cardiomyogenesis ( generation of cardiomyocytes ) had not been convincingly demonstrated in the adult mammalian heart until very recently ; the potential for myocardial regeneration was only recognized in organisms such as fish and amphibians .
however , carefully designed and performed studies have produced compelling evidence for the existence of cardiomyocytes in the adult heart , that were formed well after birth , in rodents and even humans .
although the various studies do not agree about the rate of cardiomyocyte renewal in adults , this is clearly low and inadequate to replenish the substantial losses of cells after major injuries such as a myocardial infarction . in addition
, controversy surrounds the putative cellular sources of postnatal cardiomyogenesis : do new myocytes arise from proliferation of pre - exisiting ones or from cardiomyogenic differentiation of endogenous progenitors ? in this review , we will present the evidence supporting the contribution of these two mechanisms in adult cardiomyogenesis and discuss their relative importance in different settings , such as normal ageing and post - myocardial injury .
we will also critically present the existing methodologies that allowed the investigation of these mechanisms , with emphasis on their strengths and limitations .
while lower vertebrates ( such as newts and zebrafish ) and neonatal mice possess a robust ability for myocardial regeneration , the ability of the mammalian heart to generate myocytes beyond the early neonatal period has been controversial . during the 20th century , the postnatal mammalian heart was viewed as a post - mitotic static organ , in which increases in mass occur exclusively through myocyte hypertrophy ( i.e. increase in cell size ) , rather than hyperplasia ( increase in cell number ) .
the first studies hinting towards postnatal cardiomyogenesis in the human heart can be traced back to the 1970s . by employing histopathological , biochemical and cytophotometric techniques ( to measure dna content and nuclear ploidy ) , adler et al .
they found that while normal hearts contained 2 billion cardiomyocytes , hearts with pathologic hypertrophy contained up to 4.8 billion cardiomyocytes .
these findings suggest that adult human hearts may generate new cardiomyocytes during pathologic hypertrophy . in 1998 ,
the anversa group examined explanted human hearts obtained from end - stage heart failure patients and from control subjects ( who died of non - cardiovascular causes ) .
fluorescent immunohistochemistry ( ihc ) , for sarcomeric proteins and propidium iodide ( pi , a dna dye which labels the cell nucleus ) , demonstrated the presence of cardiomyocytes undergoing mitosis ( either nuclear division [ karyokinesis ] or cell division [ cytokinesis ] ) in control hearts ( 14 mitotic myocytes / million myocytes ) . in end - stage heart failure hearts ,
the number of mitotic cardiomyocytes increased 10-fold ( 140 mitotic myocytes / million myocytes ) .
these rates of myocyte mitosis ( if they translate into genuine proliferation ) project to an annual turnover of 10% in the healthy adult human heart and 107% in the failing human heart .
fluorescent ihc for sarcomeric proteins , pi and ki67 ( a protein expressed in the nucleus of cells in the active phases of the cell cycle [ late g1 , s , g2 , and m phase ] ) in human hearts obtained from patients who died 412 days post myocardial infarction demonstrated ki67 expression in 4% of myocyte nuclei in the infarct border zone and in 1% of myocytes in the remote myocardium .
mitosis ( karyokinesis or cytokinesis ) was observed in 0.08% of myocytes ( 800 mitotic myocytes / million myocytes ) in the border zone and 0.03% of myocytes ( 300 mitotic myocytes / million myocytes ) in the remote myocardium . if such exceptionally high rates of myocyte cell cycling could be persisted and resulted in genuine proliferation ( while no significant myocyte loss occurred post - myocardial infarction beyond the initial ischemic insult ) , then all myocytes lost after an infarct affecting 30% of the left ventricle could be replaced within as little as 18 days .
more recently , the anversa group investigated the rates of myocyte turnover in the aging human heart .
fluorescent ihc for sarcomeric proteins , ki67 , phosphorylated histone h3 ( h3p , a marker of karyokinesis ) and aurora - b - kinase ( a marker of cytokinesis ) was performed in explanted hearts from human subjects who died of non - cardiovascular causes .
the investigators reported that myocyte turnover increases with age , and that female hearts possess a higher regenerative capacity compared to male hearts .
