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IMPORTANCE: Dapagliflozin reduces the risk of hospitalizations for heart failure and the progression of chronic kidney disease in patients with and without type 2 diabetes (T2D), whereas the effects on reducing atherosclerotic events appear less clear. OBJECTIVE: To explore whether N-terminal pro–B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin T (hsTnT) levels can identify a subset of patients with T2D at higher risk and who might benefit more from dapagliflozin with regard to atherosclerotic events. DESIGN, SETTING, AND PARTICIPANTS: This was a secondary analysis of the DECLARE-TIMI 58 trial, a randomized clinical trial of dapagliflozin in patients with T2D and either multiple risk factors for atherosclerotic cardiovascular disease (ASCVD; approximately 60%) or established ASCVD (approximately 40%). All patients with available blood samples at randomization were included in these analyses. Data were collected from May 2013 to September 2018, and data were analyzed from May 2019 to June 2022. INTERVENTIONS: Dapagliflozin vs placebo. MAIN OUTCOMES AND MEASURES: Major adverse cardiovascular events (MACE), the composite of myocardial infarction, ischemic stroke, or cardiovascular death, which was one of dual primary outcomes of the main trial. RESULTS: Of 14 565 included patients, 9143 (62.8%) were male, and the mean (SD) age was 63.9 (6.8) years. When tested individually in a multivariable model for MACE risk, NT-proBNP and hsTnT were each significantly associated with the risk of MACE (adjusted hazard ratio [aHR] per 1 SD in log-transformed biomarker: NT-proBNP, 1.62; 95% CI, 1.49-1.76; hsTnT: 1.59; 95% CI, 1.46-1.74). The magnitude of the association was similar in patients with ASCVD (NT-proBNP: aHR, 1.60; 95% CI, 1.45-1.77; hsTnT: aHR, 1.62; 95% CI, 1.45-1.81) and multiple risk factors for ASCVD (NT-proBNP: aHR, 1.62; 95% CI, 1.40-1.88; hsTnT: aHR, 1.51; 95% CI, 1.29-1.77). Moreover, both biomarkers remained independently associated with MACE when both were included in the multivariable model (NT-proBNP: aHR, 1.46; 95% CI, 1.34-1.60; hsTnT: aHR, 1.39; 95% CI, 1.26-1.53). Modeled as a continuous variable, baseline biomarker levels did not modify the relative treatment effect of dapagliflozin vs placebo with MACE. However, the relative risk reduction numerically grew with higher biomarker levels, as did the baseline risk. Thus, MACE event rates were nominally lower in dapagliflozin-treated vs placebo-treated patients with biomarker concentrations in the top quartile (NT-proBNP: HR, 0.83; 95% CI, 0.71-0.97; absolute risk reduction [ARR], 2.4%; hsTnT: HR, 0.85; 95% CI, 0.72-0.99; ARR, 2.7%), whereas there was no significant treatment effect in patients with biomarkers levels in quartiles 1 to 3 (NT-proBNP: HR, 1.02; 95% CI, 0.88-1.18; ARR, 0%; hsTnT: HR, 0.97; 95% CI, 0.84-1.13; ARR, 0.2%). CONCLUSIONS AND RELEVANCE: In this study, NT-proBNP and hsTnT levels were associated with the risk for future cardiovascular events in both primary and secondary prevention patients with T2D. Both cardiac biomarkers were helpful to identify patients at very high risk for atherosclerotic events that may derive reduction in risk of MACE with dapagliflozin. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01730534

IMPORTANCE: Studies have demonstrated an association between single measures of high-sensitivity troponin (hsTn) and future cardiovascular events in patients with chronic coronary syndromes. However, limited data exist regarding the association between changes in serial values of hsTn and subsequent cardiovascular events in this patient population. OBJECTIVE: To evaluate the association between changes in high-sensitivity troponin T (hsTnT) and subsequent cardiovascular events in patients stabilized after acute coronary syndrome (ACS). DESIGN, SETTING, AND PARTICIPANTS: This is a secondary analysis from the Improved Reduction of Outcomes: Vytorin Efficacy International Trial (IMPROVE-IT), a randomized clinical trial of ezetimibe vs placebo on a background of simvastatin in 18 144 patients hospitalized for an ACS across 1147 sites in 39 countries. The current biomarker substudy includes the 6035 participants consenting to the biomarker substudy with available hsTnT at months 1 and 4. Data were collected from October 26, 2005, through July 8, 2010, with the database locked October 21, 2014. Data were analyzed from February 28, 2021, through August 14, 2022. MAIN OUTCOMES AND MEASURES: The outcomes of interest were cardiovascular death, myocardial infarction (MI), stroke, or hospitalization for heart failure (HHF). Associations of absolute and relative changes in hsTnT between month 1 and month 4 as a function of the starting month 1 hsTnT and the composite outcome were examined using landmark analyses. RESULTS: Of 6035 patients in this analysis (median [IQR] age, 64 [57-71]), 1486 (24.6%) were female; 361 (6.0%) were Asian; 121 were (2.0%) Black; 252 (4.2%) were Spanish descent; 4959 were (82.2%) White; and 342 (5.7%) reported another race (consolidated owing to small numbers), declined to respond, or were not asked to report race owing to regulatory prohibitions. Most patients (4114 [68.2%]) had stable hsTnT values (change <3 ng/L), with 1158 (19.2%) and 763 (12.6%) having changes of 3 to less than 7 ng/L and 7 ng/L or more, respectively. After adjustment for clinical risk factors and stratification by the starting month 1 hsTnT level, an absolute increase in hsTnT of 7 ng/L or more was associated with a more than 3-fold greater risk of the composite outcome (adjusted hazard ratio [aHR], 3.33; 95% CI, 1.99-5.57; P < .001), whereas decreases of 7 ng/L or more were associated with similar to lower risk (aHR, 0.51; 95% CI, 0.26-1.03; P = .06) compared with stable values. There was a stepwise association moving from larger absolute decreases (aHR, 0.51; 95% CI, 0.26-1.03) to larger absolute increases (aHR, 3.33; 95% CI, 1.99-5.57) in hsTnT with future risk of the composite outcome (P trend <.001). A similar association was observed when analyzed on the basis of relative percent and continuous change. CONCLUSIONS AND RELEVANCE: Among stable patients post-ACS, changes in hsTnT were associated with a gradient of risk of subsequent cardiovascular events across the range of starting hsTnT values. Serial assessment of hsTnT may refine risk stratification with the potential to guide therapy decisions in this patient population. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00202878

