis via Domain Adaptation.

Reliable motion estimation and strain analysis using 3D+ time echocardiography (4DE) for localization and characterization of myocardial injury is valuable for early detection and targeted interventions. However, motion estimation is difficult due to the low-SNR that stems from the inherent image properties of 4DE, and intelligent regularization is critical for producing reliable motion estimates. In this work, we incorporated the notion of domain adaptation into a supervised neural network regularization framework. We first propose a semi-supervised Multi-Layered Perceptron (MLP) network with biomechanical constraints for learning a latent representation that is shown to have more physiologically plausible displacements. We extended this framework to include a supervised loss term on synthetic data and showed the effects of biomechanical constraints on the network's ability for domain adaptation. We validated the semi-supervised regularization method on in vivo data with implanted sonomicrometers. Finally, we showed the ability of our semi-supervised learning regularization approach to identify infarct regions using estimated regional strain maps with good agreement to manually traced infarct regions from postmortem excised hearts.

A Vectorial, ER-Mitochondria Link to Energy Homeostasis in the Vascular Endothelium.

The precise mechanisms of free fatty acid (FFA) uptake in the vascular endothelium are unclear. In this issue of Cell Metabolism, Ibrahim et al. (2020) discover that FFA uptake is partially mediated by a vectorial, ER-mitochondria link, in which mitochondrial ATP production is locally used for the acyl-CoA synthetase activity of the ER-located fatty acid transport protein 4.

Overfeeding and Substrate Availability, But Not Age or BMI, Alter Human Satellite Cell Function.

Satellite cells (SC) aid skeletal muscle growth and regeneration. SC-mediated skeletal muscle repair can both be influenced by and exacerbate several diseases linked to a fatty diet, obesity, and aging. The purpose of this study was to evaluate the effects of different lifestyle factors on SC function, including body mass index (BMI), age, and high-fat overfeeding. For this study, SCs were isolated from the vastus lateralis of sedentary young (18-30 years) and sedentary older (60-80 years) men with varying BMIs (18-32 kg/m2), as well as young sedentary men before and after four weeks of overfeeding (OVF) (55% fat/ + 1000 kcal, n = 4). The isolated SCs were then treated in vitro with a control (5 mM glucose, 10% fetal bovine serum (FBS)) or a high substrate growth media (HSM) (10% FBS, 25 mM glucose, and 400 µM 2:1 oleate-palmitate). Cells were assessed on their ability to proliferate, differentiate, and fuel substrate oxidation after differentiation. The effect of HSM was measured as the percentage difference between SCs exposed to HSM compared to control media. In vitro SC function was not affected by donor age. OVF reduced SC proliferation rates (-19% p < 0.05) but did not influence differentiation. Cellular proliferation in response to HSM was correlated to the donor's body mass index (BMI) (r2 = 0.6121, p < 0.01). When exposed to HSM, SCs from normal weight (BMI 18-25 kg/m2) participants exhibited reduced proliferation and fusion rates with increased fatty-acid oxidation (p < 0.05), while SCs from participants with higher BMIs (BMI 25-32 kg/m2) demonstrated enhanced proliferation in HSM. HSM reduced proliferation and fusion (p < 0.05) in SCs isolated from subjects before OVF, whereas HSM exposure accelerated proliferation and fusion in SCs collected following OVF. These results indicated that diet has a greater influence on SC function than age and BMI. Though age and BMI do not influence in vitro SC function when grown in controlled conditions, both factors influenced the response of SCs to substrate challenges, indicating age and BMI may mediate responses to diet.

Regional myocardial strain analysis via 2D speckle tracking echocardiography: validation with sonomicrometry and correlation with regional blood flow in the presence of graded coronary stenoses and dobutamine stress.