in the female heart , myocyte turnover occurs at an annual rate of 10% , 14% , and 40% at 20 , 60 , and 100 years of age , respectively .
the corresponding values in the male heart are 7% , 12% , and 32% per year .
the investigators calculated that from 20 to 100 years of age , the myocyte compartment is replaced 15 times in women and 11 times in men .
performed fluorescent immunocytochemistry for h3p in dissociated myocytes isolated from explanted hearts from human subjects who died of non - cardiovascular causes .
fluorescent immunohistochemistry ( in tissue sections ) for mitotic kinesin - like protein was employed for investigation of myocyte cytokinesis .
the investigators reported a decrease in myocyte turnover with age : annual myocyte turnover is 100% during the first year of life , decreases to 1.9% at 20 years of age and drops to 0.04% in subjects older than 40 years .
the investigators calculated that during the first two decades of life , the total number of myocytes in the left ventricle increases 3.4 fold .
however , it needs to be noted that while cardiomyocyte karyokinesis was detectable throughout life , no instances of myocyte cytokinesis were observed beyond 20 years of age .
while the aforementioned studies demonstrate that human myocytes can re - enter the cell cycle and may possess some ability to proliferate beyond the early postnatal period , the calculated turnover rates need to be interpreted with caution .
estimations of the total number of myocytes per heart ( as performed by adler et al . ) are complicated , require numerous assumptions and can be confounded by problematic discrimination of myocyte versus non myocyte nuclei ( which in those studies was performed based on nuclear size and morphology ) .
quantification of cardiomyocyte cycling with histology ( as performed by the anversa group ) is prone to sampling error and can be complicated by the fact that conventional histology has been shown to be problematic for identification of cardiomyocyte nuclei ( this will be discussed later ) .
finally , estimation of turnover rates based on exceedingly rare and brief ( mitosis in adult rat cardiomyocytes lasts 1.8  h in vitro ) events that may not represent instances of genuine myocyte division ( mitotic events involving in karyokinesis and myocyte multinucleation , but not cytokinesis and generation of daughter cells ) leaves significant room for error . ideally , slow processes ( like myocyte regeneration )
need to be quantified over time , rather than based on a single  snapshot  . to that end , a more reliable approach to study birth of new myocytes over prolonged periods of time is the  pulse - chase  approach . in 
pulse - chase  experiments cells ( in our case cardiac myocytes ) are exposed to a labeled compound (  pulse  ) , and then are followed for a period when the labeled compound is no longer administered (  chase  ) . however , while  pulse - chase  approaches can be readily implemented in experimental animals ( where pulsing is typically performed through administration of nucleoside analogues ) , extraordinary circumstances are required for this approach with humans .
examples of such extraordinary circumstances were the above - ground nuclear testing during the cold war , which resulted in the temporary release of large quantities of c into the atmosphere between 1955 and 1963 ( c  pulse  ) .
after the limited nuclear test ban treaty in 1963 , atmospheric c concentration dropped exponentially (  chase  ) , resulting in unique  pulse - chase  conditions . in a seminal study , bergmann et al . hypothesized that postnatally generated cardiomyocytes
the concentration of c in dna of myocytes can be used to retrospectively establish a date mark for when myocytes were born ( by identifying the year that atmospheric c levels were similar to c levels in myocyte dna ) .
this can be achieved since : a ) c levels in the human body reflect those of the atmospheric air ( c reacts with oxygen in the atmosphere to form co2 , which then enters the biotope through photosynthesis ) ; and b ) dna remains stable following the last cell division . by measuring c concentration ( with accelerator mass spectrometry ) in dna extracted from fluorescence - activated - sorted myocyte ( troponin positive ) nuclei isolated from explanted hearts ( 12 hearts total , 10 from subjects who died of non - cardiovascular causes and 2 from patients who died due to an acute myocardial infarction ) , bergmann et al
mathematical modeling demonstrated that postnatal cardiomyogenesis decreases with age : cardiomyocyte turnover is 1%/year at the age of 25 and gradually decreases at 0.45%/year at the age of 75 , resulting in an exchange of 45% of myocytes during a 50-year span . a more recent study by the anversa group employed similar methods but yielded strikingly different results .
