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Features of cardiomyocyte proliferation and its potential for cardiac regeneration: Stem Cells Review Series
Journal of Cellular and Molecular Medicine
Abstract
The human heart does not regenerate. Instead, following injury, human hearts scar. The loss of contractile tissue contributes significantly to morbidity and mortality. In contrast to humans, zebrafish and newts faithfully regenerate their hearts. Interestingly, regeneration is in both cases based on cardiomyocyte proliferation. In addition, mammalian cardiomyocytes proliferate during foetal development. Their proliferation reaches its maximum around chamber formation, stops shortly after birth, and subsequent heart growth is mostly achieved by an increase in cardiomyocyte size (hypertrophy). The underlying mechanisms that regulate cell cycle arrest and the switch from proliferation to hypertrophy are unclear. In this review, we highlight features of dividing cardiomyocytes, summarize the attempts to induce mammalian cardiomyocyte proliferation, critically discuss methods commonly used for its detection, and explore the potential and problems of inducing cardiomyocyte proliferation to improve function in diseased hearts.
... MI is defined as an area that has reduced or absent oxygen perfusion, which can cause the initiation of necrosis, apoptosis and autophagy leading to death of myocardial cells [2][3][4]. It is generally accepted that the mammalian heart cannot regenerate after ischemia or injury [5]. Thus, the compensatory mechanism after ischemia or injury, are assumed to favor fibrotic healing and hypertrophy instead of regeneration and cardiomyocyte proliferation [2,5]. ...
... It is generally accepted that the mammalian heart cannot regenerate after ischemia or injury [5]. Thus, the compensatory mechanism after ischemia or injury, are assumed to favor fibrotic healing and hypertrophy instead of regeneration and cardiomyocyte proliferation [2,5]. Unfortunately, fibrotic healing may result in loss of heart contractility, leading to various complications and symptoms such as arrhythmias that also contribute to an increase in morbidity and mortality [5]. ...
... Thus, the compensatory mechanism after ischemia or injury, are assumed to favor fibrotic healing and hypertrophy instead of regeneration and cardiomyocyte proliferation [2,5]. Unfortunately, fibrotic healing may result in loss of heart contractility, leading to various complications and symptoms such as arrhythmias that also contribute to an increase in morbidity and mortality [5]. Today, only heart transplantation and palliative medication is offered as treatment of MI. ...
Myocardial infarction (MI) is a worldwide condition that affects millions of people. This is mainly caused by the adult human heart lacking the ability to regenerate upon injury, whereas zebrafish have the capacity through cardiomyocyte proliferation to fully regenerate the heart following injury such as apex resection (AR). But a systematic overview of the methods used to evidence heart regrowth and regeneration in the zebrafish is lacking. Herein, we conducted a systematical search in Embase and Pubmed for studies on heart regeneration in the zebrafish following injury and identified 47 AR studies meeting the inclusion criteria. Overall, three different methods were used to assess heart regeneration in zebrafish AR hearts. 45 out of 47 studies performed qualitative (37) and quantitative (8) histology, whereas immunohistochemistry for various cell cycle markers combined with cardiomyocyte specific proteins was used in 34 out of 47 studies to determine cardiomyocyte proliferation qualitatively (6 studies) or quantitatively (28 studies). For both methods, analysis was based on selected heart sections and not the whole heart, which may bias interpretations. Likewise, interstudy comparison of reported cardiomyocyte proliferation indexes seems complicated by distinct study designs and reporting manners. Finally, six studies performed functional analysis to determine heart function, a hallmark of human heart injury after MI. In conclusion, our data implies that future studies should consider more quantitative methods eventually taking the 3D of the zebrafish heart into consideration when evidencing myocardial regrowth after AR. Furthermore, standardized guidelines for reporting cardiomyocyte proliferation and sham surgery details may be considered to enable inter study comparisons and robustly determine the effect of given genes on the process of heart regeneration.
... Subsequent "ballooning" of the chambers and growth of the heart are achieved by proliferation of contracting cardiomyocytes (109). After birth, mammalian cardiomyocytes exit the cell cycle and stop proliferating ( Fig. 1) (107,115). ...
... Cardiomyocyte cell cycle activity does not necessarily equate with proliferation as it can also reflect pathological hypertrophy, polyploidization, or polynucleation (115,129). Therefore, it is important to be careful with the interpretation of data from cell cycle assays (Table 1). Misinterpretation of cell cycle assays might provide an explanation for the controversies in the field of cardiac regeneration, as to the extent of cardiomyocyte proliferation. ...
... However, DNA synthesis occurs not only during semiconservative DNA replication during S phase but also during DNA repair. Moreover, semiconservative DNA replication is merely an indicator of S-phase cell cycle progression (115,129). Thus, a disadvantage of this assay is that it does not predict whether a cell will divide or undergo G2/M arrest, polyploidization, or polynucleation. Pulse-chase experiments permit the identification of colony formation and thus, by deduction, cell division, as the label will be reduced by 50% per each cell division. ...
The newt and the zebrafish have the ability to regenerate many of their tissues and organs including the heart. Thus, a major goal in experimental medicine is to elucidate the molecular mechanisms underlying the regenerative capacity of these species. A wide variety of experiments have demonstrated that naturally occurring heart regeneration relies on cardiomyocyte proliferation. Thus, major efforts have been invested to induce proliferation of mammalian cardiomyocytes in order to improve cardiac function after injury or to protect the heart from further functional deterioration. In this review, we describe and analyze methods currently used to evaluate cardiomyocyte proliferation. In addition, we summarize the literature on naturally occurring heart regeneration. Our analysis highlights that newt and zebrafish heart regeneration relies on factors that are utilized for cardiomyocyte proliferation during mammalian fetal development. Most of these factors have, however, failed to induce adult mammalian cardiomyocyte proliferation. Finally, our analysis of mammalian neonatal heart regeneration indicates experiments that could resolve conflicting results in the literature, such as binucleation assays and clonal analysis. Collectively, cardiac regeneration based on cardiomyocyte proliferation is a promising approach to improve adult human cardiac function after injury. Yet, it is important to elucidate the mechanisms arresting mammalian cardiomyocyte proliferation after birth and to utilize better assays to determine formation of new muscle mass.
Copyright © 2015, American Journal of Physiology - Heart and Circulatory Physiology.
... Still, the putative causal relationship between cell cycle exit and nuclear envelope MTOC formation is unclear. Notably, while ncMTOC formation and cell cycle exit occur prior to contraction in skeletal muscle, fetal cardiomyocytes contract and at the same time exhibit a centrosomal MTOC and proliferate [4,245]. ...
... Considering that cardiovascular diseases are among the leading causes of death worldwide, research in recent years was primarily focussed on inducing cardiomyocyte cell cycle re-entry to increase muscle mass [249]. Despite the importance of understanding the mechanisms underlying the postmitotic state of mammalian cardiomyocytes for heart regenerative approaches, few studies have tried to unveil these mechanisms [245,250,251]. Zebrowski and colleagues reported that postnatal cardiomyocytes not only establish a nuclear envelope MTOC but also lose centrosome integrity resulting in centriole splitting [4]. ...
Distinctly organized microtubule networks contribute to the function of differentiated cell types such as neurons, epithelial cells, skeletal myotubes, and cardiomyocytes. In striated (i.e. skeletal and cardiac) muscle cells, the nuclear envelope acts as the dominant microtubule-organizing center (MTOC) and the function of the centrosome—the canonical MTOC of mammalian cells—is attenuated, a common feature of differentiated cell types. We summarize the mechanisms known to underlie MTOC formation at the nuclear envelope, discuss the significance of the nuclear envelope MTOC for muscle function and cell cycle progression, and outline potential mechanisms of centrosome attenuation.
... To assess cardiomyocyte renewal, investigators performed colorimetric assays and immunostaining for proliferation markers on heart tissue sections. One major challenge is that cardiomyocytes that undergo bi-/polynucleation or endoreduplication stain positive for commonly used proliferation markers such as Ki67 or phospho-Histon3 in the absence of true proliferation [94]. This limitation can be overcome by utilizing elegant transgenic mouse models, which can unequivocally identify cardiomyocytes that underwent cell division [81,85]. ...
Myocardial injury often leads to heart failure due to the loss and insufficient regeneration of resident cardiomyocytes. The low regenerative potential of the mammalian heart is one of the main drivers of heart failure progression, especially after myocardial infarction accompanied by large contractile muscle loss. Preclinical therapies for cardiac regeneration are promising, but clinically still missing. Mammalian models represent an excellent translational in vivo platform to test drugs and treatments for the promotion of cardiac regeneration. Particularly, short-lived mice offer the possibility to monitor the outcome of such treatments throughout the life span. Importantly, there is a short period of time in newborn mice in which the heart retains full regenerative capacity after cardiac injury, which potentially also holds true for the neonatal human heart. Thus, in vivo neonatal mouse models of cardiac injury are crucial to gain insights into the molecular mechanisms underlying the cardiac regenerative processes and to devise novel therapeutic strategies for the treatment of diseased adult hearts. Here, we provide an overview of the established injury models to study cardiac regeneration. We summarize pioneering studies that demonstrate the potential of using neonatal cardiac injury models to identify factors that may stimulate heart regeneration by inducing endogenous cardiomyocyte proliferation in the adult heart. To conclude, we briefly summarize studies in large animal models and the insights gained in humans, which may pave the way toward the development of novel approaches in regenerative medicine.
... Significant morphological changes occur in the myocardium, characterized by a decrease in the proliferative activity of CMCs, an increase in their ploidy, size, and differentiation [19,. CMCs go through the last cell cycle without completing cytokinesis, which leads to an increase in the number of binucleated terminally differentiated CMCs at the G 0 [28,29,[34][35][36][52][53][54]. ...
The myocardium of children with tetralogy of Fallot (TF) undergoes hemodynamic overload and hypoxemia immediately after birth. Comparative analysis of changes in the ploidy and morphology of the right ventricular cardiomyocytes in children with TF in the first years of life demonstrated their significant increase compared with the control group. In children with TF, there was a predominantly diffuse distribution of Connexin43-containing gap junctions over the cardiomyocytes sarcolemma, which redistributed into the intercalated discs as cardiomyocytes differentiation increased. The number of Ki67-positive cardiomyocytes varied greatly and amounted to 7.0–1025.5/106 cardiomyocytes and also were decreased with increased myocytes differentiation. Ultrastructural signs of immaturity and proliferative activity of cardiomyocytes in children with TF were demonstrated. The proportion of interstitial tissue did not differ significantly from the control group. The myocardium of children with TF under six months of age was most sensitive to hypoxemia, it was manifested by a delay in the intercalated discs and myofibril assembly and the appearance of ultrastructural signs of dystrophic changes in the cardiomyocytes. Thus, the acceleration of ontogenetic growth and differentiation of the cardiomyocytes, but not the reactivation of their proliferation, was an adaptation of the immature myocardium of children with TF to hemodynamic overload and hypoxemia.
... Considering that Pcnt S is upregulated when cardiomyocyte cell cycle progression is arrested, we wondered whether Pcnt S overexpression inhibits cell cycle progression. As cardiomyocyte proliferation cannot be maintained in fetal cardiomyocytes and postnatal cardiomyocytes quickly establish a nuclear MTOC and cell cycle arrest [17,32], we overexpressed Pcnt S in proliferating C2C12 myoblasts. C2C12 myoblasts, like cardiomyocytes, establish a nuclear MTOC during differentiation [33]. ...
Induction of cardiomyocyte proliferation is a promising option to regenerate the heart. Thus, it is important to elucidate mechanisms that contribute to the cell cycle arrest of mammalian cardiomyocytes. Here, we assessed the contribution of the pericentrin (Pcnt) S isoform to cell cycle arrest in postnatal cardiomyocytes. Immunofluorescence staining of Pcnt isoforms combined with SiRNA-mediated depletion indicates that Pcnt S preferentially localizes to the nuclear envelope, while the Pcnt B isoform is enriched at centrosomes. This is further supported by the localization of ectopically expressed FLAG-tagged Pcnt S and Pcnt B in postnatal cardiomyocytes. Analysis of centriole configuration upon Pcnt depletion revealed that Pcnt B but not Pcnt S is required for centriole cohesion. Importantly, ectopic expression of Pcnt S induced centriole splitting in a heterologous system, ARPE-19 cells, and was sufficient to impair DNA synthesis in C2C12 myoblasts. Moreover, Pcnt S depletion enhanced serum-induced cell cycle re-entry in postnatal cardiomyocytes. Analysis of mitosis, binucleation rate, and cell number suggests that Pcnt S depletion enhances serum-induced progression of postnatal cardiomyocytes through the cell cycle resulting in cell division. Collectively, our data indicate that alternative splicing of Pcnt contributes to the establishment of cardiomyocyte cell cycle arrest shortly after birth.
... When comparing RhoA and Ki-67 expression, some discrepancies can be observed, especially in unstimulated CMC (Hyp-CMC), which show a high Ki-67 expression after 24 and 48 h. This may be attributed due to higher endoreduplication, as it has been described that CMC without any stimulation do not significantly replicate after ischemia (Van Amerongen and Engel, 2008). When reviewing expression changes of HIF-1α, Ki-67, and RhoA and the proliferation assay, the general pro-survival and proliferation inducing effect of MSC secretome can be concluded. ...
Introduction
Despite major leaps in regenerative medicine, the regeneration of cardiomyocytes after ischemic conditions remains to elucidate. It is crucial to understand hypoxia induced cellular mechanisms to provide advanced treatment options, including the use of stem cell paracrine factors for myocardial regeneration.
Materials and Methods
In this study, the regenerative potential of hypoxic human cardiomyocytes (group Hyp-CMC) in vitro was evaluated when co-cultured with human bone-marrow derived MSC (group Hyp-CMC-MSC) or stimulated with the secretome of MSC (group Hyp-CMC-SMSC). The secretome of normoxic MSC and CMC, and the hypoxic CMC was analyzed with a cytokine panel. Gene expression changes of HIF-1α, proliferation marker Ki-67 and cytokinesis marker RhoA over different reoxygenation time periods of 4, 8, 24, 48, and 72 h were analyzed in comparison to normoxic CMC and MSC. Further, the proinflammatory cytokine IL-18 protein expression change, metabolic activity and proliferation was assessed in all experimental setups.