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Chromosome 13q deletion [del(13q)], harboring the miR-15a/16-1cluster, is one of the most common genetic alterations in mature B-cell malignancies, which originate from germinal center (GC) and post-GC B cells. Moreover, miR-15a/16 expression is frequently reduced in lymphoma and multiple myeloma (MM) cells without del(13q), suggesting important tumor-suppressor activity. However, the role of miR-15a/16-1in B-cell activation and initiation of mature B-cell neoplasms remains to be determined. We show that conditional deletion of the miR-15a/16-1cluster in murine GC B cells induces moderate but widespread molecular and functional changes including an increased number of GC B cells, percentage of dark zone B cells, and maturation into plasma cells. With time, this leads to development of mature B-cell neoplasms resembling human extramedullary plasmacytoma (EP) as well as follicular and diffuse large B-cell lymphomas. The indolent nature and lack of bone marrow involvement of EP in our murine model resembles human primary EP rather than MM that has progressed to extramedullary disease. We corroborate human primary EP having low levels of miR-15a/16 expression, with del(13q) being the most common genetic loss. Additionally, we show that, although the mutational profile of human EP is similar to MM, there are some exceptions such as the low frequency of hyperdiploidy in EP, which could account for different disease presentation. Taken together, our studies highlight the significant role of the miR-15a/16-1cluster in the regulation of the GC reaction and its fundamental context-dependent tumor-suppression function in plasma cell and B-cell malignancies.

Chromosome 13q deletion [del(13q)], harboring the miR-15a/16-1 cluster, is one of the most common genetic alterations in mature B-cell malignancies, which originate from germinal center (GC) and post-GC B cells. Moreover, miR-15a/16 expression is frequently reduced in lymphoma and multiple myeloma (MM) cells without del(13q), suggesting important tumor-suppressor activity. However, the role of miR-15a/16-1 in B-cell activation and initiation of mature B-cell neoplasms remains to be determined. We show that conditional deletion of the miR-15a/16-1 cluster in murine GC B cells induces moderate but widespread molecular and functional changes including an increased number of GC B cells, percentage of dark zone B cells, and maturation into plasma cells. With time, this leads to development of mature B-cell neoplasms resembling human extramedullary plasmacytoma (EP) as well as follicular and diffuse large B-cell lymphomas. The indolent nature and lack of bone marrow involvement of EP in our murine model resembles human primary EP rather than MM that has progressed to extramedullary disease. We corroborate human primary EP having low levels of miR-15a/16 expression, with del(13q) being the most common genetic loss. Additionally, we show that, although the mutational profile of human EP is similar to MM, there are some exceptions such as the low frequency of hyperdiploidy in EP, which could account for different disease presentation. Taken together, our studies highlight the significant role of the miR-15a/16-1 cluster in the regulation of the GC reaction and its fundamental context-dependent tumor-suppression function in plasma cell and B-cell malignancies.

IMPORTANCE: The 2018 American Heart Association/American College of Cardiology (AHA/ACC) cholesterol management guidelines identified 2 distinct groups of patients with atherosclerotic cardiovascular disease (ASCVD) prompting different treatment recommendations. OBJECTIVE: To investigate whether the addition of high-sensitivity troponin (hsTn) testing to guideline-derived ASCVD risk can improve risk classification and downstream treatment recommendations. DESIGN, SETTING, AND PARTICIPANTS: A prospective cohort biomarker substudy was performed that included 8635 patients enrolled in the Prevention of Cardiovascular Events in Patients with Prior Heart Attack Using Ticagrelor Compared to Placebo on a Background of Aspirin–Thrombolysis in Myocardial Infarction 54 (PEGASUS-TIMI 54) trial. Patients were assigned to risk groups of either very high-risk ASCVD or lower-risk ASCVD based on their cardiovascular history and comorbidities, in line with the 2018 AHA/ACC cholesterol management guidelines criteria. Patients were also classified on the basis of hsTnI level (ARCHITECT assay; Abbott) using cut points of 2 ng/L (limit of detection) and 6 ng/L (risk threshold), followed by joint classification on the basis of clinical features and hsTnI level. The setting was a nested prospective cohort study in a completed multinational trial. Participants were all patients who had a myocardial infarction 1 to 3 years before enrollment, were at least 50 years of age, and had at least 1 high-risk feature. The study dates were October 2010 to December 2014. The dates of analysis were June 2019 to January 2020. MAIN OUTCOMES AND MEASURES: The primary end point was a composite of cardiovascular death, myocardial infarction, or stroke. RESULTS: Among 8635 patients enrolled in the PEGASUS-TIMI 54 trial, the median age was 65 years (interquartile range, 58-71 years), and 6614 (76.6%) were men; 8340 (96.6%) were White individuals and 176 (2.0%) were Black individuals. Patients meeting clinical criteria for the very high-risk ASCVD group had a primary end point 3-year event rate of 8.8% compared with 5.0% in the lower-risk ASCVD group (hazard ratio, 2.01; 95% CI, 1.58-2.57; P < .001). When patients in the very high-risk ASCVD group were further risk stratified by hsTnI level, 614 of 6789 patients (9.0%) with an undetectable hsTnI level had a 3-year event rate of 2.7% (<1% per year), which was less than the overall rate in the lower-risk ASCVD group. Analogously, in the lower-risk ASCVD group, 417 of 1846 patients (22.6%) with an hsTnI level exceeding 6 ng/L had an event rate of 9.1%, comparable to the overall rate in the very high-risk ASCVD group. The addition of hsTnI to guideline-derived ASCVD risk led to a net reclassification index at event rate of 0.15 (95% CI, 0.10-0.21). Overall, use of hsTnI reclassified 1031 of 8635 patients (11.9%) (1 in 11 with very high-risk ASCVD and 1 in 4 with lower-risk ASCVD). CONCLUSIONS AND RELEVANCE: The findings of this cohort substudy suggest that a strategy incorporating hsTn into a guideline-derived ASCVD risk algorithm provides enhanced risk stratification and reclassifies 11.9% of patients into a more appropriate risk group. This application of hsTn testing might be used to optimize the care of patients with ASCVD.