performed carbon dating with accelerator mass spectrometry in dna extracted from myocytes obtained from 19 healthy hearts and 17 hearts with cardiomyopathy .
it should be noted that in this study myocytes were isolated based on density centrifugation , rather than fluorescence - activated cells sorting of troponin positive nuclei ( as in the study by bergmann et al . ) .
the investigators found that the healthy adult human heart replaces its entire myocyte compartment 8 times between 20 and 78 years of age .
somewhat unexpectedly , the turnover rate of myocytes was found to be similar to that of endothelial cells and cardiac fibroblasts .
it has been argued that staining of myocyte nuclei for troponin ( as performed by bergmann et al . ) only identifies nuclei of senescent myocytes ; this could result in significant underestimation of myocyte turnover .
another unique circumstance allowing for implementation of a  pulse - chase  approach to measure myocyte turnover in the human heart is the use of radiosensitizing nucleoside analogues for therapeutic purposes in cancer patients .
nucleoside analogues are incorporated into newly - synthesized dna of cycling cells and can therefore serve as markers of dna synthesis .
investigated incorportation of the thymidine analogue iododeoxyuridine ( idu ) into dna of myocytes by fluorescent immunohistochemistry in hearts explanted from 8 cancer patients that had previously ( 8 days  4 years before death and heart explantation ) received infusions of the radiosensitizing agent .
the investigators found idu labeling ( i.e. dna synthesis ) in 2.546% of myocyte nuclei , which projects to an annual myocyte turnover of 22% .
concerns have been raised that such high turnover rates are difficult to reconcile with high labeling indices of idu after long chase periods : rapid cell turnover would presumably translate into death of a significant portion of idu - labeled myocytes as well as into substantial dilution of idu ( to undetectable levels ) due to rapid cell proliferation during the chase period ; both processes would result in significantly lower myocyte labeling indices of idu at the time of death .
a perhaps more logical explanation for the high rates of idu myocyte labeling in that study is that a substantial portion of the measured dna synthesis may represent instances of abortive cell - cyle re - entry , resulting in polypolidization or binucleation , rather than genuine myocyte proliferation ( more on that later ) .
while the investigators attempted to rule out polyploidization as a significant confounding factor ( they surprisingly reported that > 80% of human myocyte nuclei are diploid , in contrast to several other studies suggesting that the majority is polyploidy ) , no effort was undertaken to quantify to the potential contribution of bi / multinucleation to the measured rates of dna synthesis . the estimated rates of myocyte turnover measured in adult mammalian hearts ( human and mouse ) are depicted schematically in figure 1 .
taken together , even though reported rates of myocyte turnover vary wildly ( by more than 1 order of magnitude ) , the aforementioned studies convincingly establish that the human heart can generate new myocytes beyond the early postnatal period .
however , studies in human subjects can not provide insight into the cellular origins of postnatal mammalian cardiomyogenesis : do newly - generated myocytes arise from division of pre - existing myocytes or from cardiomyogenic differentiation of endogenous progenitors ?
a landmark study by soonpaa and field in 1998 demonstrated convincingly that preformed cardiomyocytes can actively cycle in the adult mouse heart . 
pulse - chase  experiments were performed in transgenic mice , in which a nuclear - localized -galactosidase reporter gene was expressed in cardiomyocytes ( driven by the -myosin heavy chain [ mhc ] promoter ) .
adult mice received injections of h - thymidine and were sacrificed 4  h after the last injection .
heart sections were processed for x - gal reaction ( to identify cardiomyocyte nuclei ) and autoradiography ( to identify incorporation of h - thymidine into the dna , i.e. dna synthesis ) .
normal adult hearts had a myocyte labeling index of 0.0006% ( 1/180000 cardiomyocyte nuclei had incorporated h - thymidine ) .
this projects to an annual myocyte turnover of 1% ( if dna synthesis is accompanied by genuine cell division ) .
