CARD- BOARD REVIEW

Physiology/Biochemistry

1. At the completion of a cycle of excitation-contraction coupling, cytosolic Ca2+ is

sequestered in the sarcoplasmic reticulum by what adenosine triphosphatase?

Choices
A. glyceraldehyde phosphate dehydrogenase
B. sarcoplasmic-endoplasmic reticulum calcium ATPase type 2 (SERCA2)
C. adenylyl cyclase
D. V-adenosine triphosphatase
E. L-type calcium channels

Answer
B. SERCA2.

2. In myocardium from patients with CHF, the SERCA2 to phospholamban ratio has
been shown to be

Choices
A. increased
B. unchanged
C. decreased

Answer
C. Decreased.

3. Downregulation of signaling along the adrenergic pathway in failing myocardium is

due to all the below, except:

Choices
A. overexpression of beta-adrenoreceptor (β-AR) kinase
B. downregulation of the β2-receptor
C. phosphodiesterase inhibitors
D. beta-blocker therapy
E. ACE inhibitors

Answer
D. Intracellular calcium (Ca2+) plays an integral role in contraction and relaxation in
cardiac myocytes, a process tightly controlled by mechanisms that regulate its rise
and fall. During depolarization, Ca2+ entry through the L-type Ca2+ channels
triggers an exponential release of Ca2+ from the sarcoplasmic reticulum through
ryanodine receptors, resulting in activation of contractile proteins. At the completion
of a cycle of excitation-contraction coupling, cytosolic Ca2+ is sequestered in the SR
by the SR-Ca2+ adenosine triphosphatase (SERCA2a) pump (~75%) or exported
extracellularly via the Na/Ca exchanger (~25%) located on the sarcolemmal
membrane. Cardiomyocytes isolated from humans with CHF are characterized by
contractile dysfunction as evidenced by decreased systolic force generation,
prolonged relaxation, and elevated diastolic force. Abnormalities in Ca2+
homeostasis, including reduced SR Ca2+ release, elevated diastolic Ca2+ levels,
and a reduced rate of Ca2+ removal parallel the contractile dysfunction seen in the

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 failing myocardium. Furthermore, a reduction in frequency-dependent systolic force
and Ca2+ can be witnessed in failing human myocytes. Key components in the
development of the derangements in contraction and relaxation observed in CHF
have been shown to be SERCA2a and its regulatory protein, phospholamban.
SERCA2a controls function of Ca2+ re-uptake after myocytes contraction and
serves to regulate Ca2+ transients initiating diastolic relaxation. Phospholamban
exerts an inhibitory effect on SERCA2a functioning, reducing its ability to assist in
removal of cytosolic Ca2+ after contraction, a mechanism believed to result in the
diastolic dysfunction seen in CHF patients. The ratio of SERCA2a: phospholamban
has been demonstrated to be decreased in patients with CHF, resulting in the
derangements described previously. With this improved understanding of calcium
homeostasis in failing hearts, interests have pointed toward methods of ameliorating
these dysfunctional mechanisms. Heart failure results in dramatic changes in certain
neurotransmitter and hormone receptors. The majority of the changes occur in the
heart and generally can be classified as regulatory phenomena that withdraw the
failing heart from adrenergic stimulation. However, these changes also can result in
alterations of excitation-contraction coupling and, ultimately contribute to CHF.
Derangements in β-adrenergic signaling, including β-AR receptor downregulation, β-
AR uncoupling from second messenger systems, and upregulation β-AR kinase,
have been demonstrated as significant components of heart failure.
Phosphodiesterase inhibitors, such as milrinone, are used to increaseβ-adrenergic
signaling by bypassing the β-AR. ACE-Is have no effect on β-adrenergic signaling.
Beta-blockers increase β-adrenergic signaling by increasing the density of β-AR
on the cardiomyocyte cell surface.