MYD88 L265P is the most common mutation in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) and one of the most frequent in poor-prognosis subtypes of diffuse large B-cell lymphoma (DLBCL). Although inhibition of the mutated MYD88 pathway has an adverse impact on LPL/WM and DLBCL cell survival, its role in lymphoma initiation remains to be clarified. We show that in mice, human MYD88L265P promotes development of a non-clonal, low-grade B-cell lymphoproliferative disorder with several clinicopathologic features that resemble human LPL/WM, including expansion of lymphoplasmacytoid cells, increased serum immunoglobulin M (IgM) concentration, rouleaux formation, increased number of mast cells in the bone marrow, and proinflammatory signaling that progresses sporadically to clonal, high-grade DLBCL. Murine findings regarding differences in the pattern of MYD88 staining and immune infiltrates in the bone marrows of MYD88 wild-type (MYD88WT) and MYD88L265P mice are recapitulated in the human setting, which provides insight into LPL/WM pathogenesis. Furthermore, histologic transformation to DLBCL is associated with acquisition of secondary genetic lesions frequently seen in de novo human DLBCL as well as LPL/WM-transformed cases. These findings indicate that, although the MYD88L265P mutation might be indispensable for the LPL/WM phenotype, it is insufficient by itself to drive malignant transformation in B cells and relies on other, potentially targetable cooperating genetic events for full development of lymphoma.

MYD88L265Pis the most common mutation in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) and one of the most frequent in poor-prognosis subtypes of diffuse large B-cell lymphoma (DLBCL). Although inhibition of the mutated MYD88 pathway has an adverse impact on LPL/WM and DLBCL cell survival, its role in lymphoma initiation remains to be clarified. We show that in mice, human MYD88L265Ppromotes development of a non-clonal, low-grade B-cell lymphoproliferative disorder with several clinicopathologic features that resemble human LPL/WM, including expansion of lymphoplasmacytoid cells, increased serum immunoglobulin M (IgM) concentration, rouleaux formation, increased number of mast cells in the bone marrow, and proinflammatory signaling that progresses sporadically to clonal, high-grade DLBCL. Murine findings regarding differences in the pattern of MYD88 staining and immune infiltrates in the bone marrows of MYD88wild-type (MYD88WT) and MYD88L265Pmice are recapitulated in the human setting, which provides insight into LPL/WM pathogenesis. Furthermore, histologic transformation to DLBCL is associated with acquisition of secondary genetic lesions frequently seen in de novo human DLBCL as well as LPL/WM-transformed cases. These findings indicate that, although the MYD88L265Pmutation might be indispensable for the LPL/WM phenotype, it is insufficient by itself to drive malignant transformation in B cells and relies on other, potentially targetable cooperating genetic events for full development of lymphoma.

Intradialytic hypotension (IDH) is a common complication at the initiation of hemodialysis (HD) therapy, is associated with greater mortality, and may be related to relatively rapid shifts in plasma osmolality. This study sought to evaluate the effect of an intervention to minimize intradialytic changes in plasma osmolality on the occurrence of IDH.

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Growth differentiation factor-15 (GDF-15) is a stress-inducible cytokine and member of the transforming growth factor-β cytokine superfamily that refines prognostic assessment in subgroups of patients with heart failure (HF). We evaluated its role in HF patients with chronic kidney disease (CKD, estimated glomerular filtration rate <60 mL/min/1.73 m2).

Supplemental Digital Content is available in the text.

IMPORTANCE: Elevated fibroblast growth factor 23 (FGF-23) concentrations are associated with myocardial fibrosis and renin-angiotensin system upregulation, potentially providing prognostic information distinct from standard cardiovascular (CV) biomarkers. OBJECTIVE: To evaluate the association of FGF-23 with recurrent CV events in patients after an acute coronary syndrome (ACS). DESIGN, SETTING, AND PARTICIPANTS: C-terminal FGF-23 was measured in plasma samples using an established enzyme-linked immunosorbent assay system for 4947 patients within 30 days of ACS (median, 14 days) and with 1 additional CV risk factor in the Stabilization of Plaques Using Darapladib-Thrombolysis in Myocardial Infarction 52 (SOLID-TIMI 52) trial of the lipoprotein-associated phospholipase A2 inhibitor darapladib vs placebo performed from December 1, 2009, to April 24, 2014 (median follow-up, 2.5 years). Analyses were adjusted for clinical risk factors, renal function, and established cardiorenal biomarkers. This secondary analysis was performed from September 25, 2014, to October 1, 2017. EXPOSURE: The FGF-23 concentration at baseline. MAIN OUTCOMES AND MEASURES: The primary end point for this post hoc analysis was the composite of CV death or hospitalization for heart failure. RESULTS: In this study, baseline FGF-23 concentrations were available for 4947 patients (median age, 64.0 years; interquartile range, 59.0-71.0 years; 1276 [25.8%] female). Patients with higher FGF-23 concentrations were older and more likely female, with a greater proportion of hypertension, diabetes, and previous myocardial infarction. After multivariable adjustment for baseline clinical characteristics and established biomarkers (high-sensitivity troponin I, brain-type natriuretic peptide, and high-sensitivity C-reactive protein), FGF-23 concentration in the top quartile was independently associated with an increased risk of CV death or heart failure hospitalization (adjusted hazard ratio [HR], 2.35; 95% CI, 1.82-3.02; P &lt; .001) and its individual components. Elevated FGF-23 concentration was also associated with an increased risk of all-cause mortality (adjusted HR, 2.27; 95% CI, 1.73-2.97; P &lt; .001) and CV death, myocardial infarction, or stroke (adjusted HR, 1.42; 95% CI, 1.17-1.71; P &lt; .001). When analyses were stratified by patient sex, the association between FGF-23 and CV risk, including CV death or heart failure, appeared to be attenuated in women (adjusted HR, 1.11; 95% CI, 0.70-1.76; P = .67) compared with men (HR, 3.11; 95% CI, 2.29-4.22; P &lt; .001; P &lt; .001 for interaction). CONCLUSIONS AND RELEVANCE: In patients stabilized after ACS, elevated FGF-23 concentrations may be associated with recurrent major CV events and all-cause mortality, providing information independent of established clinical risk factors and cardiorenal biomarkers. A potential sex difference in these findings deserves further study.

Circulating thyroglobulin antibodies (TgAb) can confound measurement of serum thyroglobulin and impair thyroid cancer surveillance. Few data exist on the significance of TgAb in pediatric thyroid cancer.