after myocardial injury ( focal cauterization ) , myocyte cycling in the border zone increased 14-fold ( labeling index of 0.0083% [ 3/36000 cardiomyocyte nuclei had incorporated h - thymidine ] ) . with regard to the cellular origins of cycling cardiomyocytes ,
identification of morphologically mature cardiomyocytes that have synthesized their dna within 4  h after a single injection of a nucleoside analogue ) can only be attributed to dna synthesis in pre - existing myocytes ( as differentiation of progenitors to mature myocytes would take longer ) .
more recently , an elaborate study by the lee group investigated the contribution of resident myocyte proliferation to postnatal cardiomyogenesis by a combination of genetic fate - mapping , stable isotope labelling and multi - isotope imaging mass spectrometry .
the investigators employed an inducible fate mapping approach utilizing bitransgenic mhc- mercremer / zeg mice . in these mice ,
induction of cre recombinase activity ( driven by mhc promoter ) by 4-oh - tamoxifen results in efficient ( 80% ) permanent genetic labeling of myocytes ( and their progeny ) by green fluorescent protein ( gfp ) . 4-oh - tamoxifen - pulsed adult , healthy bitransgenic mice underwent pulsing with n - thymidine over a 10-week period .
multi - isotope imaging mass spectrometry ( which has a resolution capacity of <  1 m and thus can readily distinguish cardiomyocyte nuclei from adjacent non - cardiomyocyte nuclei ) in sections from explanted hearts revealed a n myocyte labeling index of 0.8% over 10 weeks ; this projects to an annual rate of myocyte dna replication of 4.4% .
the investigators undertook extensive efforts to calculate the contribution of abortive cell cycle re - entry ( resulting in polyploidization and/or multinucleation , rather than genuine cell division ) to the measured dna synthesis .
perhaps not surprisingly , they found that the majority of dna synthesis occurred in polyploid and/or multinucleated myocytes .
however 17% of the measured dna synthesis could not be explained away by these confounding factors , leaving generation of new myocytes as the only likely explanation and suggesting an annual myocyte turnover of 0.74% in the healthy adult mouse heart . with regard to the cellular origins of postnatal cardiomyogenesis , n+ cardiomyocytes expressed gfp at a similar frequency as n- myocytes , suggesting that in the normal heart new myocytes are generated mainly through proliferation of pre - existing myocytes , rather than differentiation of endogenous progenitors .
similar experiments performed in adult mice post - myocardial infarction showed a 20-fold increase in myocyte division in the infarct border zone during the first 8 weeks following injury , which was also attributed to proliferation of pre - existing myocytes rather than contributions of endogenous progenitors to the myocyte pool . in a parallel study at the marbn laboratory , we attempted to quantify postnatal cardiomyogenesis and trace its cellular origins using a combination of genetic fate mapping with long - term pulsing with the nucleoside analogue bromodeoxyuridine ( brdu ) .
healthy adult bitransgenic mhc - mercremer / zeg mice were pulsed with 4-oh - tamoxifen to genetically label pre - existing cardiomyocytes with gfp .
figure 2 depicts two genetically labeled pre - existing cardiomyocytes ( pseudocolored in green ) that have incorporated brdu ( pseudocolored in white ) .
measurement of dna synthesis ( brdu incorporation ) in pre - exisiting ( gfp+ ) cardiomyocytes by different methods ( flow cytometry of whole cells , flow cytometry of isolated myocyte nuclei , fluorescent immunocytochemistry of dissociated cardiomyocytes and fluorescent immuhistochemistry in cardiac sections ) demonstrated that 0.4% of pre - existing ( gfp+ ) myocytes synthesized dna during 5 weeks of brdu pulsing , suggesting an annual rate of dna replication in resident myocytes of 4% .
actively cycling cardiomyocytes were smaller and more - often mononucleated compared to non - cycling myocytes .
quantification of the contributions of polyploidization and multinucleation to the measured rates of brdu incorporation demonstrated that abortive cell - cycle re - entry without genuine cell division could explain 69% of the observed dna synthesis ; thus the calculated annual rate of myocyte turnover due to cardiomyocyte proliferation in the adult healthy mouse heart was 1.3% .
myocardial infarction resulted in a 2-fold increase in the total number of proliferating myocytes in the left ventricle during the first 5 weeks post - injury ; this increase was attributed to an upregulation in proliferation of pre - existing myocytes in the border zone ( 10 fold compared to normal heart ) , but not in the remote myocardium .
