Association of Aortic Stiffness and Pressure Pulsatility With Noninvasive Estimates of Hepatic Steatosis and Fibrosis: The Framingham Heart Study.

BACKGROUND: Arterial stiffening may contribute to the pathogenesis of metabolic dysfunction-associated steatotic liver disease. We aimed to assess relations of vascular hemodynamic measures with measures of hepatic steatosis and fibrosis in the community. METHODS: Our sample was drawn from the Framingham Offspring, New Offspring Spouse, Third Generation, Omni-1, and Omni-2 cohorts (N=3875; mean age, 56 years; 54% women). We used vibration-controlled transient elastography to assess controlled attenuation parameter and liver stiffness measurements as measures of liver steatosis and liver fibrosis, respectively. We assessed noninvasive vascular hemodynamics using arterial tonometry. We assessed cross-sectional relations of vascular hemodynamic measures with continuous and dichotomous measures of hepatic steatosis and fibrosis using multivariable linear and logistic regression. RESULTS: In multivariable models adjusting for cardiometabolic risk factors, higher carotid-femoral pulse wave velocity (estimated ß per SD, 0.05 [95% CI, 0.01-0.09]; P=0.003), but not forward pressure wave amplitude and central pulse pressure, was associated with more liver steatosis (higher controlled attenuation parameter). Additionally, higher carotid-femoral pulse wave velocity (ß=0.11 [95% CI, 0.07-0.15]; P<0.001), forward pressure wave amplitude (ß=0.05 [95% CI, 0.01-0.09]; P=0.01), and central pulse pressure (ß=0.05 [95% CI, 0.01-0.09]; P=0.01) were associated with more hepatic fibrosis (higher liver stiffness measurement). Associations were more prominent among men and among participants with obesity, diabetes, and metabolic syndrome (interaction P values, <0.001-0.04). Higher carotid-femoral pulse wave velocity, but not forward pressure wave amplitude and central pulse pressure, was associated with higher odds of hepatic steatosis (odds ratio, 1.16 [95% CI, 1.02-1.31]; P=0.02) and fibrosis (odds ratio, 1.40 [95% CI, 1.19-1.64]; P<0.001). CONCLUSIONS: Elevated aortic stiffness and pressure pulsatility may contribute to hepatic steatosis and fibrosis.

Digital Peripheral Arterial Tonometry and Cardiovascular Disease Events: The Framingham Heart Study.

Background and Purpose: Novel noninvasive measures of vascular function are emerging as subclinical markers for cardiovascular disease (CVD) and may be useful to predict CVD events. The purpose of our prospective study was to assess associations between digital peripheral arterial tonometry (PAT) measures and first-onset major CVD events in a sample of FHS (Framingham Heart Study) participants. Methods: Using a fingertip PAT device, we assessed pulse amplitude in Framingham Offspring and Third Generation participants (n=3865; mean age, 55±14 years; 52% women) at baseline and in 30-second intervals for 4 minutes during reactive hyperemia. The PAT ratio (relative hyperemia index) was calculated as the post-to-pre occlusion pulse signal ratio in the occluded arm, relative to the same ratio in the control (nonoccluded) arm, and corrected for baseline vascular tone. Baseline pulse amplitude and PAT ratio during hyperemia are measures of pressure pulsatility and microvascular function in the finger, respectively. We used Cox proportional hazards regression to relate PAT measures in the fingertip to incident CVD events. Results: During follow-up (median, 9.2 years; range, 0.04­10.0 years), 270 participants (7%) experienced new-onset CVD events (n=270). In multivariable models adjusted for cardiovascular risk factors, baseline pulse amplitude (hazard ratio [HR] per 1 SD, 1.04 [95% CI, 0.90­1.21]; P=0.57) and PAT ratio (HR, 0.95 [95% CI, 0.84­1.08]; P=0.43) were not significantly related to incident composite CVD events, including myocardial infarction or heart failure. However, higher PAT ratio (HR, 0.76 [95% CI, 0.61­0.94]; P=0.013), but not baseline pulse amplitude (HR, 1.15 [95% CI, 0.89­1.49]; P=0.29), was related to lower risk for incident stroke. In a sensitivity analysis by stroke subtype, higher PAT ratio was related to lower risk of incident ischemic stroke events (HR, 0.68 [95% CI, 0.53­0.86]; P=0.001). Conclusions: Novel digital PAT measures may represent a marker of stroke risk in the community.