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IMPORTANCE: Sarcomere mutations and left ventricular (LV) hypertrophy (LVH) are cardinal features of hypertrophic cardiomyopathy (HCM). However, little is known about the full spectrum of phenotypic manifestations or how LVH influences disease expression. OBJECTIVES: (1) To characterize and assess phenotypic burden in sarcomere mutation carriers (genotype positive [G+]) and (2) to investigate the correlation between LV wall thickness (LVWT) and other disease features in mutation carriers. DESIGN, SETTING, AND PARTICIPANTS: This investigation was a cross-sectional, multicenter observational study in the setting of the HCMNet network of HCM clinical centers. Mutation carriers with LVH (G+/LVH+), mutation carriers without LVH (G+/LVH−), and healthy related control individuals (G−/LVH−) were enrolled through HCMNet sites. A total of 193 participants were enrolled and underwent study procedures. Participants were enrolled between April 9, 2010, and January 30, 2012. Study analysis was performed between June 2015 and May 2016. EXPOSURES: The primary stratifying variables were the presence of a sarcomere mutation and measures of LVWT. MAIN OUTCOMES AND MEASURES: Variables from standardized exercise testing, echocardiography, cardiac magnetic resonance imaging, serum biomarker measurement, and electrocardiography were compared across study cohorts. RESULTS: Analyses were performed in 178 participants, including 81 G+/LVH+ (mean [SD] age at baseline, 27 [14] years), 55 G+/LVH− (20 [10] years), and 42 G−/LVH− (18 [8] years). All mutation carriers had smaller LV cavity, higher ratio of LVWT to diastolic diameter, and higher echocardiographic LV ejection fraction than controls. A phenotypic burden score was evaluated as the cumulative number of 7 traits (changes on electrocardiography; decreased LV systolic, diastolic diameter, or septal E′ velocity; higher ratio of LVWT to diastolic diameter; serum troponin level; and natriuretic peptide level) in each individual. The mean (SE) phenotypic burden was 4.9 (0.2) phenotypes per individual in G+/LVH+, 2.4 (0.2) in G+/LVH−, and 1.3 (0.2) in controls (P &lt; .001). Classification and regression tree analysis identified an LV end-diastolic dimension z score less than −1.85 or the combination of an LV end-diastolic dimension z score of −1.85 or higher and a septal E′ velocity z score less than −0.52 as having 74% accuracy in discriminating G+/LVH− participants from controls. In mutation carriers, clinical variables demonstrated a continuous correlation with LVWT, generally without a clear cutoff signifying pathologic transition. CONCLUSIONS AND RELEVANCE: G+/LVH− individuals demonstrated altered cardiac dimensions and function and a higher burden of early phenotypes than healthy G− controls. Two methods discriminated phenotypic subgroups, namely, a sum across 7 traits and a regression tree–based rule that identifies constellations of distinguishing factors. Greater LVWT is associated with more prominent cardiac abnormalities in a continuous, although not always linear, manner. A single value of LVWT could not dichotomize the presence or absence of disease.

This subgroup analysis of the FAVORIT trial including 1131 patients shows that high B-type natriuretic peptide and cardiac troponin I are associated with death, dialysis-dependent kidney failure, and cardiovascular outcomes, suggesting that these biomarkers could be used to identify candidates for targeted risk reduction. Supplemental digital content is available in the text.

IMPORTANCE: Treatment decisions in atrial fibrillation (AF) are based on clinical assessment of risk. The CHA2DS2-VASc (cardiac failure or dysfunction, hypertension, age 65-74 [1 point] or ≥75 years [2 points], diabetes mellitus, and stroke, transient ischemic attack or thromboembolism [2 points]–vascular disease, and sex category [female]) risk score is pragmatic and widely used but has only moderate discrimination. OBJECTIVE: To develop and test a cardiovascular biomarker score for indication of risk in patients with AF. DESIGN, SETTING, AND PARTICIPANTS: The ENGAGE AF-TIMI 48 trial was a randomized, double-blind, double-dummy clinical trial comparing 2 once-daily edoxaban dose regimens with warfarin in 21 105 patients with AF at moderate to high risk of stroke. This prespecified subanalysis was performed in 4880 patients enrolled at randomization in the biomarker substudy. Cardiac troponin I, N-terminal pro-B-type natriuretic peptide, and d-dimer levels were measured at baseline. A multimarker risk score was developed to determine the probability of stroke, systemic embolic events, or death by assigning tiered points for higher concentrations of the biomarkers. MAIN OUTCOMES AND MEASURES: Risk score and clinical outcomes based on cardiac troponin I, N-terminal pro-B-type natriuretic peptide, and d-dimer levels at baseline. RESULTS: Of the 5002 patients enrolled in the biomarker substudy of the ENGAGE AF-TIMI 48 trial, 4880 patients (97.6%) had all 3 biomarkers available at randomization (1820 [37.3%] were women; median [interquartile range] age, 71 [64-77] years). After adjustment for the CHA2DS2-VASc score, each biomarker was associated with a 2.8-fold to 4.2-fold gradient of risk comparing the highest vs lowest concentrations across groups of increasing concentrations (P &lt; .001 for trend for each). The multimarker risk score identified a more than 15-fold gradient of risk after adjustment for CHA2DS2-VASc score. When added to the CHA2DS2-VASc score, the biomarker score significantly enhanced prognostic accuracy by improving the C statistic from 0.586 (95% CI, 0.565-0.607) to 0.708 (95% CI, 0.688-0.728) (P &lt; .001) and reclassification with a net reclassification improvement of 59.4% (P &lt; .001). CONCLUSIONS AND RELEVANCE: A prototype multimarker risk score significantly enhanced risk assessment for stroke, systemic embolic events, or death compared with traditional clinical risk stratification. Incorporation of biomarkers into clinical decision making to define therapeutic management in AF warrants consideration. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00781391

