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
Basic Myocardial
Structure
Cardiac Myofilaments
Cardiac Tissue
Cardiac Myofibril
Cardiac Myocyte
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
Basic Cardiac Electrophysiology
Superior Vena
Cava
Left Atrium
Bundle of His
Bundle
Branches
Sinoatrial Node
Left
Ventricle
Right
Atrium
Purkinje
Fibers
Atrioventricular
Node
Papillary
Muscle
Right Ventricle
Purkinje Fibers
Ion Channels and Transporters in Cardiac
Myocytes
Decrease in voltage triggers
Ca2+ in, K+ out, and Na+ out
Ca2+
in slow
mitochondrion
Na+
in fast
K+
out fast
Cellular Adaptation
Adapted Cell (hypertrophy)
Normal Cardiac
Myocyte
Reversible
Cell Injury
Irreversible Cell Injury
Figure adapted from Robbins Pathologic Basis of Disease, Cotran, et al.
Cell
Hypoxia
Cell
Death
Cardiac Myocyte Adaptation
Normal myocardium
Cardiomyopathy
Myocardial degeneration
Myocardial necrosis
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
Rotenone
X
Antimycin A
X
X
Cyanide
and carbon
monoxide
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)
R
T
P
Q
S
QT Interval
U
– Blockage of multiple ionic channels that may
lead to syncope and ventricular fibrillation
(torsade de pointes)
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
1997
Chlorpromazine (Thorazine®) – anti-psychotic
Arsenic trioxide (Trisenox®) – anti-cancer/leukemia
Erythromycin (Erythrocin®) – antibiotic
Fluoxetine (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
Dose
(ng/kg)
0
3
10
22
46
100
100
stop
10
12
22
25
32
36
22
0
30
100
175
300
550
1000
1000
stop
9
16
17
16
24
28
32
15
• PCB126
Dose
(ng/kg)
Normal heart
Cardiomyopathy
Cardiac Toxicity observed in
90-days exposure to Bis(2chloroethoxy)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
Histopathological findings in the heart in rats treated
for 3 months with CEM (n=10). ( ) severity
Dose (mg/kg/day)
Histopathological findings
0
50
100
200
400
600 *
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)
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)
Males
Females
*
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
Heart of a female rat treated with 600 mg/kg of CEM (R-right
ventricle; M-interventricular septa; L- left ventricle)
L
M
R
Myofiber vavuolation and mononuclear cell
infitration
Myofiber cytoplasmic vacuolization
16-day cardiac toxicity of bis(2-chlorethoxy)methane
H&E staining
Control
2-D
3-D
5-D
16-day cardiac toxicity of bis(2-chloroethoxy)methane
masson’s trichrome staining
Control
2-D
3-D
5-D
16-day cardiac toxicity of bis(2-chlorethoxy)methane
troponin immunostaining
Control
2-D
3-D
5-D
16-day cardiac toxicity of bis(2-chlorethoxy)methane
TUNEL immunostaining
Control
2-D
3-D
5-D
Proposed Mechanism of Heart Toxicity
CI – CH2 – CH2 – O – CH2 – O – CH2 – CH2 – CI
Bis(2-chloroethoxy)methane
HOOC – CH2 – S – CH2 – COOH
Mitochondrial damage
Thiodiglycolic acid
Apoptotic signals
Damage to myocytes apoptosis
Cell death
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
N
H
H
H
N
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
APOPTOTIC
BODIES
MACROPHAGES
MACROPHAGES
APOPTOTIC
BODIES
Rat Heart Trimming Procedure
The heart is sampled for:
- Histopathology
- Electron Microscopy
- Toxicogenomic evaluation
Julie F. Foley 3/12/05
Dorsal
On the dorsal aspect of the heart, place
the blade below the pulmonary artery
and cut a 3 mm transverse section.
PA
LA
V
Make first cut below this
point. Make second cut
3 mm below first cut.
Dorsal
Top
Middle
Bottom
The heart is now divided into 3 sections.
• Top
• Middle
• Bottom
Dorsal
Middle
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.
Cut surface down
in mega-cassette.
Remove the middle transverse section and place
the bottom cut surface down in a mega-cassette
lined 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.
Index card
LV
RV
Mega-cassette
Fix the tissue overnight in 10% neutral buffered
formalin. Remove the paper from the cassette before
processing.
Dorsal
Top
LA
PA
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.
Bottom
1 2
RV
3
LV
Representative cubes to submit for EM studies.
1) Right ventricular wall
2) Interventricular septum
3) Left ventricular wall
Discard or use for RNA studies.
Longitudinal section bisecting the top of the heart.
RA
LA
RV
LV
RA
LV
RV
LA
RA
V
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.