Incorporation of Novel Vascular Measures into Clinical Management: Recent Insights from the Framingham Heart Study.

PURPOSE OF REVIEW: The review discusses evidence from the Framingham Heart Study that supports the assessment and utility of novel vascular and blood pressure measures to inform clinical management of blood pressure-related cardiovascular disease. RECENT FINDINGS: Recent Framingham Heart Study investigations provide new insights into the associations of novel and traditional vascular and blood pressure measures, such as measures of aortic stiffness, components of blood pressure waves, and orthostatic change in blood pressure, with cardiovascular disease events and brain structure and function. Novel vascular measures provide opportunities for additional investigation and potential development of new interventions that are more precisely targeted at underlying pathophysiology. Inclusion of novel vascular measures should be considered in clinical practice to screen for early, subclinical disease and to stratify high-risk individuals for targeted therapies.

Components of hemodynamic load and cardiovascular events: the Framingham Heart Study.

BACKGROUND: Elevated blood pressure is the leading modifiable risk factor for cardiovascular disease (CVD) and premature death. The blood pressure waveform consists of discrete hemodynamic components, derived from measured central pressure and flow, which may contribute separately to risk for an adverse outcome. However, pressure-flow measures have not been studied in a large, community-based sample. METHODS AND RESULTS: We used proportional hazards models to examine the association of incident CVD with forward pressure wave amplitude, mean arterial pressure, and global reflection coefficient derived from wave separation analysis and echocardiography in 2492 participants (mean age 66±9 years, 56% women) in the Framingham Heart Study. During follow-up (0.04-6.8 years), 149 participants (6%) had a CVD event. In multivariable models adjusting for age, sex, antihypertensive therapy, body mass index, heart rate, total and high-density lipoprotein cholesterol concentrations, smoking, and the presence of diabetes mellitus, forward pressure wave amplitude (hazard ratio, 1.40; 95% confidence interval, 1.16-1.67; P=0.0003) was associated with incident CVD, whereas mean arterial pressure (hazard ratio, 1.10; 95% confidence interval, 0.94-1.29; P=0.25) and global wave reflection (hazard ratio, 0.93; 95% confidence interval, 0.78-1.12; P=0.58) were not. After adding systolic blood pressure and carotid-femoral pulse wave velocity to the model, forward pressure wave amplitude persisted as a correlate of events (hazard ratio, 1.33; 95% confidence interval, 1.05-1.68; P=0.02). CONCLUSIONS: Higher forward pressure wave amplitude (a measure of proximal aortic geometry and stiffness) was associated with increased risk for incident CVD, whereas mean arterial pressure and relative wave reflection (correlates of resistance vessel structure and function) were not associated with increased risk for incident CVD.

Hyperphosphorylation of RyRs underlies triggered activity in transgenic rabbit model of LQT2 syndrome.