Results and Conclusion
HIF-1α was persistently overexpressed in Hyp-CMC-SMSC as compared to Hyp-CMC (except at 72 h). Hyp-CMC-MSC showed a weaker HIF-1α expression than Hyp-CMC-SMSC in most tested time points, except after 8 h. The Ki-67 expression showed the strongest upregulation in Hyp-CMC after 24 and 48 h incubation, then returned to baseline level, while a temporary increase in Ki-67 expression in Hyp-CMC-MSC at 4 and 8 h and at 48 h in Hyp-CMC-SMSC could be observed. RhoA was increased in normoxic MSCs and in Hyp-CMC-SMSC over time, but not in Hyp-CMC-MSC. A temporary increase in IL-18 protein expression was detected in Hyp-CMC-SMSC and Hyp-CMC. Our study demonstrates timely dynamic changes in expression of different ischemia and regeneration-related genes of CMCs, depending from the culture condition, with stronger expression of HIF-1α, RhoA and IL-18 if the hypoxic CMC were subjected to the secretome of MSCs.
... 2 For example, nearly 20 years of research aimed at harnessing the alleged endogenous regenerative potential of mammalian heart by generating de novo cardiomyocytes from an elusive resident cardiac stem cell population appears to have now been debunked with accruing evidence, not only showing lack of endogenous regeneration from stem cells but no cardiomyocyte replacement from any noncardiomyocyte source. 3,4 Mammalian ACMs are terminally differentiated cells with minimal endogenous proliferative capacity following myocardial infarction (MI), 5 whereas several lower organisms such as zebrafish, newts, and even neonatal mice can regenerate damaged myocardium through the proliferation of preexisting cardiomyocytes. [6][7][8] Therefore, the development of novel therapeutic interventions to regenerate ischemic myocardium by inducing proliferation of preexisting ACMs has become an even more critical area of research. ...
Background
Myocardial infarction results in a large‐scale cardiomyocyte loss and heart failure due to subsequent pathological remodeling. Whereas zebrafish and neonatal mice have evident cardiomyocyte expansion following injury, adult mammalian cardiomyocytes are principally nonproliferative. Despite historical presumptions of stem cell–mediated cardiac regeneration, numerous recent studies using advanced lineage‐tracing methods demonstrated that the only source of cardiomyocyte renewal originates from the extant myocardium; thus, the augmented proliferation of preexisting adult cardiomyocytes remains a leading therapeutic approach toward cardiac regeneration. In the present study we investigate the significance of suppressing cell cycle inhibitors Rb1 and Meis2 to promote adult cardiomyocyte reentry to the cell cycle.
Methods and Results
In vitro experiments with small interfering RNA –mediated simultaneous knockdown of Rb1 and Meis2 in both adult rat cardiomyocytes, isolated from 12‐week‐old Fischer rats, and human induced pluripotent stem cell–derived cardiomyocytes showed a significant increase in cell number, a decrease in cell size, and an increase in mononucleated cardiomyocytes. In vivo, a hydrogel‐based delivery method for small interfering RNA –mediated silencing of Rb1 and Meis2 is utilized following myocardial infarction. Immunofluorescent imaging analysis revealed a significant increase in proliferation markers 5‐ethynyl‐2′‐deoxyuridine, PH 3, KI 67, and Aurora B in adult cardiomyocytes as well as improved cell survivability with the additional benefit of enhanced peri‐infarct angiogenesis. Together, this intervention resulted in a reduced infarct size and improved cardiac function post–myocardial infarction.
Conclusions
Silencing of senescence‐inducing pathways in adult cardiomyocytes via inhibition of Rb1 and Meis2 results in marked cardiomyocyte proliferation and increased protection of cardiac function in the setting of ischemic injury.
... However, briefly after birth, CMs initiate a final cell cycle completing only karyokinesis, but fail to complete cytokinesis. This uncoupling of cytokinesis from karyokinesis results in an increased number of binucleated CMs that have exited the cell cycle and became terminally differentiated (G 0 ) Elhelaly et al. 2016;Li et al. 1996;Paradis et al. 2014;Rumyantsev 1977;Soonpaa et al. 1996;van Amerongen and Engel 2008). In rodents, binuclearity occurs only within the first 14 days of life as a consequence of mitosis without cell division, instead of fusion of two individual mononucleated CMs Li et al. 1996). ...
Considerable effort has gone into investigating mechanisms that underlie the developmental transition in which mammalian cardiomyocytes (CMs) switch from being able to proliferate during development, to essentially having lost that ability at maturity. This problem is interesting not only for scientific curiosity, but also for its clinical relevance because controlling the ability of mature CMs to replicate would provide a much-needed approach for restoring cardiac function in damaged hearts. In this review, we focus on the propensity of mature mammalian CMs to be multinucleated and polyploid, and the extent to which this may be necessary for normal physiology yet possibly disadvantageous in some circumstances. In this context, we explore whether the concept of the myonuclear domain (MND) in multinucleated skeletal muscle fibers might apply to cardiomyocytes, and whether cardio-MND size might be related to the transition of CMs to become multinuclear. Nuclei in CMs are almost certainly integrators of not only biochemical, but also—because of their central location within the myofibrils—mechanical information, and this multimodal, integrative function in adult CMs—involving molecules that have been extensively studied along with newly identified possibilities—could influence both gene expression as well as replication of the genome and the nuclei themselves.
... The neonatal mammalian heart harbors a tremendous potential to promote cardiomyocyte proliferation to facilitate repair and/or regeneration 12 . In the neonatal mouse, the cardiomyocyte proliferative capacity diminishes rapidly within a 1-week period following birth [12][13][14] . In contrast, only limited cardiomyocyte turnover occurs in the adult mammalian heart, a capacity that is insufficient to repair or regenerate the injured heart 15,16 . ...
The mammalian heart has a limited regenerative capacity and typically progresses to heart failure following injury. Here, we defined a hedgehog (HH)-Gli1-Mycn network for cardiomyocyte proliferation and heart regeneration from amphibians to mammals. Using a genome-wide screen, we verified that HH signaling was essential for heart regeneration in the injured newt. Next, pharmacological and genetic loss- and gain-of-function of HH signaling demonstrated the essential requirement for HH signaling in the neonatal, adolescent, and adult mouse heart regeneration, and in the proliferation of hiPSC-derived cardiomyocytes. Fate-mapping and molecular biological studies revealed that HH signaling, via a HH-Gli1-Mycn network, contributed to heart regeneration by inducing proliferation of pre-existing cardiomyocytes and not by de novo cardiomyogenesis. Further, Mycn mRNA transfection experiments recapitulated the effects of HH signaling and promoted adult cardiomyocyte proliferation. These studies defined an evolutionarily conserved function of HH signaling that may serve as a platform for human regenerative therapies.
... Binucleation in CMs has been described to take place by regression of the cleavage furrow 28 . The midbody was reported to form normally in such a scenario and therefore, to provide no indication, whether a cell will divide or become binucleated 29 . Herein we show that both, the position of the midbody and the distance between the daughter nuclei are discriminatory parameters. ...
Rationale:
New strategies in the field of cardiac regeneration are directed at identifying proliferation-inducing substances to induce regrowth of myocardium. Current screening assays utilize neonatal cardiomyocytes and markers for cytokinesis, such as Aurora B-kinase. However, detection of cardiomyocyte division is complicated because of cell cycle variants, in particular, binucleation.
Objective:
To analyze the process of cardiomyocyte binucleation to identify definitive discriminators for cell cycle variants and authentic cardiomyocyte division.
Methods and results:
Herein, we demonstrate by direct visualization of the contractile ring and midbody in Myh6 (myosin, heavy chain 6)-eGFP (enhanced green fluorescent protein)-anillin transgenic mice that cardiomyocyte binucleation starts by formation of a contractile ring. This is followed by irregular positioning of the midbody and movement of the 2 nuclei into close proximity to each other. In addition, the widespread used marker Aurora B-kinase was found to also label binucleating cardiomyocytes, complicating the interpretation of existing screening assays. Instead, atypical midbody positioning and the distance of daughter nuclei on karyokinesis are bona fide markers for cardiomyocyte binucleation enabling to unequivocally discern such events from cardiomyocyte division in vitro and in vivo.
Conclusions:
The 2 criteria provide a new method for identifying cardiomyocyte division and should be considered in future studies investigating cardiomyocyte turnover and regeneration after injury, in particular in the postnatal heart to prevent the assignment of false positive proliferation events.
... Repair of damaged myocardium for cardiovascular applications is limited due to the low proliferative rate of cardiomyocytes in adults [162] while insufficient angiogenesis has implications in vascular disease. Scaffold-based miRNA delivery for cardiovascular-related tissue has garnered a reasonable amount of interest in recent years as it can limit the risk of undesired offtarget miRNA effects on multiple mRNAs in several different tissues and is currently a growing area of research as documented here. ...
microRNA-based therapies are an advantageous strategy with applications in both regenerative medicine (RM) and cancer treatments. microRNAs (miRNAs) are an evolutionary conserved class of small RNA molecules that modulate up to one third of the human nonprotein coding genome. Thus, synthetic miRNA activators and inhibitors hold immense potential to finely balance gene expression and reestablish tissue health. Ongoing industry-sponsored clinical trials inspire a new miRNA therapeutics era, but progress largely relies on the development of safe and efficient delivery systems. The emerging application of biomaterial scaffolds for this purpose offers spatiotemporal control and circumvents biological and mechanical barriers that impede successful miRNA delivery. The nascent research in scaffold-mediated miRNA therapies translates know-how learnt from studies in antitumoral and genetic disorders as well as work on plasmid (p)DNA/siRNA delivery to expand the miRNA therapies arena. In this progress report, the state of the art methods of regulating miRNAs are reviewed. Relevant miRNA delivery vectors and scaffold systems applied to-date for RM and cancer treatment applications are discussed, as well as the challenges involved in their design. Overall, this progress report demonstrates the opportunity that exists for the application of miRNA-activated scaffolds in the future of RM and cancer treatments.
... 1,2 Although a number of studies have detected modest numbers of replicating CMs after cardiac injury, suggesting an attempt at myocardial regeneration, 3 the repair typically occurs through a scarring process leading to tissue fibrosis and loss of function. 4 Heart transplantation continues to be the gold-standard treatment for end-stage HF. 5,6 However, complications including the limited availability of donated organs, donor−patient compatibility, immune rejection, and hospitalization costs limit widespread clinical availability. 7,8 In light of the limited efficacy of current treatments, direct injection of exogenous cells has been used to repair damaged myocardium. ...
The ability of the adult heart to regenerate cardiomyocytes (CMs) lost after injury is limited, generating interest in developing efficient cell-based transplantation therapies. Rigid carbon nanotubes (CNTs) scaffolds have been used to improve CMs viability, proliferation, and maturation, but they require undesirable invasive surgeries for implantation. To overcome this limitation, we developed an injectable reverse thermal gel (RTG) functionalized with CNTs (RTG-CNT) that transitions from a solution at room temperature to a three-dimensional (3D) gel-based matrix shortly after reaching body temperature. Here we show experimental evidence that this 3D RTG-CNT system supports long-term CMs survival, promotes CMs alignment and proliferation, and improves CMs function when compared with traditional two-dimensional gelatin controls and 3D plain RTG system without CNTs. Therefore, our injectable RTG-CNT system could potentially be used as a minimally invasive tool for cardiac tissue engineering efforts.
... D uring embryogenesis and fetal life, the heart grows by rapid mitotic divisions of cardiomyocytes, producing hyperplastic growth. In the perinatal period, cell division ceases, cardiomyocytes withdraw from the cell cycle, and the further increase in cardiac mass is achieved through increase in cell size (hypertrophy) (1)(2)(3). The switch from hyperplastic to hypertrophic growth is characterized by extensive binucleation of cardiomyocytes. ...
Significance
Here, we demonstrate that mice lacking GAS2L3, a cytoskeleton-associated protein that interacts with actin filaments and tubulin, develop cardiomyopathy and heart failure after birth. During embryogenesis, cardiomyocytes rapidly divide. In the perinatal and neonatal period, cardiomyocytes withdraw from the cell cycle, binucleate, and the further increase in cardiac mass is achieved by hypertrophy. Germ-line deletion of Gas2l3 results in decreased cardiomyocyte proliferation and in cardiomyocyte hypertrophy. Embryonal cardiomyocytes from Gas2l3 -deficient mice exhibit increased expression of the cell cycle inhibitor p21 and display premature binucleation of cardiomyocytes due to defects in cytokinetic abscission. Together these results suggest that GAS2L3 plays a central role in cardiomyocyte proliferation and cytokinesis during development.
... Cardiovascular disease claims over 17 million lives each year worldwide and for decades has been the biggest killer in the United States [1][2][3][4]. The major factor contributing to this is the terminally differentiated nature of the adult mammalian heart and the inability of cardiomyocytes to proliferate [5][6][7]. While there are reports about limited turnover of cardiomyocytes in an adult heart, it is still insufficient to replenish the loss of cardiomyocytes following a myocardial injury [8]. ...
In mammals, proliferative capacity of cardiomyocytes is lost soon after birth, while zebrafish and other lower organisms like newts are known to regenerate injured hearts even at an adult age. Here, we show that miR-1825 can induce robust proliferation of adult rat cardiomyocytes and can improve cardiac function in-vivo post myocardial infarction. Rat adult cardiomyocytes transfected with miR-1825 showed a significant increase in DNA synthesis, mitosis, cytokinesis, and an increase in cell number when compared to cel-miR-67 transfected control. We also observed a reduction in mitochondrial number and a decrease in ROS and DNA-damage. RNA-sequencing data identified NDUFA10, a key gene involved in the mitochondrial electron transport chain to be a direct target of miR-1825. SiRNA mediated silencing of NDUFA10 showed a significant increase in cardiomyocyte proliferation indicating its role downstream of miRNA-1825. In addition, microRNA microarray results identified miR-1825 to regulate expression of a known proliferation inducing miRNA, miR-199a. We also identified the direct targets of miR-199a, namely p16, Rb1, and Meis2 to be downregulated following miR-1825 transfection. However, miR-199a alone did not have similar proliferation inducing effects as miR-1825, indicating that miR-1825 works through multiple pathways and is a master regulator of cardiomyocyte proliferation. In addition, our in-vivo analysis in animal models of LAD ligation and intra-cardiac miRNA delivery showed proliferation of endogenous cardiomyocytes in the peri-infarcted region and an improvement in heart function. These findings establish miR-1825 as a potential therapeutic agent for induction of cardiomyocyte proliferation and cardiac regeneration, with a significant translational potential.
