Table 1.

Key characteristics (KCs) of cardiovascular (CV) toxicants: relevant assays and biomarkers and representative agents.

KC	In vitro/ex vivo	Relevant assays and biomarkers		Representative chemical and other agents
		Animal	Human	Pharmaceutical	Environmental
Mainly cardiac
1. Impairs regulation of cardiac excitability	Patch-clamp recordings in heterologous expression systems, isolated myocytes, or human induced pluripotent stem cell-derived cardiomyocytes (blockade of Na or K ion currents, enhancement of late Na ion current); microelectrode array recordings or optical mapping in ex vivo heart preparation or monolayers of stem cell-derived cardiomyocytes (action potential duration and heterogeneity, conduction velocity), intracellular calcium imaging/measurements.	ECG recordings (QRS duration, QTc intervals), electrophysiologic studies (HV intervals, effective refractory period, and cardiac arrhythmia inducibility), ambulatory ECG recordings (occurrences of torsade de pointes ventricular arrhythmias and sudden cardiac death).	ECG recordings (QRS duration, QTc intervals), electrophysiologic studies (HV intervals, effective refractory period, and cardiac arrhythmia inducibility), cardiac implantable electronic device interrogation (occurrences of ventricular arrhythmias), development of torsade de pointes ventricular arrhythmias, and sudden cardiac death.	Anti-arrhythmic drugs (sotalol, dofetilide, ibutilide, quinidine, procainamide, disopyramide); anti-malarial drug (chloroquine); antibiotics (clarithromycin, erythromycin, azithromycin); tyrosine kinase inhibitors (nilotinib, dasatinib, and sunitinib); antipsychotics (thioridazine, haloperidol); antidepressants (amitriptyline, imiprmaine, fluoxetine, desipramine, paroxetine); anticonvulsants (felbamate and fosphenytoin); gastric motility drug (cisapride).	Tetrodotoxin, saxitoxin, batrachotoxin, and conotoxin (naturally occurring toxins); lead, alcohol, BPA.
2. Impairs cardiac contractility and relaxation	Contractile measurements via edge detection or sarcomere detection, impedance-based contractility, force transducer, pressure–volume catheter or balloon catheter. Measure the above in isolated cardiomyocytes, stem cell-derived cardiomyocytes, isolated muscle fibers, intact heart preparations.	Pressure–volume catheter; ejection fraction on echocardiography.	Ejection fraction on echocardiography, cardiac CT and MRI; blood pressure and cardiac catheterization.	Glycosides (e.g., digoxin); beta-adrenergic antagonists (e.g., metoprolol, atenolol, carvedilol); calcium sensitizer (e.g., levosimendan); adrenergic agonists (e.g., dobutamine, isoproterenol); haloanesthetics (e.g., halothane, isoflurane); chemotherapeutics (e.g., arsenic trioxide).	Metals (e.g., barium, cadmium, cobalt, lead, nickel); ethanol; BPA.
3. Induces cardiomyocyte injury and death	Cytotoxicity (troponin release, ATP production, nuclear integrity, mitochondrial integrity) in isolated or induced pluripotent stem cell-derived cardiomyocytes; cytochrome complex release, loss of mitochondrial membrane potential.	Cardiac biomarkers (e.g., troponin). Histopathological evaluation (hypertrophy, hyalinization, necrosis, vacuolation, fibrosis).	Cardiac biomarkers (e.g., troponin). Histopathological evaluation (hypertrophy, hyalinization, necrosis, vacuolation, fibrosis).	Anthracyclines (e.g., doxorubicin); sympathomimetics (e.g., isoproterenol); cardiac calcitropes (e.g., milrinone); imatinib mesylate; trastuzumab.	Ethanol, air pollution; diethanolamine; ephedrine; methyl bromide; monochloroacetic acid; 3,3′,4′,4′,5 pentachlorobiphenyl (PCB 126); 2,3′,7,8-tetrachlorodibenzo-$p$-dioxin; urethane, cadmium.