IMPORTANCE: Cardiac biomarkers provide insights into pathophysiologic processes and offer an attractive strategy for the assessment of cardiovascular risk. OBJECTIVE: To assess the incremental prognostic value of biomarkers that reflect different pathophysiologic processes in patients with type 2 diabetes. DESIGN, SETTING, AND PARTICIPANTS: The Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus (SAVOR)–Thrombolysis in Myocardial Infarction (TIMI) 53 is a randomized, double-blind, placebo-controlled clinical trial that evaluated the safety of saxagliptin vs placebo in 16 492 outpatients with type 2 diabetes with overt cardiovascular disease (CVD) or multiple risk factors. In this secondary analysis, widely used biomarkers were evaluated to ascertain whether they would provide incremental prognostic value in the risk stratification. Median follow-up was 2.1 years (interquartile range, 1.8-2.3 years). The study was performed from May 10, 2010, to June 15, 2013. INTERVENTIONS: Randomization to saxagliptin vs placebo in addition to standard care. MAIN OUTCOMES AND MEASURES: Concentrations of high-sensitivity troponin T, N-terminal pro–B-type natriuretic peptide, and high-sensitivity C-reactive protein were analyzed continuously and by established cut points. Cardiovascular death, myocardial infarction, ischemic stroke, and hospitalization for heart failure (HF) were adjudicated by a blinded events committee. RESULTS: Of the 16 492 patients, 5455 (33.1%) were female and 11 037 (66.9%) were male. Mean (SD) age was 65.0 (8.5) years (range, 39-99 years). Baseline biomarkers were measured in 12 310 patients. Elevated levels of each biomarker were associated significantly with increased risk for all cardiovascular end points. When added to clinical variables, biomarkers significantly improved the discrimination and appropriate reclassification of risk. Elevated high-sensitivity troponin T was associated with an increased risk of cardiovascular death (adjusted hazard ratio [AHR], 3.07; 95% CI, 2.35-4.02; P &lt; .001), myocardial infarction (AHR, 2.13; 95% CI, 1.69-2.67; P &lt; .001), and hospitalization for HF (AHR, 3.85; 95% CI, 2.82-5.27; P &lt; .001). Elevated N-terminal pro–B-type natriuretic peptide was also associated with an increased risk of cardiovascular death (AHR, 3.09; 95% CI, 2.46-3.89; P &lt; .001), myocardial infarction (AHR, 1.95; 95% CI, 1.51-2.53; P &lt; .001), and hospitalization for HF (AHR, 3.92; 95% CI, 3.11-4.92; P &lt; .001). Elevated high-sensitivity C-reactive protein was more weakly associated with an increased risk of cardiovascular death (AHR, 1.49; 95% CI, 1.22-1.82; P &lt; .001) and hospitalization for HF (AHR, 1.47; 95% CI, 1.20-1.81; P &lt; .001). Consistent results were seen in patients with or without established CVD. CONCLUSIONS AND RELEVANCE: A substantial proportion of patients with stable type 2 diabetes with established CVD or multiple clinical risk factors have evidence of ongoing myocardial injury, hemodynamic stress, or systemic inflammation. Biomarker risk stratification thus challenges the traditional differentiation between primary and secondary prevention based simply on clinical history. Strategies to improve risk stratification in patients with type 2 diabetes, with or without CVD, should consider incorporation of biomarker data into standard risk algorithms. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01107886

Population-level SARS-CoV-2 immunological protection is poorly understood but can guide vaccination and non-pharmaceutical intervention priorities. Our objective was to characterise cumulative infections and immunological protection in the Dominican Republic.

Introduction:Circulating cardiovascular (CV) biomarkers, including N-terminal pro-B type natriuretic peptide (NT-proBNP) and high-sensitivity troponin T (hsTnT), are associated with risk of heart failure (HF) events in patients (pts) with type 2 diabetes (T2D). Less is known about the prognostic significance of changes in these biomarkers over time or the early effects of the SGLT2i dapagliflozin (dapa) on these markers.Methods:DECLARE-TIMI 58 was a randomized, placebo-controlled trial of dapa in 17,160 pts with T2D (median f/u = 4.2y). NT-proBNP & hsTnT (Roche) were measured at baseline & 6 mo. The outcome of interest was CV death (CVD) or hosp for HF (HHF). Outcome analyses were performed from a landmark of the 6-mo study visit with pts categorized by change in each biomarker over the first 6 mo. Hazard ratios were adjusted for baseline biomarker value, randomized treatment, age, sex, race, smoking, eGFR, prior HF, BMI, T2D duration, insulin use, CAD, prior MI, ischemic stroke, PAD, dyslipidemia, & hypertension. Linear mixed models were used to assess the effect of dapa on log-transformed NT-proBNP & hsTnT.Results:Serial NT-proBNP & hsTnT values were available in 13,459 pts (78%). Among pts allocated to placebo (n=6,698), NT-proBNP was more dynamic than hsTnT (≥20% change from baseline in 71% vs. 33% of pts; p<0.001). In the full cohort, independent of randomized treatment, there was a stepwise graded relationship between change in NT-proBNP & hsTnT and risk of CVD/HHF, whereby increases in either biomarker were associated with higher risk and decreases in either biomarker were associated with lower risk (p-trend for adj-HR <0.001 for each). Considered together, changes in the 2 markers were complementary (Fig; p<0.001). Dapa significantly reduced NT-proBNP (relative LS mean change, -6% [-4% to -8%]; p<0.001) but not hsTnT (0% [-1% to +1%]; p=0.92) over 6 mo.Conclusions:Early changes in hsTnT & NT-proBNP are associated with subsequent risk of CVD/HHF in pts with T2D.

Background and Aims:Intermittent claudication (IC) in patients with peripheral artery disease (PAD) is thought to be caused by reduced lower extremity macrovascular blood flow. However, the ankle-brachial index (ABI) (a macrovascular measure) does not correlate well with patient-reported outcome measures. We aimed to study microvascular and metabolic skeletal muscle responses to exercise in patients with IC using positron emission tomography (PET).Methods:We enrolled 19 patients with IC (ABI &lt; 0.90 and imaging/angiographic evidence of PAD) and 12 healthy controls (HCs) (normal ABI). All subjects underwent rest perfusion imaging of the legs with 13N-ammonia or 11C-acetate PET followed by a graded treadmill test. Subjects exercised until limited by claudication or the end of the 20-minute protocol. Subjects then underwent immediately post-exercise PET imaging of the legs. Skeletal muscle blood flow (SMBF) was quantified in each leg at rest and immediately post-exercise. Skeletal muscle oxygen utilization (SMVO2) normalized to SMBF was also quantified for patients who underwent imaging with 11C-acetate. Peripheral artery questionnaire (PAQ) summary score was obtained for all subjects.Results:We found that SMBF was increased post-exercise in all subjects, and stress/rest SMBF was higher in the legs of subjects with PAD than in the legs of HCs (Figure 1A). We also found that stress SMVO2/SMBF was higher in legs of patients with PAD than in the legs of HCs (Figure 1B). There was a moderate, significant correlation between limb stress/rest SMBF and PAQ score (Figure 1C) and a strong, significant correlation between limb stress SMVO2/SMBF and PAQ score (Figure 1D).Conclusions:In conclusion, we found that SMBF increased in all subjects after treadmill exercise and paradoxically to the greatest degree in patients with IC. We also found that PET-derived markers of post-exercise microvascular perfusion and metabolism correlated with patient-reported outcomes.

