Post-genomic cardiology

896 Pages·2014·73.729 MB·English

by  José Marín-García, Alexander Akhmedov, Gordon W Moe

P -G c
ost enomic ardioloGy
P -G c
ost enomic ardioloGy
Second Edition
By
JOSÉ MARÍN-GARCÍA, M.D.
Director, The Molecular Cardiology and Neuromuscular Institute, Highland Park,
New Jersey, USA
Formerly Professor of Pediatrics and Director of Pediatric Cardiology at UMDNJ,
Newark, NJ, USA
With contributions by
ALEXANDER AKHMEDOV, PH.D. AND VITALYI RYBIN, PH.D.
Senior Research Scientist, The Molecular Cardiology and Neuromuscular
Institute, Highland Park, New Jersey, USA
GORDON W. MOE, M.D.
Professor, Department of Medicine, University of Toronto,
St. Michael Hospital, Toronto, Canada
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Preface
Although the convergence of genomics and clinical research subcellular structures (e.g., sarcomere, cytoskeleton, and
is an established and exciting paradigm, diagnostic meth- channels), metabolic regulatory enzymes (e.g., the renin-
ods and therapies based on genomic information have only angiotensin system and the cholesterol metabolic pathway),
recently emerged. New methods of mutation screening and intracellular signaling pathways (e.g., calcineurin,
are evolving, both for the genome as well as for the “epig- CaMK, and TNFα).
enome,” and better understanding of the genetic mutations Bioinformatic methods employed to search within con-
underlying cardiovascular diseases and adverse drug reac- stantly growing databases with the routine use of reverse
tions is within our reach. In this second edition of Post- genetics techniques are providing subsequent cloning of
Genomic Cardiology, new and developing technologies novel genes/cDNAs of interest, followed by character-
in molecular cardiology such as translational genomic, ization of the spatial–temporal patterns of specific gene
next-generation sequencing (NGS), genome-wide asso- expression. Inclusion of both transcriptomic and proteomic
ciation studies (GWAS), bioinformatics, and systems biol- methodologies are helpful to determine the function of gene
ogy will be discussed in light of their therapeutic potential. products by defining their precise role in pathogenesis, elu-
Discussions of HapMap—the largest international effort to cidating their interaction with other proteins in the subcel-
date aiming to define differences between our individual lular pathways, and potentially enabling their application as
genomes—will be presented. Understanding of genome clinical markers of specific CVDs.
organization and function in model organisms with sim- This volume will also cover the mechanisms govern-
pler and therefore better characterized genetics, has been ing early specification of cardiac chambers in the develop-
extremely valuable in deciphering the complex mechanisms ing heart tube, which have not yet been precisely delineated
underlying human diseases. but are thought to involve novel cell-to-cell signaling among
This new edition of Post-Genomic Cardiology has been migrating cells and triggering of chamber-specific gene-
written with great care and completeness. Several new expression programs, mediated by specific transcription
chapters have been added, including thorough discussions factors and growth factors. Other areas expanded in this
of mitochondria dynamics and their role in cardiac dysfunc- second edition include discussions of the role of signaling
tion and translational studies, genomics of cell death, genet- molecules (e.g., Wnts) using conditional gene knockouts in
ics and epigenetics of the cardiovascular system, and gene a variety of genetic backgrounds and accessing their inter-
and stem cells therapy. All other chapters have been care- action with critical transcription factors such as dHAND,
fully and completely updated. NKX2-5, GATA4, and TBX. Similar approaches may also
Although fundamental questions still remain regard- prove informative in probing the origins of the cardiac con-
ing the basic underlying mechanisms and pathophysiology duction system and in deciphering the role of signaling sys-
of most cardiovascular diseases (CVDs), including con- tems as participants in vascular formation in endothelial
genital and acquired heart defects, several breakthroughs cells, focusing on the interaction of VEGF, angiopoietin,
in molecular genetic technology are being applied that TGF, and the Notch pathway.
