Cardiomyocyte Culture - An Update On The in Vitro Cardiovascular Model and Future Challenges

985
INVITED REVIEW
Cardiomyocyte culture an update on the in vitro cardiovascular
model and future challenges
Sreejit Parameswaran, Sujeet Kumar, Rama Shanker Verma, and Rajendra K. Sharma
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by McMaster University on 12/10/13
Abstract: The success of any work with isolated cardiomyocytes depends on the reproducibility of cell isolation, because the
cells do not divide. To date, there is no suitable in vitro model to study human adult cardiac cell biology. Although embryonic
stem cells and induced pluripotent stem cells are able to differentiate into cardiomyocytes in vitro, the efciency of this process
is low. Isolation and expansion of human cardiomyocyte progenitor cells from cardiac surgical waste or, alternatively, from fetal
heart tissue is another option. However, to overcome various issues related to human tissue usage, especially ethical concerns,
researchers use large- and small-animal models to study cardiac pathophysiology. A simple model to study the changes at the
cellular level is cultures of cardiomyocytes. Although primary murine cardiomyocyte cultures have their own advantages and
drawbacks, alternative strategies have been developed in the last two decades to minimise animal usage and interspecies
differences. This review discusses the use of freshly isolated murine cardiomyocytes and cardiomyocyte alternatives for use in
cardiac disease models and other related studies.
Key words: cardiomyocytes, myocardial ischemia, perfusion, primary culture, cardiac cell lines, cardiac differentiation, cardio-
toxicity.
Rsum : Le succs de toute exprience ralise avec des cardiomyocytes isols dpend de la reproductibilit de l'isolement
cellulaire car ces cellules ne se divisent pas. Jusqu'a prsent, il n'existe aucun modle in vitro convenable qui permette d'tudier
For personal use only.
la biologie cellulaire de la cellule cardiaque adulte. Mme si les cellules souches embryonnaires et les cellules pluripotentes
induites peuvent se diffrencier en cardiomyocytes in vitro, l'efcacit de ce processus est faible. L'isolement et la croissance des
cellules souches humaines de cardiomyocytes a partir de dchets chirurgicaux ou de tissu cardiaque ftal constituent une autre
option. Toutefois, an de contourner les problmes de diffrents ordres relis a l'utilisation de tissu humain, notamment les
proccupations thiques, les chercheurs utilisent des modles animaux de grande ou de petite taille an d'tudier la patho-
physiologie cardiaque. Les cultures de cardiomyocytes constituent un modle simple permettant d'tudier les changements au
niveau cellulaire. Mme si les cultures de cardiomyocytes de souris possdent leurs propres avantages et inconvnients, des
stratgies alternatives se sont dveloppes au cours des deux dernires dcennies an de minimiser l'utilisation d'animaux et les
diffrences inter-espces. Cet article de revue discute de l'utilisation des cardiomyocytes de souris frais et d'alternatives aux
cardiomyocytes comme modles de maladies cardiaques et d'autres maladies relies. [Traduit par la Rdaction]
Mots-cls : cardiomyocytes, ischmie myocardique, perfusion, culture primaire, lignes cellulaires cardiaques, diffrenciation
cardiaque, cardiotoxicit.
Introduction cular diseases (CVDs) remain the biggest cause of deaths world-
The menace of cardiac diseases is rapidly increasing throughout wide, and in 2008 alone, more than 17 million people succumbed
the world. Recent scientic advances have helped in providing a to CVDs. More than 3 million of these deaths occurred before the
greater understanding of the underlying causes of cardiac diseases. age of 60 and could have been prevented. The percentage of prema-
Various models used by different groups are, however, focused ei- ture deaths from CVDs ranges from 4% in high-income countries to
ther on the pathophysiology of the disease condition or on the ther- 42% in low-income countries, leading to growing inequalities in the
apeutic applications. In comparison with other organ systems, the occurrence and outcome of CVDs between countries and popula-
research performed at the cellular level for cardiac conditions has tions (Mendis et al. 2011). Though CVDs usually affect adults in later
not yet provided a signicant breakthrough due to the difculty in stages of life, the precursors such as atherosclerosis and diabetes
replicating the conditions of the human heart. Ethical issues prevent notably begin earlier, making primary prevention efforts necessary
the use of human tissue at almost all stages of research. Murine from childhood (McGill et al. 2008).
animals have become the easiest and most reliable source of primary The heart is a myogenic organ that pumps blood throughout
cardiomyocyte cultures. The minimal cellular manipulation re- the blood vessels by repeated, rhythmic contraction and is princi-
quired for murine cardiomyocyte isolation and maintenance pally composed of cardiac muscle and connective tissue. Cardiac
makes them ideal for research and possible therapeutic purposes. muscle is an involuntary striated muscle tissue found only in the
heart and is responsible for the ability of the heart to pump blood
Cardiac diseases and the heart (Barrett et al. 2010; Hall and Guyton 2011). The mammalian heart is
Cardiovascular or heart diseases are a class of diseases that a four-chambered organ derived from embryonic mesoderm germ
involve the heart or blood vessels (arteries and veins). Cardiovas- layer cells that differentiate after gastrulation into the mesothelium,
Received 25 April 2013. Accepted 8 August 2013.

