Proteomics of the heart.

Mass spectrometry-based proteomics is a sophisticated identification tool specializing in portraying protein dynamics at a molecular level. Proteomics provides biologists with a snapshot of context-dependent protein and proteoform expression, structural conformations, dynamic turnover, and protein-protein interactions. Cardiac proteomics can offer a broader and deeper understanding of the molecular mechanisms that underscore cardiovascular disease, and it is foundational to the development of future therapeutic interventions. This review encapsulates the evolution, current technologies, and future perspectives of proteomic-based mass spectrometry as it applies to the study of the heart. Key technological advancements have allowed researchers to study proteomes at a single-cell level and employ robot-assisted automation systems for enhanced sample preparation techniques, and the increase in fidelity of the mass spectrometers has allowed for the unambiguous identification of numerous dynamic posttranslational modifications. Animal models of cardiovascular disease, ranging from early animal experiments to current sophisticated models of heart failure with preserved ejection fraction, have provided the tools to study a challenging organ in the laboratory. Further technological development will pave the way for the implementation of proteomics even closer within the clinical setting, allowing not only scientists but also patients to benefit from an understanding of protein interplay as it relates to cardiac disease physiology.

High-Field Asymmetric Waveform Ion Mobility Spectrometry: Practical Alternative for Cardiac Proteome Sample Processing.

Heart tissue sample preparation for mass spectrometry (MS) analysis that includes prefractionation reduces the cellular protein dynamic range and increases the relative abundance of nonsarcomeric proteins. We previously described "IN-Sequence" (IN-Seq) where heart tissue lysate is sequentially partitioned into three subcellular fractions to increase the proteome coverage more than a single direct tissue analysis by mass spectrometry. Here, we report an adaptation of the high-field asymmetric ion mobility spectrometry (FAIMS) coupled to mass spectrometry, and the establishment of a simple one step sample preparation coupled with gas-phase fractionation. The FAIMS approach substantially reduces manual sample handling, significantly shortens the MS instrument processing time, and produces unique protein identification and quantification approximating the commonly used IN-Seq method in less time.

Targeting Endothelial HIF2α/ARNT Expression for Ischemic Heart Disease Therapy.

Ischemic heart disease (IHD) is a major cause of mortality and morbidity worldwide, with novel therapeutic strategies urgently needed. Endothelial dysfunction is a hallmark of IHD, contributing to its development and progression. Hypoxia-inducible factors (HIFs) are transcription factors activated in response to low oxygen levels, playing crucial roles in various pathophysiological processes related to cardiovascular diseases. Among the HIF isoforms, HIF2α is predominantly expressed in cardiac vascular endothelial cells and has a key role in cardiovascular diseases. HIFß, also known as ARNT, is the obligate binding partner of HIFα subunits and is necessary for HIFα's transcriptional activity. ARNT itself plays an essential role in the development of the cardiovascular system, regulating angiogenesis, limiting inflammatory cytokine production, and protecting against cardiomyopathy. This review provides an overview of the current understanding of HIF2α and ARNT signaling in endothelial cell function and dysfunction and their involvement in IHD pathogenesis. We highlight their roles in inflammation and maintaining the integrity of the endothelial barrier, as well as their potential as therapeutic targets for IHD.

A Novel HIF-2α/ARNT Signaling Pathway Protects Against Microvascular Dysfunction and heart failure After Myocardial Infarction.