RATIONALE: Loss-of-function mutations in human ether go-go (HERG) potassium channels underlie long QT syndrome type 2 (LQT2) and are associated with fatal ventricular tachyarrhythmia. Previously, most studies focused on plasma membrane-related pathways involved in arrhythmogenesis in long QT syndrome, whereas proarrhythmic changes in intracellular Ca(2+) handling remained unexplored. OBJECTIVE: We investigated the remodeling of Ca(2+) homeostasis in ventricular cardiomyocytes derived from transgenic rabbit model of LQT2 to determine whether these changes contribute to triggered activity in the form of early after depolarizations (EADs). METHODS AND RESULTS: Confocal Ca(2+) imaging revealed decrease in amplitude of Ca(2+) transients and sarcoplasmic reticulum Ca(2+) content in LQT2 myocytes. Experiments using sarcoplasmic reticulum-entrapped Ca(2+) indicator demonstrated enhanced ryanodine receptor (RyR)-mediated sarcoplasmic reticulum Ca(2+) leak in LQT2 cells. Western blot analyses showed increased phosphorylation of RyR in LQT2 myocytes versus controls. Coimmunoprecipitation experiments demonstrated loss of protein phosphatases type 1 and type 2 from the RyR complex. Stimulation of LQT2 cells with ß-adrenergic agonist isoproterenol resulted in prolongation of the plateau of action potentials accompanied by aberrant Ca(2+) releases and EADs, which were abolished by inhibition of Ca(2+)/calmodulin-dependent protein kinase type 2. Computer simulations showed that late aberrant Ca(2+) releases caused by RyR hyperactivity promote EADs and underlie the enhanced triggered activity through increased forward mode of Na(+)/Ca(2+) exchanger type 1. CONCLUSIONS: Hyperactive, hyperphosphorylated RyRs because of reduced local phosphatase activity enhance triggered activity in LQT2 syndrome. EADs are promoted by aberrant RyR-mediated Ca(2+) releases that are present despite a reduction of sarcoplasmic reticulum content. Those releases increase forward mode Na(+)/Ca(2+) exchanger type 1, thereby slowing repolarization and enabling L-type Ca(2+) current reactivation.

Redox modification of ryanodine receptors by mitochondria-derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts.

Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca(2+)) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca(2+) handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5-9 months) and old (4-6 years) rabbit hearts to test the hypothesis that changes in Ca(2+) homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) by mitochondria-derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca(2+) handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca(2+) dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age-related changes in ROS production and mitochondria membrane potential using a ROS-sensitive dye and a mitochondrial voltage-sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca(2+) load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR-mediated SR Ca(2+) leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under ß-adrenergic stimulation, Ca(2+) transient amplitude, SR Ca(2+) load, and latency of pro-arrhythmic spontaneous Ca(2+) waves (SCWs) were decreased while RyR-mediated SR Ca(2+) leak was increased in cardiomyocytes from old rabbits. Additionally, with ß-adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, which reduced SR Ca(2+) leak, ablated most SCWs, and increased latency to levels comparable to young. These data indicate that an age-associated increase of ROS production by mitochondria leads to the thiol-oxidation of RyRs, which underlies the hyperactivity of RyRs and thereby shortened refractoriness of Ca(2+) release in cardiomyocytes from the ageing heart. This mechanism probably plays an important role in the increased incidence of arrhythmia and sudden death in the ageing population.

Longitudinal Hemodynamic Correlates of and Sex Differences in the Evolution of Blood Pressure Across the Adult Lifespan: The Framingham Heart Study.

Background Systolic blood pressure increases with age after midlife, particularly in women, and contributes to development of wide pulse pressure hypertension in middle-aged and older adults. Relative contributions of aortic stiffness and premature wave reflection to increases in pulse pressure remain controversial. Methods and Results We evaluated visit-specific values and change in key correlates of pulse pressure, aortic characteristic impedance, forward and backward wave amplitude, and global reflection coefficient, at 3 sequential examinations of the Framingham Generation 3 (N=4082), Omni-2 (N=410), and New Offspring Spouse (N=103) cohorts (53% women). Data were analyzed using repeated-measures linear mixed models adjusted for age, sex, and risk factor exposures. Pulse pressure increased markedly with age after midlife (age and age-squared terms, P<0.0001), particularly in women (age slope 3.1±0.2 mm Hg/decade higher in women, P<0.0001). In sex-specific models, change in pulse pressure was closely related (all P<0.0001) to baseline (6.7±0.2 and 7.3±0.2 mm Hg/SD in men and women, respectively) and change (11.8±0.1 and 11.7±0.1 mm Hg/SD) in forward wave amplitude, whereas relations with baseline (2.1±0.15 and 2.0±0.14 mm Hg/SD) and change (4.0±0.13 and 3.4±0.11 mm Hg/SD) in global reflection coefficient were weaker. Global reflection coefficient fell as aortic characteristic impedance increased (P<0.0001), consistent with the hypothesis that impedance matching reduces relative wave reflection in the arterial system. Conclusions Proximal aortic stiffening, as assessed by higher aortic characteristic impedance and larger forward wave amplitude, is strongly associated with longitudinal increase in pulse pressure, especially in women, whereas wave reflection has more modest relations.

