1. By Snaa Hussain

2.  Growth of the heart is dynamic, occurring during:
 Embryogenesis
 Postnatal development
 Maturity
 Senescence
 Environmental & pathological conditions
 Physiological growth of the heart in general mediated by:
 Developmental programmes
 Mechanical load
 Locally derived/circulating growth factors
 Pathological cardiovascular growth is generally mediated by:
 Similar factors as physiological growth
 Superimposed myocardial injury

3.  Cardiovascular ageing is a growing concern
due to the population of the UK ageing
Fig 2: Population by age, UK, 1983, 2008 and 2033(www.Direct.gov.co.uk)

4.  In 1983, there were just over 600,000 people in the UK
aged 85 and over
 Since then, the numbers have more than doubled
reaching 1.3 million in 2008
 By 2033 this figure is to rise again reaching 3.2 million,
accounting for 5% of the total population
 Cardiovascular ageing is a continuous process; the rate
varies amongst individuals, altered by different influences:
 Physiological changes
 Previous disease
 Surgery in younger life
 Individual life style

5.  Main morphological change:
 Structure of cardiac tissue & chambers
 Conduction system
 Coronary arteries
 Most obvious change is an increase in left ventricular wall thickness
 Increase in systolic pressure
 Corresponding increase in arterial stiffness and delayed progress
 Similarly, coronary blood flow hemodynamics & coronary vascular
resistance become impaired as a function of age
 With these properties continue to deteriorate;
 Leading first to depressed cardiac functional reserve
 and subsequently to overt failure & death

6.  The heart is made up of cardiomyocytes,
 Contractile muscle cells
 75% of the total volume of the myocardium
 Make contact to neighbouring myocytes via intercalated disks
 Discs run longitudinally to provide lateral contact
 Intercalated disks consist of three main specialised components:
 Gap junctions
 Adherens junctions
 Desmosomes
 Desmosomes are another type of adherens junctions;
 “Integrators of mechanical integrity”
 Provide insertion sites for desmin-containing intermediate filaments
 Leading to formation of a transcellular cytoskeletal network
 Promotes longitudinal force transmission
 Therefore desmin is found in all contracting cardiomyocytes

7.  With age cardiomyocytes reduce in number
 Attributable to apoptosis as well as necrosis
 Partial compensatory increase in the size of
myocytes that survive
 Observations in humans and animals suggest
that myocyte maturation & ageing are
characterised by loss of replicative potential,
telomeric shortening & the expression of age-
associated proteins p16INK4a and p53

8.  When the heart undergoes immense pressure/workload,
ventricular myocytes grow in response to this, and is
known as hypertrophy
 Initial excessive mechanical stress is corrected towards towards
normal
 Laplace Law
  increased wall thickness decreases wall stress
 Hyperplasia is the increase in cell number
 Principle feature of cardiac growth during foetal & neonatal
periods
 First 3 to 4 weeks of life, cardiac myocytes double in number
 During the late gestation period, dividing cells rapidly decrease
in number
 Thereafter, normal growth of heart is via hypertrophy

9.  Paradigm that the heart is a postmitotic organ
incapable of regenerating parenchymal cells was
established 1970’s
 Therefore the only response for cardiomyocytes
to stress is hypertrophy and apoptosis
 This is now being questioned by studies carried
out within the last 3 decades to show that there
is also evidence of regeneration via stem cells

10.  Replicating cardiomyocytes resemble small
amplifying cells that have originated by activation of
progenitor cells
 Progenitor cells:
▪ Relatively undifferentiated cell types that are derived from
asymmetric stem cells division and lack the capacity to self renew
 Stem cells
 Cells that have not yet taken on the identity of any specific
cell type
 No dedicated function
 Proliferation is dependant on a functional telomere
▪ Telomeres are chromatin structures composed of tandem G-
repeats bound to an array of proteins that cap the ends of
chromosomes

11.  The presence of stem cells resident in the heart itself was first
reported by Beltrami et al 2003
 c-Kit receptor found on bone marrow stem/progenitor cells have
recently been identified on cardiac stem cells
 c-kit:
 Proto-oncogene member of the receptor tyrosine kinase family
 Closely related to platelet derived growth factors
 Regulates a variety of biological responses
▪ Chemotaxis
▪ Cell proliferation
▪ Apoptosis
▪ Adhesion
 Plays a major role in regulating the myocardial balance of angiogenic
cytokines and therefore controls cardiomyocyte regeneration and
repair

12.  Do old hearts possess c-Kit+ cells, a marker of
progenitor cells & this ability for hyperplasia
to occur in the old heart?
 Additionally does the amount of c-Kit
expressed change with age?