S. Parameswaran, S. Kumar, and R.K. Sharma. Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada;
Cancer Research Cluster, Room 4D40, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada.
R.S. Verma. Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai, TN 600036, India.
Corresponding author: Rajendra K. Sharma (e-mail: rajendra.sharma@usask.ca).
Can. J. Physiol. Pharmacol. 91: 985998 (2013) dx.doi.org/10.1139/cjpp-2013-0161 Published at www.nrcresearchpress.com/cjpp on 26 August 2013.
986 Can. J. Physiol. Pharmacol. Vol. 91, 2013
endothelium, and myocardium. The mesothelial pericardium diomyocytes. It is also suggested that postnatal cardiomyocyte
forms the outer lining of the heart. The inner lining of the heart differentiation and cell cycle withdrawal are distinct processes.
and the lymphatic and blood vessels develop from the endothe- Therefore, terminal differentiation may not be due to altered ex-
lium (Markwald and Wessels 2001; van Wijk et al. 2012). The heart pression of genes that regulate the cell cycle but could involve
also includes a network of cardiac neurons that are involved in the specically induced epigenetic changes (Naqvi et al. 2009).
intrinsic electrical conduction of the heart, allowing electrical
propagation by the generation of an action potential (Barrett et al. Importance of cardiomyocyte culture
2010; Hall and Guyton 2011). Animal models have been used as tools to understand cardiac
physiology and disease (Dhein et al. 2005; Gross 2009). Most initial
Cardiomyocyte studies have involved surgical and pharmacological interventions
The cells that comprise cardiac muscle are called cardiomyo- in the intact heart (Dhein et al. 2005). In the late 19th century, a
cytes. Cardiomyocytes contain myobrils that are long chains of number of investigators were working on perfecting an isolated
sarcomeres and are the contractile units of the cell (Bird et al. heart model, but it was Oscar Langendorff who, in 1895, pioneered
2003). Cardiomyocytes show similar patterns to skeletal muscle the isolated perfused mammalian heart (Dhein et al. 2005; Gross
cells, but unlike multinucleated skeletal cells, these cells contain 2009). The Langendorff preparation has continuously evolved and
only one or two, and very rarely three or four, cell nuclei (Olivetti provided a wealth of data underpinning our understanding of the
et al. 1996). A healthy adult cardiomyocyte has a cylindrical shape fundamental physiology of the heart, its contractile function, cor-
that is approximately 100 m long and 1025 m in diameter. onary blood ow regulation, and cardiac metabolism, as well as
Previous studies on the cellular morphology of cardiac tissue re- the pathophysiology of ischemiareperfusion and disease states
vealed a complex cellular architecture at the sinoatrial (SA) node (Bell et al. 2011). Though the perfused heart model is still used in
that may determine the electrical activity of sinoatrial pacemaker understanding cardiac physiology following both pharmacologi-
cells (Wu et al. 2001). It is also observed that the cellular morphol- cal and genetic manipulations, advances in cell culture led to the
ogy determines the electrical properties of the atrial myocytes as use of intact muscle preparations, which allowed direct experi-
atrioventricular nodal cells contain ovoid and rod-shaped cardio- mental manipulation of the muscle (Gross 2009). Techniques for
myocytes with different action potentials and ionic current pro- cardiomyocyte isolation were rst developed about 40 years ago
les (Munk et al. 1996). Cell shape- and size-dependent differences and were soon followed by in vitro cardiomyocyte culture and
in the ionic currents may be correlated with the regional differ- maintenance (Louch et al. 2011). Experiments on single myocytes
ences in the electrical activity within the SA node and elsewhere have shown great insight into their cellular and subcellular phys-
in the heart, suggesting that the different shapes and sizes may iology. These studies have used a variety of techniques, including
not have the same electrical activity (Honjo et al. 1996). The elec- electrophysiology, calcium imaging, cell mechanics, immunohis-
trochemical gradient generated in the heart produces an action tochemistry, and protein biochemistry (Gross 2009). The advent of
potential that results in the contraction and relaxation of car- gene transfer technology has further enhanced the importance of
diomyoctes. The simultaneous contraction and relaxation of the cardiomyocyte culture. At present, the use of cell cultures is in-
cardiomyocytes in a sequential mode leads to the functional beat- creasing due to the versatility, economy, and convenience of the
ing of the heart (Barrett et al. 2010; Hall and Guyton 2011). methodology as compared with whole-animal experiments.
Cardiomyocytes are highly fatigue resistant owing to the large The study of isolated cardiomyocytes rather than the whole
number of mitochondria in these cells, which enables continuous heart eliminates other cell types, especially endothelial cells and
aerobic respiration via oxidative phosphorylation. The presence broblasts, which signicantly reduces the inuence of other cell
of numerous myoglobins (oxygen-storing pigment) and a good types (Diaz and Wilson 2006). For various studies ranging from
blood supply provides constant nutrition and oxygen. The heart cell signaling involved in preconditioning to subcellular compo-
relies on utilizing aerobic metabolism to an extent that it is un- nents such as the cell membrane versus mitochondria, a pure
able to pump sufciently under ischaemic conditions. At basal collection of cardiomyocytes is preferred rather than the various
metabolic rates, only about 1% of energy is derived from anaerobic cell types present in the whole heart (Dhein et al. 2005; Gross
metabolism, which may rise to 10% under moderately hypoxic 2009). Microscopic visualization, which plays an important role in
conditions. However, under more severe hypoxic conditions, not cardiomyocyte studies, provides another advantage of the use of
enough energy can be produced by lactate production to sustain isolated cardiomyocytes. It is even possible to use the cardiomyo-
ventricular contractions (Barrett et al. 2010; Hall and Guyton 2011). cyte as its own control through repeated visualization of the cells,
Though the heart is known as an organ containing terminally especially those adhering to a plate in a primary cell culture.
differentiated cells, recent data suggest, however, that there is an There is substantial cell-to-cell variability in many parameters
inherent regeneration capability in cardiac tissue (Bergmann studied, especially as a result of size differences, and therefore,
et al. 2009; Porrello et al. 2011). Heart regeneration is well docu- cardiomyocytes may be studied either when freshly isolated, with
mented in sh, amphibian, and developing mammals (Ausoni and the limitation that repeated observations cannot be made on the
Sartore 2009; Porrello et al. 2011). After birth, however, human same cell, or in culture without this limitation (Diaz and Wilson
heart regeneration becomes limited to the very slow cardiomyo- 2006). Cell culture offers the additional advantages that targeted
cyte replacement. This could also be the reason why only a few cell manipulation of gene expression can be achieved and a variety of
lines with a cardiac phenotype exist and tumors of the heart are imaging techniques can be applied (Dhein et al. 2005). Isolated
rare. heart cells are becoming increasingly advocated as a supplement
Mammalian cardiomyocytes withdraw from the cell cycle soon and sometimes as a replacement for whole-heart or whole-animal
after birth through a process called terminal differentiation that cardiotoxicity studies. Isolated cardiomyocytes have the advan-
is marked by the absence of mitosis and cytokinesis (Naqvi et al. tage of enabling mechanistic and comparative studies of com-
2009). It was observed that following stimulation to divide, cell pounds that are toxic to cardiomyocytes (Remio et al. 2001).
division is prevented by the activation of various cell cycle check- In humans, myocardial ischemia is mostly localised in nature,
points in terminally differentiated adult mammalian cardiomyo- affecting particular cardiac regions correlated to the blood sup-
cytes. Ploidy and multinucleation are observed in adult mammalian ply, and this condition has been widely studied in intact animals.
cardiomyocytes through endoreduplication (Herget et al. 1997; Currently, the treatment of acute ischemic heart disease has en-
Soonpaa and Field 1997; Soonpaa et al. 1996). Thus a multilayered tered a new era in which mortality can be approximately halved
mechanism to prevent cytokinesis exists in adult mammalian car- by procedures that allow the rapid return of blood ow to the
Published by NRC Research Press