Electromechanical and structural alterations in the aging rabbit heart and aorta.

Aging increases the risk for arrhythmias and sudden cardiac death (SCD). We aimed at elucidating aging-related electrical, functional, and structural changes in the heart and vasculature that account for this heightened arrhythmogenic risk. Young (5-9 mo) and old (3.5-6 yr) female New Zealand White (NZW) rabbits were subjected to in vivo hemodynamic, electrophysiological, and echocardiographic studies as well as ex vivo optical mapping, high-field magnetic resonance imaging (MRI), and histochemical experiments. Aging increased aortic stiffness (baseline pulse wave velocity: young, 3.54 ± 0.36 vs. old, 4.35 ± 0.28 m/s, P < 0.002) and diastolic (end diastolic pressure-volume relations: 3.28 ± 0.5 vs. 4.95 ± 1.5 mmHg/ml, P < 0.05) and systolic (end systolic pressure-volume relations: 20.56 ± 4.2 vs. 33.14 ± 8.4 mmHg/ml, P < 0.01) myocardial elastances in old rabbits. Electrophysiological and optical mapping studies revealed age-related slowing of ventricular and His-Purkinje conduction (His-to-ventricle interval: 23 ± 2.5 vs. 31.9 ± 2.9 ms, P < 0.0001), altered conduction anisotropy, and a greater inducibility of ventricular fibrillation (VF, 3/12 vs. 7/9, P < 0.05) in old rabbits. Histochemical studies confirmed an aging-related increased fibrosis in the ventricles. MRI showed a deterioration of the free-running Purkinje fiber network in ventricular and septal walls in old hearts as well as aging-related alterations of the myofibrillar orientation and myocardial sheet structure that may account for this slowed conduction velocity. Aging leads to parallel stiffening of the aorta and the heart, including an increase in systolic stiffness and contractility and diastolic stiffness. Increasingly, anisotropic conduction velocity due to fibrosis and altered myofibrillar orientation and myocardial sheet structure may contribute to the pathogenesis of VF in old hearts. The aging rabbit model represents a useful tool for elucidating age-related changes that predispose the aging heart to arrhythmias and SCD.

Association of Aortic Stiffness and Pressure Pulsatility With Global Amyloid-ß and Regional Tau Burden Among Framingham Heart Study Participants Without Dementia.

