BACKGROUND: Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant.

METHODS: We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals).

RESULTS: TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced "late" cardiac sodium current (INa), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak INa, and that reduced PDGF receptor (PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late INa. Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval (P<0.001).

CONCLUSIONS: These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor-mediated PI3K signaling.

Proteomics has been used to study type 2 diabetes, but the majority of available data are from White participants. Here, we extend prior work by analyzing a large cohort of self-identified African Americans in the Jackson Heart Study (n = 1,313). We found 325 proteins associated with incident diabetes after adjusting for age, sex, and sample batch (false discovery rate q < 0.05) measured using a single-stranded DNA aptamer affinity-based method on fasting plasma samples. A subset was independent of established markers of diabetes development pathways, such as adiposity, glycemia, and/or insulin resistance, suggesting potential novel biological processes associated with disease development. Thirty-six associations remained significant after additional adjustments for BMI, fasting plasma glucose, cholesterol levels, hypertension, statin use, and renal function. Twelve associations, including the top associations of complement factor H, formimidoyltransferase cyclodeaminase, serine/threonine-protein kinase 17B, and high-mobility group protein B1, were replicated in a meta-analysis of two self-identified White cohorts-the Framingham Heart Study and the Malmö Diet and Cancer Study-supporting the generalizability of these biomarkers. A selection of these diabetes-associated proteins also improved risk prediction. Thus, we uncovered both novel and broadly generalizable associations by studying a diverse population, providing a more complete understanding of the diabetes-associated proteome.

Recent advances in proteomic technologies have made high-throughput profiling of low-abundance proteins in large epidemiological cohorts increasingly feasible. We investigated whether aptamer-based proteomic profiling could identify biomarkers associated with future development of type 2 diabetes (T2DM) beyond known risk factors. We identified dozens of markers with highly significant associations with future T2DM across 2 large longitudinal cohorts (n = 2839) followed for up to 16 years. We leveraged proteomic, metabolomic, genetic, and clinical data from humans to nominate 1 specific candidate to test for potential causal relationships in model systems. Our studies identified functional effects of aminoacylase 1 (ACY1), a top protein association with future T2DM risk, on amino acid metabolism and insulin homeostasis in vitro and in vivo. Furthermore, a loss-of-function variant associated with circulating levels of the biomarker WAP, Kazal, immunoglobulin, Kunitz, and NTR domain-containing protein 2 (WFIKKN2) was, in turn, associated with fasting glucose, hemoglobin A1c, and HOMA-IR measurements in humans. In addition to identifying potentially novel disease markers and pathways in T2DM, we provide publicly available data to be leveraged for insights about gene function and disease pathogenesis in the context of human metabolism.

BACKGROUND: We used a large-scale, high-throughput DNA aptamer-based discovery proteomic platform to identify circulating biomarkers of cardiac remodeling and incident heart failure (HF) in community-dwelling individuals.

METHODS: We evaluated 1895 FHS (Framingham Heart Study) participants (age 55±10 years, 54% women) who underwent proteomic profiling and echocardiography. Plasma levels of 1305 proteins were related to echocardiographic traits and to incident HF using multivariable regression. Statistically significant protein-HF associations were replicated in the HUNT (Nord-Trøndelag Health) study (n=2497, age 63±10 years, 43% women), and results were meta-analyzed. Genetic variants associated with circulating protein levels (pQTLs) were related to echocardiographic traits in the EchoGen (n=30 201) and to incident HF in the CHARGE (n=20 926) consortia.

RESULTS: Seventeen proteins associated with echocardiographic traits in cross-sectional analyses (false discovery rate <0.10), and 8 of these proteins had pQTLs associated with echocardiographic traits in EchoGen (P<0.0007). In Cox models adjusted for clinical risk factors, 29 proteins demonstrated associations with incident HF in FHS (174 HF events, mean follow-up 19 [limits, 0.2-23.7] years). In meta-analyses of FHS and HUNT, 6 of these proteins were associated with incident HF (P<3.8×10-5; 3 with higher risk: NT-proBNP [N-terminal proB-type natriuretic peptide], TSP2 [thrombospondin-2], MBL [mannose-binding lectin]; and 3 with lower risk: ErbB1 [epidermal growth factor receptor], GDF-11/8 [growth differentiation factor-11/8], and RGMC [hemojuvelin]). For 5 of the 6 proteins, pQTLs were associated with echocardiographic traits (P<0.0006) in EchoGen, and for RGMC, a protein quantitative trait loci was associated with incident HF (P=0.001).