Parameswaran et al. 987
ischemic zone of the myocardium, i.e., reperfusion therapy ischemia by pelleting and also to assess the level of ischemic
(Ferdinandy et al. 2007). Reperfusion, however, may lead to fur- injury (Armstrong et al. 1994; Armstrong and Ganote 1994). One of
ther complications such as diminished cardiac contractile func- the earliest studies of ischemic preconditioning in isolated cardi-
tion (stunning) and arrhythmia. Moreover, there is experimental omyocytes used human ventricular cardiomyocytes in primary
evidence that irreversible cell injury leading to necrosis and apo- culture (Ikonomidis et al. 1994).
ptosis may be precipitated by reperfusion (Ferdinandy et al. 2007). Preconditioning of cardiomyocytes protected the cells from a
It should be noted that in other cardiac conditions such as chronic subsequent prolonged period of ischemia and reperfusion (Shirai
myocardial ischemia, left ventricular hypertrophy, congestive et al. 1998). Genetic manipulation is another tool used to study
heart failure, arrhythymias, and atrial brillation, animal cell cul- preconditioning mechanisms as it bypasses the nonspecicity and
ture based models have also been developed (Dhein et al. 2005). undesirable side effects of pharmacological manipulations (Diaz
Modication of an in vitro cell culture model for load-induced and Wilson 2006; Feldman et al. 2011; Horikawa et al. 2011; Sun
cardiac ischemia developed from neonatal rat heart cells cultured 2004). Cardiomyocytes are isolated from knockout mice in
in a sealed ask has been used for studying chronic myocardial which the production of functional copies of a particular protein
ischemia (Holotnakova et al. 2008; Takahashi et al. 2001). Simi- have been substantially reduced or eliminated because cell viabil-
larly, a model for hypertrophy was developed by treating in vitro ity is considered as the end point (Feldman et al. 2011; Sun 2004;
cultured cardiomyocytes with angiotensin II and endothelin-1 to Takeishi and Walsh 2001; Tsutsui et al. 2010). Another approach is
promote hypertrophic responses (Watkins et al. 2011). Disease to transfect cardiomyocytes in cell culture with a recombinant
models for dilated cardiomyopathy, congestive heart failure, and virus that will express one or more mutants of the gene of interest
cardiac arrhythmias are being developed and are computationally (Horikawa et al. 2011; Sun 2004). Efforts are being directed towards
modeled in primary cardiomyocyte cultures, e.g., re-entry ar- understanding the mechanism of ischemic preconditioning by
rhythmias (McCain et al. 2012; van de Stolpe and den Toonder linking the triggers and mediators of preconditioning with the
2013). In addition to biochemical stimuli, mechanical cues, elec- end effectors. Studies on such areas will increasingly require the
trical stimulation, and stiffness, parameters are being worked use of isolated cardiomyocytes. Tracing of signaling pathways in
upon primary cardiomyocyte cultures to develop the models for cardiomyocytes can reveal various interactions with multiprotein
cardiac diseases (van de Stolpe and den Toonder 2013). signaling complexes and key end effector proteins using tech-
Though a large number of animal models have been used for niques such as immunoblotting and immunoprecipitation com-
cardiovascular research, in recent years, molecular biologists bined with immunocytochemical visualization (Diaz and Wilson
have provided physiologists with new animal models of cardiovas- 2006).
cular disease by altering the mammalian genome through the Cardiotoxicity, by denition, is a recognised chemotherapy-
introduction or modication of genes (Verdouw et al. 1998). The induced adverse event caused by a number of known chemother-
mouse has become the animal of choice because its genome is apeutic agents. Cardiotoxicity results in the formation of reactive
well characterized and its maintenance cost is relatively low. A oxygen species (ROS), apoptosis, altered contractibility, change in
large animal population can be generated in a short period by cardiac rhythm, and altered cardiac gene expression, which can
standardised breeding methods. The production of myocardial be life threatening or may lead to long-term alterations of cardio-
ischemia and infarction in small animals by occlusion of coronary vascular functions (Deshmukh et al. 2012). In the pharmaceutical
arteries in vivo was developed in 1946 by Heimburger and was industry, the cardiotoxicity test models are based on cell lines,
later improved upon by other groups (Curtis et al. 1987). In animal cardiomyocytes, and models of small or large animals
1990, Vander Heide and colleagues developed an in vitro model (Braam et al. 2010; Dorr et al. 1988; Mandenius et al. 2011; O'Brien
of myocardial ischemia using freshly isolated rat cardiomyo- 2008). U.S. Food and Drug Administration guidelines on cardiotox-
cytes (Vander Heide et al. 1990). icity studies suggest the use of both in vitro and in vivo assays,
Isolated myocytes possess several advantages that make them which includes classical in vitro assays on a molecular level (afn-
uniquely suited for the study of certain aspects of myocardial ity binding assays, heterologous human ether-a-go-go-related
ischemic injury (Vander Heide et al. 1990). In contrast to in vivo gene (hERG) channel expression), on a cellular level (hERG chan-
systems, isolated myocytes possess the inherent advantages of an nel block in native cardiac myocytes), on a tissue level (repolari-
in vitro model system that allows precise control of experimental sation assays on papillary muscle or Purkinje bres), or on an
conditions and manipulations. Myocyte preparations are nearly organ level (Langendorff heart) (Deshmukh et al. 2012). The more
homogenous populations, which allows accurate measurements complex an in vitro system gets, the more parameters can be
of metabolic parameters free from the inuence of diffusion prob- monitored and the closer the correlation will be with the in vivo
lems or the presence of other cell types in the intact preparations. assays. However, a higher degree of complexity also increases
Finally, isolated myocytes provide a model of ischemia in which it costs and decreases the potential for automation, miniaturiza-
is uniquely possible to test for cell viability without introducing tion, and high-throughput formats (Deshmukh et al. 2012; Meyer
new forms of cell injury or accelerating pre-existing cell injury. et al. 2007). Primary explant cultures are heterogeneous and often
Though a variety of techniques have been used to simulate isch- produce inconsistent results and low reproducibility in toxicity
emia in culture, severe hypoxia using nitrogen concentrations testing (Astashkina et al. 2012). Similarly, live-animal models may
(between 95% and 100%) has been used consistently in various not mimic the human physiology, can raise ethical and (or) ani-
models (Dhein et al. 2005; Diaz and Wilson 2006; Gross 2009). mal welfare concerns, and are expensive to maintain (Deshmukh
Experiments in isolated cardiomyocytes have generally repro- et al. 2012). The failure of animal tissue model systems to respond
duced the results on cardiomyocyte cell death in whole hearts of a to drugs in a similar manner as human tissue has motivated some
wide variety of interventions exploring cellular mechanisms in pharmaceutical companies to use human embryonic stem cells
preconditioning (Diaz and Wilson 2006). However, the simulation (ESCs) and induced pluripotent stem cells (iPSCs) derived cardio-
of ischemia is more challenging in cell culture, as only a small myocytes for cardiotoxicity testing (Braam et al. 2010). Cardiomy-
fraction of cardiomyocytes from a heart is available on culture ocytes from ESCs and iPSCs are functional in vitro and have
plates compared with using the fresh isolate. This could be a responded to the drugs in a similar manner as fetal cardiomyo-
practical limitation for immunoblotting and immunoprecipita- cytes (Kong et al. 2010).
tion assays. Ischemic preconditioning in isolated cardiomyocytes Another bottleneck of current cardiac safety pharmacology is
was rst studied by Armstrong and Ganote (1994). Armstrong was the lack of healthy adult human ventricular cardiomyocytes
able to precondition an in vitro simulated ischemic model either (Meyer et al. 2007). It is crucial to work with cardiac preparation as
by incubation in the absence of glucose or by brief simulated human as possible as the mechanism of repolarisation can differ

enormously between species, thus excluding typical laboratory Table 1. Advantages and limitations of using murine species in the
animals such as rat and mouse (Astashkina et al. 2012). The use of study of cardiovascular disease.
the human version of the hERG channel in noncardiomyocyte Advantages Limitations
heterologeous expression systems (HEK, CHO) or fully integrated
primary cardiomyocytes is controversial as the modied cells may 1. Small size and relatively Small heart size limits regional
not be of human origin and sometimes not even mammalian. low maintenance costs biochemical and physiological
Research on ESCs and iPSCs promises to enhance drug discovery analysis
2. Rapid gestation period and Species-dependent cardiac protein
and development of a new chemical entity (NCE) or a new biolog-
large litter size isoforms expression
ical entity (NBE) as therapeutic agents by providing simple, repro-
3. Genome extensively Potential species-dependent
ducible, and cost-effective tools for toxicity testing of drugs under
characterized phenotypic consequences of
development and, on the other hand, for studying the disease
transgene expression
mechanisms and pathways (Deshmukh et al. 2012). Cardiomyo- 4. Multiple inbred lines Higher basal heart rate and
cytes derived from human ESCs and iPSCs may offer an option to infection susceptibility
obtain human cardiomyocytes with a ventricular phenotype following surgical procedures
(Jonsson et al. 2012; Mandenius et al. 2011). Although initial studies 5. Germ line transmission only Lack of functional collaterals and
do look promising, the yields of cells obtained from classical dif- possible in this species unique electrophysiology
ferentiation methods may not yet meet the demands for higher
throughput screening (Astashkina et al. 2012). In addition to this
bottleneck, the differentiation status of the cells needs to be close
Walsh 2001; Takeishi and Walsh 2001). The small size, low main-
to an adult ventricular phenotype, and thus, much more optimi-
tenance cost, rapid gestation period (21 days), and large litter sizes
sation in the cardiogenic differentiation of the cells is needed
are relatively advantageous compared with the larger rat, which is
(Meyer et al. 2007). Recent data using basic electrophysiology and
slightly more aggressive in handling. In addition, compared with
extensive testing demonstrated that maturation of the electrical
rats, there is extensive knowledge of the mouse genome, with
phenotype is a prerequisite for future implementation of the over 10 000 genetic markers, including in excess of 6000 micro-
model in arrhythmogenic safety testing (Jonsson et al. 2012). satellite sequences that are critical for manipulation in genetic
studies (Guan et al. 2008). The entire mouse genome has been
Animal models for cardiomyocyte culture
sequenced, and there are hundreds of inbred strains that facilitate
Since the 1980s, murine animals have become popular for the study of the important and increasingly recognized inuence
studying myocardial ischemia and infarction (Curtis et al. 1987).
of the genetic background on gene expression. Also, germline