BACKGROUND: Quantitative regional strain analysis by speckle tracking echocardiography (STE) may be particularly useful in the assessment of myocardial ischemia and viability, although reliable measurement of regional strain remains challenging, especially in the circumferential and radial directions. We present an acute canine model that integrates a complex sonomicrometer array with microsphere blood flow measurements to evaluate regional myocardial strain and flow in the setting of graded coronary stenoses and dobutamine stress. We apply this unique model to rigorously evaluate a commercial 2D STE software package and explore fundamental regional myocardial flow-function relationships. METHODS: Sonomicrometers (16 crystals) were implanted in epicardial and endocardial pairs across the anterior myocardium of anesthetized open chest dogs (n = 7) to form three adjacent cubes representing the ischemic, border, and remote regions, as defined by their relative locations to a hydraulic occluder on the mid-left anterior descending coronary artery (LAD). Additional cardiac (n = 3) and extra-cardiac (n = 3) reference crystals were placed to define the cardiac axes and aid image registration. 2D short axis echocardiograms, sonometric data, and microsphere blood flow data were acquired at baseline and in the presence of mild and moderate LAD stenoses, both before and during low-dose dobutamine stress (5 µg/kg/min). Regional end-systolic 2D STE radial and circumferential strains were calculated with commercial software (EchoInsight) and compared to those determined by sonomicrometry and to microsphere blood flow measurements. Post-systolic indices (PSIs) were also calculated for radial and circumferential strains. RESULTS: Low-dose dobutamine augmented both strain and flow in the presence of mild and moderate stenoses. Regional 2D STE strains correlated moderately with strains assessed by sonomicrometry (Rradial = 0.56, p < 0.0001; Rcirc = 0.55, p < 0.0001) and with regional flow quantities (Rradial = 0.61, Rcirc = 0.63). Overall, correspondence between 2D STE and sonomicrometry was better in the circumferential direction (Bias ± 1.96 SD: - 1.0 ± 8.2% strain, p = 0.06) than the radial direction (5.7 ± 18.3%, p < 0.0001). Mean PSI values were greatest in low flow conditions and normalized with low-dose dobutamine. CONCLUSIONS: 2D STE identifies changes in regional end-systolic circumferential and radial strain produced by mild and moderate coronary stenoses and low-dose dobutamine stress. Regional 2D STE end-systolic strain measurements correlate modestly with regional sonomicrometer strain and microsphere flow measurements.

Serial Assessment of Coronary Flow Reserve by Rubidium-82 Positron Emission Tomography Predicts Mortality in Heart Transplant Recipients.

OBJECTIVES: This study aimed to evaluate the long-term prognostic value of serial assessment of coronary flow reserve (CFR) by rubidium Rb 82 (82Rb) positron emission tomography (PET) in heart transplantation (HT) patients. BACKGROUND: Cardiac allograft vasculopathy is a major determinant of late mortality in HT recipients. The long-term prognostic value of serial CFR quantification by PET imaging in HT patients is unknown. METHODS: A total of 89 patients with history of HT (71% men, 7.0 ± 5.7 years post-HT, age 57 ± 11 years) scheduled for dynamic rest and stress (dipyridamole) 82Rb PET between March 1, 2008 and July 31, 2009 (PET-1) were prospectively enrolled in a single-center study. PET myocardial perfusion studies were reprocessed using U.S. Food and Drug Administration-approved software (Corridor 4DM, version 2017) for calculation of CFR. Follow-up PET (PET-2) imaging was performed in 69 patients at 1.9 ± 0.3 years following PET-1. Patients were categorized based on CFR values considering CFR ≤1.5 as low and CFR >1.5 as high CFR. RESULTS: Forty deaths occurred during the median follow-up time of 8.6 years. Low CFR at PET-1 was associated with a 2.77-fold increase in all-cause mortality (95% confidence interval [CI]: 1.34 to 5.74; p = 0.004). CFR decreased over time in patients with follow-up imaging (PET-1: 2.11 ± 0.74 vs. PET-2: 1.81 ± 0.61; p = 0.003). Twenty-five patients were reclassified based on PET-1 and PET-2 (high to low CFR: n = 18, low to high CFR: n = 7). Overall survival was similar in patients reclassified from high to low as patients with low to low CFR, whereas patients reclassified from low to high had similar survival as patients with high to high CFR. In multivariate Cox regression of patients with PET-2, higher baseline CFR (hazard ratio [HR] for a 0.73 unit (one SD) increase: 0.36, 95% CI: 0.16 to 0.82) and reduction in CFR from PET-1 to PET-2 (HR for a 0.79 unit (one SD) decrease: 1.50 to 7.84) were independent predictors of all-cause mortality. CONCLUSIONS: Serial assessment of CFR by 82Rb PET independently predicts long-term mortality in HT patients.