13.  Western Blotting
 Freeze thaw
 ProteinAssay analysis
 ECL
 48hrs
 C-Kit Antibody(Santa Cruz)
 Desmin Antibody(Dako)
 Used desmin antibody to detect if there was protein in the assay
and for complete transfer of protein to membrane, as desmin is
not as costly as the c-Kit antibody
 The ECL was carried out for three different timings, 5, 7 & 10
minutes.

16.  The results show that there is more c-kit expression in the
young rat samples than the old rat samples but never the
less there is still expression of c-kit in the old samples.
 The t-test showed that there is a significant difference in
the expression of c-kit with age where p<0.05 and df=9.
 The results obtained were as expected and that the c-kit
expression was positive in both young and old samples but
with a significant difference with the increase in age.
 Both the desmin and c-kit results showed that there was a
decrease in expression in the older rat samples.

17.  In this study it was found that c-kit was expressed in
both young and old tissue samples.
 The use of western blotting on c-kit expression has
not been carried out before with the c-kit antibody on
old tissue, therefore the known concentration of the
primary antibody to use was a trial and error theory
for most of this study as to see whether the
concentration being used was enough for the c-kit to
be expressed.
 The results showed that with an increase in age the
amount of c-kit expression was decreased but still
showed quite strong bands with a 1:200 dilution of
antibody.

18.  Cell regeneration would be anticipated to be enhanced in the
presence of injury in an attempt to attenuate organ damage
and restore its physiological function.
 This was not considered feasible in the myocardium until
recently.
 The recent paradigm shift in cardiac biology toward the
heart as an organ capable of self-renewal and repair has
created new opportunities for treatment of heart disease.
 These replicating myocytes may reside in the heart or
represent the committed progeny of circulating primitive
cells that homed to the myocardium.

19.  In the clinical investigation by Dimmeler et al., it states
that the local injection of bone marrow cells to the
infracted mouse heart results in significant reconstruction
of the necrotic myocardium and remarkable improvement
in ventricular function.
 With research like this one cardiac stem cell therapy can
have a positive result on the ageing heart as there is a
rapid increase in the older population in the UK and other
western areas.
 Ageing affects decrease in the functional reserve of the
heart and loss of myocytes contributes to the attenuation
of the response of the old heart to sudden changes in
ventricular loading

20.  A study carried out by Fransioli et al has revealed that the use of genetic
engineering and conventional immunochemistry allowed them to
validate the identity of c-Kit expressing cells using both the transgene
and c-Kit protein expression as markers.
 Evidence presented shows the association of c-Kit expressing cells in
postnatal development, response to myocardial infarction, and the
commitment of these cells to cardiogenic lineages, thereby supporting a
role for c-Kit cells in myocardial growth and repair following injury.
 For further study opportunities would be to look at c-kit expression via
immunochemistry, perfusions, and genetic engineering and looking at c-
Kit expression in the hypertrophied heart, and comparing it to neonatal
cell growth, foetal, adolescence and senescence cell growth of the
myocardium.
 Also to look at what stem cell therapy can bring in the future in regards
to myocardial repair and regeneration.

21.  The discovery that stem cells reside in the heart and constantly
give rise to a myocyte progeny has changed dramatically our
interpretation of the aetiology of heart failure of multiple causes
and offered novel therapeutic options for the management of this
devastating disease.
 With increasing acceptance of c-Kit+ cells as an authentic source
for cellular-based myocardial repair, the challenge is to enhance
the potential of these cells to mediate regenerative processes in
the damaged heart.
 GFP tagging of the c-Kit population as done by Fransioli et al., will
be a valuable approach for in vivo tracking of cells following injury.
 Focusing future studies on this type of study, with manipulations
to enhance the survival and growth of stem cells with cytokines
and paracrine factors, may boost cellular repair processes in the
damaged heart.

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 www.Direct.gov.co.uk. Viewed online on Thursday 27th August 2009.

23. ANY QUESTIONS