... In humans, acute myocardial infarction (MI) causes the death of cardiac muscle cells due to ischaemia and ischaemia/reperfusion (Heusch and Schulz, 2012). In surviving patients, the necrotic myocardium will be replaced by collagenrich non-contractile scar tissue leading to pathologic hypertrophy (Rubart and Field, 2006;van Amerongen and Engel, 2008).Because of the inability of cardiomyocytes to re-enter the cell cycle to restore optimal cardiac performance in humans, the heart is often more susceptible to future MI events (Azevedo et al., 2005). In contrast, the zebrafish heart has been shown to be able to regenerate within a month after experimental induced injuries, such as ventricular resection and cryocautherisation (Poss et al., 2002;Raya et al., 2003;Schnabel et al., 2011). ...
Background:
Current treatments for congenital heart defects often require surgery and implantation of a synthetic patch or baffle that becomes a fibrous scar and leads to a high number of reoperations. Previous studies in rats have shown that a pre-vascularized scaffold can integrate into the heart and result in regions of vascularized and muscularized tissue. However, increasing the thickness of this scaffold for use in human hearts requires a method to populate the thick scaffold and mature it under physiologic flow and electrical conditions.
Experiment:
We developed a bioreactor system that can perfuse up to six 7-mm porous scaffolds with tunable gravity-mediated flow and chronic electrical stimulation. Three polymers which have been reported to be biocompatible were evaluated for effects on the viability of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM). Bioreactor flow and electrical stimulation functions were tested, and the bioreactor was operated for up to 7 days to ensure reliability and lack of leaks in a 37C, humidified incubator. Height and flow relationships were measured for perfusion through an electrospun polycaprolactone (PCL) and gelatin scaffold previously reported by our laboratory. Culture with cells was evaluated by plating human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (hDF) on top of the scaffolds in both static and flow conditions for 2,5 and 7 days. As a proof-of concept, scaffolds were cryosectioned and cell infiltration was quantified using immunofluorescence staining.
Results:
Neither MED610 (Stratasys), Vero (Stratasys), nor FORMLAB materials affected the viability of iPSC derived cardiomyocytes, and MED610 was chosen for manufacture due to familiarity of 3D printing from this material. The generation of electrical field stimulation from 0 to 5 volts and physiological ranges of pump capacities were verified. The relationship between height and flow was calculated for scaffolds with and without cells. Finally, we demonstrated evaluation of cell depth and structure in scaffolds cultured for 2, 5, and 7 days.
Conclusion:
The gravity-mediated flow bioreactor system we developed can be used as a platform for 3D cell culture particularly designed for perfusing vascularized tissue constructs with electrical stimulation for cardiac maturation.
Exosomes are vesicles with a size range of 50 to 200 nm and released by different cells, which are essential for the exchange of information between cells. They have attracted a lot of interest from medical researchers. Exosomal non-coding RNAs play an important part in pathological cardiac remodelings, such as cardiomyocyte hypertrophy, cardiomyocyte apoptosis, and cardiac fibrosis. This review summarizes the origins and functions of exosomes, the role of exosomal non-coding RNAs in the process of pathological cardiac remodeling, as well as their theoretical basis for clinical application.
Cardiovascular disease is one of the leading causes of morbidity and mortality worldwide, with myocardial infarctions being amongst the deadliest manifestations. Reduced blood flow to the heart can result in the death of cardiac tissue, leaving affected patients susceptible to further complications and recurrent disease. Further, contemporary management typically involves a pharmacopeia to manage the metabolic conditions contributing to atherosclerotic and hypertensive heart disease, rather than regeneration of the damaged myocardium. With modern healthcare extending lifespan, a larger demographic will be at risk for heart disease, driving the need for novel therapeutics that surpass those currently available in efficacy. Transdifferentiation and cellular reprogramming have been looked to as potential methods for the treatment of diseases throughout the body. Specifically targeting the fibrotic cells in cardiac scar tissue as a source to be reprogrammed into induced cardiomyocytes remains an appealing option. This review aims to highlight the history of and advances in cardiac reprogramming and describe its translational potential as a treatment for cardiovascular disease.
Transcription factors play a fundamental role in cardiovascular adaptation to stress. Nuclear receptor subfamily 4 group A member 2 (NR4A2; NURR1) is an immediate-early gene and transcription factor with a versatile role throughout many organs. In the adult mammalian heart, and particularly in cardiac myocytes, NR4A2 is strongly up-regulated in response to beta-adrenergic stimulation. The physiologic implications of this increase remain unknown. In this study, we aimed to interrogate the consequences of cardiac NR4A2 up-regulation under normal conditions and in response to pressure overload. In mice, tamoxifen-dependent, cardiomyocyte-restricted overexpression of NR4A2 led to cardiomyocyte hypertrophy, left ventricular dilation, heart failure, and death within 40 days. Chronic NR4A2 induction also precipitated cardiac decompensation during transverse aortic constriction (TAC)-induced pressure overload. Mechanistically, NR4A2 caused adult cardiac myocytes to return to a fetal-like phenotype, with a switch to glycolytic metabolism and disassembly of sarcomeric structures. NR4A2 also re-activated cell cycle progression and stimulated DNA replication and karyokinesis but failed to induce cytokinesis, thereby promoting multinucleation of cardiac myocytes. Activation of cell cycle checkpoints led to induction of an apoptotic response which ultimately resulted in excessive loss of cardiac myocytes and impaired left ventricular contractile function. In summary, myocyte-specific overexpression of NR4A2 in the postnatal mammalian heart results in increased cell cycle re-entry and DNA replication but does not result in cardiac myocyte division. Our findings expose a novel function for the nuclear receptor as a critical regulator in the self-renewal of the cardiac myocyte and heart regeneration.
The high organ specification of the human heart is inversely proportional to its functional recovery after damage. The discovery of induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) has accelerated research in human heart regeneration and physiology. Nevertheless, due to the immaturity of iPSC‐CMs, they are far from being an representative model of the adult heart physiology. Therefore, number of laboratories strive to obtain a heart tissues by engineering methods by structuring iPSC‐CMs into complex and advanced platforms. By using the iPSC‐CMs and arranging them in 3D cultures it is possible to obtain a human heart muscle with physiological capabilities potentially similar to the adult heart, while remaining in vitro. Here, we attempt to describe existing examples of heart muscle either in vitro or ex vivo models and discuss potential options for the further development of such structures. This will be a crucial step for ultimate derivation of complete heart tissue‐mimicking organs and their future use in drug development, therapeutic approaches testing, pre‐clinical studies, and clinical applications. This review particularly aims to compile available models of advanced human heart tissue for scientists considering which model would best fit their research needs.
Cardiovascular diseases (CVDs) comprises disorders of blood vessels and heart. Multiple cells in the heart suggests that hetero-cellular communication, which is an important aspect in heart functioning and there is a need to elucidate the way in which this inter-cellular communication occurs. Now a days, exosomal research has gained much attention. Exosomes, nano-shuttles, are EVs with diameters ranging from 40 to 160 nm (average 100 nm), secreted by body cells. These vesicles act as cell-to-cell communicators and are carriers of important biomolecules such as RNAs, miRNAs, Proteins and lipids. Exosomes can change the gene expression of the recipient cells, thereby, changes the cellular characteristics. Exosomes have known to play an essential role in protection as well as progression of various cardiovascular diseases. In the present review, role of exosomes in various CVDs have been discussed.
Cardiac tissue engineering is a promising strategy to generate human cardiac tissues for modeling cardiac diseases, screening for therapeutic drugs, and repairing the injured heart. Yet, several issues remain to be resolved including the generation of tissues with high cardiomyocyte density. Here, it is shown that the integration of the glycogen synthase kinase‐3β inhibitor CHIR99021 in collagen I hydrogels promotes proliferation of human‐induced pluripotent stem cell‐derived (hiPSC) cardiomyocytes post‐fabrication improving contractility of and calcium flow in engineered 3D cardiac microtissues. CHIR99021 has no effect on the gelation kinetics or the mechanical properties of collagen I hydrogels. Analysis of cell density and proliferation based on Ki‐67 staining indicates that integration of CHIR99021 together with external CHIR99021 stimulation increases hiPSC‐cardiomyocyte number by ≈2‐fold within 7 d post‐fabrication. Analysis of the contractility of engineered cardiac tissues after another 3 d in the absence of external CHIR99021 shows that CHIR99021‐induced hiPSC‐cardiomyocyte proliferation results in synchronized calcium flow, rhythmic beating, increased speed of contraction and contraction amplitude, and reduced peak‐to‐peak time. The CHIR99021‐stimulated engineered cardiac microtissues exhibit spontaneous rhythmic contractions for at least 35 d. Collectively, the data demonstrate the potential of induced cardiomyocyte proliferation to enhance engineered cardiac microtissues by increasing cardiomyocyte density.
Ischemic heart disease is one of the leading causes of deaths worldwide. A major hindrance to resolving this challenge lies in the mammalian hearts inability to regenerate after injury. In contrast, zebrafish retain a regenerative capacity of the heart throughout their lifetimes. Apex resection (AR) is a popular zebrafish model for studying heart regeneration, and entails resecting 10–20% of the heart in the apex region, whereafter the regeneration process is monitored until the heart is fully regenerated within 60 days. Despite this popularity, video tutorials describing this technique in detail are lacking. In this paper we visualize and describe the entire AR procedure including anaesthesia, surgery, and recovery. In addition, we show that the concentration and duration of anaesthesia are important parameters to consider, to balance sufficient levels of sedation and minimizing mortality. Moreover, we provide examples of how zebrafish heart regeneration can be assessed both in 2D (immunohistochemistry of heart sections) and 3D (analyses of whole, tissue cleared hearts using multiphoton imaging). In summary, this paper aims to aid beginners in establishing and conducting the AR model in their laboratory, but also to spur further interest in improving the model and its evaluation.
Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The postmitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression. The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.
Neuregulin‐1 (NRG‐1)/erythroblastic leukaemia viral oncogene homologues (ErbB) pathway activation plays a crucial role in regulating the adaptation of the adult heart to physiological and pathological stress. In the present study, we investigate the effect of recombined human NRG‐1 (rhNRG‐1) on mitochondrial biogenesis, mitochondrial function, and cell survival in neonatal rat cardiac myocytes (NRCMs) exposed to hypoxia/reoxygenation (H/R). The results of this study showed that, in the H/R‐exposed NRCMs, mitochondrial biogenesis was impaired, as manifested by the decrease of the expression of peroxisome proliferator‐activated receptor gamma coactivator‐1 alpha (PGC‐1α) and mitochondrial membrane proteins, the inner membrane (Tim23), mitofusin 1 (Mfn1), and mitofusin 2 (Mfn2). RhNRG‐1 pretreatment effectively restored the expression of PGC‐1α and these membrane proteins, upregulated the expression of the anti‐apoptosis proteins Bcl‐2 and Bcl‐xL, preserved the mitochondrial membrane potential, and attenuated H/R‐induced cell apoptosis. Blocking PGC‐1 expression with siRNA abolished the beneficial role of rhNRG‐1 on mitochondrial function and cell survival. The results of the present study strongly suggest that NRG‐1/ErbB activation enhances the adaption of cardiomyocytes to H/R injury via promoted mitochondrial biogenesis and improved mitochondrial homeostasis.
Significance of the Study
The results of this research revealed for the first time the relationship between neuregulin‐1 (NRG‐1)/erythroblastic leukaemia viral oncogene homologues (ErbB) activation and mitochondrial biogenesis in neonatal cardiomyocytes and verified the significance of this promoted mitochondrial biogenesis in attenuating hypoxia/reoxygenation injury. This finding may open a new field to further understand the biological role of NRG‐1/ErbB signalling pathway in cardiomyocyte.
Heart failure (HF) remains the major cause of morbidity and mortality in the world with an alarming rapid rise in heart diseases. Myocardial infarction (MI), depriving of blood flow to the heart muscle, causes myocardial necrosis and apoptosis resulting in decrease of myocardial quantity and ventricular remodeling, which is the main cause of HF. As mature cardiomyocytes (CMs) are incapable of differentiation and proliferation, cardiomyocytes damage is not likely to be reversed. Recent research has advanced to the point where damaged CMs or myocardium can be repaired by exogenous cells or artificial tissue. This review provides the most up-to-date information on the current status of cell therapy and tissue three-dimensional (3D) bioprinting for the treatment of HF. Requirements on 3D printing of biomaterials will be provided including biophysical properties, biocompatibility and biodegradable absorption in vivo that are key factors on the survival and proliferation of transplanted cells in the MI area. Also discussed are the current challenges in cell-based HF therapies and future perspectives in clinical applications.
Aim: Characterizing the response to myocardial infarction (MI) in the regenerative sheep fetus heart compared to the post-natal non-regenerative adolescent heart may reveal key morphological and molecular differences that equate to the response to MI in humans. We hypothesized that the immediate response to injury in (a) infarct compared with sham, and (b) infarct, border, and remote tissue, in the fetal sheep heart would be fundamentally different to the adolescent, allowing for repair after damage.
Methods: We used a sheep model of MI induced by ligating the left anterior descending coronary artery. Surgery was performed on fetuses (105 days) and adolescent sheep (6 months). Sheep were randomly separated into MI (n = 5) or Sham (n = 5) surgery groups at both ages. We used magnetic resonance imaging (MRI), histological/immunohistochemical staining, and qRT-PCR to assess the morphological and molecular differences between the different age groups in response to infarction.