Computed Tomographic Angiography Assessment of Epicardial Coronary Vasoreactivity for Early Detection of Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: The vascular endothelium is a novel target for the detection, management, and prevention of doxorubicin (DOX)-induced cardiotoxicity. OBJECTIVES: The study aimed to: 1) develop a methodology by computed tomography angiography (CTA) to evaluate stress-induced changes in epicardial coronary diameter; and 2) apply this to a chronic canine model of DOX-induced cardiotoxicity to assess vascular toxicity. METHODS: To develop and validate quantitative methods, sequential retrospectively gated coronary CTAs were performed in 16 canines. Coronary diameters were measured at prespecified distances during rest, adenosine (ADE) (280 µg/kg/min), rest 30 min post-ADE, and dobutamine (DOB) (5 µg/kg/min). A subgroup of 8 canines received weekly intravenous DOX (1 mg/kg) for 12 to 15 weeks, followed by rest-stress CTA at cumulative doses of ∼4-mg/kg (3 to 5 mg/kg), ∼8-mg/kg (7 to 9 mg/kg), and ∼12-mg/kg (12 to 15 mg/kg) of DOX. Echocardiograms were performed at these timepoints to assess left ventricular ejection fraction and global longitudinal strain. RESULTS: Under normal conditions, epicardial coronary arteries reproducibly dilated in response to ADE (left anterior descending coronary artery [LAD]: 12 ± 2%, left circumflex coronary artery [LCx]: 13 ± 2%, right coronary artery [RCA]: 14 ± 2%) and DOB (LAD: 17 ± 3%, LCx: 18 ± 2%, RCA: 15 ± 3%). With DOX, ADE vasodilator responses were impaired after ∼4-mg/kg (LAD: -3 ± 1%, LCx: 0 ± 2%, RCA: -5 ± 2%) and ∼8-mg/kg (LAD: -3 ± 1%, LCx: 0 ± 1%, RCA: -2 ± 2%). The DOB dilation response was preserved at ∼4-mg/kg of DOX (LAD: 18 ± 4%, LCx: 11 ± 3%, RCA: 11 ± 2%) but tended to decrease at ∼8-mg/kg of DOX (LAD: 4 ± 2%, LCx: 8 ± 3%, RCA: 3 ± 2%). A significant left ventricular ejection fraction reduction was observed only at 12 to 15 mg/kg DOX (baseline: 63 ± 2%, 12-mg/kg: 45 ± 3%). Global longitudinal strain was abnormal at ∼4-mg/kg of DOX (p = 0.011). CONCLUSIONS: CTA can reliably assess epicardial coronary diameter in response to pharmacological stressors, providing a noninvasive functional index of coronary vasoreactivity. Impaired epicardial vasodilation occurs early in DOX-induced cardiotoxicity.

Dual Angiotensin Receptor-Neprilysin Inhibition With Sacubitril/Valsartan Attenuates Systolic Dysfunction in Experimental Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: Doxorubicin (DOX) induces cardiotoxicity in part by activation of matrix metalloproteinases (MMPs). Sacubitril/valsartan (Sac/Val) exerts additive cardioprotective actions over renin-angiotensin-aldosterone inhibitors in preclinical models of myocardial infarction and in heart failure patients. We hypothesized that Sac/Val would be more cardioprotective than Val in a rodent model of progressive DOX-induced cardiotoxicity, and this benefit would be associated with modulation of MMP activation. OBJECTIVES: We sought to investigate the efficacy of Sac/Val for the treatment of anthracycline-induced cardiotoxicity. METHODS: Male Wistar rats received DOX intraperitoneally (15 mg/kg cumulative) or saline over 3 weeks. Following the first treatment, control animals were gavaged daily with water (n = 25), while DOX-treated animals were gavaged daily with water (n = 25), Val (31 mg/kg; n = 25) or Sac/Val (68 mg/kg; n = 25) for either 4 or 6 weeks. Echocardiography was performed at baseline, and 4 and 6 weeks after DOX initiation. In addition, myocardial MMP activity was assessed with 99mTc-RP805, and cardiotoxicity severity was assessed by histology at these time points in a subgroup of animals. RESULTS: Left ventricular ejection fraction decreased by 10% at 6 weeks in DOX and DOX + Val rats (both p < 0.05), while this reduction was attenuated in DOX + Sac/Val rats. MMP activity was increased at 6 weeks by 76% in DOX-alone rats, and tended to increase in DOX + Val rats (36%; p = 0.051) but was similar in DOX + Sac/Val rats as compared with time-matched control animals. Both therapies attenuated histological evidence of cellular toxicity and fibrosis (p < 0.05). CONCLUSIONS: Sac/Val offers greater protection against left ventricular remodeling and dysfunction compared with standard angiotensin receptor blocker therapy in a rodent model of progressive DOX-induced cardiotoxicity.

Serum endotoxin, gut permeability and skeletal muscle metabolic adaptations following a short term high fat diet in humans.

