CARDIAC TOXICOLOGY

James Swenberg

University of North Carolina

Basic Cardiovascular Function:

The heart, in concert with elastic blood vessels, maintains precise control of blood pressure and critical tissue perfusion

Cardiac Myocyte

Basic Myocardial Structure

Cardiac Tissue

Cardiac Myofibril

Cardiac Myofilaments

Basic Myocardial Structure• Cardiac muscle is striated, BUT :

– single nucleus – under autonomic control – all cardiac cells contract together

(skeletal - selective recruitment of motor units)

– high oxygen demand – structure permits coordinated contraction

(acts as a syncytium)– Cardiac myocytes do not regenerate

Superior Vena Cava Left Atrium

Bundle Branches

Papillary Muscle

Sinoatrial Node

Right Atrium

Atrioventricular Node

Right VentriclePurkinje Fibers

Purkinje Fibers

Left Ventricle

Bundle of His

Basic Cardiac Electrophysiology

Ion Channels and Transporters in Cardiac Myocytes

Na+ in fast

Decrease in voltage triggers Ca2+ in, K+ out, and Na+ out

Ca2+ in slow

K+ out fast

mitochondrion

Cellular Adaptation

Normal Cardiac Myocyte

Cell Death

Irreversible Cell Injury

Adapted Cell (hypertrophy)

Reversible Cell Injury

Figure adapted from Robbins Pathologic Basis of Disease, Cotran, et al.

Cell Hypoxia

Cardiac Myocyte Adaptation

Normal myocardium Myocardial degeneration

Myocardial necrosisCardiomyopathy

Hypertrophic Cardiomyopathy

Ischemic Injury

Manifestations of Cardiac Dysfunction

• Arrhythmias (flutters, fibrillations)

• Cardiomyopathies

• Organism effects– poor tissue oxygenation/perfusion (heart,

other)– accumulation of body fluids in inappropriate

locations (lungs, abdominal cavity, legs)– organ failure (kidney, liver, lungs)– in the extreme …….death

Measures of Toxicity:Alterations in Cardiovascular Function

• Physiologic function– Electrocardiograms (EKG)

• most sensitive early indicator of cardiac toxicity

– Heart rate• tail cuff method with photosensor (noninvasive)• implanted telemetry devices

– Systemic arterial blood pressure/blood flow• electromagnetic or doppler ultrasonic techniques

– Cardiac output• transthoracic echocardiography

Measures of Toxicity:Alterations in Cardiovascular Function

• Clinical Chemistry– Electrolyte disturbances/imbalances

• sodium, potassium, calcium, magnesium, zinc

– Blood gas (acid/base balance)– Proteins

• plasma albumin, myoglobin, fibrinogen

– Lipids• plasma total cholesterol and triglycerides• plasma lipoproteins, total lipids, phospholipids

Measures of Toxicity:Alterations in Cardiovascular

Structure• Clinical Chemistry

– enzyme release (short half-life)– creatine phosphokinase (CPK)

• hybrid dimer specific for cardiac muscle (CPK-MB)

– lactate dehydrogenase (LDH) -hydroxybutyrate dehydrogenase (-HBDH)– serum glutamic-oxaloacetic transaminase

(SGOT, AST)

Measures of Toxicity:Alterations in Cardiovascular

Function

• Anatomic Pathology– Direct cardiotoxicity– Thrombosis & infarction– Inflammation (myositis, endocarditis,

vasculitis)– Downstream tissue/organ effects

Compound-Induced Toxicity

• Toxicants that alter aerobic metabolism

• Toxicants that alter myocardial conduction

• Toxicants that alter cell membrane function

• Toxicants that directly damage myocardium

• Toxicants that induce vascular changes

Toxicants That Interfere With Aerobic Metabolism

• High energy demands of the heart make it susceptible to toxicants that interfere with:

– oxygen availability (e.g., nitrite, carbon monoxide)

– carbohydrate metabolism (e.g., fluoroacetate), or

– oxidative phosphorylation (e.g., dinitrophenols)

• rotenone

• antimycin A

• cyanide and carbon monoxide

• Toxicity may result in myocardial necrosis

Direct Cardiotoxicity: Myocardial Degeneration & Myocytolysis

Toxicant Interference With Oxidative Phosphorylation

RotenoneX

Antimycin AX

Cyanide and carbon monoxide

X

Toxicants That Alter Myocardial Conduction

• Alter impulse formation and cause arrhythmias– Toxicants that cause acidosis and hyperkalemia