4. Induces proliferation of valve stroma	In vitro activation of $5{\text{HT}}_{2\mathrm{B}}$ receptors; DNA synthesis induction in cultured interstitial cells from human cardiac values via $5{\text{-HT}}_{2\mathrm{B}}$ activation; $5{\text{-HT}}_{2\mathrm{B}}$ receptor activation and/or in silico screening incorporated to de-select compounds during drug development.	Valve leaflet fibroplasia and thickening in mice and rats; increased 5-HT levels in whole blood; echocardiogram assessment showing cardiac valve regurgitation.	Valve leaflet fibroplasia and thickening; echocardiogram assessment showing cardiac valve regurgitation.	Fenfluramine, pergolide, cabergoline, ergotamines, MDMA.	None identified.
Mainly vascular
5. Impacts endothelial and vascular function	Measurement of binding affinity, functional potency, or expression of vascular receptors and enzymes. Functional effects in isolated vascular tissue preparations segments (human and animal); enzymatic or biochemical effects in endothelial cell culture (e.g., nitric oxide synthase activity, endothelin). Intracellular calcium imaging/measurements.	Blood pressure; regional blood flow measurement (Doppler, ultrasonic transit time, microspheres); vascular resistance determinations.	Blood pressure; cutaneous blood flow assessment (laser Doppler); brachial flow-mediated dilation; arterial stiffness (pulse wave velocity).	Phenylephrine, sunitinib, sodium nitroprusside, prazosin, minoxidil; calcium channel blockers (e.g., verapamil, nifedipine, diltiazem).	PCBs, BPA, malathion, DDT, air pollution, cigarette smoke, arsenic, cadmium, lead.
6. Alters hemostasis	Platelet aggregation; platelet activation and function (e.g., surface and cytoplasmic markers and EVs by flow cytometry). Altered coagulation and fibrinolysis (e.g., ACT, PT, APTT; assays of global coagulation; levels of coagulation factors). Endothelial cell anti-aggregation and coagulation function.	Blood cell and platelet counts, MPV; platelet aggregation; platelet activation and function, tail vein bleeding time. Serum antibodies (e.g., anti-PF4 in HIT, lupus anticoagulants). Altered coagulation and fibrinolysis (e.g., ACT, PT, APTT; coagulation component levels; thrombus formation in blood vessels, tissue ischemia).	Blood cell and platelet count, MPV; platelet activation and function. Serum antibodies (e.g., anti-PF4 in HIT, lupus anticoagulants). Altered coagulation and fibrinolysis (e.g., ACT, PT, APTT; assays of global coagulation). Vitamin K and vitamin K epoxide levels in serum or plasma (warfarin).	Ibuprofen, quinine, oxaliplatin (immune-mediated thrombocytopenia); heparin (HIT); warfarin (interferes with fibrin clot formation by vitamin K deficiency); procainamide, chlorpromazine, and hydralazine (may induce lupus anticoagulants.	Air pollution (${\mathrm{PM}}_{2.5}$), arsenic, cadmium.
7. Causes dyslipidemia	Altered gene expression of lipid-related genes and altered synthesis and secretion of VLDL in cultured hepatocytes.	Altered plasma levels of lipids in rodents; altered gene expression of lipid- related genes in liver specimens.	Altered plasma levels of lipids in occupational and epidemiological studies.	Human immunodeficiency virus protease inhibitors; antipsychotic drugs.	PCBs, PFAS, BPA, phthalates, cadmium and lead.