taken together , the aforementioned studies suggest that mammalian myocytes retain a limited but measurable capacity to proliferate in the healthy adult heart , and that myocyte proliferation increases modestly in the border zone following myocardial infarction .
during the past decade several studies supported the notion that the adult mammalian heart contains its own reservoir of stem cells .
numerous populations of putative adult endogenous cardiomyocyte progenitors have been proposed ( including c - kit cells , sca-1 cells , side population cells , cardiosphere - forming cells , ssea-1 cells , pdgfr cells and neural - crest derived cells ) largely based on expression of surface markers or functional properties that have been used to mark progenitors in other organs .
the high number of distinct populations of putative endogenous myocyte progenitors is difficult to reconcile with the limited regenerative capacity of the adult mammalian heart .
importantly , while several cell types have been shown to express cardiac proteins in vitro or after delivery into recipient hearts following ex vivo expansion , their physiologic importance and contribution to cardiomyocyte replenishment in the normal or injured adult heart remains controversial .
genetic fate mapping is a powerful tool that enables study of cardiac regeneration in vivo .
perhaps the most compelling evidence indicating postnatal contribution of endogenous progenitors to the adult myocyte pool comes from a landmark study performed at the lee laboratory . using mhc - mercremer / zeg adult bitransgenic mice , hsieh et al .
achieved inducible genetic labeling of 80% of preexisting myocytes with gfp , without any detectable labeling of progenitor - like cells ( which in their inactive state presumably do not express mhc ) . in the normal heart ,
the percentage of gfp+ myocytes remained unchanged over 1 year of follow - up , indicating that progenitor cells do not contribute significantly to myocyte renewal during normal aging .
in contrast , when mice were subjected to myocardial infarction the percentage of gfp+ cardiomyocytes decreased from 83% to 68% in the border zone and to 77% in the remote myocardium over a 3-month follow - up period
. following pressure - overload , the percentage of gfp+ myocytes decreased from 83% to 76% over 3 months .
these results indicate that post - cardiac injury , unlabeled progenitors undergo cardiomyogenic differentiation , resulting in dilution of gfp+ myocytes by gfp     myocytes ( generated from unlabeled precursors ) ( figure 3a ) .
alternatively , the observed dilution of the labeled myocyte pool could be attributed to intrinsic differences between gfp+ and gfp- myocytes , i.e. increased proliferative capacity of gfp- myocytes post - injury or increased susceptibility of gfp+ myocytes to injury ( even though the latter hypothesis has not been shown to occur ) .
brdu pulsing revealed increased incorporation of brdu in gfp- myocytes ( compared to gfp+ myocytes ) post - injury , a finding also compatible with generation of new myocytes from progenitors .
based on these findings , the contribution of endogenous precursors to the myocyte pool post - injury appears to be quite substantial : over a 3-month period , 15% of myocytes in the border zone and 6% of all myocytes in the left ventricle arise from cardiomyogenic differentiation of progenitors .
the results of this study ( particularly the increased brdu labeling of gfp- myocytes ) do not fully agree with a more recent study from the same group ( described earlier ) , in which no increased n incorporation into gfp     myocytes could be detected by multi - isotope imaging mass spectrometry , suggesting no significant contribution of endogenous precursors to the myocyte pool post - injury .
this discrepancy may be a result of the very small number of mononucleated / diploid n+ myocytes ( 16 n+ myocytes/ 7063 myocytes analyzed ) detected in injured hearts by multi - isotope imaging mass spectrometry ( an extremely time - consuming method that precludes analysis of large amounts of tissue ) ; this number may be too small to detect differences in the rate of n incorporation in gfp+ and gfp     myocytes . at the marbn lab
after inducible genetic labeling of cardiomyocytes by gfp , adult bitrangenic mhc - mercremer / zeg mice received daily brdu injections for 5 weeks .
comparison of brdu incorporation in gfp+ and gfp     cardiomyocytes ( by flow cytometry , immunocytochemistry and histology ) revealed similar rates of brdu labeling in both myocyte subsets in healthy mice .