Importance: Aortic stiffness is associated with clinical hallmarks of Alzheimer disease and related dementias and could be a modifiable target for disease prevention. Objective: To assess associations of aortic stiffness and pressure pulsatility with global amyloid-ß plaques and regional tau burden in the brain of middle-aged and older adults without dementia. Design, Setting, and Participants: The sample for this cross-sectional study was drawn from the Framingham Heart Study Third Generation Cohort at examination 3 (N = 3171; 2016-2019), of whom 3092 successfully underwent comprehensive hemodynamic evaluations. In a supplemental visit (2015-2021), a subset of 270 participants without dementia who represented the spectrum of vascular risk also underwent positron emission tomography. Thirteen participants were excluded for missing covariate data. The final sample size was 257 participants. Exposures: Three measures of aortic stiffness and pressure pulsatility (carotid-femoral pulse wave velocity, central pulse pressure [CPP], and forward wave amplitude [FWA]) were evaluated using arterial tonometry. Main Outcomes and Measures: Global amyloid-ß plaques and regional tau were assessed using 11C-Pittsburgh compound B and 18F-flortaucipir positron emission tomography tracers, respectively. Results: The mean (SD) age of the 257 participants was 54 (8) years, and 126 were women (49%). All participants were White Western European race. In multivariable models, higher CPP (ß per SD = 0.17; 95% CI, 0.00-0.35; P = .045) and FWA (ß per SD = 0.16; 95% CI, 0.00-0.31; P = .04) were associated with greater entorhinal tau burden. In similar models, higher CPP (ß per SD = 0.19; 95% CI, 0.02-0.36; P = .03) and FWA (ß per SD = 0.17; 95% CI, 0.01-0.32; P = .03) were associated with greater rhinal tau burden. Aortic stiffness and pressure pulsatility measures were not associated with amygdala, inferior temporal, precuneus tau burden, or global amyloid-ß plaques. Associations for entorhinal and rhinal tau outcomes were more prominent in older participants (≥60 years). For example, higher levels of all aortic stiffness and pressure pulsatility measures (ß per SD = 0.40-0.92; P = .001-.02) were associated with higher entorhinal tau burden among older but not younger participants in stratified analyses. Conclusions and Relevance: In this cross-sectional study, abnormal central vascular hemodynamics were associated with higher tau burden in specific brain regions. Findings suggest that aortic stiffness, which is potentially modifiable, may be a probable independent target for prevention of tau-related pathologies.

Relations of postural change in blood pressure with hypertension-mediated organ damage in middle-aged adults of the Framingham heart study: A cross-sectional study.

Background: Dysregulation of compensatory mechanisms to regulate blood pressure (BP) upon postural change is a phenotype of BP variability and an emerging risk factor for cardiovascular outcomes. Materials and methods: We assessed postural change in BP (starting 2 min after standing from a supine position), carotid-femoral pulse wave velocity (cfPWV), and markers of hypertension-mediated organ damage (HMOD) in the heart, kidney, and brain in Framingham Third Generation, Omni-2, and New Offspring Spouse Cohort participants. We related vascular measures (postural change in BP measures and cfPWV) with HMOD in 3,495 participants (mean age 47 years, 53% women) using multivariable logistic and linear regression models. Results: In multivariable-adjusted models, we did not observe significant associations of vascular measures with presence of left ventricular hypertrophy, albuminuria, covert brain infarcts, or white matter hyperintensities (Bonferroni-adjusted P-values > 0.05/20 > 0.0025). In multivariable models, greater cfPWV (est. ß = 0.11 ± 0.03; P < 0.001), but not postural change in BP measures (Bonferroni-adjusted P-values > 0.05/20 > 0.0025), was associated with higher white matter free water using brain magnetic resonance imaging. In multivariable models, greater postural change in pulse pressure was associated with higher urinary albumin-creatinine ratio (est. ß = 0.07 ± 0.02; P < 0.001). No other postural change in BP measure was associated with urinary albumin-creatinine ratio (Bonferroni-adjusted P-values > 0.05/20 > 0.0025). In sex-specific analyses, higher cfPWV was associated with higher urinary albumin-creatinine ratio in men (est. ß: 0.11 ± 0.04; P = 0.002) but not in women (est. ß: 0.03 ± 0.03; P = 0.44). We also observed marginal to strong effect modification by above vs. at/below median postural change in BP for the association of cfPWV with urinary albumin-creatinine ratio (Bonferroni-adjusted interaction P < 0.001-0.01). Vascular measures were not related to left ventricular mass index or fractional anisotropy (Bonferroni-adjusted P-values > 0.05/20 > 0.0025). Conclusion: Baroreflex dysfunction is associated with greater subclinical kidney damage. Additionally, relations of higher aortic stiffness with greater kidney damage may be modified by associated baroreflex dysregulation.

Discrepancies in Observed and Predicted Longitudinal Change in Central Hemodynamic Measures: The Framingham Heart Study.

[Figure: see text].

Intrinsic Frequencies of Carotid Pressure Waveforms Predict Heart Failure Events: The Framingham Heart Study.