(e.g., ethylene glycol)• enhance slow current activity• increase automaticity and promote arrhythmia

– Cardiotoxic divalent ions (e.g., barium, strontium)• replace calcium in slow-current channels• alter efflux of potassium from myocardial cells

hypokalemia and arrhythmias

Toxicants That Alter Myocardial Conduction

• Alter impulse formation and cause arrhythmias– Toxicants that cause prolongation of the QT

interval (e.g., seldane)

– Blockage of multiple ionic channels that may lead to syncope and ventricular fibrillation (torsade de pointes)

PQ S

R

TU

QT Interval

Toxicants That Cause Prolongation of the QT Interval

• Over 100 marketed pharmaceutical agents cause interference in ventricular repolarization

• QT prolongation is mentioned in the FDA-approved labeling as a known action of the drug

• e.g.Terfenadine (Seldane®) – antihistamine/removed in 1997Chlorpromazine (Thorazine®) – anti-psychoticArsenic trioxide (Trisenox®) – anti-cancer/leukemiaErythromycin (Erythrocin®) – antibioticFluoxetine (Prozac®, Sarafem®) – anti-depressant Haloperidol (Haldol®) – anti-psychotic/schizophrenia

Toxicants That Alter Myocardial Conduction

• Alter impulse formation and cause arrhythmias– Halogenated hydrocarbons (e.g., chloroform)

• suppress SA node (AV node becomes pacemaker)• sensitizes myocardium to arrhythmogenic effects of

sympathomimetic amines (catecholamines)

– Cardiac glycosides (e.g., digitalis)• inhibit the sodium-potassium exchange mechanism

decreased intracellular potassium, increased intracellular sodium catecholamine sensitivity

• increase refractory period of the AV node

Toxicants That Alter Cell Membrane Function

• Alter cell membrane control of ion movement and affect cardiac contraction– Cardiac glycosides and catecholamines– Chemical ionophores (e.g., monensin)

• facilitates the passage of sodium, potassium, or calcium• monensin: alters Ca2+ and Na+ transport increased

intracellular calcium changes myocardial contractility• excessive calcium accumulation impairs mitochondrial

oxidative phosphorylation myocardial necrosis

Toxicants That Alter Cell Membrane Function

• Alter cell membrane control of ion movement and affect cardiac contraction– Toxicants that bind to phospholipids (e.g.,

gossypol)• effect potassium transport hyperkalemia

arrhythmias

– Toxicants that selectively block sodium channels• tetrodotoxin, saxitoxin• decreased intracellular Na+ depression of normal

pacemaker function and conduction arrhythmias

Toxicants That Directly Damage Myocardium

• Damage the pumping effectiveness by reducing the number of active myocytes– Toxicants that cause oxidative damage and

lipid peroxidation (e.g., doxorubicin, ethanol)• redox cycling of doxorubicin semiquinone and

superoxide radicals

• ethanol metabolism lipid peroxidation of myocytes

• results in cell swelling, altered Ca2+ homeostasis, and irreversible myocyte injury

Toxicants That Directly Damage Myocardium

• Damage the pumping effectiveness by reducing the number of active myocytes– Toxicants that cause sarcolemmal injury and

calcium alterations (e.g., catecholamines)• endogenous: epinephrine and norepinephrine

• exogenous: isoproterenol (> toxicity than above)

• sarcolemmal damage through lipid peroxidation

• increased calcium uptake impaired mitochondrial function and activation of neutral proteases and phospholipases myocyte dysfunction and toxicity

Cardiovascular changes following chronic rodent exposure to

dioxin-like compounds

Reference: Jokinen MP, Walker NJ, Brix AE, Sells DM , Haseman JK, Nyska A (2003). Cardiovascular Pathology

in Female Sprague-Dawley Rats Following Chronic Treatment with Dioxin-like Compounds. Cardiovascular