will allow identification of many genes involved in both The identification of molecular regulators that control
the primary etiology of CVD and as risk factors partici- cardiomyocyte proliferation is another area of interest that
pating in the development of cardiovascular pathology. has been updated. Cardiomyocytes are mitotically active
Identification of genes responsible for rare familial forms during embryogenesis and generally cease proliferation
of CVD has proved to be informative in the study of non- shortly after birth. Understanding the molecular basis of
syndromic patients with cardiac pathology. Some common cardiomyocyte proliferation could greatly impact on our
cardiovascular anomalies (e.g., cardiomyopathy, congeni- clinical efforts to repair a damaged heart. The mechanism
tal heart disease, atherosclerosis, hypertension, and cardiac of cell growth regulation is being investigated by care-
dysrhythmias) seem to be concordant by association with ful comparison of comprehensive gene expression profiles
distinctive subsets of genes, including genes responsible for of embryonic and postnatal myocytes, as well as by the
ix
x Preface
generation of myocyte cell culture lines with the capacity to Research on animal models of aging have begun to pro-
respond to proliferative inducers. An alternative approach, vide new insights into the functioning of the genes involved
which can be applied to restore myocyte number in dis- in determining longevity as well as into genes involved in
eased or ischemia-damaged hearts is cell transplantation. aging-related diseases, particularly CVD.
New research efforts are needed to further define the opti- As the role of genetic screening in cardiology is
mal conditions necessary for cardiomyocyte differentiation strengthened and as research on the multiple signaling path-
and proliferation and for the fully functional integration ways involved in cardiac organogenesis and pathology pro-
of stem cells in the myocardium as well as to determine gresses, this second edition of Post-Genomic Cardiology
the ability of transplanted stem cells to repair defects in will further attempt to integrate known facts, what is devel-
the young and adult heart. For example, it will be criti- oping, and what is becoming known. New areas of inter-
cal to learn whether heart failure secondary to myocardial est to cardiologists and researchers in diverse fields may
ischemia or dilated cardiomyopathy, myocardial infarct include systems biology, the constructive cycle of compu-
in adults, or children with Kawasaki disease and myocar- tational model building, and experimental verification capa-
dial damage or with ARVD can be treated with cell-based ble of providing the input for exciting new discoveries and
therapy. hope, including the management of diseases in a “personal-
Progress in cell engineering may bring an end to many ized” way.
of the cardiac abnormalities that weaken human life and
bankrupt the health care system. New insights into the car- As witness of the transition,
diovascular consequences of abnormal gene function and From the past, to the present,
expression that may ultimately impact the development of And tomorrow future,
targeted therapeutic strategies and disease management is The truth is.... There! Everywhere!
analyzed; in this way, less effective treatment modalities
directed solely at rectifying structural cardiac defects and J. Marín-García
temporal improvement of function have been replaced. Highland Park, 2013
1
CHAPTER 
Introduction to the Molecular Biology
of the Cell
NUCLEIC ACIDS, GENES, CHROMATIN, The gene is the fundamental unit of inheritance and rep-
AND CHROMOSOMES resents a region of DNA that carries genetic information for
a polypeptide and/or RNA in a form of the linear sequence
The central dogma of molecular genetics—DNA → of nucleotides. The organism’s total DNA content, the
RNA → protein—defines a principal flow of genetic infor- sum of all genetic information, represents its genome. The
mation in all living organisms (Figure 1.1). It also intro- genome size of prokaryote Escherichia coli is 4.6 × 106 bp
duces the key macromolecules of the cell—nucleic acids and lower eukaryotic unicellular organism Sacharomyces
(deoxyribonucleic and ribonucleic acids; DNA and RNA, cerevisiae is 12.1 × 106 bp, whereas the human genome
respectively) and proteins—which define all unique fea- contains 3.2 × 109 bp.