Intrinsic frequencies (IFs) derived from arterial waveforms are associated with cardiovascular performance, aging, and prevalent cardiovascular disease (CVD). However, prognostic value of these novel measures is unknown. We hypothesized that IFs are associated with incident CVD risk. Our sample was drawn from the Framingham Heart Study Original, Offspring, and Third Generation Cohorts and included participants free of CVD at baseline (N=4700; mean age 52 years, 55% women). We extracted 2 dominant frequencies directly from a series of carotid pressure waves: the IF of the coupled heart and vascular system during systole (ω1) and the IF of the decoupled vasculature during diastole (ω2). Total frequency variation (Δω) was defined as the difference between ω1 and ω2. We used Cox proportional hazards regression models to relate IFs to incident CVD events during a mean follow-up of 10.6 years. In multivariable models adjusted for CVD risk factors, higher ω1 (hazard ratio [HR], 1.14 [95% CI], 1.03-1.26]; P=0.01) and Δω (HR, 1.16 [95% CI, 1.03-1.30]; P=0.02) but lower ω2 (HR, 0.87 [95% CI, 0.77-0.99]; P=0.03) were associated with higher risk for incident composite CVD events. In similarly adjusted models, higher ω1 (HR, 1.23 [95% CI, 1.07-1.42]; P=0.004) and Δω (HR, 1.26 [95% CI, 1.05-1.50]; P=0.01) but lower ω2 (HR, 0.81 [95% CI, 0.66-0.99]; P=0.04) were associated with higher risk for incident heart failure. IFs were not significantly associated with incident myocardial infarction or stroke. Novel IFs may represent valuable markers of heart failure risk in the community.

Kidney Function and Aortic Stiffness, Pulsatility, and Endothelial Function in African Americans: The Jackson Heart Study.

RATIONALE & OBJECTIVE: The relation of vascular stiffness, endothelial function, and kidney function is incompletely elucidated in African Americans. Our hypothesis was that increased vascular stiffness and endothelial dysfunction are associated with low estimated glomerular filtration rate (eGFR) and albuminuria in African Americans. STUDY DESIGN: Cross-sectional cohort analysis of data from the Jackson Heart Study. SETTINGS & PATIENTS: 2,244 Jackson Heart Study participants (2012-2017 after Exam 3) who had undergone noninvasive hemodynamic assessment using arterial tonometry. PREDICTORS: Baseline carotid-femoral pulse wave velocity, pulsatile hemodynamics forward wave amplitude, and hyperemic brachial artery flow were measured. Reduced eGFR was defined as eGFR between 15 and 60 mL/min/1.73 m2. OUTCOMES: Prevalent albuminuria, urinary albumin-creatinine ratio. ANALYTICAL APPROACH: 2-sample t test for continuous variables and χ2 test for categorical variables in addition to logistic and linear regression models to assess the risk for chronic kidney disease with each proposed hemodynamic variable. RESULTS: Among 2,244 participants, mean age was 66 ± 11 years and 64% were women. Reduced eGFR was present in 233 (10.4%), and elevated urinary albumin-creatinine ratio, in 232 (10.4%). In multivariable-adjusted analyses, higher carotid-femoral pulse wave velocity was associated with the presence of reduced eGFR (OR, 1.37 [95% CI, 1.08-1.75] per SD; P = 0.01) and with prevalent albuminuria (OR, 1.66 [95% CI, 1.32-2.11]; P < 0.001). Higher forward wave amplitude was significantly associated with prevalent albuminuria (OR, 1.37 [95% CI, 1.14-1.65]; P = 0.001). LIMITATIONS: Cross-sectional analyses cannot inform causality. CONCLUSIONS: Higher arterial stiffness and pulsatility are associated with higher odds of reduced eGFR in African Americans. Future studies should focus on whether improving arterial stiffness contributes to kidney protection in African Americans.

Characterization of an archaeal medium-chain acyl coenzyme A synthetase from Methanosarcina acetivorans.

Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated Macs(Ma), utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PP(i) were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PP(i). These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The Macs(Ma) structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys(256), Cys(298), Gly(351), Trp(259), Trp(237), and Trp(254), were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme.

Clinical Associations of Vascular Stiffness, Microvascular Dysfunction, and Prevalent Cardiovascular Disease in a Black Cohort: The Jackson Heart Study.

Background Measures of vascular dysfunction are related to adverse cardiovascular disease (CVD) outcomes in non-Hispanic, White populations; however, data from Black individuals are limited. We aimed to investigate the associations between novel hemodynamic measures and prevalent CVD in a sample of Black individuals. Methods and Results Among older Black participants of the Jackson Heart Study, we assessed noninvasive vascular hemodynamic measures using arterial tonometry and Doppler ultrasound. We assessed 5 measures of aortic stiffness and wave reflection (carotid-femoral pulse wave velocity, pulse wave velocity ratio, forward pressure wave amplitude, central pulse pressure, and augmentation index), and 2 measures of microvascular function (baseline and hyperemic brachial flow velocity). Using multivariable logistic regression models, we examined the relations between vascular hemodynamic measures and prevalent CVD. In models adjusted for traditional CVD risk factors, higher carotid-femoral pulse wave velocity (odds ratio [OR],1.25; 95% CI, 1.01-1.55; P=0.04), lower augmentation index (OR, 0.84; 95% CI, 0.70-0.99; P=0.05), and lower hyperemic brachial flow velocity (OR, 0.77; 95% CI, 0.65-0.90; P=0.001) were associated with higher odds of CVD. After further adjustment for hypertension treatment, lower augmentation index (OR, 0.84; 95% CI, 0.70-0.99; P=0.04) and hyperemic brachial flow velocity (OR, 0.79; 95% CI, 0.67-0.94; P=0.006), but not carotid-femoral pulse wave velocity (OR, 1.23; 95% CI, 0.99-1.051; P=0.06), were associated with higher odds of CVD. Conclusions In a sample of older Black individuals, more severe microvascular damage and aortic stiffness were associated with prevalent CVD. Further research on hemodynamic mechanisms that contribute to cardiovascular risk among older Black individuals is merited.

The 2.1 A crystal structure of an acyl-CoA synthetase from Methanosarcina acetivorans reveals an alternate acyl-binding pocket for small branched acyl substrates.

The acyl-AMP forming family of adenylating enzymes catalyze two-step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X-ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140 degrees to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl-binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl-binding pocket.

Enhancing Autophagy Diminishes Aberrant Ca2+ Homeostasis and Arrhythmogenesis in Aging Rabbit Hearts.

AIM: Aging in humans is associated with a 10-40-fold greater incidence of sudden cardiac death from malignant tachyarrhythmia. We have reported that thiol oxidation of ryanodine receptors (RyR2s) by mitochondria-derived reactive oxygen species (mito-ROS) contributes to defective Ca2+ homeostasis in cardiomyocytes (CMs) from aging rabbit hearts. However, mechanisms responsible for the increase in mito-ROS in the aging heart remain poorly understood. Here we test the hypothesis that age-associated decrease in autophagy is a major contributor to enhanced mito-ROS production and thereby pro-arrhythmic disturbances in Ca2+ homeostasis. METHODS AND RESULTS: Ventricular tissues from aged rabbits displayed significant downregulation of proteins involved in mitochondrial autophagy compared with tissues from young controls. Blocking autophagy with chloroquine increased total ROS production in primary rabbit CMs and mito-ROS production in HL-1 CMs. Furthermore, chloroquine treatment of HL-1 cells depolarized mitochondrial membrane potential (Δψm) to 50% that of controls. Blocking autophagy significantly increased oxidation of RyR2, resulting in enhanced propensity to pro-arrhythmic spontaneous Ca2+ release under ß-adrenergic stimulation. Aberrant Ca2+ release was abolished by treatment with the mito-ROS scavenger mito-TEMPO. Importantly, the autophagy enhancer Torin1 and ATG7 overexpression reduced the rate of mito-ROS production and restored both Δψm and defective Ca2+ handling in CMs derived from aged rabbit hearts. CONCLUSION: Decreased autophagy is a major cause of increased mito-ROS production in the aging heart. Our data suggest that promoting autophagy may reduce pathologic mito-ROS during normal aging and reduce pro-arrhythmic spontaneous Ca2+ release via oxidized RyR2s.