Toxicology. ; 3(4): 299-310

Cardiomyopathy

• Myocardial fiber degeneration/necrosis, inflammation, fibrosis

• Common spontaneous degenerative change of myocardium in old rats– Cause unknown but is affected by diet and

stress

Incidences of Cardiomyopathy

• TCDD

• PCB126

Dose

(ng/kg)0 3 10 22 46 100 100

stop

10 12 22 25 32 36 22

Dose

(ng/kg)0 30 100 175 300 550 1000 1000

stop

9 16 17 16 24 28 32 15

Normal heart

Cardiomyopathy

Cardiac Toxicity observed in90-days exposure to Bis(2-

chloroethoxy)methane in rats and mice

Bis (2-chloroethoxy)methane (CEM) is a synthetic organic compound used as the

starting compound to produce polysulfide elastomers used extensively in a variety of

sealant applications.

Histopathologic definitions of cardiac lesions

Myocyte vacuolization

- Widespread accumulation of multiple, round, variably sized, primarily small, and clear vacuoles, located within the myocyte sarcoplasm

- Vacuoles, often similar in appearance, present in the interstitium are interpreted as a background change

Histopathologic definitions of cardiac lesions (Cont.)

Myocyte necrosis - Small areas containing fragmented,

angular, brightly eosinophilic myofibers with dark, shrunken nuclei

Atrial thrombosis - A mature, antemortem blood clot present

within the lumen of the atrium, consisting of alternating areas of fibrin and layered cellular elements

Dose (mg/kg/day)Histopathological findings

0 50 100 200 400 600 *

Males

Cardiomyopathy 10(1) 8(1.3) 10(1) 10(1) 1(1) 1(1)

Fibrosis 0 0 0 0 0 1(1)

Infiltration cell, mononuclear 0 0 1(1) 0 7(1) 10(1.8)

mineralization 1(1) 0 0 0 0 0

Myocardium, necrosis 0 0 0 0 0 7(3.7)

Myocardium, vacuolization, cytoplasmic

0 0 0 0 6(1.3) 9(1.9)

Atrium - thrombosis 0 0 0 0 0 3(1.4)

Females

Cardiomyopathy 8 (1) 6(1) 8 (1) 4(1) 3(1) 0

Infiltration cell, mononuclear 0 1(1) 0 7(1) 6(1.2) 10(2)

mineralization 0 0 0 1(1) 0 0

Myocardium, necrosis 0 0 0 1(1) 1(1) 6(1.7)

Mypcardium, vacuolization, cytoplasmic

0 0 0 2(1) 5(1.4) 8(1.6)

Histopathological findings in the heart in rats treatedfor 3 months with CEM (n=10). ( ) severity

* all male and female animals treated with the 600 mg/kg/day and two females treated with the 400 mg/kg/day died before the end of the study.

Atrial thrombosis

RR

MMLL

Heart of a female rat treated with 600 mg/kg of CEM (R-right ventricle; M-interventricular septa; L- left ventricle)

Myofiber vavuolation and mononuclear cell infitration

Myofiber cytoplasmic vacuolization

16-day cardiac toxicity of bis(2-chlorethoxy)methane H&E staining

ControlControl 2-D2-D

3-D3-D 5-D5-D

16-day cardiac toxicity of bis(2-chloroethoxy)methane masson’s trichrome staining

ControlControl 2-D2-D

3-D3-D 5-D5-D

16-day cardiac toxicity of bis(2-chlorethoxy)methane troponin immunostaining

ControlControl 2-D2-D

3-D3-D 5-D5-D

16-day cardiac toxicity of bis(2-chlorethoxy)methane TUNEL immunostaining

ControlControl 2-D2-D

3-D3-D 5-D5-D

Apoptotic signals

CI – CH2 – CH2 – O – CH2 – O – CH2 – CH2 – CIBis(2-chloroethoxy)methane

HOOC – CH2 – S – CH2 – COOHThiodiglycolic acid

Mitochondrial damage

Damage to myocytes - apoptosis

Cell death

Proposed Mechanism of Heart Toxicity

MITOCHONDRIAL CARDIOMYOPATHY IN 3'AZIDO-3'DEOXYTHYMIDINE (AZT)/ 3TC MULTIGENERATIONAL REPRODUCTIVE

ASSESSMENT BY CONTINUOUS BREEDING WHEN ADMINISTERED TO CD-1® MICE BY

GAVAGE AZT (Zidovudine) and 3TC

(Lamivudine) – are nucleoside reverse transcriptase inhibitors for

HIV-1 infection and AIDS-

AZT - Introduction• AZT is known to cause mitochondrial

myopathy in human and animals

• The mechanism of cardiomyopathy from AZT is not completely understood, but suggested to be related to depletion mtDNA replication, resulting in impaired synthesis of mitochondrial enzymes that generate ATP