RATIONALE: Cardiac microvascular leakage and inflammation are triggered during myocardial infarction (MI) and contribute to heart failure. Hypoxia-inducible factor 2α (Hif2α) is highly expressed in endothelial cells (ECs) and rapidly activated by myocardial ischemia, but whether it has a role in endothelial barrier function during MI is unclear. OBJECTIVE: To test our hypothesis that the expression of Hif2α and its binding partner aryl hydrocarbon nuclear translocator (ARNT) in ECs regulate cardiac microvascular permeability in infarcted hearts. METHODS AND RESULTS: Experiments were conducted with mice carrying an inducible EC-specific Hif2α-knockout (ecHif2α-/-) mutation, with mouse cardiac microvascular endothelial cells (CMVECs) isolated from the hearts of ecHif2α-/- mice after the mutation was induced, and with human CMVECs and umbilical-vein endothelial cells transfected with ecHif2α siRNA. After MI induction, echocardiographic assessments of cardiac function were significantly lower, while measures of cardiac microvascular leakage (Evans blue assay), plasma IL6 levels, and cardiac neutrophil accumulation and fibrosis (histology) were significantly greater, in ecHif2α-/- mice than in control mice, and RNA-sequencing analysis of heart tissues from both groups indicated that the expression of genes involved in vascular permeability and collagen synthesis was enriched in ecHif2α-/- hearts. In cultured ECs, ecHif2α deficiency was associated with declines in endothelial barrier function (electrical cell impedance assay) and the reduced abundance of tight-junction proteins, as well as an increase in the expression of inflammatory markers, all of which were largely reversed by the overexpression of ARNT. We also found that ARNT, but not Hif2α, binds directly to the IL6 promoter and suppresses IL6 expression. CONCLUSIONS: EC-specific deficiencies in Hif2α expression significantly increase cardiac microvascular permeability, promote inflammation, and reduce cardiac function in infarcted mouse hearts, and ARNT overexpression can reverse the upregulation of inflammatory genes and restore endothelial-barrier function in Hif2α-deficient ECs.

Endothelial Aryl Hydrocarbon Receptor Nuclear Translocator Mediates the Angiogenic Response to Peripheral Ischemia in Mice With Type 2 Diabetes Mellitus.

Hypoxia-inducible factors (HIFs) are the master regulators of angiogenesis, a process that is impaired in patients with diabetes mellitus (DM). The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF1ß) has been implicated in the development and progression of diabetes. Angiogenesis is driven primarily by endothelial cells (ECs), but both global and EC-specific loss of ARNT-cause are associated with embryonic lethality. Thus, we conducted experiments in a line of mice carrying an inducible, EC-specific ARNT-knockout mutation (Arnt Δ EC, ERT2) to determine whether aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. Mice were first fed with a high-fat diet to induce diabetes. Arnt Δ EC, ERT2 mice were then adminstrated with oral tamoxifen to disrupt Arnt and peripheral angiogenesis was evaluated by using laser-Doppler perfusion imaging to monitor blood flow after hindlimb ischemia. The Arnt Δ EC, ERT2 mice had impaired blood flow recovery under both non-diabetic and diabetic conditions, but the degree of impairment was greater in diabetic animals. In addition, siRNA-mediated knockdown of ARNT activity reduced measurements of tube formation, and cell viability in human umbilical vein endothelial cells (HUVECs) cultured under high-glucose conditions. The Arnt Δ EC, ERT2 mutation also reduced measures of cell viability, while increasing the production of reactive oxygen species (ROS) in microvascular endothelial cells (MVECs) isolated from mouse skeletal muscle, and the viability of Arnt Δ EC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with an ROS inhibitor. Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice, and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production.

Binge Alcohol Exposure in Adolescence Impairs Normal Heart Growth.

Background Approximately 1 in 6 adolescents report regular binge alcohol consumption, and we hypothesize it affects heart growth during this period. Methods and Results Adolescent, genetically diverse, male Wistar rats were gavaged with water or ethanol once per day for 6 days. In vivo structure and function were assessed before and after exposure. Binge alcohol exposure in adolescence significantly impaired normal cardiac growth but did not affect whole-body growth during adolescence, therefore this pathology was specific to the heart. Binge rats also exhibited signs of accelerated pathological growth (concentric cellular hypertrophy and thickening of the myocardial wall), suggesting a global reorientation from physiologic to pathologic growth. Binge rats compensated for their smaller filling volumes by increasing systolic function and sympathetic stimulation. Consequently, binge alcohol exposure increased PKA (protein kinase A) phosphorylation of troponin I, inducing myofilament calcium desensitization. Binge alcohol also impaired in vivo relaxation and increased titin-based cellular stiffness due to titin phosphorylation by PKCα (protein kinase C α). Mechanistically, alcohol inhibited extracellular signal-related kinase activity, a nodal signaling kinase activating physiology hypertrophy. Thus, binge alcohol exposure depressed genes involved in growth. These cardiac structural alterations from binge alcohol exposure persisted through adolescence even after cessation of ethanol exposure. Conclusions Alcohol negatively impacts function in the adult heart, but the adolescent heart is substantially more sensitive to its effects. This difference is likely because adolescent binge alcohol impedes the normal rapid physiological growth and reorients it towards pathological hypertrophy. Many adolescents regularly binge alcohol, and here we report a novel pathological consequence as well as mechanisms involved.