4. All of the delivery vectors listed below allow for the possibility of stable integration of
genetic material into chromosomal DNA except :

Choices
A. adeno-associated virus
B. adenovirus
C. plasmid DNA
D. liposomes

Answer
B. Adenovirus does not allow for the possibility of stable integration.
Replication-defective adenoviral vectors have emerged as the primary modality for
gene transfer in a variety of preclinical and clinical studies of gene therapy. A number
of properties have resulted in the popularity of these vectors for cardiovascular gene
therapy. Adenoviral vectors are rendered replication incompetent by deleting the
early (E1A and E1B) genes responsible for viral gene expression fro the genome
an are st bly integrated into the host cells in an extrachromosomal form. This
decreases the risk of integration into the host cell genome and mutagenesis.
Adenoviral vectors have been shown to result in transient expression of therapeutic
genes in vivo, peaking at 7 days and lasting approximately 4 weeks. Unlike
replication-defective adenoviral vectors, adeno-associated virus vectors do not
express any viral gene products, rendering them significantly less immunogenic. This
vector demonstrated efficient and stable integration of its transgene with a minimal
inflammatory response. Multiple studies have demonstrated the feasibility ofin vivo
gene transfer into myocardial cells by direct injection of plasmid DNA. These vectors
have the enticing qualities of being relatively nonimmunogenic and nonpathogenic,

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 with the potential to stably integrate in the cellular genome, resulting in long-term
gene expression in postmitotic cells in vivo . Furthermore, plasmid DNA is rapidly
degraded in the blood stream; therefore, the chance of transgene expression in
distant organ systems is negligible. The use of bacteriophage for gene transfer is
presently theoretical gene transfer is presently theoretical and not under
consideration for clinical use at this time.

5. All of the delivery vectors listed below allow for the possibility of stable integration of
genetic material into chromosomal DNA, except:

Choices
A. adeno-associated virus
B. adenovirus
C. plasmid DNA
D. liposomes

Answer
B. Adenovirus does not allow for the possibility of stable integration. Replication-
defective adenoviral vectors have emerged as the primary modality for gene transfer
in a variety of preclinical and clinical studies of gene therapy. A number of properties
have resulted in the popularity of these vectors for cardiovascular gene therapy.
Adenoviral vectors are rendered replication incompetent by deleting the early (E1A
and E1B) genes responsible for viral gene expression from the genome and are
stably integrated into the host cells in an extrachromosomal form. This decreases the
risk of integration into the host cell genome and mutagenesis. Adenoviral vectors
have been shown to result in transient expression of therapeutic genes in vivo,
peaking at 7 days and lasting approximately 4 weeks. Unlike replication-defective
adenoviral vectors, adeno-associated virus vectors do not express any viral gene
products, rendering them significantly less immunogenic. This vector demonstrated
efficient and stable integration of its transgene with a minimal inflammatory
response. Multiple studies have demonstrated the feasibility of in vivo gene transfer
into myocardial cells by direct injection of plasmid DNA. These vectors have the
enticing qualities of being relatively nonimmunogenic and nonpathogenic, with the
potential to stably integrate in the cellular genome, resulting in long-term gene
expression in postmitotic cells . Furthermore plasmid DNA is rapidly degraded in the
blood stream; therefore, the chance of transgene expression in distant organ
systems is negligible. The use of bacteriophage for gene transfer is presently
theoretical and not under consideration for clinical use at this time.

6. Arterial thrombosis after plaque rupture is initiated by

Choices
A. tissue plasminogen activator
B. factor XIII
C. protein C
D. activated protein C
E. tissue factor

Answer
E. Tissue factor.

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 Tissue factor binding to factor VII is the initiating event for the extrinsic blood
coagulation cascade. The complex can also cleave factor IX and contribute to
activation of the intrinsic cascade as well. Tissue factor is normally not expressed in
the vasculature, but, in atherosclerotic vessels, tissue factor is expressed by
macrophages and smooth muscle cells. Tissue factor expression is increased in the
lesions of patients who present with unstable angina. On plaque rupture the
exposure of tissue factor to blood borne coagulation factors leads to thrombus
formation.

7. Blood-borne markers of inflammation that have been shown to predict the presence
of CAD or ACS include the following, except

Choices
A. high-sensitivity C-reactive protein
B. interleukin-6
C. myeloperoxidase
D. interferon alpha
E. serum amyloid A

Answer
D. Interferon alpha has not been shown to predict the presence of CAD or ACS.
Increased levels of each of these circulating markers have been found in patients
with CAD compared to the levels found in control populations.