Introduction:Hypocretin/orexin system has been shown to play a role in heart failure. Whether this system also influences all-cause mortality in patients after myocardial infarction (MI) is unknown. The aim of the present study was to evaluate the effect of rs7767652 allele variants, upstream of hypocretin/orexin receptor-2, on prognosis of patients with MI.Methods:Data from a single-center prospectively designed AMBITION registry of consecutive patients hospitalized for MI at a large tertiary cardiology center were analyzed. Patients with type I MI, without previous history of MI or heart failure were included. A random population sample including 1,953 individuals was used to compare allele frequencies in the population.Results:Out of 1,593 patients in the registry, 1,009 fulfilled the inclusion criteria. In total, 6.1% patients were homozygotes and 39.4% heterozygotes for the minor allele. Allele frequencies in patients with MI did not differ from the general population (χ2p=0.62), suggesting no influence on the MI risk. During the median follow-up of 27 months (IQR 13-41), mortality rate was 8.4%. Homozygotes for the rs7767652 minor allele had a higher mortality risk as compared to heterozygotes (p=0.001) and homozygotes for the major allele (p=0.001) (Figure). After multivariate adjustment, minor allele homozygotes remained at increased mortality risk (HR 2.83, 95% CI 1.55-5.19). Minor allele homozygotes experienced more often ventricular fibrillation (12.9% vs. 4.8%, p=0.01) and required more often CPR (16.1% vs. 7.0%, p=0.02). Among patients with EF<40% at hospital discharge, minor allele homozygotes had a lower increase in EF during the follow-up (2.5±11.0 vs. 8.4±9.4, p=0.04).Conclusions:Our Mendelian randomization study shows that hypocretin/orexin system influences prognosis of patients with MI. This effect may be partially explained by the increased arrhythmic risk and the effect on the left ventricular systolic function recovery.

Supplemental Digital Content is available in the text.

Supplemental Digital Content is available in the text.

Background: Newer troponin assays offer the ability to quantify circulating troponin levels at an order of magnitude lower than contemporary assays, fueling continued debate over the prognostic implications of very low-level increases in concentration. We evaluated the prognostic implications of low-level increases in cardiac troponin I (cTnI) using an investigational single-molecule high-sensitivity assay in patients with acute coronary syndrome (ACS).Methods: We measured cTnI using both a high-sensitivity troponin I (hsTnI) assay (Erenna, Singulex, 99thpercentile 9 pg/ml) and a current generation sensitive assay (TnI-Ultra, Siemens, 99thpercentile 40 pg/ml) at baseline in 1807 patients with non-ST elevation ACS and compared their prognostic ability for adverse cardiovascular events at 30 days and one year.Results: Among patients with TnI-Ultra<99thpercentile, patients with elevated hsTnI (≥9 pg/ml) had a significantly higher risk than patients with hsTnI<9 pg/ml: cardiovascular death (CVD) or myocardial infarction (MI) at one year (7.0% vs 3.8%; p<0.001, hazard ratio (HR) 2.05, confidence interval (CI) 1.23–3.41); including a higher risk of CVD (3.5% vs 1.5%, p<0.001) and MI (5.0% vs 2.8%, p<0.001) individually. This higher risk of CVD/MI was independent of clinical risk stratification using the TIMI Risk Score (adj. HR 1.76, CI 1.05–2.90). Moreover, hsTnI showed a trend toward a gradient of risk even below the hsTnI 99 percentile.Conclusions: Low-level cardiac troponin detected using a single-molecule technique, below the cutpoint of a contemporary sensitive assay, identified a significant gradient of risk. These findings support the prognostic relevance of low-level cardiac troponin elevation with increasingly sensitive assays in patients with ACS.

The aim of this study was to evaluate the association of fibroblast growth factor (FGF)-23 with clinical and laboratory findings, the prognostic value of FGF-23, and the relationship between angiotensin-converting enzyme inhibitor (ACEi) therapy, FGF-23 levels, and outcomes in patients with chronic systolic heart failure (HF).

New oral anticoagulants for stroke prevention in atrial fibrillation were developed to be given in fixed doses without the need for the routine monitoring that has hindered usage and acceptance of vitamin K antagonists. A concern has emerged, however, that measurement of drug concentration or anticoagulant activity might be needed to prevent excess drug concentrations, which significantly increase bleeding risk. In the ENGAGE AF-TIMI 48 trial, higher-dose and lower-dose edoxaban were compared with warfarin in patients with atrial fibrillation. Each regimen incorporated a 50% dose reduction in patients with clinical features known to increase edoxaban drug exposure. We aim to assess whether adjustment of edoxaban dose in this trial prevented excess drug concentration and the risk of bleeding events.

Background: Rapid diagnosis of acute coronary syndrome is a clinical and operational priority in busy emergency departments (ED). We examined the performance of an investigational troponin I (TnI) assay with 10–100-times greater sensitivity than current commercial assays.Methods: Among patients with non-traumatic chest pain enrolled in the BWH-TIMI ED Chest Pain Study, we measured TnI (n=381) at baseline, 4–6 h, and 12–24 h with an investigational assay (S-TnI; Singulex, detection-limit 0.0002 µg/l, 99th percentile 0.009 µg/l) and a contemporary sensitive assay (TnI-Ultra; Siemens, detection-limit 0.006 µg/l, 99th percentile 0.04 µg/l). Final diagnosis was adjudicated using all diagnostic data and local hospital-based cardiac TnI (Siemens), blinded to investigational cardiac Tn.Results: The adjudicated diagnosis was myocardial infarction (MI) in 96 patients, unstable angina in 41, and acute non-coronary cardiovascular conditions in 50 patients. Baseline S-TnI was highly sensitive for MI (97%, 95% CI 91–99%) with specificity 81% (95% CI 76–86%) and positive predictive value 63% (95% CI 55–71%). The negative predictive value with S-TnI was 99% (95% CI 96–100%). S-TnI had better diagnostic accuracy than the local assay (area under the curve 0.976 vs. 0.916, p=0.003). Among 20 patients with negative baseline TnI and diagnosis of MI, 19 had elevated baseline S-TnI. Compared to TnI-Ultra, S-TnI trended toward higher sensitivity (97 vs. 94%, p=NS) but did not differ significantly in negative predictive value (99 vs. 98%) or area under the curve (p=0.29).Conclusion: Current and investigational Tn assays substantially increased clinical sensitivity and improved diagnostic accuracy for MI, despite a decline in specificity. A contemporary sensitive assay delivered similar overall accuracy to the investigational test, suggesting that we have reached a point of maximum diagnostic return with increasing analytical sensitivity.