The choice of species for any experiment depends to a large extent transmission has been reliably achieved with mouse embryonic
on the goals of the study undertaken. It should be noted that all stem cells (Gertsenstein et al. 2010; Ward et al. 2002). This latter
species have their perceived advantages and disadvantages, and it approach allows more targeted alterations of the mammalian ge-
is not the intention of this article to promote the use of any nome that are possible using microinjection.
particular species as the issue of clinical relevance has not been
resolved. Instead, we have emphasised the advantages and disad- Cardiomyocyte isolation and culture
vantages and the use and abuse of murine preparations, especially It is difcult to stress the intact myocardium in vitro without
as models for irreversible ischemia, which is associated with se- introducing potentially complicating inuences. In vitro perfused
vere ventricular arrhythmias and infarction (Table 1). Murine an- anoxic hearts can circumvent some of these problems, but they
imal models show better resolution of the early (024 h) natural cannot be considered as a model of ischemia as the cells have
history of acute myocardial ischemia in man, especially those of access to unlimited extracellular space. Cultured isolated cardio-
younger age groups. Neonatal murine cardiomyocyte cultures myocytes offer the advantage of looking directly at myocyte
have also been used to study the toxicity and transport of drugs changes without the inuence of other contaminating tissues. In
(Dhein et al. 2005). Cultures of neonatal murine cardiomyocytes addition, it is easy to acutely stress isolated cardiomyocytes by
are widely used today for patch clamp experiments, for experi- varying the incubation conditions. Isolated adult murine cardio-
ments on propagation of action potentials using optical dyes, and myocytes offer signicant advantages in studying myocardial
for investigation of regulation of protein synthesis using Western ischemia (Vander Heide et al. 1990). For isolated cardiomyocytes to
blots or polymerase chain reactions (PCR) (Dhein et al. 2005; be a useful model, it must rst be established that the cells in vitro
Herron et al. 2012; Liu and Olson 2002; Nuss and Marban 1994; Sun respond to ischemia similarly to ischemic cells in vivo.
2004). Moreover, these cultures may be used for histological or Although isolation of adult murine cardiomyocytes has been
immunohistological experiments (VanWinkle et al. 1995). conducted for nearly 40 years (Powell and Twist 1976), there re-
Rats are the animal of choice for the small-animal cardiac mains no single universal method that can be easily employed to
model (Curtis et al. 1987). The rat heart is the most commonly used produce a large yield of high-quality, viable cells without some
model for isolation of ventricular cardiomyocytes for two reasons adjustments. The protocols used by different laboratories vary
(Dhein et al. 2005). First, rats are easy to handle, the animal itself and may depend not only on the species from which cells are
is not expensive, the isolation of cardiomyocytes normally gives isolated, but also on the type of experiment (Dhein et al. 2005;
reasonable cell numbers that can be used within the next day, and Louch et al. 2011). Interestingly, even laboratories that routinely
the size of the normal rat heart is appropriate to be used with isolate cells may experience difculties with the isolation. In gen-
commercially available perfusion systems. Second, the rat is the eral, the heart is quickly removed from the animal, connective
most commonly used animal model in cardiac biology; therefore, tissue is trimmed away, and the heart is connected to a Langen-
any data acquired at the single-cell level can be compared with a dorff perfusion system and perfused retrograde with a buffer to
broad eld of data available in the literature. Finally, myocytes remove any blood (Bell et al. 2011; Louch et al. 2011). The whole
from in vivo variations, i.e., spontaneously hypertensive rats, ge- heart is then treated with collagenase for a certain length of time
netically modied rats, or rats receiving surgery before use, can in the absence of exogenous calcium (Ca2+). This step allows the
also be investigated. However, mouse is increasingly becoming disruption of connections of individual cells with the extracellu-
the preferred mammalian system for cardiac studies, especially lar network and the dissociation of Ca2+-dependent desmosome
genetic manipulation, for a variety of reasons previously de- structures. Commercially puried collagenase is preferred over
scribed in other reports (Hedrich and Bullock 2004; Hoit and crude and partially puried preparations to avoid lot-to-lot

variations, thus providing better and more reproducible results. Fig. 1. Summary of the procedure and time for murine adult
Thereafter, the heart is removed from the perfusion system, and cardiomyocyte culture.
the ventricle is cut from the rest of the heart and minced into
small pieces. These are further treated with collagenase, and the
isolated cells are separated from the larger tissue pieces by ltra-
tion through a nylon mesh (mesh size 200 m). The isolated cells
are then re-exposed to physiological Ca2+ concentrations by in-
creasing the Ca2+ concentrations stepwise and then separated
from noncardiomyocytes by centrifugation (Dhein et al. 2005;
Vander Heide et al. 1990). These steps are summarized in Fig. 1.
The nal cell pellet can then be resuspended in cell culture me-
dium and cultured.

Isolated adult cardiomyocytes possess several advantages that
make them uniquely suited to the study of certain aspects of
myocardial ischemic injury (Vander Heide et al. 1990). First, iso-
lated myocytes possess the inherent advantages of an in vitro
model system that allows more precise control of experimental
conditions and manipulations compared with in vivo systems
such as whole-organ or whole-animal models. The myocyte prep-
arations are nearly pure populations of cardiomyocyte, thereby
allowing exact measurement of various metabolites free from
inuences of diffusion or the presence of other cell types in intact
preparations. Finally, isolated cardiomyocytes can be used to
model acutely stressed ischemic cardiomyocytes (cell swelling)
without the potential complications of no-reow, oxygen-induced
injury and oxygen free radical production present in other in vivo
ischemia models (Hearse et al. 1980; Kloner et al. 1974; McCord
1985). Besides ischemia, adult cardiomyocytes have been used for
studies on hypoxia and reoxygenation associated with in vivo
ischemiareperfusion (Portal et al. 2013; Shang et al. 2010), cell