tures of any living cell. A typical eukaryotic protein-coding gene is composed
The central hereditary molecule, DNA, is a long, of regulatory noncoding regions, which flank the coding
unbranched polymer chain composed of four different regions. The completion of the Human Genome Project
building blocks, deoxyribonucleotides. The deoxyribonu- revealed that regulatory regions, called cis-regulatory ele-
cleotides contain the purine bases, adenine (A) and guanine ments, can be located not only near the coding regions but
(G), and the pyrimidine bases, cytosine (C) and thymine many thousands of bases away from them, sometimes in
(T). The bases are attached to the sugar (deoxyribose)- introns of neighboring genes. In addition, it has also been
phosphate chain, in which the 5’ carbon of one deoxyribose discovered that the human genome is full of overlapping
group is linked by a phosphodiester bond to the 3’ carbon genes. Cis-regulatory elements provide the binding sites for
of the next (Figure 1.2). DNA is a very long molecule; trans-acting regulators, transcription factors, and regulators.
the length of a typical mammalian DNA is approximately Cis-regulatory regions include the promoters, the DNA
3 × 109 base pairs (bp). The number of different possible sequences that are recognized and bound by the transcrip-
sequences in such molecule is very large: 43 × 109! tion machinery to transcribe the coding region, and termi-
DNA is composed of two such antiparallel strands that nators, the regions at the 3’ end of the gene to terminate the
entwine, forming a right-handed helical structure with the movement of the transcription machinery. Additional cis-
sugar-phosphate backbone on the outside and the bases on regulatory elements include enhancers and silencers, which
the inside of the double helix. A vital characteristic of the can significantly modulate the rate of gene transcription. In
DNA molecule is complementary base pairing between humans, most of the coding regions of the protein-coding
two strands: a larger purine base A or G on one strand pairs genes are composed of exons, which encode the fragments
via hydrogen bonds with a smaller pyrimidine base T or of polypeptide chains, interspersed with noncoding introns.
C, respectively, on the other strand (Figure 1.2). Pairing Surprisingly, it has recently been demonstrated that pro-
between A and T involves two hydrogen bonds, whereas tein-coding exons from one genome region combine with
pairing between G and C is slightly stronger and involves exons from another distant region located hundreds of thou-
three hydrogen bonds. sands of bases away, with multiple other genes between
RNA is also a polymer composed of a linear sequence them.1–3 The Human Genome Project demonstrated that
of four nucleotides; however, unlike DNA, T is replaced exons account for only approximately 2% of the genome,
by uracil (U), and the sugar–phosphate backbone contains whereas introns account for 8–10% of the genome.4 Thus
ribose instead of deoxyribose. Moreover, in contrast to the vast majority (up to 90%) of the genome appears not
DNA, RNA is a single-stranded molecule; but it contains to be essential; however, emerging evidence strongly sug-
regions that form double-helical structures via complemen- gests that a significant fraction of this so-called junk DNA
tary base pairing, in which A pairs with U instead of T. is transcribed generating several types of regulatory RNAs,
Post-Genomic Cardiology, 2e © 22001144 Elsevier Inc.
DOI: http://dx.doi.org/10.1016/B978-0-12-404599-6.00001-9 3 All rights reserved.
4 SECTION I l Post-Genomic Cardiology
Cytosol
DNA
mRNA translation
DNA transcription
pre-mRNA
RNA processing
protein
mRNA Ribosome
nuclear
Nucleus envelope
plasma membrane
FIGURE 1.1 Schematic representation of a fundamental flow
of genetic information in all living organisms: DNA → RNA →
protein. Transfer of genetic information from DNA to RNA, DNA FIGURE 1.2 Molecular structure of a fragment of DNA double
transcription, and maturation of precursor RNA (pre-mRNA) occur helix. Complementary base pairing (cytosine–guanine and adenine–
inside the cell nucleus. Mature messenger RNA (mRNA) is exported thymine) between two DNA strands, located inside of the double
into the cytosol, where it is translated on the specialized organelle, helix, is schematically shown (hydrogen bonds are depicted as red
ribosome, composed in human cells of 60 S and 40 S subunits, to pro- dotted lines). The sugar–phosphate backbones on the outside of the
duce a polypeptide chain. DNA double helix are marked by grey panels.