CONCLUSIONS: A large-scale proteomics approach identified new predictors of cardiac remodeling and incident HF. Future studies are warranted to elucidate how biological pathways represented by these proteins may mediate cardiac remodeling and HF risk and to assess if these proteins can improve HF risk prediction.

In addition to their fundamental role in clearance, the kidneys release select molecules into the circulation, but whether any of these anabolic functions provides insight on kidney health is unknown. Using aptamer-based proteomics, we characterized arterial (A)-to-renal venous (V) gradients for >1,300 proteins in 22 individuals who underwent invasive sampling. Although most of the proteins that changed significantly decreased from A to V, consistent with renal clearance, several were found to increase, the most significant of which was testican-2. To assess the clinical implications of these physiologic findings, we examined proteomic data in the Jackson Heart Study (JHS), an African-American cohort (n = 1,928), with replication in the Framingham Heart Study (FHS), a White cohort (n = 1,621). In both populations, testican-2 had a strong, positive correlation with estimated glomerular filtration rate (eGFR). In addition, higher baseline testican-2 levels were associated with a lower rate of eGFR decline in models adjusted for age, gender, hypertension, type 2 diabetes, body mass index, baseline eGFR, and albuminuria. Glomerular expression of testican-2 in human kidneys was demonstrated by immunohistochemistry, immunofluorescence, and electron microscopy, while single-cell RNA sequencing of human kidneys showed expression of the cognate gene, SPOCK2, exclusively in podocytes. In vitro, testican-2 increased glomerular endothelial tube formation and motility, raising the possibility that its secretion has a functional role within the glomerulus. Taken together, our findings identify testican-2 as a podocyte-derived biomarker of kidney health and prognosis.

Activin type II receptor (ActRII) ligands have been implicated in muscle wasting in aging and disease. However, the role of these ligands and ActRII signaling in the heart remains unclear. Here, we investigated this catabolic pathway in human aging and heart failure (HF) using circulating follistatin-like 3 (FSTL3) as a potential indicator of systemic ActRII activity. FSTL3 is a downstream regulator of ActRII signaling, whose expression is up-regulated by the major ActRII ligands, activin A, circulating growth differentiation factor-8 (GDF8), and GDF11. In humans, we found that circulating FSTL3 increased with aging, frailty, and HF severity, correlating with an increase in circulating activins. In mice, increasing circulating activin A increased cardiac ActRII signaling and FSTL3 expression, as well as impaired cardiac function. Conversely, ActRII blockade with either clinical-stage inhibitors or genetic ablation reduced cardiac ActRII signaling while restoring or preserving cardiac function in multiple models of HF induced by aging, sarcomere mutation, or pressure overload. Using unbiased RNA sequencing, we show that activin A, GDF8, and GDF11 all induce a similar pathologic profile associated with up-regulation of the proteasome pathway in mammalian cardiomyocytes. The E3 ubiquitin ligase, Smurf1, was identified as a key downstream effector of activin-mediated ActRII signaling, which increased proteasome-dependent degradation of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), a critical determinant of cardiomyocyte function. Together, our findings suggest that increased activin/ActRII signaling links aging and HF pathobiology and that targeted inhibition of this catabolic pathway holds promise as a therapeutic strategy for multiple forms of HF.