cycle analysis (Walsh et al. 2010), and diabetic cardiomyopathy
(Caglayan et al. 2008). Like other models, there are limitations to
the adult cardiomyocyte model that may limit its applicability
(Vander Heide et al. 1990). The isolation of cells from each other
makes it impossible to study function and functional recovery
during and after ischemia or the modulating effects that mechan- endothelial and immune cells, which can be used for other studies
ical motion may have on ischemic injury. Furthermore, being an (Louch et al. 2011; Nickson et al. 2007; Song et al. 2000; Sreejit et al.
in vitro system, it lacks the possible modulating effects that vas- 2008; Sreejit and Verma 2011). A summarised protocol has been
cular tissue, the glycocalyx, neurohumoral substances, and leuko- provided in Fig. 2.
cytes and platelets may contribute to the overall mechanism of Various groups working with adult murine cardiomyocyte cul-
ischemic injury (Kammermeier and Rose 1988; Reimer et al. 1985). tures have elaborated on several issues. The issues are mainly in
Although these factors undoubtedly play a role in various cardiac nding and formulating conditions for proper tissue dissociation
diseases, isolated adult cardiomyocytes, when properly utilized, such as enzymes and the duration of enzyme treatment and in
still provide a valuable model for the study of cardiac conditions. selecting various growth supplements and appropriate serum
The neonatal murine cardiomyocyte culture was rst described concentrations for cell attachment and also appropriate attach-
by Harary and Farley (1963a, 1963b); since then, several modica- ment surfaces, especially for long-term cell cultures (Chlopclkova
tions have been developed and reported in the past 50 years et al. 2001; Claycomb 1981; Dhein et al. 2005; Kruppenbacher et al.
(Dhein et al. 2005; Fu et al. 2005; Nickson et al. 2007; Song et al. 1993; Nag et al. 1990). These ndings have also been applied in
2000; Sreejit et al. 2008). Whole hearts from decapitated neonates fetal and neonatal murine cardiomyocyte cultures. Fetal cardio-
are excised and immediately transferred into prechilled phosphate- myocyte isolation from embryonic age 15 or age 18 (E15 or E18)
buffered saline (PBS). The blood is gently squeezed out of the heart murine ventricles by serial enzymatic digestions has been per-
using sterile forceps to prevent clot formation inside the lumen of formed by many groups (Liu et al. 1996; McKoy et al. 2007;
the heart. The excised hearts are again washed with chilled PBS Nakamura et al. 1993; Zaruba et al. 2010). The number of foetus
and buffered salt solution. The ventricles are excised carefully, sacriced to generate a sufcient number of fetal cardiomyocytes
and the tissues are minced with a sterile scalpel blade into small for experiments is too high. Though fetal cardiomyocytes can
pieces. The myocardial cells are dispersed by incubation with di- divide and be maintained for few passages, the properties are
gestion enzymes such as collagenase, pancreatin, dispase, pro- slightly different from the neonatal and adult cardiomyocytes and
teases, trypsin, or a mix of 2 or more enzymes along with agitation have been used mainly for transplantation studies (Li et al. 1996;
or tissue-disrupting materials such as glass beads (varies with Sakai et al. 1999). Like fetal cells, neonatal cardiomyocytes also
groups), and the cell-containing solutions are collected and neu- have the advantage that the cells grow and divide (at least several
tralised. The digestion steps are repeated until the tissue is com- times) provided that the neonates used are not older than 96 h.
pletely dissolved, and then the cell suspension from each These cells form spontaneously beating individual cells or clusters
digestion is pooled and pelleted. Cell pellets are immediately re- within about 23 days (Dhein et al. 2005).
suspended in culture medium and seeded onto plates precoated Fetal and neonatal murine cardiomyocytes have limited us-
with adhesion enhancers such as gelatin, bronectin, lamnin, or age as they lack many adult cardiomyocyte characteristics.
even synthetic peptides. The differential attachment has been Also, cardiomyocyte cultures are overgrown by nonmyocytes
suggested to enhance the purity of the cardiomyocyte cultures by after a few days, and genetic manipulation is difcult after the
removing noncardiomyocyte cells such as cardiac broblasts and neonatal period as the cells cease to divide (White et al. 2004).

Fig. 2. Summary of the procedure and time for murine neonatal The developed strategies are directed mostly towards develop-
cardiomyocyte culture. ing immortalised cell lines, including cardiomyocyte cell lines,
cardiomyocyte-like cardiomyogenic cell lines, and cell lines with
few cardiomyogenic characters. The development of an immortal-
ized, cardiac muscle cell line that proliferates in culture while
maintaining a differentiated phenotype provides researchers
with a tool for understanding and probing the intricacies of car-
diomyocyte functions (White et al. 2004). The rst of the immor-
talized cardiomyocyte cells are AT-1 cells, which are derived from
an atrial tumor growing in a transgenic mouse. However, AT-1
cells need to be maintained by serial propagation as ectopic grafts
in syngeneic mice and cannot be passaged in culture or recovered

from frozen stocks and thus must be used as primary cells
(Delcarpio et al. 1991; Lanson et al. 1992).
Claycomb et al. (1998) derived the HL-1 cell line from AT-1 car-
diac myocytes; this HL-1 cell line could be serially passaged while
maintaining a differentiated phenotype and could also be revived
from cryopreserved stocks. HL-1 cardiomyocytes, upon character-
ization by microscopic and immunohistochemical, electrophysi-
ological, and pharmacological methods, have revealed an adult
cardiomyocyte-like prole with expression of cardiac-specic
functional receptors and intracellular signaling proteins. Over the
years, HL-1 cardiomyocytes have been used for studying many
aspects of cardiac biology, including hypoxia, hyperglycemia, cel-
lular signalling, electrophysiology, calcium regulation, cell cycle
regulation, apoptosis, genetic manipulation, and toxicological ef-
fects (Strigun et al. 2012; White et al. 2004). The HL-1 cell line,
being a relatively undifferentiated, transformed tumor cell line,
has characteristics that are not suited to study cardiac conditions.
However, recent proteomic analysis of HL-1 in comparison with

patient samples using uorescence 2-dimensional difference gel
electrophoresis (DIGE) reveals HL-1 as a suitable ischemic cardiac
in vitro model for studying cellular response to acute ischemia
and regeneration (Haas et al. 2012). Subsequently, additional HL
cell lines (HL-1P, HL-2, and HL-5) were derived from cultured AT-1
Although a certain set of experiments can be performed with few cells. Morphological, genetic, and immunohistochemical analyses
cells, signicant changes occur in isolated cardiomyocyte struc- of HL cell lines showed that they all exhibit essentially the same
ture, protein composition, and electrical and contractile proper- adult cardiomyocyte phenotype as HL-1 cells (White et al. 2004).
ties following isolation, especially in long-term cultures (Louch Most of the cardiac-specic genes are expressed in these HL cell
et al. 2011; Vander Heide et al. 1990). Under these conditions, when lines. The HL-5 cell line has already been used to study intracellu-
using isolated cardiomyocytes as a model of ischemia, the cells lar processing of the atrial natriuretic peptide by using RNA inter-
ideally are to be used within a few hours of isolation to ensure that ference technology, besides characterizing apoptotic signaling
they reect the normal state of cellular protein composition dur- mechanisms in an in vitro model of ischemiareperfusion
ing the experiment. Therefore, in experiments involving genetic (Cicconi et al. 2003; Wu et al. 2002).
modications and protein expression or long-term drug treat- The myoblast cell line H9c2, derived from embryonic rat heart,
ment, it is essential to compare the results with those of control has been used as an in vitro model for both skeletal and cardiac
cultures under matching conditions. Attempts have been made muscles as the cells show electrophysiological and biochemical
to delay the changes occurring during culture by using electri- properties of both skeletal and cardiac tissues (Kimes and Brandt
cal pacing (Holt et al. 1997), drugs such as adrenergic agonists 1976; Sardao et al. 2007). H9c2 cells lack the ability to beat but
(Akuzawa-Tateyama et al. 2006), growth factors or hormones such show many similarities to primary cardiomyocytes and therefore
as insulin (FGF-2) (Sreejit et al. 2008), and other preconditioning have been used for investigating the molecular and cellular pro-
approaches (Diaz and Wilson 2006). However, none of these ap- cesses involved in myocardial hypertrophy, apoptosis, differenti-
proaches completely inhibits or reverses the changes in cardiomy- ation, and toxicology (Koekemoer et al. 2009; Pereira et al. 2011;
ocytes following culture over time. Although the phenotype of Watkins et al. 2011). H9c2 cells have the ability to divide compared
cultured neonatal cardiomyocytes is highly stable (Yamashita with terminally differentiated cardiomyocytes, and many studies
et al. 1994), it is essential that shorter culture duration and appro- have used the H9c2 cardiomyocyte cell line as an animal-free al-
priate control experiments are included in obtaining physiologi- ternative (Kimes and Brandt 1976; Koekemoer et al. 2009; Zordoky
cally relevant information from cultured cardiomyocytes (Louch and El-Kadi 2007). Cardiomyocyte cell lines are a valid in vitro
et al. 2011). The fetal and neonatal murine cardiomyocytes models system for understanding cardiac diseases. The current and
are appropriate for the study of different aspects of heart disease emerging cardiomyocyte cell lines hold much promise for in vitro
when adult cardiomyocytes are not easily available. Ideally, the analysis of cardiomyocyte function for studies in the future.
results obtained from fetal and (or) neonatal cardiomyocytes P19 embryonic carcinoma (EC) cells, isolated from an experi-
should be validated with similarly designed experiments using mental embryo-derived teratocarcinoma in mice, are the most
adult cardiomyocytes. commonly used cardiomyocyte-like cardiomyogenic cell lines
(van der Heyden and Deze 2003). These cells are multipotent and
Alternate strategies can differentiate into cell types of all three germ layers, including
To address these issues, various groups have developed suit- cardiac lineage. P19 EC cells are very useful in isolating clonal
able cell culture systems, which has proven to be challenging. sublines, with or without prior mutagenesis, and the cloned