Results: Magnetic resonance imaging showed no difference in fetuses for key functional parameters; however there was a significant decrease in left ventricular ejection fraction and cardiac output in the adolescent sheep heart at 3 days post-infarction. There was no significant difference in functional parameters between MRI sessions at Day 0 and Day 3 after surgery. Expression of genes involved in glucose transport and fatty acid metabolism, inflammatory cytokines as well as growth factors and cell cycle regulators remained largely unchanged in the infarcted compared to sham ventricular tissue in the fetus, but were significantly dysregulated in the adolescent sheep. Different cardiac tissue region-specific gene expression profiles were observed between the fetal and adolescent sheep.
Conclusion: Fetuses demonstrated a resistance to cardiac damage not observed in the adolescent animals. The manipulation of specific gene expression profiles to a fetal-like state may provide a therapeutic strategy to treat patients following an infarction.
Over 5 million people in the United States suffer from heart failure, due to the limited ability to regenerate functional cardiac tissue. One potential therapeutic strategy is to enhance proliferation of resident cardiomyocytes. However, phenotypic screening for therapeutic agents is challenged by the limited ability of conventional markers to discriminate between cardiomyocyte proliferation and endoreplication (e.g. polyploidy and multinucleation). Here, we developed a novel assay that combines automated live-cell microscopy and image processing algorithms to discriminate between proliferation and endoreplication by quantifying changes in the number of nuclei, changes in the number of cells, binucleation, and nuclear DNA content. We applied this assay to further prioritize hits from a primary screen for DNA synthesis, identifying 30 compounds that enhance proliferation of human induced pluripotent stem cell-derived cardiomyocytes. Among the most active compounds from the phenotypic screen are clinically approved L-type calcium channel blockers from multiple chemical classes whose activities were confirmed across different sources of human induced pluripotent stem cell-derived cardiomyocytes. Identification of compounds that stimulate human cardiomyocyte proliferation may provide new therapeutic strategies for heart failure.
Although the contractile function of the heart is universally conserved, the organ itself varies in structure across species. This variation includes the number of ventricular chambers (one, two, or an incompletely divided chamber), the structure of the myocardial wall (compact or trabeculated), and the proliferative capacity of the resident cardiomyocytes. Whereas zebrafish is capable of comparatively high rates of constitutive cardiomyocyte proliferation, humans and rodents are not. However, for most species, the capacity to generate new cardiomyocytes under homeostatic conditions remains unclear. Here, we investigate cardiomyocyte proliferation in the lizard Eublepharis macularius, the leopard gecko. As for other lizards, the leopard gecko heart has a partially septated ventricular lumen with a trabeculated myocardial wall. To test our hypothesis that leopard gecko cardiomyocytes routinely proliferate, we performed 5‐bromo‐2′‐deoxyuridine incorporation and immunostained for the mitotic marker phosphorylated histone H3 (pHH3) and the DNA synthesis phase (S phase) marker proliferating cell nuclear antigen (PCNA). Using double immunofluorescence, we co‐localized pHH3 or PCNA with the cardiomyocyte marker myosin heavy chain (MHC). We found that ~0.5% of cardiomyocytes were mitotically active (pHH3+/MHC+), while ~10% were in S phase (PCNA+/MHC+). We also determined that cell cycling by gecko cardiomyocytes is not impacted by caudal autotomy (tail loss), a dramatic form of self‐amputation. Finally, we show that populations of cardiac cells are slow cycling. Overall, our findings provide predictive evidence that geckos may be capable of spontaneous cardiac self‐repair and regeneration following a direct injury. We determined that leopard gecko cardiomyocytes routinely proliferate under homeostatic conditions. Cardiomyocyte proliferation is not altered in response to tail loss, suggesting geckos are resilient to injury‐mediate systemic activation.
Background:
Extracellular vesicles (EVs) and exosomes are nano-sized, membrane-bound vesicles shed by most eukaryotic cells studied to date. EVs play key signaling roles in cellular development, cancer metastasis, immune modulation and tissue regeneration. Attempts to modify exosomes to increase their targeting efficiency to specific tissue types are still in their infancy. Here we describe an EV membrane anchoring platform termed "cloaking" to directly embed tissue-specific antibodies or homing peptides on EV membrane surfaces ex vivo for enhanced vesicle uptake in cells of interest. The cloaking system consists of three components: DMPE phospholipid membrane anchor, polyethylene glycol spacer and a conjugated streptavidin platform molecule, to which any biotinylated molecule can be coupled for EV decoration.
Results:
We demonstrate the utility of membrane surface engineering and biodistribution tracking with this technology along with targeting EVs for enhanced uptake in cardiac fibroblasts, myoblasts and ischemic myocardium using combinations of fluorescent tags, tissue-targeting antibodies and homing peptide surface cloaks. We compare cloaking to a complementary approach, surface display, in which parental cells are engineered to secrete EVs with fusion surface targeting proteins.
Conclusions:
EV targeting can be enhanced both by cloaking and by surface display; the former entails chemical modification of preformed EVs, while the latter requires genetic modification of the parent cells. Reduction to practice of the cloaking approach, using several different EV surface modifications to target distinct cells and tissues, supports the notion of cloaking as a platform technology.
Cardiomyocyte proliferation is crucial for cardiac growth, patterning and regeneration; however, few studies have investigated the behavior of dividing cardiomyocytes in vivo Here, we use time-lapse imaging of beating hearts in combination with the FUCCI system to monitor the behavior of proliferating cardiomyocytes in developing zebrafish. Confirming in vitro observations, sarcomere disassembly, as well as changes in cell shape and volume, precede cardiomyocyte cytokinesis. Notably, cardiomyocytes in zebrafish embryos and young larvae mostly divide parallel to the myocardial wall in both the compact and trabecular layers, and cardiomyocyte proliferation is more frequent in the trabecular layer. While analyzing known regulators of cardiomyocyte proliferation, we observed that the Nrg/ErbB2 and TGFβ signaling pathways differentially affect compact and trabecular layer cardiomyocytes, indicating that distinct mechanisms drive proliferation in these two layers. In summary, our data indicate that, in zebrafish, cardiomyocyte proliferation is essential for trabecular growth, but not initiation, and set the stage to further investigate the cellular and molecular mechanisms driving cardiomyocyte proliferation in vivo.
Statement of significance:
Cardiovascular disease remains a leading cause of death in the United States and a major health-care burden. Myocardial infarction (MI) is a main cause of death in cardiovascular diseases. MI occurs as a consequence of sudden blocking of blood vessels supplying the heart. When occlusions in the coronary arteries occur, an immediate decrease in nutrient and oxygen supply to the cardiac muscle, resulting in permanent cardiac cell death. Eventually, scar tissue formed in the damaged cardiac muscle that cannot conduct electrical or mechanical stimuli thus leading to a reduction in the pumping efficiency of the heart. The therapeutic options available for end-stage heart failure is to undergo heart transplantation or the use of mechanical ventricular assist devices (VADs). However, many patients die while being on a waiting list, due to the organ shortage and limitation of VADs, such as surgical complications, infection, thrombogenesis, and failure of the electrical motor and hemolysis. Ultimately, 3D bioprinting strategy aims to create clinically applicable tissue constructs that can be immediately implanted in the body. To date, the focus on replicating complex and heterogeneous tissue constructs continues to increase as 3D bioprinting technologies advance. In this study, we demonstrated the feasibility of 3D bioprinting strategy to bioengineer the functional cardiac tissue that possesses a highly organized structure with unique physiological and biomechanical properties similar to native cardiac tissue. This bioprinting strategy has great potential to precisely generate functional cardiac tissues for use in pharmaceutical and regenerative medicine applications.
Recent epidemiologic studies evidence a dramatic increase of cardiovascular diseases, especially associated with the aging of the world population. During aging, the progressive impairment of the cardiovascular functions results from the compromised tissue abilities to protect the heart against stress. At the molecular level, in fact, a gradual weakening of the cellular processes regulating cardiovascular homeostasis occurs in aging cells. Atherosclerosis and heart failure are particularly correlated with aging-related cardiovascular senescence, that is, the inability of cells to progress in the mitotic program until completion of cytokinesis. In this review, we explore the intrinsic and extrinsic causes of cellular senescence and their role in the onset of these cardiovascular pathologies. Additionally, we dissect the effects of aging on the cardiac endogenous and exogenous reservoirs of stem cells. Finally, we offer an overview on the strategies of regenerative medicine that have been advanced in the quest for heart rejuvenation.
Key messages:
MiRNA-210 transfected adult rat CMs show proliferation and reduced cell death in vitro. Cell cycle inhibitor APC is a target of miR-210. MiR-210 overexpressing (210-TG) mouse hearts show CMs cell cycle re-entry and survival post myocardial injury. 210-TG mice show significant neovascularization and angiogenic potential post myocardial infarction. 210-TG hearts show reduced infarct size following ischemic injury.
Mammalian cardiomyocytes substantially lose proliferative capacity immediately after birth, limiting adult heart regeneration after injury. However, clinical myocarditis appears to be self-limiting with tissue-reparative properties. Here, we investigated the molecular mechanisms underlying the recovery from myocarditis with regard to cardiomyocyte proliferation using an experimental autoimmune myocarditis (EAM) model. Three weeks after EAM induction (EAM3w), cardiac tissue displayed infiltration of inflammatory cells with cardiomyocyte apoptosis. However, by EAM5w, the myocardial damage was remarkably attenuated, associated with an increase in cardiomyocytes that were positively stained with cell cycle markers at EAM3w. Cardiomyocyte fate mapping study revealed that the proliferating cardiomyocytes primarily derived from pre-existing cardiomyocytes. Signal transducer and activator of transcription 3 (STAT3) was robustly activated in cardiomyocytes during inflammation, accompanied by induction of interleukin-6 family cytokines. Cardiomyocyte-specific ablation of STAT3 gene suppressed the frequency of cycling cardiomyocytes in the recovery period without influencing inflammatory status, resulting in impaired tissue repair and cardiac dysfunction. Finally, microarray analysis revealed that the expression of regeneration-related genes, metallothioneins and clusterin, in cardiomyocytes was decreased by STAT3 gene deletion. These data show that adult mammalian cardiomyocytes restore regenerative capacity with cell cycle reentry through STAT3 as the heart recovers from myocarditis-induced cardiac damage.
Objective:
The purpose of this article was to review the molecular mechanisms of low-level laser irradiation (LLLI) preconditioning for heart cell therapy.
Background data:
Stem cell transplantation appears to offer a better alternative to cardiac regenerative therapy. Previous studies have confirmed that the application of LLLI plays a positive role in regulating stem cell proliferation and in remodeling the hostile milieu of infarcted myocardium. Greater understanding of LLLI's underlying mechanisms would be helpful in translating cell transplantation therapy into the clinic.
Methods:
Studies investigating LLLI preconditioning for cardiac regenerative therapy published up to 2015 were retrieved from library sources and Pubmed databases.
Results:
LLLI preconditioning stimulates proliferation and differentiation of stem cells through activation of cell proliferation signaling pathways and alteration of microRNA expression. It also could stimulate paracrine secretion of stem cells and alter cardiac cytokine expression in infarcted myocardium.
Conclusions:
LLLI preconditioning provides a promising approach to maximize the efficacy of cardiac cell-based therapy. Although many studies have reported possible molecular mechanisms involved in LLLI preconditioning, the exact mechanisms are still not clearly understood.
Severe heart diseases such as myocarditis and cardiomyopathy are often characterized by progressive damages of contractile heart tissue which ultimately can lead to terminal heart failure. There is a need for relevant in vitro cultures of human cardiomyocytes to study pathogenic processes and to perform pharmacological testing of new heart drugs. By using the upcyte/EPCC (enhanced primary cell culture) approach for direct multiplication of organ-specific cells, we established proliferating human cardiomyocyte cultures derived from atrial appendages. For qualitative cardiac expression profiling we established a comprehensive set of multiplex PCR assays, selected from a panel of 32 genes, to rapidly screen changes at the transcriptional level in human ventricular and atrial cardiomyocytes. Our multiplex PCR approach revealed some donor variability of native atrial heart tissue that need to be confirmed by further studies with more samples. Our initial studies further indicated that characteristic heart muscle cell markers such as MLC-2a, MLC-2v, CHRM2, ADRB1, DES, EDRNB, Cx40 and KCNA5 were down-regulated when isolated cardiomyocytes were taken into primary cell culture. Compared to native heart tissue, proliferating atrial cardiomyocytes lacked expression of those cardiac markers but still expressed MYCD, GATA-4, Cx43, SERCA2, BNP, Tbx5, EDNRA and ACTB . Surprisingly, atrium-derived cardiomyocytes started to express NFAc4 in passage three, and cardiomyocyte marker expressions of Cx43 and BNP were even increased over cultivation time. In conclusion, our novel multiplex PCR assays should be useful for expression profiling of native heart tissues from patients with different disease conditions and for characterization of in vitro cardiomyocyte cultures.
http://content.iospress.com/articles/journal-of-cellular-biotechnology/jcb15025
Heart diseases are a major social and economic burden, as the human heart cannot regenerate after injury. Therefore, there is an urgent need for new therapeutic approaches to minimize, prevent, or reverse cardiac damage. In recent years stem cell-based approaches have gained considerable attention from scientists as well as the public. A publication by Orlic and coworkers describing that “locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease” initiated the area of stem cell-based cardiac therapy. However, subsequent work of several independent groups revealed that neither bone marrow stem cells nor other stem cells can significantly contribute to restore lost myocardium by differentiating into cardiomyocytes. Instead, Gnecchi and coworkers have suggested that the beneficial effect of stem cell-based therapies is predominantly due to bioactive molecules secreted by the transplanted stem cells. This hypothesis has been substantiated in the last decade by accumulating evidence that factors of the stem cell secretome promote cardiomyocyte survival and proliferation, modulate the immune system, have beneficial effects on cardiac metabolism, reduce cardiac remodeling, and induce angiogenesis. These findings raise the question if stem cells are needed or whether a detailed understanding of the stem cell secretome will allow a cell-free therapy for heart diseases.