Both cardiac and vascular
8. Impairs mitochondrial function	Mitochondrial oxygen consumption determination; mitochondrial ROS measurement; mitochondrial ${\mathrm{Ca}}^{2+}$ imaging; mitochondrial biogenesis, and mitochondrial content determination; mitochondrial membrane polarization measurements; mitochondrial DNA oxidation measurements; ultrastructure imaging. Measurement of the above in isolated cardiomyocytes; submitochondrial preparations; intact heart preparations; human induced pluripotent stem cell-derived cardiomyocytes.	8-OHdG adducts of mitochondrial DNA; mitochondrial oxidative damage (e.g., protein carbonyls and malondialdehyde); histopathological, immunohistochemical, and mitochondrial ultrastructure examination; cardiac contractility–ejection fraction, diastolic relaxation, instrumented LV pressures, QA interval.	Blood mitochondrial DNA methylation; cardiac magnetic resonance.	Chemotherapeutics (e.g., anthracyclines, cisplatin, arsenic trioxide); antiviral compounds (e.g., azidothymidine); anti-diabetics (e.g., rosiglitazone).	Air pollution; metals (e.g., arsenic, mercury, cadmium and lead); diphenylmethane derivatives (e.g., BPA); ethanol, chlorinated hydrocarbons (e.g., PCBs).
9. Modifies autonomic nervous system activity	Measurement of binding affinity or functional potency at autonomic receptors (e.g., alpha and beta-adrenergic; muscarinic subtypes) and transporters (e.g., norepinephrine). Assessment of sympathetic/parasympathetic receptor-mediated function (e.g., cAMP levels, protein phosphorylation) in isolated tissues (heart or vascular tissues). Assess membrane currents/action potentials in isolated neurons/nerves that control CV function. Measure electrical and mechanical activity in co-cultures of cardiomyocytes with para/sympathetic neurons.	Direct measures of sympathetic nerve activity using electrodes or implantable telemetry (membrane currents, action potentials); heart rate variability, baroreflex sensitivity chemoreceptor sensitivity, with linkage to functional and biochemical measures of CV function (e.g., echocardiography, blood pressure and ECG telemetry, pressure–volume catheter, plasma and urinary catecholamines) in rodents and/or dogs.	Heart rate variability, baroreflex sensitivity, chemoreceptor sensitivity, Valsalva maneuver, isometric handgrip test, deep breathing test, cold pressor test, mental arithmetic, orthostatic test, head-up tilt test, plasma and urinary catecholamines, noradrenaline spillover rate, microneurography (e.g., muscle sympathetic nerve activity), sudomotor function (responses of sweat glands to stimuli), and linkage to measures of CV function (e.g., echocardiography, ECG, blood pressure, and plasma and urinary catecholamines).	Beta-adrenergic agonists (e.g., dobutamine), beta-adrenergic antagonists (atenolol and esmolol), alpha-adrenergic agonists (e.g., clonidine), alpha-adrenergic antagonists (prazosin), muscarinic antagonists (atropine).	Ambient particulate matter air pollution, heavy metals (lead, mercury), cigarette smoke, BPA.
10. Induces oxidative stress	Increased ROS generation in macrophages, endothelial cells, cardiomyocytes, fibroblasts, human induced pluripotent stem cell-derived cardiomyocytes; increased lipid peroxidation in liposomes.	Increased lipid peroxidation in rats and mice (e.g., malondialdehyde, 8-isoprostanes, hydroxyeicosatetraenoates, hydroxyoctadecadienoates); decreased paraoxonase 1 activity in mice and glutathione peroxidase in rats; oxidative changes in plasma lipoproteins of hyperlipidemic mice resulting in proatherogenic LDL and dysfunctional pro-inflammatory high-density lipoprotein.	Increased lipid peroxidation and decreased paraoxonase 1 activity in plasma; decreased glutathione peroxidase, decreased superoxide dismutase in blood; Increased NOX in blood.	Anthracyclines.	Air pollution, (${\mathrm{PM}}_{2.5}$, ultrafine particles); diesel exhaust; gasoline exhaust; PAHs; arsenic; cadmium; lead; mercury; pesticides (organophosphates); insecticides (carbamates, fenitrothion), tobacco cigarette.