sublines include P19 S18, P19 D3, P19 RAC65, and P19 Cl6 cells, through a pluripotent intermediate and thereby reducing the
which, on induction, can further differentiate and develop cell time for differentiation (Efe et al. 2011).
lines (Edwards et al. 1983; Jones-Villeneuve et al. 1983). Following The scale of applications of ESC- and iPSC-derived cardiomyo-
long-term culture under conditions promoting mesoderm differ- cytes is diverse and includes drug discovery, high-throughput tox-
entiation and in the presence of inducing agents such as DMSO, icology assays, and regenerative medicine (Astashkina et al. 2012;
5-azacytidine, dynorphin B, and oxytocin, P19 Cl6 differentiated Braam et al. 2010; Chavakis et al. 2010; Deshmukh et al. 2012; Dorr
into cardiac muscle (Chavakis et al. 2010; Gong et al. 2012; et al. 1988; Jonsson et al. 2012; Kong et al. 2010; Mandenius et al.
Habara-Ohkubo 1996; Paquin et al. 2002; van der Heyden and 2011; Meyer et al. 2007; O'Brien 2008; Yoshida and Yamanaka 2011).
Deze 2003). The differentiated cardiomyocyte-like cells are com- However, most of the studies using ESCs and iPSCs have been
parable with embryonal and neonatal cardiac cells, lacking only performed with human cells to avoid interspecies differences
the muscarinic cholinoceptor response (Wobus et al. 1994). Be- both in regenerative medicine and in toxicology (Andersson et al.
cause of their ability to form cardiomyocytes, they have been used 2010; Wobus and Lser 2011; Yoshida and Yamanaka 2011). How-
to dissect the role of cardiac-specic transcription factors and ever, studies of pluripotent mouse ESCs have led to the develop-
upstream signalling pathways in cardiac cell differentiation and ment of in vitro models of cardiomyocyte differentiation (Boheler
recently for assays to evaluate biomaterial compatibility (Dawson et al. 2002). Morphological, electrophysiological, and molecular
et al. 2009; van der Heyden and Deze 2003; van der Heyden et al. techniques show that the in vitro differentiation process repeats
2003). P19 cell lines and their derivatives being transformed ide- the developmental pattern of early cardiogenesis. Genetic manip-
ally should not be used as models to study heart diseases other ulations demonstrate how signaling molecules, transcription fac-
than the rare cardiac tumors. tors, ECM components, and calcium-handling proteins interact
Murine embryonic stem cells (ESCs) derived from different and affect differentiation. It is indeed a fact that much of what we
sources of blastocysts and related structures have been used prior know about the differentiation of ESCs and iPSCs to cardiomyo-
to studies with P19 cells (Boheler et al. 2002; Doetschman et al. cytes in vitro has been learned from studies with murine cells.
1985). Murine ESCs are useful models for studying both cardiomy- ESCs and iPSCs are important advances in the pursuit towards
ocyte development and differentiation (Hescheler et al. 1997; Klug establishing a human cardiomyocyte cell culture model to study
et al. 1996; Maltsev et al. 1993, 1994; Sachinidis et al. 2003). ESCs cardiac diseases. However, a lot more research is required to uti-
form multicellular aggregates called embryoid bodies (EBs) by lize the full potential of these innovations.
the hanging drop method (Martin 1981; Sachinidis et al. 2003). Taking clues from the strategies used for ESC differentiation,
Embryoid bodies can differentiate spontaneously or with the help of bone marrow derived stem cells (BMSCs) and stem cells derived
inducing agents, which include chemicals, endogenous substances,
from other sources following induction by various factors have
and niche or environmental components such as 5-azacytidine, reti-

differentiated into cells of cardiomyogenic lineage (Fukuda 2001,
noic acid, ascorbic acid, nitric oxide, bone morphogenetic protein-2
2003; Lobon et al. 2009; Madonna et al. 2008; Makino et al. 1999;
(BMP-2), electrical stimulation, genetic manipulation, ECM compo-
Prat-Vidal et al. 2007; Zhu et al. 2008). Spontaneous beating has
nents, co-culture with broblastic cells, high-density culture, and
been observed in differentiated BMSCs following induction with
nano-patterning (Baharvand et al. 2005; Becker et al. 2011; Boyang
5-azacytidine. Recently, BMSCs grown on cardiac ECM differenti-
et al. 2011; Hakuno et al. 2005; Lee et al. 2011; Pucat 2008; Sachinidis
ated into cardiomyogenic lineage, even in the absence of any
et al. 2006). Pure or highly enriched populations of cardiomyocytes
chemical induction, including 5-azacytidine (Sreejit and Verma
were obtained by differentiating genetically altered ESCs and cell
2013a). The use of cardiac ECM will create a microenvironment
sorting (Klug et al. 1996; Mller et al. 2000).
similar to that of the heart and thus can potentially be used as a
Induced pluripotent stem cells (iPSCs), the most innovative dis-
cardiac cell model with 3-dimensional architecture besides being
covery in stem cell biology in the last decade, were generated
used in regenerative applications (Sreejit and Verma 2013b). Fol-
directly from somatic cells by the introduction of dened tran-
lowing the report that cardiomyocyte differentiation also oc-
scription factors (Takahashi and Yamanaka 2006). iPSCs allow re-
curred in BMSC transplanted into infarct tissue after myocardial
searchers to obtain pluripotent stem cells with the ability to
infarction (Orlic et al. 2001), the focus of utilising differentiated
generate almost all cell types, which makes them important in
research, and they have potential therapeutic applications BMSCs has been mostly towards regenerative therapy (Ge et al.
without the controversial use of embryos. Like ESCs, iPSCs can 2009; Heng et al. 2004; Huang et al. 2012; Rasmussen and
self-renew indenitely and can differentiate into the cellular Simonsen 2012; Tomita et al. 2007; Yang et al. 2009). It should also
derivatives of all three germ layers (Murata et al. 2010; Takahashi be noted that the mechanisms involved in the differentiation are
and Yamanaka 2006). Several groups have reported cardiac differ- vague, and conicting ndings on differentiation have been re-
entiation in mouse iPSCs using either EBs or stroma cell co- ported (Liu et al. 2003; Ramirez et al. 2005; Wan Safwani et al.
culture (Fujiwara et al. 2011; Mauritz et al. 2008; Mller et al. 2011; 2012). Because the other models are well accepted and are easy to
Narazaki et al. 2008; Schenke-Layland et al. 2008). The most com- develop and maintain, not much work has been done using car-
monly used method is the EBs in suspension culture, which has diomyocytes obtained from differentiated stem cells (Meyer et al.
been also applied to iPSCs, which can also differentiate in 2007). However, with the high probability of using stem cells and
5-azacytidine, bone morphogenetic proteins, ascorbic acid, vascu- differentiated cells in regenerative therapy, work on these cells
lar endothelial growth factor, and genetic manipulation (Fujiwara will become a necessity to understand the post-transplantation
et al. 2011; Mller et al. 2011; Murata et al. 2010). A list of car- response in cardiac diseases (Li and Deng 2011; Sreejit and Verma
diomyogenic differentiation activators used in ESCs, iPSCs, and 2013b; Swain et al. 2012).
MSCs cultures has been provided in Table 2. However, the ef-
ciency of cardiac differentiation and proliferation of differenti- Possible strategies
ated cardiomyocytes is lower in iPSCs than in ESCs (Mauritz et al. According to Curtis et al. (1987), an ideal model for cardiac
2008). By manipulating pluripotent stem cells, dened cardiomy- disease would (i) completely mimic one or more of the various
ocyte subtypes can be generated for toxicologicalpharmacological clinical conditions, (ii) respond to drugs in a manner that corre-
screenings or regenerative medicine. However, a recent study has sponded exactly with the clinical response, (iii) have sufcient
pointed out that total pluripotency may not be critical for trans- precision and accuracy to function as a bioassay, (iv) permit a
differentiation. A brief reactivation of reprogramming factors in variety of responses to be measured, and (v) be undemanding in
embryonic and adult broblasts has been used to rapidly generate terms of cost, time, and expertise. Although whole-animal heart is
contracting cardiomyocytes almost certainly without going still the preferred choice, small-animal cardiomyocyte cultures,