In recent years, there has been a dramatic increase in research aimed at regenerating the mammalian heart by promoting endogenous cardiomyocyte proliferation. Despite many encouraging successes, it remains unclear if we are any closer to achieving levels of mammalian cardiomyocyte proliferation for regeneration as seen during zebrafish regeneration. Further, current cardiac regenerative approaches do not clarify whether the induced cardiomyocyte proliferation is an epiphenomena or responsible for the observed improvement in cardiac function. Moreover, due to the lack of standardized protocols to determine cardiomyocyte proliferation in vivo, it remains unclear if one mammalian regenerative factor is more effective than another. Here, we discuss current methods to identify and evaluate factors for the induction of cardiomyocyte proliferation and challenges therein. Addressing challenges in evaluating adult cardiomyocyte proliferation will assist in determining 1) which regenerative factors should be pursued in large animal studies, 2) if a particular level of cell cycle regulation presents a better therapeutic target than another (e.g. mitogenic receptors vs. cyclins), and 3) which combinatorial approaches offer the greatest likelihood of success. As more and more regenerative studies come to pass, progress will require a system that not only can evaluate efficacy in an objective manner but can also consolidate observations in a meaningful way.
Recent evidence has demonstrated that cardiac progenitor cells play an essential role in the induction of angiomyogenesis in infarcted myocardium. We and others have shown that engraftment of c-kit + cardiac stem cells (CSCs) into infarcted hearts led to myocardium regeneration and neovascularization, which was associated with an improvement of ventricular function. The purpose of this study is aimed at investigating the functional role of transcription factor (TF) Oct3/4 in facilitating CSCs to promote myocardium regeneration and preserve cardiac performance in the post-MI heart.
c-kit + CSCs were isolated from adult hearts and re-introduced into the infarcted myocardium in which the mouse MI model was created by permanent ligation of the left anterior descending artery (LAD). The Oct3/4 of CSCs was inhibited by transfection of Oct3/4 siRNA, and transfection of CSCs with control siRNA serves as control groups. Myocardial functions were evaluated by echocardiographic measurement. Histological analysis was employed to assess newly formed cardiogenesis, neovascularization, and cell proliferations. Terminal deoxynucleotidyltransferase (TdT) nick-end labeling (TUNEL) was carried out to assess apoptotic cardiomyocytes. Real time polymerase chain reaction and Western blot were carried out to evaluate the level of Oct 3/4 in CSCs.
Two weeks after engraftment, CSCs increased ventricular functional recovery as shown by a serial echocardiographic measurement, which is concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Suppression of Oct 3/4 of CSCs abrogated functional improvements and mitigated the hypertrophic response and cardiac remodeling. Transplantation of c-kit + CSCs into MI hearts promoted cardiac regeneration and neovascularization, which were abolished with the knockdown of Oct3/4. Additionally, suppression of Oct3/4 abrogated myocyte proliferation in the CSC-engrafted myocardium.
Our results indicate that CSCs-derived cardiac regeneration improves the restoration of cardiac function and is mediated through Oct 3/4.
Cardiovascular diseases are among the leading causes of death worldwide. In addition, congenital heart disease is the most common type of birth defect, affecting almost 1% of newborns. The primary cause of most heart diseases is the irreversible loss of heart muscle cells, the cardiomyocytes. Thus, it is critically important to develop novel strategies to treat heart disease. One promising strategy is cardiac tissue engineering (CTE). In this chapter, we review the advances of CTE in recent years, the special requirements for heart repair and the remaining challenges. Subsequently, we discuss the suitability of silk fibroin for CTE.
In mammals, cardiomyocytes rapidly proliferate in the fetus and continue to do so for a few more days after birth. These cardiomyocytes then enter into growth arrest but the detailed molecular mechanisms involved have not been fully elucidated. We have addressed this issue by comparing the transcriptomes of 2-day-old (containing dividing cardiomyocytes) with 13-day-old (containing growth arrested cardiomyocytes) postnatal mouse hearts. We performed comparative microarray analysis on the heart tissues and then conducted Functional annotation, Gene ontology, KEGG pathway and Gene Set enrichment analyses on the differentially expressed genes. The bioinformatics analysis revealed that gene ontology categories associated with the “cell cycle”, “DNA replication”, “chromosome segregation” and “microtubule cytoskeleton” were down-regulated. Inversely, “immune response”, “extracellular matrix”, “cell differentiation” and “cell membrane” were up-regulated. Ingenuity Pathways Analysis (IPA) has revealed that GATA4, MYH7 and IGF1R were the key drivers of the gene interaction networks. In addition, Regulator Effects network analysis suggested that TASP1, TOB1, C1orf61, AIF1, ROCK1, TFF2 and miR503-5p may be acting on the cardiomyocytes in 13-day-old mouse hearts to inhibit cardiomyocyte proliferation and G1/S phase transition. RT-qPCR was used to validate genes which were differentially expressed and genes that play a prominent role in the pathways and interaction networks that we identified. In sum, our integrative analysis has provided more insights into the transcriptional regulation of cardiomyocyte exit from the cell cycle during postnatal heart development. The results also pinpoint potential regulators that could be used to induce growth arrested cardiomyocytes to proliferate in the infarcted heart
The growth of the heart during mammalian embryonic development is primarily dependent on an increase in the number of cardiomyocytes (CM). However, shortly following birth, CMs cease proliferating and further growth of the myocardium is achieved via hypertrophic expansion of the existing CM population. The cyclin-dependent kinase inhibitor 2A (Cdkn2a) locus encodes overlapping genes for two tumor suppressor proteins, p16INK4a and p19 alternative reading frame (ARF). To determine whether decreased Cdkn2a gene expression results in improved cardiac regeneration in vitro and in vivo following cardiac injury, the proliferation of CMs isolated from Cdkn2a knockout (KO) and wild‑type (WT) mice in vitro and in vivo were evaluated following generation of ischemia reperfusion (IR) injury. The KO mice demonstrated enhanced CM proliferation not only in vitro, but also in vivo. Furthermore, heart function was improved and scar size was decreased in the KO mice compared with that of the WT mice. The results also indicated that microRNA (miR)‑1 and miR‑195 expression levels associated with cell proliferation were reduced following IR injury in KO mice compared with those of WT mice. These results suggested that the inactivation of INK4a and ARF stimulated CM proliferation and promoted cardiac repair.
Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk) disassembly precedes that of titin (M-line), suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble) and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1) induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.
MicroRNA (miRNA) directs post-transcriptional regulation of a network of genes by targeting mRNA. Although relatively recent in development, many miRNAs direct differentiation of various stem cells including induced pluripotent stem cells (iPSCs), a major player in regenerative medicine. An effective and safe delivery of miRNA holds the key to translating miRNA technologies. Both viral and nonviral delivery systems have seen success in miRNA delivery, and each approach possesses advantages and disadvantages. A number of studies have demonstrated success in augmenting osteogenesis, improving cardiogenesis, and reducing fibrosis among many other tissue engineering applications. A scaffold-based approach with the possibility of local and sustained delivery of miRNA is particularly attractive since the physical cues provided by the scaffold may synergize with the biochemical cues induced by miRNA therapy. Herein, we first briefly cover the application of miRNA to direct stem cell fate via replacement and inhibition therapies, followed by the discussion of the promising viral and nonviral delivery systems. Next we present the unique advantages of a scaffold-based delivery in achieving lineage-specific differentiation and tissue development.
Copyright © 2015. Published by Elsevier B.V.
In the United States, each year over 700,000 people suffer from a heart attack and over 25% of deaths are related to heart disease, making it the leading cause of death. Following ischemic injury a part of the heart muscle is replaced by a scar tissue, reducing its functioning capacity. Recent advancements in surgical intervention and pharmacotherapy only provide symptomatic relief and do not address the root cause of the problem which is the massive loss of cardiomyocytes (CM). Therefore, the development of novel therapeutic intervention for the repair and regeneration of ischemic myocardium remains an area of intense research. While existing CM in zebra fish and neonatal mice are known to proliferate and replenish the infarcted heart, it has been shown that adult mammalian CM lose this ability, thus preventing regeneration of the scar tissue. There have been many attempts to facilitate regeneration of ischemic heart but have met with limited success. Micro-RNAs (miRNAs) are one of the promising candidates towards this goal as they are known to play important regulatory roles during differentiation and tissue regeneration, and regulate genetic information by post-transcriptional modification as well as regulation of other miRNAs. While previous work by Eulalio et al., showed miRNAs inducing proliferation in neonatal CM (NCM), we here identify miRNAs inducing proliferation of rat adult-CM (ACM). This commentary while analyses recent work by Eulalio et al ([1]) also shows some new data with microRNAs in rat adult-CMs. Further work into the mechanism of these miRNAs can determine their therapeutic potential towards regenerating cardiac tissue post ischemic injury.
The present article investigates the use of a novel electrospun fibrous blend of poly(glycerol sebacate) (PGS) and poly(butylene succinate-co-butylene dilinoleate) (PBS-DLA) as a candidate for cardiac tissue engineering. Random electrospun fibers with various PGS/PBS-DLA compositions (70/30, 60/40, 50/50, and 0/100) were fabricated. In order to examine the suitability of these fiber blends for heart patches, their morphology, as well as their physical, chemical, and mechanical properties were measured prior to examining their biocompatibility through cell adhesion. The fabricated fibers were bead-free and exhibited a relatively narrow diameter distribution. The addition of PBS-DLA to PGS resulted in an increase of the average fiber diameter, while increasing the amount of PBS-DLA decreased the hydrophilicity and the water uptake of the nanofibrous scaffolds to values that approached those of neat PBS-DLA nanofibers. Moreover, the addition of PBS-DLA significantly increased the elastic modulus. Initial toxicity studies with C2C12 myoblast cells up to 72 hours confirmed non-toxic behavior of the blends. Immunofluorescence analyses and scanning electron microscopy analyses confirmed that C2C12 cells showed better cell attachment and proliferation on electrospun mats with higher PBS-DLA content. However, immunofluorescence analyses of the 3-days old rat cardiomyocytes cultured for 2 and 5 days demonstrated better attachment on the 70/30 fibers containing well-aligned sarcomeres and expressing high amounts of connexin 43 in cellular junctions indicating efficient cell-to-cell communication. It can be concluded, therefore, that the fibrous PGS/PBS-DLA scaffolds showed promising characteristics as a biomaterial for cardiac patch applications.
MicroRNAs (miRNAs) have emerged as potent modulators of mammalian gene expression, thereby broadening the spectrum of molecular mechanisms orchestrating human physiological and pathological cellular functions. Growing evidence suggests that these small non-coding RNA molecules are pivotal regulators of cardiovascular development and disease. Importantly, multiple miRNAs have been specifically implicated in the onset and progression of heart failure, thus providing a new platform for battling this multi-faceted disease. This review introduces the basic concepts of miRNA biology, describes representative examples of miRNAs associated with multiple aspects of HF pathogenesis, and explores the prognostic, diagnostic and therapeutic potential of miRNAs in the cardiology clinic.
During the maturation of the cardiac myocyte, a transition occurs from hyperplastic to hypertrophic growth. The factors that control this transition in the developing heart are unknown. Proto-oncogenes such as c-myc have been implicated in the regulation of cellular proliferation and differentiation, and in the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc can influence myocyte proliferation or differentiation, we examined the in vivo effect of increasing c-myc expression during embryogenesis and of preventing the decrease in c-myc mRNA expression that normally occurs during cardiac development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. In these transgenic mice, increased c-myc mRNA expression was found to be associated with both atrial and ventricular enlargement. This increase in cardiac mass was secondary to myocyte hyperplasia, with the transgenic hearts containing more than twice as many myocytes as did nontransgenic hearts. The results suggest that in the transgenic animals there is additional hyperplastic growth during fetal development. However, this additional proliferative growth is not reflected in abnormal myocyte maturation, as assessed by the expression of the cardiac and skeletal isoforms of alpha-actin. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth and suggest a regulatory role for this proto-oncogene in cardiac myogenesis.
Early in neonatal development, differentiated myocardial cells lose their ability to proliferate, and further enlargement of the heart occurs through hypertrophy of existing cardiac muscle cells. To study the process of myocardial growth and hypertrophy we have recently utilized a neonatal rat myocardial cell model (Lee, H. R., Henderson, S. A., Reynolds, R., Dunnmon, P., Yuan, D., and Chien, K. R. (1988) J. Biol. Chem. 263, 7352-7538). The present study was designed to determine if the expression of SV40 large T antigen would be capable of restoring the proliferative capacity of terminally differentiated neonatal rat myocardial cells. Utilizing a replication-defective recombinant human adenovirus which contains an SV40 early T antigen insert, maximal expression of T antigen was achieved at 24-48 h postinfection, with over 85-90% of the cells displaying positive T antigen staining. Furthermore, the expression of the T antigen-induced proliferation of the myocardial cells without the loss of expression of certain differentiated properties, including myosin light chain expression and assembly into organized myofibrils, spontaneous contractile activity, and a chronotropic response to adrenergic agonists. These results demonstrate the utility of recombinant human adenoviruses to achieve high efficiency transient expression of foreign genes in differentiated myocardial cells and suggest that the expression of T antigen may provide a suitable model to study the biochemical events which are required to maintain the proliferative capacity of myocardial cells.