which control the expression of the coding genes.5,6 The represent the diploid number (2 N), whereas germ cells con-
precise roles and the mechanism of action of these regula- tain the haploid number (N), 23 chromosomes.
tory RNAs are largely unknown. In light of this high com- Genotype can be defined as the actual genetic makeup
plexity, which was not anticipated, developing a precise, of an organism. Expression of the genotype defines the
single definition of a “gene” is a challenging task.7,8 physical appearance and features of an organism, which
The human cell contains approximately 2 m of genomic are termed the phenotype. A variant form of a given gene
DNA, which is packaged in a highly compact configura- is called an allele. If a diploid organism carries two iden-
tion inside the cell nucleus. Eukaryotic genomic DNA is tical alleles of a gene, this organism is homozygous for
organized into a DNA–protein complex called chroma- that gene, whereas an organism with two different alleles
tin.9 The long genomic DNA chain is arranged in arrays is termed heterozygous with respect to that gene. In a het-
of nucleosomes, the basic structural units of chromatin. erozygous organism, when one allele determines the phe-
Each nucleosome contains 147 bp of DNA, wrapped in 1.7 notype irrespective of the presence of the other allele, the
superhelical turns around a core histone octamer, consisting former allele is called dominant and the latter is called
of the histones H2A, H2B, H3, and H4, and is connected recessive.
by 20–50 bp of linker DNA with neighboring nucleo- The ends of each chromosome are composed of repeated
somes.10–12 The nucleosomes form a “beads on a string” DNA sequences associated with specialized proteins called
structure, also known as the 10 nm fiber. The linker histones telomeres.14,15 These structures play a role of protective
H1 and H5 interact with linker DNA and contribute to less caps for chromosomes that prevent the chromosome ends
characterized, higher-order chromatin compaction, forming from being recognized as DNA damage and being end-
the so-called 30 nm chromatin fiber. Multiple nonhistone to-end fused.16 Human telomeric DNA is typically 5–15 kb
proteins assist in further chromatin folding into the more in length and consists of the 3’ or G-rich strand composed
condensed structure of mitotic chromosomes (Figure 1.3). of 5’ [TTAGGG] tracts and the complementary 5’ or C-rich
n
The human genome comprises 23 pairs of chromo- strand composed of 5’ [CCCTAA] tracts. Single-stranded
n
somes: 22 pairs of autosomes and one pair (X and Y) of sex DNA overhangs the G-strand and forms a telomeric loop
chromosomes. Males have one X and one Y chromosome, (t-loop); telomeric proteins facilitate t-loop formation.17–19
whereas females have a pair of X chromosomes. During Conventional DNA polymerases are unable to com-
cell division, each chromosome is composed of two daugh- pletely replicate telomere ends.20–22 Thus after many
ter chromatids, which are held together by a centromere, rounds of DNA replication associated with cell division
the structure essential for proper chromosome segregation. telomeres are gradually shortened and eventually pre-
The total set of 46 human chromosomes is visible at the vent further division and induce cell senescence.23 A
metaphase stage of mitosis and is called the human karyo- specialized enzyme known as telomerase catalyzes the
type. The 46 chromosomes present in human somatic cells addition of telomeric DNA tracts onto the chromosome
CHAPTER 1 l Introduction to the Molecular Biology of the Cell 5
involved in that genome’s replication, transcription, and
translation. Human mtDNA is a 16,569 bp, circular, neg-
atively supercoiled, double-stranded molecule, which
encodes 37 transcripts: 13 polypeptides, two ribosomal
RNAs (rRNAs), and 22 transfer RNAs (tRNAs).40,41 The
vast majority of approximately 1500 mitochondrial pro-
teins, including components of the respiratory chain, is
encoded by a nuclear genome (nDNA), synthesized on
cytosolic ribosomes, and must be imported through com-
plex pathways.42,43
DNA TRANSACTING PROCESSES:
REPLICATION, TRANSCRIPTION, AND
REPAIR
Highly organized DNA transacting processes, DNA rep-
lication, transcription, and repair, share several common
characteristics. The main molecular mechanisms of these
processes are evolutionarily conserved from bacteria to
humans. They operate not on “naked” DNA but on DNA
organized into chromatin; therefore, chromatin structure
FIGURE 1.3 DNA packaging in the cell nucleus. A long DNA
and accessibility of specific DNA regions highly affect
molecule with a diameter of 2 nm is wrapped around a core histone
octamer that consists of H2A, H2B, H3, and H4 histone proteins DNA transacting processes. In addition, all three processes
and forms a “nucleosome” with a diameter of 11 nm. The nucleo- are promoted by complex specialized machineries: mul-
some has long been assumed to be folded into 30 nm chromatin fibers tiprotein complexes with a variety of accessory proteins
before the higher-order organization of mitotic chromosomes or inter- involved. DNA replication, transcription, and repair operate
phase nuclei occurs. Only one start helix (solenoid) model is shown.