these findings indicate that in the normal adult mouse heart , myocyte turnover occurs predominantly through proliferation of resident myocytes , without any measurable progenitor - mediated myocyte formation . however , post - myocardial infarction we could detect a contribution of endogenous precursors to the myocyte pool ( 1% of myocytes in the left ventricle arose from progenitor cell differentiation over a 5-week period post - injury ) ( figure 4 ) . a different technique ( viral gene tagging ) to study endogenous cardiac regeneration was employed by the anversa group .
injected lentiviruses expressing gfp in the atria and ventricular apex ( presumably sites of cardiac stem cell niches ) of adult mice .
one to 5 months later , nested polymerase chain reaction revealed common viral integration sites in c - kit+ cells , cardiomyocytes , endothelial cells and fibroblasts isolated from the infected hearts .
since lentiviruses are semi - randomly integrated in the host genome of infected cells , these results suggest that postnatally - generated cardiomyocytes , endothelial cells and fibroblasts in the healthy adult mouse heart derive from clonal activation of endogenous stem cells .
six months after viral injection , 25% of myocytes in the mid - portion of the left ventricle ( a myocardial region away from the sites of viral injections ) were gfp+ , presumably arising from migration of gfp+ infected progenitor cells to that region and subsequent differentiation .
these results indicate a remarkable capacity of the adult mouse heart for stem - cell mediated cardiomyocyte replenishment .
however , it needs to be emphasized that viral gene tagging can not reveal the identity of the parental stem cell .
the fact that c - kit cells shared similar viral integration sites as cardiomyocytes , endothelial cells and fibroblasts could be a result of viral infection of a yet unknown progenitor that gives rise to all four celltypes .
fate mapping of resident myocytes or viral gene tagging can not reveal the identity of endogenous progenitors , as they indirectly capture the net result of their activation and differentiation .
this can only be performed through forward fate mapping experiments in which endogenous progenitors ( but not pre - existing myocytes ) are genetically labeled in a prospective manner , enabling direct visualization of their future contributions to the myocyte pool ( figure 3b ) .
such an approach was undertaken at the fukuda lab in order to investigate the contribution of neural - crest derived cells to postnatal cardiomyogenesis .
tamura et al . used bitransgenic mice in which activation of cre - recombinase ( under the control of protein-0 promoter ) induces gfp expression , resulting in genetic labeling of neural - crest derived cells by gfp . in the healthy heart , neural - crest derived ( gfp+ ) cardiomyocytes were undetectable during the first week after birth but appeared at 2 weeks postnatally and increased in number thereafter ; however , their absolute contributions to the myocyte pool were minimal ( 0.3% gfp+ cardiomyocytes in the septum , <  0.1% gfp+ cardiomyocytes in the rest of the left ventricle ) .
after myocardial infarction , small gfp+ cardiomyocytes ( presumably arising from differentiation of neural - crest - derived cells ) first appeared in the border zone 2 weeks post - injury and gradually increased in number thereafter ( comprising 3% of total cardiomyocytes in the border zone at 12 weeks post - injury ) .
while this study is limited by the fact that the activity of cre - recombinase was not temporally controlled in an inducible manner ( and thus spontaneous activation of the protein-0 promoter in resident cardiomyocytes would result in gfp labeling ) it suggests that progenitor cells may contribute to generation of new myocytes ( especially post - injury ) . using a similar forward fate mapping approach , ellison et
al recently reported that endogenous c - kit+ cardiac cells are a source of newly formed cardiomyocytes , after myocardial injury , in adult rodents .
the investigators used a model of a single , high isoproterenol dose that produces severe , albeit spontaneously resolving , myocardial injury .
endogenous c - kit+ cardiac cells were genetically labeled by exogenous administration of a lentivirus expressing cre - recombinase . under the control of a c - kit promoter , in hearts of ryp reporter mice . in this mouse model ,
expression of cre - recombinase in c - kit+ cell results in genetic labeling of infected c - kit+ cells with yellow fluorescent protein ( yfp ) .