Basic fibroblast growth factor (bFGF) is a potent mitogen for many cell lineages including fetal cardiomyocytes. Furthermore,
bFGF has been shown to modify gene expression, in vitro, in adult nonproliferative ventricular myocytes. This effect is suspected to be partly responsible for the genetic modifications
that occur in vivo under pathophysiological conditions such as ischemia or pressure overload and that lead to myocardial hypertrophy. However,
little is known about the first steps of the molecular mechanisms that take place soon after cell activation by bFGF. In this
study, using biochemical and electrophysiological approaches, we have established, on cardiomyocytes cultured from neonatal
rat ventricles, that (i) differentiated beating cells express at least two classes of bFGF-receptors having high and low affinity
(K = 10 ± 2 pM and 1 ± 0.5 nM); (ii) the stimulation of these bFGF receptors promotes an increase in the beating frequencies
of cultured cardiomyocytes (40 ± 10%); (iii) bFGF provokes the activation of poorly specific and voltage-independent calcium
channels (12pS); (iv) inositol 1,4,5-trisphosphate enhances similar bFGF-induced Ca currents and is therefore suspected to be a second messenger triggering this activation. These results support the presence,
in cultured cardiomyocytes, of new calcium channels whose activation after bFGF binding may be partly responsible for the
cell response to this growth factor.
Heart failure can result from a variety of causes, including ischemic, hypertensive, toxic, and inflammatory heart disease. However, the cellular mechanisms responsible for the progressive deterioration of myocardial function observed in heart failure remain unclear and may result from apoptosis (programmed cell death).
We examined seven explanted hearts obtained during cardiac transplantation for evidence of apoptosis. All seven patients had severe chronic heart failure: four had idiopathic dilated cardiomyopathy, and three had ischemic cardiomyopathy. DNA fragmentation (an indicator of apoptosis) was identified histochemically by in situ end-labeling as well as by agarose-gel electrophoresis of end-labeled DNA. Myocardial tissues obtained from four patients who had had a myocardial infarction one to two days previously were used as positive controls, and heart tissues obtained from four persons who died in motor vehicle accidents were used as negative controls for the end-labeling studies.
Hearts from all four patients with idiopathic dilated cardiomyopathy and from one of the three patients with ischemic cardiomyopathy had histochemical evidence of DNA fragmentation. All four myocardial samples from patients with dilated cardiomyopathy also demonstrated DNA laddering, a characteristic of apoptosis, whereas this was not seen in any of the samples from patients with ischemic cardiomyopathy. Histological evidence of apoptosis was also observed in the central necrotic zone of acute myocardial infarcts, but not in myocardium remote from the infarcted zone. Rare isolated apoptotic myocytes were seen in the myocardium from the four persons who died in motor vehicle accidents.
Loss of myocytes due to apoptosis occurs in patients with end-stage cardiomyopathy and may contribute to progressive myocardial dysfunction.
Terminal differentiation of cardiac myocyte is associated with their permanent withdrawal from the cell cycle. In adult end-stage heart failure, significant numbers of myocytes express proliferating cell nuclear antigen yet fail to progress to cell division. Cyclin dependent kinase inhibitors are powerful inhibitors of the cell cycle and may play a direct role both in myocyte development and in preventing cell division in the adult.
The expression of the CIP/KIP cyclin dependent kinase inhibitors p21, p27, p57 and the retinoblastoma protein was examined in acute (seen in brain dead transplant donors) and end-stage heart failure by Western blot analysis and compared to that seen in human and rat cardiac development. The expression of p21 showed a gradual increase during development in both rat and man, becoming maximal in adulthood p27. levels showed an initial rise with subsequent continual expression throughout life. p57 expression was detectable at only early stages in rat but persisted throughout life in man. In both acute and end-stage heart failure the levels of p21, p27 and p57 reverted to a pattern similar to that observed in human fetal heart: p21 and p27 declined while p57 expression was significantly increased. In contrast, retinoblast protein levels declined during human heart development but were unaltered in heart failure.
The expression of p21, but not p27 or p57, is consistent with a role in the gradual withdrawal of cardiac myocytes from cell cycle during development. In adult heart failure cyclin dependent kinase inhibitor expression reverts to the fetal pattern but is insufficient to initiate cell cycle activation.
Left ventricular remodeling is a major cause of progressive heart failure and death after myocardial infarction. Although neoangiogenesis within the infarcted tissue is an integral component of the remodeling process, the capillary network is unable to support the greater demands of the hypertrophied myocardium, resulting in progressive loss of viable tissue, infarct extension and fibrous replacement. Here we show that bone marrow from adult humans contains endothelial precursors with phenotypic and functional characteristics of embryonic hemangioblasts, and that these can be used to directly induce new blood vessel formation in the infarct-bed (vasculogenesis) and proliferation of preexisting vasculature (angiogenesis) after experimental myocardial infarction. The neoangiogenesis resulted in decreased apoptosis of hypertrophied myocytes in the peri-infarct region, long-term salvage and survival of viable myocardium, reduction in collagen deposition and sustained improvement in cardiac function. The use of cytokine-mobilized autologous human bone-marrow-derived angioblasts for revascularization of infarcted myocardium (alone or in conjunction with currently used therapies) has the potential to significantly reduce morbidity and mortality associated with left ventricular remodeling.
The scarring of the heart that results from myocardial infarction has been interpreted as evidence that the heart is composed of myocytes that are unable to divide. However, recent observations have provided evidence of proliferation of myocytes in the adult heart. Therefore, we studied the extent of mitosis among myocytes after myocardial infarction in humans.
Samples from the border of the infarct and from areas of the myocardium distant from the infarct were obtained from 13 patients who had died 4 to 12 days after infarction. Ten normal hearts were used as controls. Myocytes that had entered the cell cycle in preparation for cell division were measured by labeling of the nuclear antigen Ki-67, which is associated with cell division. The fraction of myocyte nuclei that were undergoing mitosis was determined, and the mitotic index (the ratio of the number of nuclei undergoing mitosis to the number not undergoing mitosis) was calculated. The presence of mitotic spindles, contractile rings, karyokinesis, and cytokinesis was also recorded.
In the infarcted hearts, Ki-67 expression was detected in 4 percent of myocyte nuclei in the regions adjacent to the infarcts and in 1 percent of those in regions distant from the infarcts. The reentry of myocytes into the cell cycle resulted in mitotic indexes of 0.08 percent and 0.03 percent, respectively, in the zones adjacent to and distant from the infarcts. Events characteristic of cell division--the formation of the mitotic spindles, the formation of contractile rings, karyokinesis, and cytokinesis--were identified; these features demonstrated that there was myocyte proliferation after myocardial infarction.
Our results challenge the dogma that the adult heart is a postmitotic organ and indicate that the regeneration of myocytes may be a critical component of the increase in muscle mass of the myocardium.
Cell proliferation and patterning must be coordinated for the development of properly proportioned organs. If the same molecules were to control both processes, such coordination would be ensured. Here we address this possibility in the Drosophila wing using the Dpp signaling pathway. Previous studies have shown that Dpp forms a gradient along the AP axis that patterns the wing, that Dpp receptors are autonomously required for wing cell proliferation, and that ectopic expression of either Dpp or an activated Dpp receptor, Tkv(Q253D), causes overgrowth. We extend these findings with a detailed analysis of the effects of Dpp signaling on wing cell growth and proliferation. Increasing Dpp signaling by expressing Tkv(Q253D) accelerated wing cell growth and cell cycle progression in a coordinate and cell-autonomous manner. Conversely, autonomously inhibiting Dpp signaling using a pathway specific inhibitor, Dad, or a mutation in tkv, slowed wing cell growth and division, also in a coordinate fashion. Stimulation of cell cycle progression by Tkv(Q253D) was blocked by the cell cycle inhibitor RBF, and required normal activity of the growth effector, PI3K. Among the known Dpp targets, vestigial was the only one tested that was required for Tkv(Q253D)-induced growth. The growth response to altering Dpp signaling varied regionally and temporally in the wing disc, indicating that other patterned factors modify the response.
Aims
Terminal differentiation of cardiac myocyte is associated with their permanent withdrawal from the cell cycle. In adult end-stage heart failure, significant numbers of myocytes express proliferating cell nuclear antigen yet fail to progress to cell division. Cyclin dependent kinase inhibitors are powerful inhibitors of the cell cycle and may play a direct role both in myocyte development and in preventing cell division in the adult.
Methods and Results
The expression of the CIP/KIP cyclin dependent kinase inhibitors p21, p27, p57 and the retinoblastoma protein was examined in acute (seen in brain dead transplant donors) and end-stage heart failure by Western blot analysis and compared to that seen in human and rat cardiac development. The expression of p21 showed a gradual increase during development in both rat and man, becoming maximal in adulthood. p27 levels showed an initial rise with subsequent continual expression throughout life. p57 expression was detectable at only early stages in rat but persisted throughout life in man. In both acute and end-stage heart failure the levels of p21, p27 and p57 reverted to a pattern similar to that observed in human fetal heart: p21 and p27 declined while p57 expression was significantly increased. In contrast, retinoblast protein levels declined during human heart development but were unaltered in heart failure.
Conclusions
The expression of p21, but not p27 or p57, is consistent with a role in the gradual withdrawal of cardiac myocytes from cell cycle during development. In adult heart failure cyclin dependent kinase inhibitor expression reverts to the fetal pattern but is insufficient to initiate cell cycle activation.
Proliferation of mammalian cardiomyocytes ceases around birth when a transition from hyperplastic to hypertrophic myocardial growth occurs. Previous studies demonstrated that directed expression of the transcription factor E2F1 induces S-phase entry in cardiomyocytes along with stimulation of programmed cell death. Here, we show that directed expression of E2F2 and E2F4 by adenovirus mediated gene transfer in neonatal cardiomyocytes induced S-phase entry but did not result in an onset of apoptosis whereas directed expression of E2F1 and E2F3 strongly evoked programmed cell death concomitant with cell cycle progression. Although both E2F2 and E2F4 induced S-phase entry only directed expression of E2F2 resulted in mitotic cell division of cardiomyocytes. Expression of E2F5 or a control LacZ-Adenovirus had no effects on cell cycle progression. Quantitative real time PCR revealed that E2F1, E2F2, E2F3, and E2F4 alleviate G0 arrest by induction of cyclinA and E cyclins. Furthermore, directed expression of E2F1, E2F3, and E2F5 led to a transcriptional activation of several proapoptotic genes, which were mitigated by E2F2 and E2F4. Our finding that expression of E2F2 induces cell division of cardiomyocytes along with a suppression of proapoptotic genes might open a new access to improve the regenerative capacity of cardiomyocytes.
Stem cells are the building blocks through which tissues are developed and maintained. We and many other groups predict that stem cells will prove tremendously useful in clinical medicine. Possible uses include systems for high- throughput drug screens, as in vitro models of disease and, eventually, in treating diseases associated with cell defi- ciency. As one of the least regenerative organs in the body, the heart stands to benefit greatly from addition of new parenchymal cells. Cardiovascular researchers have risen to this challenge and, as a result, cardiac repair is arguably the most advanced program in the emerging field of regenera- tive medicine. Progress in this field has been rapid, from humble beginnings with committed skeletal (1-3) or cardiac muscle cells (4-6), moving to multipotent adult stem cells (7-9) and, most recently, to embryonic stem cells (10-13). In this brief commentary, we review some important recent developments in stem cell-based tissue repair. (Read- ers wishing additional basic and clinical information are directed to several recent in-depth reviews (14-16) on the field.) Like other areas involving stem cell-based regeneration, the field of cardiac repair has its share of controversies. These will also be touched upon, with an aim of separating experi- mental observation, on which there is much agreement, from interpretation, which varies widely at the moment.
Overexpression of the c-mycprotooncogene in the heart of transgenic mice has been demonstrated to result in cardiac enlargement due to increased myocyte hyperplasia during the fetal period. To determine the age of completion of the proliferative phase of myocyte growth in neonatal mice with c-mycoverexpression, we used a transgenic (TG) mouse model in which c-mycoverexpression is limited to the heart. Bromodeoxyuridine (BrdU) was given to TG and wild type (WT) mice (n=3/group) at 1, 2, 3, 5, 7, 10, 14, 16, 18 and 20 days of age to identify cells in S-phase of the cell cycle. Increased cardiac mass was present in TG compared to WT mice at all time periods (P<0.05). Using computer assisted image analysis, myocardial total nuclear density (NT) in TG mice was 7–31% greater in both the left ventricle (LV) and the interventricular septum (IVS) than in WT at all ages (P<0.05), indicative of a smaller myocyte size. In WT mice, the labeling index (LI) remained almost constant at ≈11–12% until 7 days of age, and then rapidly dropped to ≈2% by 14 days and to less than 1% by 20 days. In contrast, LI in TG dropped continuously from birth to ≈4% at 7 days and ≈2% at 10 days of age (P<0.001). Thus, overexpression of the c-mycprotooncogene is associated with enhanced hyperplastic growth of the heart during fetal development, and accelerated neonatal conversion to hypertrophic myocyte growth.
A study was made of the response of adult newt ventricle to injury. The major events in the repair process after wounding involve blood clot formation, coagulation necrosis, macrophagic activity, regenerative activity of heart muscle, and connective tissue formation. Light microscopic autoradiography indicated that there was a definite area of labeled cells in trabeculae adjacent to the wound, beginning at about ten days after injury. Electron microscopic autoradiography confirmed that myocytes containing myofibrillae underwent DNA synthesis. It was concluded that adult newt myocardium is capable of reacting to injury by the proliferation of myocytes at the wound area as indicated by both DNA synthesis and mitosis.
Basierend auf einem Untersuchungsgut von etwa 720 000 zeichnerisch erfaten Zellen berichtet der Autor ber die mitotische Aktivitt des Hhnchenherzens whrend der gesamten Embryonal- und Fetalentwicklung. Die fr die einzelnen Myokard- und Endokardabschnitte ermittelten Mitosequoten knnen als ein ungefhres Ma der Wachstumsintensitt, damit auch der Schdigungsbereitschaft dieser Gewebsbezirke gelten und ermglichen durch quantitative Analyse der Wachstumsvorgnge eine differenzierte Stellungnahme zu Fragen der formalen und zeitlichen Entstehung von Herzfehlern.1.
Im Gegensatz zu frheren Ansichten erscheint die Annahme einer entscheidenden entwicklungsmechanischen Bedeutung der bulbotrunkalen Endokardkissen bei der Entstehung von Entwicklungsstrungen des arteriellen Herzendes wenig wahrscheinlich. Vielmehr lt eine Reihe von Einzelbefunden, besonders die signifikant hhere Anzahl der Kernteilungsfiguren im Myokardgewebe gegenber den im Endokard beobachteten Mitoseraten whrend der Primrperiode den Schlu zu, da der urschlich bestimmende Faktor in der Herzfehlergenese in einer, wenn auch nicht ausschlielichen, so doch berwiegenden Schdigung der myokardialen Herzanteile zu suchen ist.