in the cell nucleus, interact with each other at multiple lev-
Adapted from Maeshima et al.13 with permission from Elsevier.
els, and share several components.
ends to compensate progressive telomere shortening.24–26
DNA replication
Telomerase is a unique ribonucleoprotein minimally com-
posed of the telomerase reverse transcriptase (TERT) sub- Faithful transmission of genetic information from one gen-
unit and the integral telomerase RNA (TR) subunit. The eration to the next requires replication of an entire parental
TERT subunit contains the catalytic site and promotes genome at each cell division. DNA replication is mediated
DNA synthesis using the TR subunit as the template.27–30 by a multiprotein complex known as the replisome, which
In addition to this holoenzyme, multiple accessory proteins contains all proteins required to replicate chromosomal
are implicated in localization, regulation, and biogenesis of DNA. Three multisubunit DNA polymerases, DNA poly-
telomerase.26,30–34 merase α (Pol α), δ (Pol δ) and ε (Pol ε), constitute the
Finely tuned maintenance of telomeres is vital for heart of the replisome. In addition, the replisome includes
genome stability and human health. Upregulated telomer- the DNA helicases, enzymes that unwind duplex DNA, and
ase activity is characteristic for actively proliferating both multiple proteins that facilitate the processivity of DNA
embryonic and stem cells and also the majority of cancer synthesis promoted by the DNA polymerases.44–47
cells.19,35,36 Conversely, defects in telomerase leading to The replisome is assembled in a highly organized
undue telomere shortening can cause various telomere- stepwise fashion at multiple sites distributed along mul-
mediated diseases, including dyskeratosis congenita, aplas- tiple chromosomes, which are called replication origins.
tic anemia, and idiopathic pulmonary fibrosis.37–39 Activation of 30,000 to 50,000 replication origins at each
The presence of the nucleus, containing chromosomes cell division in humans explains how very large human
and molecular machineries responsible for replication and genomes can be entirely replicated in relatively short peri-
transcription of the chromosomal DNA, and mitochon- ods of time. At these sites, duplex DNA should be open
dria, mainly responsible for cellular energy metabolism, to allow recognition of origins and assembly of the DNA
are the major distinguishing features of eukaryotic cells. replication machinery (Figure 1.4). The replication origins
Mitochondria are the only organelles that have their own first are recognized and bound by the origin replication
compact genome (mtDNA) and multiprotein assemblies complex (ORC), which triggers loading and formation
6 SECTION I l Post-Genomic Cardiology
Transcription
The first step in gene expression is the transcription of a
DNA sequence into an RNA sequence. Like DNA replica-
tion, transcription uses DNA as a template to transfer infor-
mation to RNA, and this occurs in the nucleus. However,
in contrast to replication, in which both DNA strands of
an entire DNA molecule are copied, only the specific
regions of typically one of the DNA strands are transcribed.