after isoproterenol injury , a significant fraction of cardiomyocytes were yfp+ , indicating that they comprise newly generated cardiomyocytes arising from cardiomyogenic differentiation of infected c - kit+ cells .
while this study suggests an important role of progenitor cell - mediated cardiomyogenesis post - injury , it is in contrast with previous studies , reporting that adult c - kit+ cells have only angiogenic but not cardiomyogenic potential .
in addition , the implemented spontaneously - resolving model of heart failure may not be physiologically relevant .
finally , it needs to be emphasized that c - kit is not a specific marker of stem cells , but it is rather expressed in a variety of cells ( e.g. mast cells ) and more importantly in myocytes themselves during both proliferation and differentiation .
thus , the precise expression pattern of c - kit in forward fate mapping models has to be delineated at baseline prior to temporal assessment of possible progeny , before concrete conclusions can be reached . currently , several studies using forward fate - mapping approaches for putative markers of endogenous progenitors ( including c - kit , sca-1 and nkx2 - 5 ) are ongoing in multiple labs . taken together , the aforementioned studies suggest that endogenous progenitors may generate new myocytes in the adult heart .
while their role in postnatal cardiomyogeneis during normal ageing appears to be limited ( as most studies concur that myocyte turnover in the healthy heart most likely occurs through proliferation of pre - existing myocytes ) , multiple studies indicate progenitor cell - mediated cardiomyocyte renewal occurs following myocardial injury .
it needs to be emphasized that the two proposed mechanisms of myocyte turnover ( cardiomyocyte proliferation and differentiation of endogenous progenitors ) are not mutually exclusive .
the adult mammalian liver has an impressive capacity to regenerate ; following acute 70% partial hepatectomy , the adult liver can fully regenerate within days ( in rodents ) to weeks ( in humans ) , through division of mature hepatocytes and cholangiocytes , which re - enter the cell cycle and divide .
however , during chronic liver injuries , hepatic progenitor cells also become activated and contribute to liver regeneration .
in addition , inherent characteristics of cardiac tissue architecture and cardiomyocyte biology further complicate quantification of postnatal cardiomyogenesis and tracing of its cellular origins in the mammalian heart .
below , we review the major challenges when studying endogenous postnatal cardiac regeneration . first , since postnatal cardiomyogenesis in the mammalian heart most likely occurs at very low levels ,  pulse - chasing  approaches with long pulsing periods are preferable in experimental animals , as they maximize the chance of capturing rare events of myocyte proliferation
. however , long - term administration of radiolabeled thymidine or halogenated nucleoside analogues ( like brdu ) may be toxic and could affect the cycling rates of cardiomyocytes .
nevertheless , we did not observe any differences in the rates of actively - cycling ( ki67 + ) cardiomyocytes in mice that received brdu for up to 5 weeks compared to mice that did not receive brdu .
second , the use of conventional histology ( which typically employs confocal microscopy for analysis of cardiac sections stained for sarcomeric proteins , dna [ for nuclear identification ] and [ in some cases ] cellular borders ) to identify cardiomyocyte nuclei is problematic .
ang et al . performed a careful study to investigate the fidelity of conventional myocyte nuclear identification using confocal microscopy .
a transgenic mouse in which cardiomyocyte nuclei are genetically labelled ( by a nuclear - localized -galactosidase reporter gene driven by the mhc promoter ) was used as the gold - standard .
the investigators demonstrated that conventional histological approaches typically misidentify myocyte nuclei 10% of the time , thus significantly compromising accurate quantification of rare events ( like active cycling of myocytes ) .
the limitations of conventional microscopy can be overcome by use of transgenic lineage reporters that mark cardiomyocyte nuclei , implementation of approaches with powerful resolution capacity ( e.g. multi - isotope imaging mass spectrometry ) , or analysis of enzymatically dissociated cardiomyocytes .