Table 2. Inducing agents used for differentiating stem cells (MSCs, ESCs and iPSCs) to cardiomyogenic lineage in murine models.
Agent or factor Cell type (key references)
Chemical and growth factors
1. 5-Azacytidine MSC (Makino et al. 1999), ESC (Gao et al. 2012)
2. Dimethyl sulfoxide ESC (Dinsmore et al. 1996)
3. Retinoic acid ESC (Dinsmore et al. 1996; Wobus et al. 1997)
4. Ascorbic acid ESC (Bartsch et al. 2011), iPSC (Cao et al. 2012)
5. Reactive oxygen species and NADPH oxidase ESC (Bartsch et al. 2011; Buggisch et al. 2007)
6. Nitric oxide ESC (Bartsch et al. 2011; Danalache et al. 2007)
7. Trichostatin A ESC (Illi et al. 2005)
8. Verapamil ESC (Sachinidis et al. 2006)
9. Cyclosporine A ESC (Sachinidis et al. 2006), iPSC (Fujiwara et al. 2011)

10. Sulfonyl-hydrazone-1 iPSC (Quattrocelli et al. 2011)
11. Exogenous glucose, amino acids, and selenium ESC (Guan et al. 1999)
12. Activin-4 and apelin ESC (Wang et al. 2012)
13. Leukemia inhibitory factor ESC (Bader et al. 2000)
14. Vascular endothelial growth factor ESC (Chiriac et al. 2010)
15. Stromal-derived stem cell factor ESC (Chiriac et al. 2010)
16. Fibroblast growth factor 2 ESC (Kawai et al. 2004)
17. Insulin-like growth factor I ESC (Sachinidis et al. 2003)
18. Tumor growth factor family (TGF-1, BMP-2, and BMP-4) ESC (Behfar et al. 2002; Chiriac et al. 2010; Kawai et al. 2004;
Sachinidis et al. 2003)
19. Oxytocin and derivatives ESC (Danalache et al. 2007, 2010)
20. Transcription factors (Gata4, Mef2c, and Tbx5) iPSC (Ieda et al. 2010)
21. MEK/ERK pathway activators ESC (Kim et al. 2007)
22. Wnt/-catenin pathway antagonists ESC (Singh et al. 2007; Wang et al. 2011)
23. Activators of calcium-activated potassium channels of small iPSC (Mller et al. 2011)
and intermediate conductance
Alternate strategies
1. Targeted expression of -1,3-fucosyltransferase ESC (Sudou et al. 1997)
2. Electrical pulse ESC (Sauer et al. 1999)

3. Shear stress and mechanical loading ESC (Gwak et al. 2008; Illi et al. 2005; Shimko and Claycomb 2008)
4. Exposure to a low-frequency magnetic eld ESC (Ventura et al. 2005)
5. Flk-1 positive cardiomyogenic progenitors ESC (Chiriac et al. 2010), iPSC (Narazaki et al. 2008)
6. Aggregation through hanging drop method and mass culture ESC (Fuegemann et al. 2007; Lee et al. 2011)
7. Adhesion on surfaces ESC (Hakuno et al. 2005)
8. Extracellular matrix and components ESC (Baharvand et al. 2005), MSC (Sreejit and Verma 2013a)
9. Combinatorialhybrid matrix ESC (Gupta et al. 2011)
10. Micro- and Nano-patterning MSC, ESC, iPSC (Boyang et al. 2011)
11. Co-cultureconditioned media MSC (Condorelli et al. 2001), ESC (Mummery et al. 2002)
12. MicroRNAs ESC (Ivey et al. 2008)
especially murine, have their own value as models for cardiac gineering such as hybrid polymers containing natural compo-
diseases. Ethical issues prevent the use of human cardiomyocyte, but nents and nanoscale patterning of micrometre-sized materials
the development of ESCs and iPSCs have revolutionised the ap- can inuence the outcomes of such studies (Boyang et al. 2011;
proach of utilising human cells for cardiac disease models. Interest- Davis et al. 2005). The growth of interdisciplinary science will
ingly, a recent study showed that embryonic and adult broblasts denitely decrease the dependence on animal models and shift
could be used to rapidly generate contracting cardiomyocytes with- the focus more towards tissue and cellular levels where cardiomy-
out generating pluripotent intermediates (Efe et al. 2011). The pros- ocyte cultures will gain importance. However, ethical concerns
pect of utilising stem cells could revolutionise cardiac therapy as will continue to drive the use of murine or any other animal-based
never before expected, as personal medicine would be boosted by cardiomyocyte cultures rather than human cardiomyocytes. The
this novel approach. This approach would help us to use high- only possibility is the use of human-derived stem cells to generate
throughput technologies, and each individual would be treated as a differentiated cardiomyocytes in sufcient quantities for such
separate entity. Under this condition, each individual becomes a
studies.
separate study model, and the data obtained could be combined to
enhance our overall knowledge. Current status on drug-interaction studies
The use of primary cultures of cardiomyocytes will continue for
some time until robust models of cardiac culture such as cell lines The phenotype stability observed in neonatal murine cardiomyo-
with the ability to mimic individual or species-specic characters cyte culture models makes them ideal for drug-interaction studies
are generated. The manipulation of cell lines to phenotypically (Yamashita et al. 1994). The effect of various drugs for precondition-
mimic cardiac cells is an area with immense prospects. Ideally, ing and through ischemiareperfusion has been studied using
the studies on cell lines should be validated by performing similar this model (Armstrong et al. 1994; Armstrong and Ganote 1994;
experiments with either adult or neonatal cardiomyocytes as ne- Bell et al. 2011; Diaz and Wilson 2006; Ferdinandy et al. 2007;
cessitated. Similarly, the use of 3-dimensional cultures to mimic Ikonomidis et al. 1994; Remio et al. 2001; Shirai et al. 1998;
the cardiac niche by using biomaterials and scaffolds to grow Vander Heide et al. 1990; Yamashita et al. 1994). Toxicological
cardiomyocytes as disease models is an interesting area of study. It studies of drugs have come a long way and of late have gained
should be noted that prior studies have failed in this approach importance, especially with drugs given to patients as a part of
(Davis et al. 2006). Recent advances in biomaterial and tissue en- long-term medication (Astashkina et al. 2012; Dorr et al. 1988).