Heart failure (HF) is a major public health problem characterized by inability of the heart to maintain sufficient output of blood. The systematic characterization of circulating proteins across different stages of HF may provide pathophysiological insights and identify therapeutic targets. Here we report application of aptamer-based proteomics to identify proteins associated with prospective HF incidence in a population-based cohort, implicating modulation of immunological, complement, coagulation, natriuretic and matrix remodeling pathways up to two decades prior to overt disease onset. We observe further divergence of these proteins from the general population in advanced HF, and regression after heart transplantation. By leveraging coronary sinus samples and transcriptomic tools, we describe likely cardiac and specific cellular origins for several of the proteins, including Nt-proBNP, thrombospondin-2, interleukin-18 receptor, gelsolin, and activated C5. Our findings provide a broad perspective on both cardiac and systemic factors associated with HF development.

Background Prior studies relating proteomics markers to incident AF screened for limited numbers of proteins. Methods and Results We performed proteomics assays among participants from the Framingham Heart Study Offspring attending their fifth examination. Plasma protein levels (n=1373) were measured by the SOMAscan proteomic profiling platform. We used robust inference for the Cox proportional hazards model to relate each protein level with incident AF. In addition, we examined the association between AF-related genetic loci and levels of proteins associated with AF. Our study included 1885 participants (mean age 55±10 years, 54% women) who had proteomic profiles measured. A total of 349 participants developed AF during follow-up (mean follow-up 18.3 years). We observed that 8 proteins were significantly associated with incident AF after adjusting for age, sex, technical covariates, and correction for multiple testing ( P<0.05/1373=3.6×10-5). After additional adjustments for clinical factors associated with AF, ADAMTS13 and N-terminal pro-B-type natriuretic peptide remained significantly associated with the risk of incident AF (hazard ratio, 0.78; 95% CI, 0.70-0.88; and 1.44; 95% CI, 1.22-1.70, respectively; P<3.6×10-5 for both). None of the 8 proteins were encoded by genes at AF-related genetic loci previously identified by genome-wide association studies. Conclusions We identified 8 proteins associated with risk of incident AF after adjustment for age and sex; 2 proteins were associated with AF after adjustment for AF risk factors. Future studies are needed to replicate our findings, identify whether the markers are mechanistically related to AF development, and whether they are clinically useful for identification of future AF risk.

BACKGROUND: Emerging proteomic technologies using novel affinity-based reagents allow for efficient multiplexing with high-sample throughput. To identify early biomarkers of myocardial injury, we recently applied an aptamer-based proteomic profiling platform that measures 1129 proteins to samples from patients undergoing septal alcohol ablation for hypertrophic cardiomyopathy, a human model of planned myocardial injury. Here, we examined the scalability of this approach using a markedly expanded platform to study a far broader range of human proteins in the context of myocardial injury.

METHODS: We applied a highly multiplexed, expanded proteomic technique that uses single-stranded DNA aptamers to assay 4783 human proteins (4137 distinct human gene targets) to derivation and validation cohorts of planned myocardial injury, individuals with spontaneous myocardial infarction, and at-risk controls.

RESULTS: We found 376 target proteins that significantly changed in the blood after planned myocardial injury in a derivation cohort (n=20; P<1.05E-05, 1-way repeated measures analysis of variance, Bonferroni threshold). Two hundred forty-seven of these proteins were validated in an independent planned myocardial injury cohort (n=15; P<1.33E-04, 1-way repeated measures analysis of variance); >90% were directionally consistent and reached nominal significance in the validation cohort. Among the validated proteins that were increased within 1 hour after planned myocardial injury, 29 were also elevated in patients with spontaneous myocardial infarction (n=63; P<6.17E-04). Many of the novel markers identified in our study are intracellular proteins not previously identified in the peripheral circulation or have functional roles relevant to myocardial injury. For example, the cardiac LIM protein, cysteine- and glycine-rich protein 3, is thought to mediate cardiac mechanotransduction and stress responses, whereas the mitochondrial ATP synthase F0 subunit component is a vasoactive peptide on its release from cells. Last, we performed aptamer-affinity enrichment coupled with mass spectrometry to technically verify aptamer specificity for a subset of the new biomarkers.

CONCLUSIONS: Our results demonstrate the feasibility of large-scale aptamer multiplexing at a level that has not previously been reported and with sample throughput that greatly exceeds other existing proteomic methods. The expanded aptamer-based proteomic platform provides a unique opportunity for biomarker and pathway discovery after myocardial injury.