third , it needs to be emphasized that dna incorporation of nucleoside analogues or nuclear expression of cell - cycle proteins , while demonstrating cell - cycle activity , does not necessarily translate into genuine cell division and proliferation ; important confounding factors that need to be accounted for include polyploidization ( dna proliferation without karyokinesis and cytokinesis ) and bi / multinucleation ( dna proliferation and karyokinesis without cytokinesis ) , cell fusion and dna repair ( figure 5 ) . while cell fusion and dna damage and repair are exceptionally rare events that can not account for the magnitude of measured turnover rates , polypolidization and bi / multinucleation are particularly relevant when studying cardiomyogenesis , as abortive cell - cycle re - entry is quite prevalent in myocytes ( most myocytes in the mouse heart are diploid and binucleated , while most myocytes in the human heart are polyploid and mononucleated ) .
we and others have found that the majority of the measured myocyte cell - cycle activity in the normal or infarcted mouse heart can be attributed to polyploidization and binucleation , rather than true myocyte division .
cardiomyocyte cytokinesis is a brief ( the m phase occupies 2% of the cell cycle ) and rare event , and detection of the cleavage furrow between diving myocytes ( a hallmark of cytokinesis ) is complicated by the compact myocardial architecture as well as by division of adjacent non - myocyte cells ( which actively cycle at substantially higher rates compared to myocytes ) ; thus myocyte division is difficult to visualize convincingly and quantify accurately .
a more viable strategy may be to quantify the magnitude of abortive cell - cycle re - entry and subtract it from the total measured dna synthesis . to that end ,
measurements of ploidy levels ( by flow cytometry for dna content or by visualization of the number of sex chromosomes by fluorescence is situ hybridization ) and number of nuclei ( preferably by analysis of dissociated myocytes ) should be performed in cardiomyocytes that have incorporated nucleoside analogues . finally , while genetic fate mapping is the only reliable tool that can be used to trace the cellular origins of organ regeneration in vivo , its implementation in studies of postnatal cardiomyogenesis does not come without problems .
while several groups have used the mhc - mercremer / zeg bitrasngenic mouse for inducible marking of pre - existing myocytes , multiple studies have reported activity of the -mhc promoter in non - myocyte cells that possess characteristics compatible with endogenous cardiac progenitors . in addition
, development of new lineage - tracing models that could allow for forward fate mapping of cardiac progenitors ( in order to prospectively investigate their contributions to the myocyte pool ) is complicated by lack of specific cardiac stem cell markers and by potential re - expression of such markers in cardiomyocytes during stress - induced activation of a fetal - gene program ( figure 3 ) .
there is now ample and compelling evidence that cardiomyogenesis does occur in the adult mammalian heart , albeit at an insufficient rate to restore cardiac function after substantial cell losses such the ones that occur after a myocardial infarction .
proliferation of pre - existing cardiomyocytes appears as the dominant mechanism of generation of novel cardiomyocytes , at least during normal ageing , although after myocardial injury , differentiation of progenitor cells may also contribute to this phenomenon .
cell therapies have already reached the stage of clinical trials ; most of the cells currently under investigation ( with the exception of embryonic stem cells and induced pluripotent stem cells ) do not differentiate into functional cardiomyocytes and act through paracrine activation of endogenous reparative and regenerative pathways .
recently , insight has been provided to the mechanisms that control exit and re - entry of cardiomyocytes to the cardiac cycle , together with ways to manipulate the potential of cardiomyocytes for division and proliferation .
it is reasonable to expect that therapeutic amplification of endogenous regenerative mechanisms will bolster cardiomyocyte repopulation of injured myocardium and will result in effective therapies for cardiovascular disease and heart failure , in the not so remote future .
 in recent years , several landmark studies have provided compelling evidence that cardiomyogenesis occurs in the adult mammalian heart . 
 however , the rate of new cardiomyocyte formation is inadequate for complete restoration of the normal mass of myocardial tissue , should a significant myocardial injury occur , such as myocardial infarction . 
 the cellular origin of postnatal cardiomyogenesis in mammals remains a controversial issue and two mechanisms seem to be participating , proliferation of pre - existing cardiomyocytes and myogenic differentiation of progenitor cells . 
 we will discuss the relative importance of these two processes in different settings , such as normal ageing and post - myocardial injury , as well as the strengths and limitations of the existing experimental methodologies used in the relevant studies . 
 further clarification of the mechanisms underlying cardiomyogenesis in mammals will open the way for their therapeutic exploitation in the clinical field , with the scope of myocardial regeneration . 