BACKGROUND: Our previous work demonstrated that a short-term high fat diet (HFD) increased fasting serum endotoxin, altered postprandial excursions of serum endotoxin, and led to metabolic and transcriptional responses in skeletal muscle in young, healthy male humans. PURPOSE: The purpose of the present study was to determine if a short-term high fat diet: 1) increases intestinal permeability and, in turn, fasting endotoxin concentrations and 2) decreases postprandial skeletal muscle fat oxidation. METHODS: Thirteen normal weight young adult males (BMI 23.1 ±â€¯0.8 kg/m2, age 22.2 ±â€¯0.4 years) were fed a control diet (55% carbohydrate, 30% fat, 9% of which was saturated, 15% protein) for two weeks, followed by 5 days of an isocaloric HFD (30% carbohydrate, 55% fat, 25% of which was saturated, 15% protein, isocaloric to the control diet). Intestinal permeability (via four sugar probe test) was assessed in the fasting state. Both before and after the HFD, a high fat meal challenge (HFM, 820 kcal, 25% carbohydrate, 63% fat, 26% of which was saturated, and 12% protein) was administered. After an overnight fast, blood samples were collected before and every hour for 4 h after the HFM to assess endotoxin, and other serum blood measures. Muscle biopsies were obtained from the vastus lateralis before and 4 h after the HFM in order to assess substrate oxidation (glucose, fatty acid and pyruvate) using radiolabeled techniques. Insulin sensitivity was assessed via intravenous glucose tolerance test. Intestinal permeability, blood samples and muscle biopsies were assessed in the same manner before and following the HFD. MAIN FINDINGS: Intestinal permeability was not affected by HFD (p > 0.05), but fasting endotoxin increased two fold following the HFD (p = 0.04). Glucose oxidation and fatty acid oxidation in skeletal muscle homogenates significantly increased after the HFM before the HFD (+97%, and +106% respectively) but declined after the HFM following 5 days of the HFD (-24% and +16% respectively). Fatty acid suppressibility of pyruvate oxidation increased significantly after the HFM (+32%) but this physiological effect was abolished following 5 days of the HFD (+7%). Insulin sensitivity did not change following the HFD. CONCLUSION: These findings demonstrate that in healthy young men, consuming an isocaloric HFD for 5 days increases fasting endotoxin, independent of changes in gut permeability. These changes in endotoxin are accompanied by a broad effect on skeletal muscle substrate metabolism including increases in postprandial fat oxidation. Importantly, the latter occurs independent of changes in body weight and whole-body insulin sensitivity.

Noninvasive In Vivo Quantification of Adeno-Associated Virus Serotype 9-Mediated Expression of the Sodium/Iodide Symporter Under Hindlimb Ischemia and Neuraminidase Desialylation in Skeletal Muscle Using Single-Photon Emission Computed Tomography/Computed Tomography.

BACKGROUND: We propose micro single-photon emission computed tomography/computed tomography imaging of the hNIS (human sodium/iodide symporter) to noninvasively quantify adeno-associated virus 9 (AAV9)-mediated gene expression in a murine model of peripheral artery disease. METHODS: AAV9-hNIS (2×1011 viral genome particles) was injected into nonischemic or ischemic gastrocnemius muscles of C57Bl/6J mice following unilateral hindlimb ischemia ± the α-sialidase NA (neuraminidase). Control nonischemic limbs were injected with phosphate buffered saline or remained noninjected. Twelve mice underwent micro single-photon emission computed tomography/computed tomography imaging after serial injection of pertechnetate (99mTcO4-), a NIS substrate, up to 28 days after AAV9-hNIS injection. Twenty four animals were euthanized at selected times over 1 month for ex vivo validation. Forty-two animals were imaged with 99mTcO4- ± the selective NIS inhibitor perchlorate on day 10, to ascertain specificity of radiotracer uptake. Tissue was harvested for ex vivo validation. A modified version of the U-Net deep learning algorithm was used for image quantification. RESULTS: As quantitated by standardized uptake value, there was a gradual temporal increase in 99mTcO4- uptake in muscles treated with AAV9-hNIS. Hindlimb ischemia, NA, and hindlimb ischemia plus NA increased the magnitude of 99mTcO4- uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS. Perchlorate treatment significantly reduced 99mTcO4- uptake in AAV9-hNIS-treated muscles, demonstrating uptake specificity. The imaging results correlated well with ex vivo well counting (r2=0.9375; P<0.0001) and immunoblot analysis of NIS protein (r2=0.65; P<0.0001). CONCLUSIONS: Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated 99mTcO4- uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.