- The enzyme responsible for mtDNA replication is DNA polymerase gamma, and it was found to be inhibited by AZT

Pathological changes in the heart

• Light microscopy – not commonly seen. Using masson’s trichrome – granular cytoplasm of myofibers, but no interstitial inflammation or fibrosis

• EM – Mitochondrial swelling, with fractured, dissolution and disrupted cristae (abnormal mitochondria is defined when there is loss or dissolution of more than 25% of the cristae area)

• Clinical chemistry – increased plasma lactate (lactic acidosis - indicating disturbed oxidative metabolism)

Changes in the heart of rats exposed to Ephedrine + Caffeine

Nyska A, Murphy E, Foley JF, Collins BJ, Petranka J, Howden R, Hanlon P, Dunnick JK. Acute Hemorrhagic Myocardial Necrosis and Sudden Death of Rats Exposed to a Combination of Ephedrine and Caffeine.Toxicol Sci. 2004 Nov 10

Ephedra ('ma huang‘)• An herbal dietary supplement for weight loss or

enhanced athletic performance• Currently a matter of national controversy. • At the heart of the debate are three important

questions: (1) The identity and composition of Ephedra

products with regard to ephedrine and related alkaloids;

(2) The potential therapeutic utility of Ephedra (3) Potential health risks associated with such uses

of Ephedra, particularly in sensitive individuals or in cases of intentional abuse for its stimulant properties.

Animal (14 W old ) treated with ephedrine (25 mg/kg) and caffeine (30 mg/kg), died few hours after 1st dosing. Hemorrhage (H) and

myofiber necrosis (N) in the left ventricle

H

H

N

N

H

Animal (14 W old ) treated with ephedrine (25 mg/kg) and caffeine (30 mg/kg), Died few hours after 1st dosing. Myofiber necrosis (N)

in the myocardium of the left ventricle

APOPTOTIC BODIES

MACROPHAGES

APOPTOTIC BODIES

APOPTOTIC BODIES

MACROPHAGES

Rat Heart Trimming Procedure

Julie F. Foley 3/12/05

The heart is sampled for:- Histopathology- Electron Microscopy- Toxicogenomic evaluation

Dorsal

Dorsal

On the dorsal aspect of the heart, place the blade below the pulmonary artery and cut a 3 mm transverse section.

Aorta

LA

V

Make first cut below this point. Make second cut3 mm below first cut.

The heart is now divided into 3 sections.• Top• Middle• Bottom

Top

Middle

Bottom

PA

Dorsal

Histology Section for Morphometric Analysis

For morphometry studies, it is important to standardize tissue collection. The transverse section of the heart is collected from the same region for each animal.

Remove the middle transverse section and placethe bottom cut surface down in a mega-cassettelined with an index card.

Use of a mega-cassette will prevent compression of the tissue. A piece of index card in the bottom of the cassette will minimize curling of the tissue and ensure a flat surface.

Fix the tissue overnight in 10% neutral buffered formalin. Remove the paper from the cassette before processing.

Cut surface down in mega-cassette.

Index card

LV

RV

Mega-cassette

Middle

Dorsal

Tissue for Electron Microscopy (EM)

Cut a 3 mm transverse section through the bottom of the heart. Representative cubes will be submitted for EM studies.

1 3

Representative cubes to submit for EM studies. 1) Right ventricular wall 2) Interventricular septum 3) Left ventricular wall

Discard or use for RNA studies.

LAA

orta

Top

Bottom

LVRV

PA

2

Longitudinal section bisecting the top of the heart.

Place cut side down of both halves of the top of the heart on an index card in a mega-cassette. Again, the mega-cassette will prevent compression of the tissue. The index card will minimize folding of the tissue. Remove the index card prior to the processing of the tissue by histology.

Aorta

LA

RA

Aor

ta

LVLV

RV

RV

RARA LA

V