11. Causes inflammation	Analysis of pro-inflammatory gene expression; measurement of cytokine secretion by immune cells (e.g., macrophages); flow cytometry analysis of immune cells; immunofluorescent staining of inflammatory markers; characterization of macrophage polarization; analysis of endothelial cell function.	Analysis of circulating cytokine levels (e.g., $\text{IL-}1\mathrm{\beta }$, IL-6); flow cytometry analysis of immune cell population in blood and tissues; analysis of inflammatory gene expression in various tissues including aorta; immunostaining of key inflammatory markers in tissues; characterization of macrophage phenotypes within atherosclerotic plaques; calculation of atherosclerotic plaque stability.	Measurement of circulation inflammatory markers (e.g., IL-6, CRP); analysis of immune cells by flow cytometry or other standard methods.	Procainamide (antiarrhythmic); hydralazine (vasodilator); doxorubicin (anthracycline).	PCBs, BPA, arsenic, cadmium, lead, and air pollution (${\mathrm{PM}}_{2.5}$).
12. Alters hormone signaling	Altered contractility in isolated cardiomyocytes or intact heart preparations; whole-heart ECG; modifications of intracellular calcium imaging; changes in vascular contractility; changes in SR protein expression, or posttranslational modifications, signal transduction; pharmacological agonist/antagonist studies; adrenal-derived cell lines, increased expression of vascular endothelial growth factor and endothelial nitric oxide synthase in human primary endothelial cells.	Multiple end points in numerous experimental species (including rodents, canine, porcine, primates): ECG recordings, heart rate variability, baroreflex sensitivity, increased blood pressure, in hormone-receptor knockout rodent models; altered responses to ischemia, cardiac transcriptome; changes in fibrosis and extracellular matrix composition.	Multiple end points in epidemiological studies: modifications in blood pressure, hemostasis and vascular resistance; sex-specific lipid profiles, arrhythmia risk, increased hypertrophy, heart failure and dilated cardiomyopathy; altered ECG; increased risk for coronary and peripheral artery disease and atherosclerosis, atrial fibrillation, disturbances in cardiac output and contractility; atherogenic lipid profiles.	Amiodarone; rosiglitazone; testosterone; androgens and anabolic steroids; adrenergic agonists and antagonists; selective estrogen receptor modulators and anti-estrogens; glucocorticoids.	BPA, PCBs, arsenic, cadmium, and lead.

Note: 5-HT, 5-hydroxytryptamine (serotonin); $5{\text{-HT}}_{2\mathrm{B}}$, 5-HT subtype 2B; ACT, activated clotting time; APTT, activated partial thromboplastin time; ATP, adenosine triphosphate; BPA, bisphenol A; cAMP, cyclic adenosine monophosphate; CRP, C-reactive protein; CT, computed tomography; DDT, dichlorodiphenyltrichloroethane; ECG, electrocardiogram; EV, extracellular vesicle; HIT, heparin-induced thrombocytopenia; HV interval, conduction time through the distal His- Purkinje tissue measured from the onset of the His-bundle deflection to the earliest ventricular activation; ${\mathrm{K}}^{+}$, potassium ion; LDL, low-density lipoprotein; LV, left ventricular; MDMA, 3,4-methylenedioxymethamphetamine; MPV, mean platelet volume; MRI, magnetic resonance imaging; ${\mathrm{Na}}^{+}$, sodium ion; NOX, nicotinamide adenine dinucleotide phosphate oxidase; PAH, polycyclic aromatic hydrocarbon; PCBs, polychlorinated biphenyls; PF4, platelet factor 4; PFAS, per- and poly-fluorinated substances; ${\mathrm{PM}}_{2.5}$, particulate matter in aerodynamic diameter (fine particulate matter); PT, prothrombin time; QTc, corrected QT interval; ROS, reactive oxygen species; SR, sarcoplasmic reticulum; VLDL, very-low-density lipoprotein.