Short-Long Heart Rate Variation Increases Dispersion of Action Potential Duration in Long QT Type 2 Transgenic Rabbit Model.

The initiation of polymorphic ventricular tachycardia in long QT syndrome type 2 (LQT2) has been associated with a characteristic ECG pattern of short-long RR intervals. We hypothesize that this characteristic pattern increases APD dispersion in LQT2, thereby promoting arrhythmia. We investigated APD dispersion and its dependence on two previous cycle lengths (CLs) in transgenic rabbit models of LQT2, LQT1, and their littermate controls (LMC) using random stimulation protocols. The results show that the short-long RR pattern was associated with a larger APD dispersion in LQT2 but not in LQT1 rabbits. The multivariate analyses of APD as a function of two previous CLs (APDn = C + α1CLn-1 + α2CLn-2) showed that α1 (APD restitution slope) is largest and heterogeneous in LQT2 but uniform in LQT1, enhancing APD dispersion under long CLn-1 in LQT2. The α2 (short-term memory) was negative in LQT2 while positive in LQT1, and the spatial pattern of α1 was inversely correlated to α2 in LQT2, which explains why a short-long combination causes a larger APD dispersion in LQT2 but not in LQT1 rabbits. In conclusion, short-long RR pattern increased APD dispersion only in LQT2 rabbits through heterogeneous APD restitution and the short-term memory, underscoring the genotype-specific triggering of arrhythmias in LQT syndrome.

LITAF (Lipopolysaccharide-Induced Tumor Necrosis Factor) Regulates Cardiac L-Type Calcium Channels by Modulating NEDD (Neural Precursor Cell Expressed Developmentally Downregulated Protein) 4-1 Ubiquitin Ligase.

BACKGROUND: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. METHODS: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (ICa,L) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. RESULTS: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. CONCLUSIONS: LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.

Relations of Microvascular Function, Cardiovascular Disease Risk Factors, and Aortic Stiffness in Blacks: The Jackson Heart Study.

Background Blacks have more severe endothelial dysfunction and aortic stiffening as compared with whites. We aimed to investigate the association between aortic stiffness and microvascular function in the black community. Methods and Results We assessed the association between forearm vascular reactive hyperemia (an indicator of microvascular function) and aortic stiffness in 1458 black participants (N=965 [66% women]; mean age: 66±11 years) in the Jackson Heart Study. We evaluated 2 measures of aortic stiffness: brachial pulse pressure and carotid-femoral pulse wave velocity. Using high-resolution ultrasound and Doppler, we evaluated brachial blood flow at baseline and during reactive hyperemia after 5 minutes of forearm ischemia. Multiple cardiovascular risk factors were significantly related to baseline and hyperemic brachial flow velocity. Women had lower baseline flow across the entire age spectrum. During hyperemia, we observed a significant age-sex interaction for flow velocity ( P=0.02). Female sex was protective against microvascular dysfunction among younger participants, but older women exhibited a greater attenuation of the hyperemic flow reserve. In multivariable models that adjusted for cardiovascular disease risk factors and mean arterial pressure, higher carotid-femoral pulse wave velocity (ß=-0.106±0.033; P=0.001 was related to lower baseline flow. However, during reactive hyperemia, elevated brachial pulse pressure (ß=-0.070±0.031; P=0.03) and carotid-femoral pulse wave velocity (ß=-0.128±0.030; P<0.001) were both related to attenuated brachial flow velocity. Conclusions In a sample of blacks, higher aortic stiffness and pressure pulsatility were associated with lower flow reserve during reactive hyperemia, beyond changes attributable to traditional cardiovascular disease risk factors alone.