The purpose of this study was to compare the prognostic impact of clinical and biomarker correlates of resting heart rate (HR) and chronotropic incompetence in heart failure (HF) patients.

Circulating biomarkers can offer insight into subclinical cardiovascular stress and thus have the potential to aid in risk stratification and tailoring of therapy.

In patients with chronic kidney disease (CKD), as in other populations, elevations in cardiac biomarker levels predict increased risk of cardiovascular events. We examined the value of troponin T (TnT) and N-terminal pro–brain natriuretic peptide (NT-pro-BNP) in assessing the risk of developing end-stage renal disease (ESRD) in diabetic patients with CKD.

Introduction:Identification of factors involved in early stages of left ventricular systolic function recovery after acute myocardial infarction (AMI) can provide a basis for novel therapies. We aimed to characterize proteomic biomarkers and underlying mechanistic pathways associated with improvement of left ventricular systolic function.Hypothesis:Proteomic biomarkers and protein-protein interaction pathways differ between patients with and without systolic function recovery after AMI.Methods:Patients with ejection fraction (EF) <40% at hospital discharge were selected from a prospective registry of AMI patients (n=819). We used matched case-control design. Cases were patients with EF<40% at hospital discharge, but EF>50% at the follow-up visit 3-6 months after discharge (EF recovery group). Controls were patients with EF<40% both at hospital discharge and follow-up visit. Controls were matched for age, gender, and discharge EF. Blood samples were drawn within 24h since hospital admission. We used proximity extension assays (Olink, Uppsala) to measure plasma biomarkers and protein-protein interaction network to identify pathophysiological pathways distinguishing cases and controls.Results:In total, 41 cases and 41 controls (mean age 59±10 years, 73% males) were included. At baseline, study groups did not differ in the main clinical variables. Of the 362 biomarkers tested, and after adjustment for age, gender and baseline EF, 44 proteins were differentially expressed, of which 2 (interleukin 6 and stem cell factor) showed association with EF recovery after false discovery rate correction. In the Reactome pathway analysis, Interleukin-6 family signaling was the most significant pathway associated with systolic function recovery.Conclusions:We identified the Interleukin-6 family signaling pathway with IL-6 and leukemia inhibitory factor (LIF) as potential early factors supporting recovery of systolic function after AMI.

Introduction:The role of metformin (MET) in the treatment of patients with advanced heart failure with reduced ejection fraction (HFrEF) and type 2 diabetes mellitus (DM) is not firmly established.Hypothesis:Meformin treatment will be associated with better outcome even in DM patients with advanced HFrEF.Methods:A total of 847 stable patients with advanced HFrEF (57.4± 11.3 years, 67.7% NYHA III/IV, LVEF 23.6± 5.8%) underwent clinical and laboratory evaluation and were prospectively followed for a median of 1126 (IQRs 410; 1781) days for occurrence of death, urgent heart transplantation or mechanical circulatory support implantation. A subgroup of 380 patients (44.9%) had DM, 87 of DM patients (22.9%) were treated with MET.Results:Compared with MET-free diabetic patients, MET-treated individuals had similar fasting glucose, but worse DM control (Hb1Ac 60 mmol/mol, IQRs 50-73 vs. 52 IQRs 48-62, p=0.002), higher insulin, glucagon, β-hydroxybutyrate levels and increased insulin resistance (all p< 0.05), but better quality of life (MLHFQ-score: 36 vs. 48 points, p= 0.002). Compared to diabetics treated with any other glucose-lowering agent, MET-treated patients had better event-free survival (p=0.0005, Fig. 1), which remained significant (p= 0.035) even after adjustment for BNP, BMI and eGFR. Propensity score-matched analysis with 17 covariates yielded 81 pairs of patients and showed a significantly better survival for MET-treated subgroup (p= 0.01).Conclusions:MET treatment in patients with advanced HFrEF and DM is associated with better quality of life and improved outcome, by mechanisms beyond the improvement of blood glucose control. MET should stay among frontline drugs for treatment of HFrEF patients with DM.Supported by a grant no. NV19-02-00130 by Czech Health Research Council.

Introduction:Circulating high-sensitivity cardiac troponin T (hsTnT) reflects myocardial injury, and higher levels are associated with an increased risk of worsening heart failure (HF) and death in patients with HF with reduced ejection fraction (HFrEF). Dapagliflozin reduces the rate of worsening HF or cardiovascular death in patients with HFrEF. The effect of dapagliflozin on the risk of worsening HF or CV death in relation to baseline concentration of hsTnT in patients with HFrEF is unknown.Methods:DAPA-HF was a randomized, double-blind, placebo-controlled trial of dapagliflozin (10 mg once daily) in patients with New York Heart Association class II, III, or IV HF and a left ventricular ejection fraction ≤40%. We measured hsTnT (Roche Diagnostics) at baseline in 3,138 patients randomized in DAPA-HF and at one year among 2,500 survivors. The primary outcome was adjudicated worsening HF or cardiovascular death. Secondary outcomes included each component individually, hospitalization for HF, and death from any cause. The median follow-up was 18.2 months.Results:By the time of this presentation, data will be available for presentation of the distribution of baseline hsTnT concentration and the clinical characteristics associated with strata of hsTnT concentration. The association between baseline hsTnT and 2-year outcomes will be presented and adjusted for potential confounders, including age, sex, eGFR, history of diabetes mellitus, and principal cause of HF (ischemic or non-ischemic). The effect of dapagliflozin on the change in hsTnT concentration from baseline to one year will be presented. The effect of dapagliflozin on the primary and secondary outcomes (both absolute and relative risk reductions) stratified by baseline hsTnT concentration will be analyzed.Conclusions:Biomarkers of myocardial injury identify patients with HFrEF at heightened risk of adverse outcomes. This analysis from a large, international, randomized, placebo-controlled trial will provide a robust assessment of the effect of dapagliflozin on hsTnT and the ability of dapagliflozin to improve outcomes in relation to baseline hsTnT concentration.

Brain natriuretic peptide (BNP) is useful in diagnosing congestive heart failure (CHF) in patients presenting in the emergency department with acute dyspnea. We prospectively tested the utility of BNP for discriminating ARDS vs cardiogenic pulmonary edema (CPE).