2.
Defekte des myokardialen Septum ventriculorum sind indirekt ber eine Wachstumsstrung der seitlichen Kammerwandabschnitte entstanden zu denken. In dieser Richtung wird jedenfalls die in den ersten Tagen der Septation auffallend geringe mitotische Aktivitt im Septummyokard bei erheblich hherer Wachstumsintensitt der parietalen Kammerbezirke zu deuten sein. Damit kann gleichzeitig der von Kl. Goerttler (1958) vermutete Entstehungsmechanismus des Ventrikelseptum als passive lumenwrtige Einstlpung eines Schlauchteiles durch Krmmungen der Herzschleife direkt bewiesen werden.
3.
Die bisher nur experimentell erfolgte Festlegung einer sensiblen Phase der Herzentwicklung mit erhhter Schdigungsbereitschaft in den ersten Bebrtungstagen konnte durch Auswertung der Mitosehufigkeit substantiiert werden: hohe Mitosequoten in dieser Primrperiode sprechen fr eine besondere Empfindlichkeit und lassen uns die Entstehung von Entwicklungsstrungen verstndlich werden. In erster Linie drfte das Kammermyokard betroffen sein, weniger stark und hufig dagegen das Vorhofmyokard und die Endokardwlste im Bulbus und Ohrkanal.
4.
Jenseits der sensiblen Phase der Herzentwicklung ist eine differente Beeintrchtigung einzelner Herzteile und -gewebe weniger wahrscheinlich.
5.
Mit der Festsetzung des Mitoseindex im Normbereich wurde exemplarisch die Mglichkeit geschaffen, das Ausma einer Wachstumsverlangsamung und damit den Grad einer Schdigung durch Vergleich mit den als Normwerten ermittelten Quoten quantitativ zu erfassen. Mit gleicher Methodik ist es mglich, die Empfindlichkeit aller Organe und Gewebe des Organismus nach Magabe ihrer mitotischen Aktivitt, d.h. fr ein Kriterium der Wachstumsaktivitt, numerisch zu erfassen, die dann als Grundlage fr anzustellende Schdigungsversuche dienen kann. Fernziel dieser und hnlicher Untersuchungen mu die Aufstellung einer exakten Zeittafel mit Einarbeitung der relativen Empfindlichkeiten einzelner Organe bzw. Organteile und Gewebe sein.
MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the expression of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in mammals through targeted deletion remains unknown, and reliable prediction of mRNA targets is still problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell-cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 "seed matches" and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets.
Growth of human diploid fibroblasts in the presence of 5-bromodeoxyuridine, followed by flow cytometric analysis of DNA-specific fluorescence with Hoechst 33258 dye, allows quantitation of the proportion of cells that have not cycled, as well as those in G1 and G2 of two subsequent cell cycles. This technique allows rapid and accurate quantitation of the growth fraction and G1/S transition rate of these cells. The cell cycle kinetics of human diploid fibroblasts at all population doubling levels reveal two components: cycling cells showing a probabilistic rate of G1/S transition, and a variable proportion of noncycling cells. Both the transition probability (rate of exit from G1) and the noncycling proportion of cells change systematically as a function of serum concentration and as a function of population doubling level. The data suggest the existence of an underlying heterogeneity in the population of human diploid fibroblasts with respect to the capacity to divide in the presence of a given concentration of mitogen. Models of cell cycle kinetics must be modified to include regulation of growth by changes in the fraction of cycling cells, as well as by changes in the rate of exit from G1.
Proliferation of mammalian cardiomyocytes ceases around birth when a transition from hyperplastic to hypertrophic myocardial growth occurs. Previous studies demonstrated that directed expression of the transcription factor E2F1 induces S-phase entry in cardiomyocytes along with stimulation of programmed cell death. Here, we show that directed expression of E2F2 and E2F4 by adenovirus mediated gene transfer in neonatal cardiomyocytes induced S-phase entry but did not result in an onset of apoptosis whereas directed expression of E2F1 and E2F3 strongly evoked programmed cell death concomitant with cell cycle progression. Although both E2F2 and E2F4 induced S-phase entry only directed expression of E2F2 resulted in mitotic cell division of cardiomyocytes. Expression of E2F5 or a control LacZ-Adenovirus had no effects on cell cycle progression. Quantitative real time PCR revealed that E2F1, E2F2, E2F3, and E2F4 alleviate G0 arrest by induction of cyclinA and E cyclins. Furthermore, directed expression of E2F1, E2F3, and E2F5 led to a transcriptional activation of several proapoptotic genes, which were mitigated by E2F2 and E2F4. Our finding that expression of E2F2 induces cell division of cardiomyocytes along with a suppression of proapoptotic genes might open a new access to improve the regenerative capacity of cardiomyocytes.
The paper summarizes the results of the cardiac myogenesis and regeneration obtained by light and electron microscopical techniques, by thymidine 3H autoradiography and by the evaluation of the nuclear kinetic of myocytes. About 380 papers are cited covering following topics: 1) Differentiative Properties of Cardiac Myocytes; 2) Cell Proliferation of Cardiac Myogenesis; 3) Reactivation of Hyperplasia of Cardiac Muscle Cells and their Participation in Myocardial Regeneration; 4) Neoplastic Transformation of Cardiac Myocytes; 5) Proliferative Behavior of Cardiac Myocytes n vitro. Normal mitotic division of myocytes (without dedifferentiation) was found in the heart both during the development and in the regenerating or in the overloaded adult myocardium; the mitose being facilitated by the gradual prolongation of the mitotic cycle phases and by the specific organisation of organelles during the mitosis. The mitotic activity was found to be greater: in the immature myocardium than in the adult; in the lower vertebrates than in the mammals; and in the atria than in the ventricles. Binucleate myocytes and increased ploidy which has been found in man are explained by disturbances in the mitotic cycle and by the blockade of the cytotomy. Local injury of the ventricular myocardium induce the increased mitotic activity within the whole myocardium, including the atria in which the activity attended the maximal value. Even the specialized myocytes of the conducting system revealed increased DNA synthesis after this injury.
The regenerative response of minced cardiac muscle grafts in the adult newt was studied using autoradiography and electron microscopy. One-sixteenth to one-eighth of the newt ventricle was amputated, minced, and returned to the wounded ventricle. At five days after grafting, no reorganization of graft msucle pieces was apparent and there was degeneration of much of the muscle graft. Another, smaller population of 5-day myocytes had euchromatic nuclei and intact sarcolemmae. In 10- and 16-day grafts, continuity between ventricular and graft lumina was established and coalescence of graft pieces was apparent. Ultrastructurally, 10- and 16-day graft myocytes appeared to have fewer myofibrillae and increased amounts of rough endoplasmic reticulum, polyribosomes, Golig complexes, and dense bodies when compared to uninjured ventricular myocytes. The peak of proliferative activity of graft cells was observed at 16 days. Electron microscopic autoradiography revealed breadkdown of myofibrillar structure in labeled myocytes, whereas in myocytes in the later stages of mitosis only scattered myofilaments and no Z bands were present. By 30 days, grafts appeared as an integrated structure composed primarily of cardiac muscle. Myocytes of 30-day grafts were observed in various stages of myofibrillogenesis and contained numberous 10-nm filaments. Seventy-day graft mycoytes had numberous well organized myofibrillae and intercellular junctions similar to those seen in uninjured ventricular myocytes.
Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis.
To determine whether myocyte mitotic division occurs in the adult mammalian heart and whether this cellular process is affected by aging, we measured the percentage of myocyte nuclei showing metaphase chromosomes in myocytes isolated from the left and right ventricles of rats at 8-12, 19-24, and 28-32 months after birth. Metaphase chromosomes were found at all ages in both ventricles. However, from 8-12 to 28-32 months, the fraction of nuclei exhibiting metaphase chromosomes increased 6.3-fold and 2.3-fold in the left and right ventricles, respectively. Thus, myocyte cellular hyperplasia is present in the adult and aging myocardium as a compensatory mechanism to regenerate tissue mass and recover function, which are lost with the progression of life and senescence.
Summary Ventricular heart cultures were prepared from newborn rats and examined in Rose chambers by high resolution, phase-contrast
optics. Details of the procedure are given for obtaining a high yield of myocardial or endothelial cells. Photographic records
were obtained of living cells by using photomicrography, cinematography, and high speed filming to document the cytological
differences between endothelial and myocardial cells, details of mitosis in the two cell types, and patterns of contractility.
Myocardial cells can be distinguished from endothelial cells in the living state by the following criteria; dense cytoplasm;
giant and pleomorphic mitochondria or sarcosomes: presence of small, round nuclei; binucleation; one or two round, dense nucleoli;
a specialized Golgi apparatus around the nucleus; myofobrils; intercalated discs; myopodia; small, cell size; failure of cell
migration; spontaneous contractions; formation of synchronized networks; and flattened appearance with adhesion to other cells
during mitosis. Contracting myocardial cells are shown to undergo mitosis. Contractions become weaker at metaphase and then
cease at anaphase. Daughter cells may resume contractions. Organized myofibrils are generally not detected during the mitotic
periods when contractions are minimal or absent. It is proposed that myofibrils undergo a transient disorganization during
mid-mitosis and become reorganized in the daughter cell(s). It is suggested that there is a competition for energy at the
time of spindle changes and chromosome movements, so that priority is given to the mitotic process rather than to myofibrillar
contractions. Myofibrillogenesis is considered to be a relatively rapid process. The average mitotic time is 2.5 hr fore endothelial
cells and 6.1 hr for myocardial cells. Failure of cytokinesis frequently occurs in dividing myocardial cells and results in
the formation of binucleated cells. A sequence, is presented of a binucleated myocardial cell which undergoes an abormal multipolar
mitosis, leading to polynucleation. Myocardial cells incorporate tritiated thymidine into nuclear DNA. The question of mitosis
and differentiation of myocardial cells is reviewed. It is concluded that myocardial cells can no longer be cited in support
of the dogma, that differentiated cells do no divide. However, these is a temporary competition between the two phenomena
at the metaphase-anaphase stages.
The fine structure of mitosis in myocardial cells possessing myofibrils from embryonic chick hearts (4.5–6 days incubation age) is described. In these cells during prophase, chromosomes appear, but the nuclear membrane, Golgi complexes, and Z bands mostly disappear. In metaphase, lipid droplets and large quantities of membranous cisternae localize around the centrioles, while sarcomeric lengths of myofibrils (largely without Z bands) are found disoriented in the periphery, mainly in the polar zones. In anaphase the cisternae collect near the chromosomes, as reconstruction of the nuclear membrane commences. An intact nuclear envelope and well-formed Golgi complexes characterize telophase, but Z bands still are not present. During all of the mitotic stages, junctional complexes between dividing cells and adjacent resting cells persist.
The ultrastructure of the myocyte at all phases of mitosis as well as of early postmitotic cells has been studied in the myocardia of 14- and 18-day rat embryos and 5- and 7-day old rats. The myofibrils remain unchanged up to the late prophase. In prometaphase the majority of Z-disks in embryo myocyte myofibrils and considerable part of these disks in myofibrils of suckling rats are drastically disintegrated.
This is followed by a progressive isolation and scattering of the myofilament bundles and of the whole sarcomeres during the subsequent phases of mitosis. Thick myofilaments seem to be unchanged but thin ones become frequently poorly outlined (mainly in embryos). The sarcoplasmic reticulum, including its typically differentiated subsarcolemmal cisternae, exhibits relatively few changes during mitosis. In the early postmitotic period there is a gradual restoration of contrast-rich Z-bands, interconnecting the previously isolated sarcomeres. Patterns of this process have much in common with early stages of myofibrillogenesis (appearance of subsarcolemmal “Z-bodies”, formation of skeins of thin filaments etc.). The cleavage furrow formation is either absent or considerably retarded up to the postmitotic period.
Behaviour of some other organelles during myocyte mitosis has been described. Possible mechanisms and significance of the observed phenomena are discussed.
To better understand the mechanisms governing the proliferation of cardiac myocytes it is important to identify the factors controlling this phenomenon, and to characterize their actions. DNA synthesis was quantified in vitro in ventricular myocytes from the adult red-spotted newt, Notophthalmus viridescens. Ventricles were enzymatically separated and plated onto laminin. Myocytes were fed modified L-15 medium with 10% fetal bovine serum, and were variously treated with transforming growth factor-beta, transforming growth factor-beta combined with platelet-derived growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, 12-0-tetradecanoylphorbol-13-acetate, heparin, or conditioned medium from ventricular myocytes or non-myocytes (primarily endothelial cells). With their final feeding the cells were given 1 mu Ci/ml of tritiated thymidine, and 24 hours later were fixed and stained. Dishes were coated with photographic emulsion, exposed, and developed. The percent of cells with labeled nuclei was determined. Experimental media that significantly increased DNA synthesis included those containing acidic fibroblast growth factor (121% of control), basic fibroblast growth factor (119% of control), 12-0-tetradecanoylphorbol-13-acetate (233% of control) and conditioned medium from ventricular myocytes (230% of control) or non-myocytes (128% of control). Media significantly inhibiting DNA synthesis were those containing heparin (31% of control), transforming growth factor-beta (38% of control), non-myocyte conditioned medium and heparin (75% of control), or transforming growth factor-beta and platelet-derived growth factor (63% of control).