Chromatin structure of transcribing genes significantly
affects the process. Upon gene activation, condensed,
transcriptionally inactive chromatin is transformed into
decondensed, active state nucleosomes that are transiently
removed, allowing the transcription machinery to interact
with promoters.51
Three DNA-dependent RNA polymerases (RNAP), RNAP
I, RNAP II, and RNAP III, promote gene transcription in
FIGURE 1.4 Scheme of the eukaryotic DNA replication. At
each replication origin, the replicative DNA helicase unwinds duplex all organisms from bacteria to eukaryotes. The eukaryotic
DNA, and DNA synthesis starts with short RNA primers (shown as RNAP I, RNAP II, and RNAP III are huge 500–700 kDa
red zigzag lines) synthesized by DNA polymerase α-primase (Pol α- multisubunit enzymes composed of 14, 12, and 17 subunits,
primase). DNA synthesis always occurs in the 5′ to 3′ direction, and respectively.52,53 RNAP I and III synthesize the 25 S rRNA
one DNA strand—the leading strand (green line)—will be synthe-
precursor and short RNAs including 5 S rRNA and tRNAs,
sized continuously, whereas the other strand, the lagging strand, will
respectively. RNAP II is responsible for transcription of all
be synthesized discontinuously by short RNA-primed DNA fragments
variety of protein-coding genes, synthesizing messenger
(blue lines). The elongation of leading and lagging strands is pro-
moted by DNA polymerase ɛ (Pol ɛ) and δ (Pol δ), respectively. Many RNAs (mRNAs), and therefore is attracting the most atten-
other proteins that have been found to participate in DNA replication tion in investigators.54 Over the past two decades, crystal-
are not shown. lographic studies on RNAPs have shown a “crab claw”–like
RNAP architecture; however, the structural details of subse-
quent events, such as transcription initiation, elongation, and
of the pre-replication complex (pre-RC) and leads even- termination, remain to be determined.52
tually to assembly of the replisome progression complex Transcription is initiated with the recognition of pro-
(RPC).47–50 moter elements by the general transcription factors (TFs),
Once double-stranded DNA is unwound by the repli- which recruit and orient RNAP II to assemble the initia-
cative helicase of pre-RC, bidirectional replication starts tion complex (Figure 1.5). The TFs include TFIIA, TFIIB,
with short (~10 nucleotides) RNA primers, which are syn- TFIID, TFIIE, TFIIF, and TFIIH.54–56 TFIID, composed
thesized by Pol α-primase. Pol δ and Pol ε are recruited of TATA-binding protein (TBP), which binds upstream of
to catalyze the elongation of lagging and leading strands, the transcription start site of all promoters, and 13 TBP-
respectively (Figure 1.4). Recent crystallographic and elec- associated factors (TAFs), plays a key role in the tran-
tron microscopic studies has revealed the molecular struc- scription initiation.57,58 Moreover, the so-called Mediator,
tures of DNA polymerase catalytic subunits and highly a multisubunit complex, which can be viewed as a signal
advanced our understanding of their precise roles in DNA processor, links sequence-specific activators/repressors
replication.47 to RNAP II and the TFs to transduce the regulatory sig-
nals, both negative and positive, from enhancers/silencers
to promoters.54,59–62 The transcription initiation complex
Gene expression
also recruits chromatin remodeling complexes, including
Since Crick’s formulation of the central dogma of gene SWI/SNF, NURD, and BAF (the latter plays an especially
expression, the flow of information from DNA to RNA important role in heart development), which in turn regulate
to protein, great progress has been made not only in our the access of the transcription machinery to DNA.63–66 Thus
understanding of the complex mechanisms of individual the general transcription machinery includes core promoter
steps in the process but also in the realization of the tight recognition complexes together with coactivators/corepres-
interconnection of gene activation, transcription, RNA sors and chromatin remodelers (Figure 1.5). Over the past
processing, and translation. Therefore, all these highly decade, emerging evidence has suggested that alternative
linked and coordinated processes will be discussed in this transcription initiation complexes are assembled at diverse
subsection. core promoters, replacing canonical TFIID; this diversity