Mutations in the human SLC4A1 (AE1/band 3) gene are associated with hereditary spherocytic anaemia and with distal renal tubular acidosis (dRTA). The molecular diagnosis of AE1 mutations has been complicated by the absence of highly polymorphic genetic markers, and the pathogenic mechanisms of some dRTA-associated AE1 mutations remain unclear. Here, we characterized a polymorphic dinucleotide repeat close to the human AE1 gene and performed an immunocytochemical study of kidney tissue from a patient with inherited dRTA with a defined AE1 mutation.

It has recently been shown that the Gardos channel activity of mouse erythrocytes can be modified by endothelins, suggesting a functional linkage between endothelin receptors and the Gardos channel. Using 86Rubidium (86Rb) influx, effects were estimated of proinflammatory molecules such as platelet activator factor (PAF), endothelin-1 (ET-1), interleukin-10 (IL-10), and regulated on activation normal T cells expressed and secreted (RANTES) on the Gardos channel activity in human normal and sickle red cells. It was found that PAF (EC50: 15 ± 7 nM), RANTES (EC50, 9 ± 6 ng/mL [1.2 ± 0.8 nM]), IL-10 (EC50, 11 ± 8 ng/mL [204 ± 148 nM]), and ET-1 (EC50, 123 ± 34 nM) induce a significant increase in Gardos channel activity—between 28% and 84%—over the control. In addition, these agents modify the Gardos channel affinity for internal Ca++ (K0.5) by 2- to 6-fold. Biochemical evidence is provided for the presence of ET receptor subtype B in sickle and normal red cells. Furthermore, it was found that ET-1, PAF, RANTES, and IL-10 induce a significant increase in red cell density (P < .05). These data suggest that activation of the Gardos channel is functionally coupled to receptor motifs such as C-X-C (PAF), C-C (RANTES), and ET receptor subtype B. Thus, cell volume regulation or erythrocyte hydration states might be altered by activation of the Gardos channel by cytokines in vivo. The role of these mediators in promoting sickle cell dehydration in vivo is under investigation.

It has recently been shown that the Gardos channel activity of mouse erythrocytes can be modified by endothelins, suggesting a functional linkage between endothelin receptors and the Gardos channel. Using 86Rubidium (86Rb) influx, effects were estimated of proinflammatory molecules such as platelet activator factor (PAF), endothelin-1 (ET-1), interleukin-10 (IL-10), and regulated on activation normal T cells expressed and secreted (RANTES) on the Gardos channel activity in human normal and sickle red cells. It was found that PAF (EC50: 15 ± 7 nM), RANTES (EC50, 9 ± 6 ng/mL [1.2 ± 0.8 nM]), IL-10 (EC50, 11 ± 8 ng/mL [204 ± 148 nM]), and ET-1 (EC50, 123 ± 34 nM) induce a significant increase in Gardos channel activity—between 28% and 84%—over the control. In addition, these agents modify the Gardos channel affinity for internal Ca++(K0.5) by 2- to 6-fold. Biochemical evidence is provided for the presence of ET receptor subtype B in sickle and normal red cells. Furthermore, it was found that ET-1, PAF, RANTES, and IL-10 induce a significant increase in red cell density (P< .05). These data suggest that activation of the Gardos channel is functionally coupled to receptor motifs such as C-X-C (PAF), C-C (RANTES), and ET receptor subtype B. Thus, cell volume regulation or erythrocyte hydration states might be altered by activation of the Gardos channel by cytokines in vivo. The role of these mediators in promoting sickle cell dehydration in vivo is under investigation.

Defects involving α spectrin (Sp) are found in patients with hereditary elliptocytosis and a related disorder, hereditary pyropoikilocytosis (HPP). We have previously found that the severity of hemolysis was related to the total spectrin content of the cells and the percentage of unassembled dimeric Sp (SpD) in the membranes, which, in turn, reflected the amount of mutant Sp in the cell. However, no data are available comparing differences in the function of various α Sp mutations to clinical severity. We now report studies of nine homozygotes or double heterozygotes for four α Sp mutations: α1/74, α1/46, α1-65, and α1/61, whose red blood cells (RBCs) contained only the mutant Sp and no normal Sp. Sp α1/74, Sp α1/48, and Sp α1/65homozygotes differed strikingly in the severity of hemolysis that correlated with the severity of mutant Sp dysfunction, as reflected by the fraction of unassembled SpD in the membranes and the self-association of mutant Sp on inside-out vesicles. Homozygotes for Sp α1/74had a very severe hemolytic anemia and their SpD were virtually incapable of self-association, whereas SpD α1/46were not as severely affected. The Sp α1/65homozygotes had a relatively mild hemolytic anemia and their SpD showed the least impairment of function. Ultrastructural examination of membrane skeletons from subjects whose SpD self-association was severely impaired showed gross skeletal disruption and loss of hexagonal structure. In striking contrast, the homozygote for the mildly dysfunctional Sp α1/65had only a moderate disruption of the skeleton. Some of the homozygous or doubly heterozygous subjects also exhibited a partial deficiency of Sp that correlated with a RBC morphology characteristic of HPP, namely, marked microspherocytosis with virtual absence of elliptocytes. These data demonstrate striking differences in the function and structure of various α Sp mutants that underlie differences in clinical expression.

Oxidative injury to hemoglobin (Hb) leads to formation of methemoglobin (MetHb), reversible hemichromes (rHCRs), and irreversible hemichromes (iHCRs), iHCRs precipitate and form Heinz bodies that attach to the red cell membrane causing injury that leads to hemolysis. The molecular mechanisms of this membrane damage have not been fully elucidated. We have studied the effect of Hb oxidation products on the mechanical stability of red cell membrane skeletons and the associations of membrane skeletal proteins. Hb and MetHb stabilized the isolated membrane skeletons, whereas further oxidation to rHCRs abolished this stabilizing effect. Crude iHCRs prepared by phenylhydrazine oxidation of Hb destabilized membrane skeletons by decreasing formation of the spectrin–protein 4.1–actin complex, the effect similar to that of pure hemin. Whereas virtually no hemin was released from Hb and MetHb, high concentrations of hemin were released from crude iHCR preparations. After removal of this hemin fraction by Dowex resin, the iHCRs lost their destabilizing effect. We conclude that as the oxidation of Hb proceeds, the stabilizing effect of Hb on the membrane skeleton is gradually lost and the deleterious effect increases. The destabilization of the red cell membrane skeleton in the presence of crude iHCR is caused by release of hemin, which lowers the stability of membrane skeleton by weakening the spectrin–protein 4.1–actin interaction.