Anticancer drugs such as trastuzumab, taxanes, depsipeptides, and Conclusions and perspectives
anthracyclines, including doxorubicin (DOX) (Adriamycin), were Currently, there are many potential strategies to address the
found to be very effective but caused severe cardiotoxicity due to menace of cardiac diseases. Besides therapeutics, advancements
disruption of signalling pathways (Buzdar et al. 1985; Ng et al. 2006; in science have helped in providing a greater understanding of the
Zuppinger and Suter 2010). Consequently, drugs are now tested for underlying causes of cardiac diseases. Current studies using ani-
cardiotoxicity and the pharmacokinetic parameters are analysed to mal models have focused either on the pathophysiology of the
determine the dose-cumulative pattern and treatment-related vari- disease condition or on the therapeutic applications. The research
ables associated with cardiac-adverse events (Lebedinsky et al. 2011; performed at the cellular level has not yet provided a signicant
Soto-Matos et al. 2011). However, it should be noted that cardiotoxic- breakthrough compared with studies carried out on diseases in
ity has been evaluated in animal or clinical trials (Buzdar et al. 1985; other organs or systems. The lack of a good cellular model to
Dorr et al. 1988). Recently, cell culture based models have been used replicate the conditions of the human heart does hamper such
to study cardiac safety and the screening of drugs (Astashkina et al. studies. Currently, despite the limited access, the best in vitro
2012; Braam et al. 2010; Deshmukh et al. 2012; Jonsson et al. 2012; option to study human cardiac disease is to isolate cardiomyo-
Mandenius et al. 2011; Meyer et al. 2007). Similarly, the interest in cytes from cardiac tissue explants from patients undergoing sur-
gical procedures. However, ethical issues prevent use of human
understanding the effects of various molecules on induced stress and
tissue at almost all stages of research. Cardiac cells of human
toxicity has also increased using cell culture based models (Melchert
origin can be grown by transdifferentiation through induction of
et al. 1991; Shainberg et al. 2009; Wang and Kang 1999). Previously,
ESCs and iPSCs. The preferred strategy with minimum cellular
such studies were carried out using animal models, including trans- manipulation of primary cardiomyocyte culture would be ideal
genics (Iszard et al. 1995; Latouche et al. 2010). for research and possible therapeutic purposes. Because ethical
One of the best-studied cardiotoxic drugs is DOX, which is an issues prevent the use of human cardiac tissue, murine animals
effective anticancer drug but can lead to congestive heart failure have become the easiest and most reliable source of obtaining
(Minotti et al. 2004). DOX cardiotoxicity causes loss of myobrils primary cardiomyocyte cultures.
and vacuolization (Singal and Iliskovic 1998) due to oxidative The primary culture of the neonatal mice cardiomyocyte model
stress in studies in animals (Sun et al. 2001) but not in humans enables researchers to study and understand the morphological, bio-
(Ladas et al. 2004). This suggests that mechanisms other than chemical, and electrophysiological characteristics of the heart, be-
oxidative stress might contribute to DOX-induced heart failure sides being a valuable tool for pharmacological, toxicological, and,
(Kobayashi et al. 2010). DOX is shown to cause cardiac mitochon- recently, regeneration studies. Isolated cardiomyocytes in primary
cell cultures are an invaluable tool in the study of the cellular-level
drial injury by both oxidative and nonoxidative mechanisms

(Marcillat et al. 1989). Using cell culture, oxidative degradation of function and regulation of electrophysiology, intracellular calcium
DOX was identied as a possible salvage mechanism for diminish- uxes, contractile mechanics, and protein expression. Cardiomyo-
ing its levels and toxicity in cardiomyocytes (Menna et al. 2007). Of cytes in primary cultures offer the opportunity for repeated visual-
late, work on DOX toxicity has been carried out mostly on cardi- ization of molecular events in the same cells, thus providing an
omyocyte cultures. GATA4-inhibited DOX toxicity induced au- essential platform for cardiac disease and related studies. It is ex-
pected that this approach will pave the way for a critically important
tophagy in cultured neonatal rat cardiomyocytes (Kobayashi et al.
contribution to future research. Although such studies require a
2010). The protective role of arjunolic acid against DOX-induced
high-quality cardiomyocyte population, successful cell isolation and
intracellular ROS dependent JNK-p38 and p53-mediated cardiac
maintenance during culture remain a challenge. Because cardiomy-
apoptosis was demonstrated in cardiomyocyte culture and animal ocytes do not divide, the success of any work with isolated cardiomy-
models (Ghosh et al. 2011). It was also observed in cardiomyocyte ocytes depends entirely on the reproducibility of cell isolation and
culture and cell lines that various doses of DOX induce differential maintenance of in vitro cultures.
regulation of telomere repeat-binding factors 1 and 2 (TERF1 and
TERF2, respectively) through p53 and MAPK. This is responsible Acknowledgements
for inducing either early apoptosis or senescence and late death This work was supported by grants to RKS from the Heart and
due to mitotic catastrophe (Spallarossa et al. 2009). Interestingly, Stroke Foundation of Saskatchewan, Canada, and grants to RSV
exposing cardiomyocytes to subclinical concentrations of DOX from the Department of Science and Technology (DST), India (BT/
rapidly reduced creatine transport (Darrabie et al. 2012). PR5392/MED/14/693/2004), and the Department of Biotechnology
The current status of utilising cardiomyocyte cultures as a (DBT), India (BT/PR7951/MED/14/1193/2006). RKS and SP were involved
model for drug-interaction studies is limited by a large number of in the entire conceptualisation and preparation of the manuscript,
competing models such as derived cardiac cell lines, whole-heart, and RSV was involved in the preparation of the manuscript. The
and whole-animals models. However, the future looks promising funders had no role in the study design, data collection and analysis,
owing to two important factors: (i) economy and (ii) the potential decision to publish, or preparation of the manuscript. No competing
for modication. The economy of working with cell cultures over nancial, personal, or professional interests exist. Authors are grate-
animal models is very obvious because of the avoidance of long- ful to Dr. J.R. Dimmock, College of Pharmacy and Nutrition, Univer-
term animal care and maintenance. The ethical issues related to sity of Saskatchewan, for the reading and helpful discussion of this
manuscript.
animal experimentation and cardiomyocyte cultures are almost
similar as both require animal sacrice. The potential to modify References
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Cardiomyocyte Culture - An Update On The in Vitro Cardiovascular Model and Future Challenges

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Cardiomyocyte Culture - An Update On The in Vitro Cardiovascular Model and Future Challenges

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Received 25 April 2013. Accepted 8 August 2013.

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of the genetic background on gene expression. Also, germline

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dium and cultured.

ischemiareperfusion (Portal et al. 2013; Shang et al. 2010), cell

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in syngeneic mice and cannot be passaged in culture or recovered

However, recent proteomic analysis of HL-1 in comparison with

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and niche or environmental components such as 5-azacytidine, reti-

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9. Cyclosporine A ESC (Sachinidis et al. 2006), iPSC (Fujiwara et al. 2011)

2. Electrical pulse ESC (Sauer et al. 1999)

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drial injury by both oxidative and nonoxidative mechanisms

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Published by NRC Research Press

Published by NRC Research Press

Published by NRC Research Press

Published by NRC Research Press

Published by NRC Research Press

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