8. Potential mediators of lipid oxidation in vivo include all of the following, except :

Choices
A. myeloperoxidase
B. lipoxygenase
C. ceruloplasmin
D. catalase
E. ischemia
Answer
D. Lipoxygenase, myeloperoxidase, and ceruloplasmin are all expressed by
activated macrophages and lead to lipid oxidation. Ischemia, in particular ischemia-
reperfusion, leads to generation of free radicals and lipid peroxidation. Catalase is an
antioxidant by reacting with hydrogen peroxide and releasing water.

9. Low levels of gene expression can be detected by

Choices
A. Northern blot
B. Western blot
C. reverse transcriptase-polymerase chain reaction (RT-PCR)
D. Southern blot
E. gene transfer

Answer
C. RT-PCR. RT-PCR is capable of finding a single copy of RNA. Northern blot
analysis requires at least 5 to 10 g of total RNA. Western blot analysis is for

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 determining protein levels. Southern blot analysis is for genotyping and requires
multiple copies of DNA. Gene transfer is not a detection method.

10. DNA can be cut at sites of specific sequences using

Choices
A. hybridization
B. restriction enzymes
C. RT-PCR
D. pepsin
E. desalting column

Answer
B. Restriction enzymes.
Restriction enzymes cleave DNA at sites of specific DNA sequences. Pepsin cleaves
protein at specific sites. RT-PCR is discussed in the answer to question 9, and
hybridization refers to the process of annealing DNA to RNA or DNA.

11. Oxidized LDL can be characterized by the following, except:

Choices
A. positive charge
B. cytotoxicity
C. high malondialdehyde levels
D. recognition by the scavenger receptor
E. low vitamin E

Answer
A. Oxidized LDL has a higher electrophoretic mobility compared to native LDL due to
its negative charge. LDL does not oxidize until its vitamin E content is reduced. It is
highly cytotoxic to cells in culture. Oxidized LDL is not recognized by the LDL
receptor; rather, it is recognized by the scavenger receptor. The level of LDL
oxidation is quantified by its ability to generate high malondialdehyde levels.

12. The final common pathway of platelet aggregation is mediated through

Choices
A. adenosine diphosphate binding
B. collagen
C. thrombin
D. αvβ3 receptor
E. glycoprotein (GP)IIb/IIIa receptor

Answer
E. GPIIb/IIIa receptor. Adenosine diphosphate, collagen, and thrombin bind
independently, leading to platelet activation and, ultimately, to expression of the
GPIIb/IIIa receptor. GPIIb/IIIa receptor expression leads to platelet clumping by
binding to surrounding activated platelets. The αvβ3 receptor does not lead to platelet
aggregation.

13. Inducers of smooth muscle cell proliferation include

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 Choices
A. platelet-derived growth factor β
B. basic fibroblast growth factor (bFGF)
C. transforming growth factor β
D. thrombin
E. oxidized LDL

Answer
C. Transforming growth factor β. Platelet-derived growth factor β, bFGF, and
thrombin are all smooth muscle mitogens. Oxidized LDL causes smooth muscle cell
proliferation through the autocrine release of bFGF. Transforming growth factor β
alters the smooth muscle cell phenotype from a proliferative to a synthetic state and,
thus, is antiproliferative.

14. Apoptosis of a cell is indicated by all of the following, except :

Choices
A. phosphatidylserine in the outer leaflet
B. high caspase 3 activity
C. low annexin-V binding
D. DNA laddering
E. low cytoplasmic cytochrome C levels

Answer
C. Low annexin-V binding does not indicate apoptosis of a cell. Cellular apoptosis is
characterized by increased phosphatidylserine expression in the outer leaflet of the
plasma membrane that leads to increased annexin-V binding. Intracellular markers of
apoptosis include increased caspase 3, decreased cytochrome C levels, and
evidence of DNA laddering.

15. Inhibitors of cardiac myocyte apoptosis include

Choices
A. insulin-like growth factor-1β
B. dobutamine
C. ischemia
D. caspase 3
E. Bid cleavage

Answer
A. Insulin-like growth factor-1β . Insulin-like growth factor-1β overexpression has
been shown to be cardioprotective due to decreased apoptosis in the setting of
myocardial ischemia. Dobutamine has been shown to induce cardiomyocyte
apoptosis. Caspase 3 and Bid cleavage are cytoplasmic markers of apoptosis.