Proliferating cell nuclear antigen (PCNA) is a late growth-regulated gene that is expressed at the G1-S boundary of the cell cycle and is required for DNA synthesis and cell proliferation. Since quantitative results suggest that myocyte hyperplasia occurs in the decompensated human heart, we postulated that induction of the PCNA gene may be present in the failing heart in humans. PCNA protein was detected in myocardial samples obtained from the left and right ventricles of patients with congestive heart failure. Endomyocardial biopsies collected from donor subjects were used as control tissue. The percentage of positively stained myocyte nuclei in the ventricles was established by using PCNA monoclonal antibody and the immunoperoxidase technique. The localization of PCNA in myocytes was confirmed by alpha-sarcomeric actin antibody staining. PCNA labeling was present in left ventricular myocytes of 29 of the 32 hearts examined. In the right ventricle, 24 of the 29 samples showed positive staining. In a subset of 25 patients, the percentage of PCNA-labeled myocyte nuclei was measured and found to constitute 49 +/- 22% of left ventricular myocytes. A similar analysis for the right ventricle, conducted in 21 patients, showed that 49 +/- 19% of the myocyte nuclei exhibited PCNA protein. In addition, mitotic figures in myocytes were documented. A quantitative analysis of this cellular process revealed that 11 myocyte nuclei per 1 million cells exhibited mitotic images in chronic heart failure. Immediately after myocardial infarction, two cells per million showed mitotic division, and this phenomenon was restricted to the region adjacent to the necrotic tissue. No PCNA labeling or nuclear mitotic images were detected in the ventricular myocardium of control subjects. Thus, the observation that diffuse PCNA labeling and myocyte mitotic division are present in hearts with end-stage failure strongly suggests that adult ventricular myocytes are not terminally differentiated cells and that myocyte cellular hyperplasia may constitute a growth reserve mechanism of the diseased heart.
Fetal cardiomyocytes isolated from transgenic mice carrying a fusion gene of the alpha-cardiac myosin heavy chain promoter
with a beta-galactosidase reporter were examined for their ability to form stable intracardiac grafts. Embryonic day 15 transgenic
cardiomyocytes delivered directly into the myocardium of syngeneic hosts formed stable grafts, as identified by nuclear beta-galactosidase
activity. Grafted cardiomyocytes were observed as long as 2 months after implantation, the latest date assayed. Intracardiac
graft formation did not induce overtly negative effects on the host myocardium and was not associated with chronic immune
rejection. Electron microscopy revealed the presence of nascent intercalated disks connecting the engrafted fetal cardiomyocytes
and the host myocardium. These results suggest that intracardiac grafting might provide a useful approach for myocardial repair,
provided that the grafted cells can contribute to myocardial function.
To determine whether the molecular components implicated in the regulation of the cell cycle are activated in myocytes after infarction, the expression of cyclins E, A, and B and the levels of their associated kinase activity were measured at 1 and 7 days following surgery. The quantity of cdk2 and cdc2 and the level of their kinase activity were also determined. Myocardial infarction was characterized by an increase in cyclins E, A, and B and cdc2 proteins in the surviving myocytes at 1 and 7 days. Cyclin E, A, and B and cdk2 and cdc2 kinase activity also increased. The quantity of cyclins E and A and the level of cyclin E-associated kinase activity in myocytes after infarction were comparable with those measured in neonatal myocytes. Moreover, cdc2 protein and cdc2 kinase activity in myocytes reached levels after infarction which were similar to those in neonatal myocytes. Thus, myocytes react to myocardial infarction by activating cyclins and cyclin-dependent kinases which may be coupled with the regeneration of muscle mass and recovery of ventricular function.
To determine whether apoptotic and necrotic myocyte cell death occur acutely and chronically after infarction, the formation of DNA strand breaks and the localization of myosin monoclonal antibody labeling were analyzed in the surviving myocardium from 20 min to 1 month. DNA strand breaks in myocyte nuclei were detected as early as 3 h following coronary artery occlusion and were still present at 1 month. This cellular process was characterized biochemically by internucleosomal DNA fragmentation which produced DNA laddering on agarose gel electrophoresis. Quantitatively, 155 myocyte nuclei per 10(6) cells exhibited DNA strand breaks in the portion adjacent to the infarcted tissue at 3-12 h. This parameter increased to 704 at 1-2 days and subsequently decreased to 364 at 7 days, 188 at 14 days, and 204 at 1 month. In the remote myocardium, the number of myocyte nuclei with DNA strand breaks was 84 per 10(6) at 3-12 h and remained essentially constant up to 1 month. Programmed myocyte cell death was accompanied by a decrease in the expression of bcl-2 and an increase in the expression of bax. The changes in the expression of these genes were present at 1 and 7 days after coronary artery occlusion. In conclusion, the mechanical load produced by myocardial infarction and ventricular failure may affect the regulation of bcl-2 and bax in the viable myocytes, triggering programmed cell death and the remodeling of the ventricular wall.
We postulated that the cyclin-dependent kinase inhibitors p21 and p27 could regulate the alterations in growth potential of cardiomyocytes during left ventricular hypertrophy (LVH). LVH was induced in adult rat hearts by aortic constriction (AC) and was monitored at days 0, 1, 3, 7, 14, 21, and 42 postoperation. Relative to sham-operated controls (SH), left ventricle (LV) weight-to-body weight ratio in AC increased progressively with time without significant differences in body weight or right ventricle weight-to-body weight ratio. Atrial natriuretic factor mRNA increased significantly in AC to 287% at day 42 compared with SH (P < 0.05), whereas p21 and p27 mRNA expression in AC rats decreased significantly by 58% (P < 0.03) and 40% (P < 0.05) at day 7, respectively. p21 and p27 protein expression decreased significantly from days 3 to 21 in AC versus SH, concomitant with LV adaptive growth. Immunocytochemistry showed p21 and p27 expression in cardiomyocyte nuclei. Thus downregulation of p21 and p27 may modulate the adaptive growth of cardiomyocytes during pressure overload-induced LVH.
The cultured adult newt ventricular myocyte has been shown to undergo mitosis and cytokinesis in a fully differentiated state. Insight into its proliferation and cellular changes during the repair process involves obtaining a better understanding of the nuclear pattern (mononucleated, binucleated, or multinucleated) resulting from mitotic events. Mitosis is easily observable in cultured newt cardiac myocytes using phase-contrast microscopy.
From days 8-19 in culture, the process of mitosis in mononucleated and binucleated newt ventricular myocytes was recorded and timed by using time-lapse video microscopy. Cultured cardiac myocytes were double-stained for myosin and F-actin by using fluorescein isothiocyanate (FITC)-labeled MF20 and rhodamine phalloidin.
Mitotic, mononucleated myocytes produced mononucleated daughter cells in 80% of the cases, whereas 20% were single, binucleated myocytes, In binucleated myocytes, only 32% underwent complete cytokinesis to produce two binucleated daughter cells, whereas 68% resulted in variably nucleated myocytes. Mononucleated and binucleated myocytes undergoing mitosis had similar time intervals for the period from nuclear breakdown (prometaphase) to the start of anaphase (108.7 minutes and 94.5 minutes, respectively), but the period between anaphase and midbody formation was significantly shorter in binucleated than in mononucleated myocytes (43.5 minutes and 69.3 minutes, respectively). The myofibrillae were not as well organized in binucleated myocytes as those observed in mononucleated myocytes.
Mitosis in vitro appears to proceed more rapidly in binucleated newt cardiac myocytes, which have more poorly organized myofibrillae than mononucleated myocytes. Mitosis of cultured binucleated myocytes commonly results in variably nucleated daughter cells, whereas mononucleated myocytes produce predominantly mononucleated daughter cells.
Increases in cardiac mass during fetal life arise predominantly as a consequence of cardiomyocyte proliferation. During neonatal life, there is a transition from hyperplastic to hypertrophic growth such that further increases in myocardial mass are typically not accompanied by cardiomyocyte proliferation. In the adult myocardium, it is generally believed that the vast majority of cardiomyocytes do not proliferate. This view is supported in part by clinical observations: functionally significant myocardial regeneration has not been documented in diseases and/or injuries that result in cardiomyocyte loss. Furthermore, primary myocardial tumors are rarely observed in adults.
Although these findings suggest that the proliferative capacity of adult cardiomyocytes is quite low, they do not exclude the existence of a limited degree of hyperplastic growth in either the normal or diseased myocardium. Toward this end, a number of studies examining the proliferative capacity of cardiomyocytes in experimental animals have been reported. Because genome reduplication is a prerequisite for cell proliferation, the majority of these studies have used various methodologies to monitor cardiomyocyte DNA synthesis as a first approximation of cell division. In the present survey, issues that we consider pertinent for accurate assessment of cardiomyocyte DNA synthesis are discussed. The literature examining cardiomyocyte DNA synthesis during normal and pathological myocardial growth is then summarized.
Accurate assessment of cardiomyocyte DNA synthesis in vivo is dependent on the selection of an appropriate marker for genome reduplication as well as the criteria used for cardiomyocyte identification. These issues are considered separately below.
### Markers for DNA Synthesis
Genome reduplication is accompanied by a variety of cytological, biochemical, and molecular events, many of which can be used as either direct or indirect evidence for DNA synthesis and, by inference, proliferation. These include the presence of cytological landmarks indicative of karyokinesis, the presence of active DNA synthesis, and the expression of genes and/or protein activities …
Primary cultures of various cell types contain dividing and nondividing cells.1 2 However, nondividing cells can rest in the G phase and reenter the cell cycle on stimulation3 4 5 or become terminally differentiated and die without dividing.6 This third category of cells is not dormant in G and cannot reenter the cell cycle.6 For example, such a phenomenon is present in vivo at the tip of the villi of the small intestine,7 in the auditory hair cells of the ear,8 and in the upper layers of the epidermis.9 In contrast, hepatocytes in vivo are in a G state,10 11 12 and after partial hepatectomy or severe injury, liver regeneration is accomplished by proliferation of mature hepatocytes as well as biliary epithelial cells and fenestrated endothelial cells.12 The reconstitution of liver mass is not dependent on a reserve of stem cells, which condition regeneration of bone marrow13 and skin.14 Multipotential cells have also been identified in the central nervous system, but their ability to grow, differentiate, and ultimately survive has been problematic.15 In skeletal muscle, satellite cells can be stimulated to proliferate and develop into mature myocytes, representing an additional form of tissue regeneration.16 17 18 These mechanisms of cellular growth have not been considered possible in adult cardiac myocytes, and the concept was advanced that no proliferation of ventricular muscle cells occurs once cell division has ceased, shortly after birth in the mammalian heart.19
The dogma was introduced that adult cardiac myocytes are terminally differentiated cells and, therefore, cannot be recalled into the cell cycle.20 21 These cells are not in G, cannot be triggered into the proliferative phase, but can perform their physiological functions, undergo cellular hypertrophy, and …
In the preceding sections we have described the potential for using cell cycle regulatory molecules as targets for drug development within the cardiovascular system. Opportunities for affecting the expression and activities of selected cell cycle regulatory molecules exist in interventional cardiological procedures such as PTCA to limit specifically the intimal hyperplasia of vascular smooth muscle cells that occurs following angioplasty. In addition, the potential for targeting the cardiac myocyte cell cycle to re-initiate cell division in a controlled manner would provide a suitable approach for repairing damaged areas of myocardial tissue following an infarct. Although this approach has not been demonstrated to date in vivo, data from transgenic mouse models and in vitro studies have implicated the cell cycle as a suitable target for manipulation. The next few years will enable the feasibility of this approach to be demonstrated.
Cardiac muscle cells exhibit two related but distinct modes of growth that are highly regulated during development and disease. Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle irreversibly soon after birth, following which the predominant form of growth shifts from hyperplastic to hypertrophic. Much research has focused on identifying the candidate mitogens, hypertrophic agonists, and signaling pathways that mediate these processes in isolated cells. What drives the proliferative growth of embryonic myocardium in vivo and the mechanisms by which adult cardiac myocytes hypertrophy in vivo are less clear. Efforts to answer these questions have benefited from rapid progress made in techniques to manipulate the murine genome. Complementary technologies for gain- and loss-of-function now permit a mutational analysis of these growth control pathways in vivo in the intact heart. These studies have confirmed the importance of suspected pathways, have implicated unexpected pathways as well, and have led to new paradigms for the control of cardiac growth.
Skeletal muscle differentiation is influenced by multiple pathways, which regulate the activity of myogenic regulatory factors (MRFs)-the myogenic basic helix-loop-helix proteins and the MEF2-family members-in positive or negative ways. Here we will review and discuss the network of signals that regulate MRF function during myocyte proliferation, differentiation, and post-mitotic growth. Elucidating the mechanisms governing muscle-specific transcription will provide important insight in better understanding the embryonic development of muscle at the molecular level and will have important implications in setting out strategies aimed at muscle regeneration. Since the activity of MRFs are compromised in tumors of myogenic derivation-the rhabdomyosarcomas-the studies summarized in this review can provide a useful tool to uncover the molecular basis underlying the formation of these tumors.
Cyclin-dependent kinase 2 (cdk2) plays a critical role in the G1- to S-phase checkpoint of the cell cycle. Adult cardiomyocytes are believed to withdraw from the cell cycle. To determine whether forced overexpression of cdk2 results in altered cell-cycle regulation in the adult heart, we generated transgenic mice specifically overexpressing cdk2 in hearts. Transgenic hearts expressed high levels of both cdk2 mRNA and catalytically active cdk2 proteins. Cdk2 overexpression significantly increased the levels of cdk4 and cyclins A, D3, and E. There was an increase in both DNA synthesis and proliferating cell nuclear antigen levels in the adult transgenic hearts. The ratio of heart weight to body weight in cdk2 transgenic mice was significantly increased in neonatal day 2 but not in adults compared with that of wild-type mice. Analysis of dispersed individual adult cardiomyocytes showed a 5.6-fold increase in the proportion of smaller mononuclear cardiomyocytes in the transgenic mice. Echocardiography revealed that transgenic heart was functionally normal. However, adult transgenic ventricles expressed beta-myosin heavy chain and atrial natriuretic factor. Surgically induced pressure overload caused an exaggerated maladaptive hypertrophic response in transgenic mice but did not change the proportion of mononuclear cardiomyocytes. The data suggest that overexpression of cdk2 promotes smaller, less-differentiated mononuclear cardiomyocytes in adult hearts that respond in an exaggerated manner to pressure overload.
Myocardial infarction leads to loss of tissue and impairment of cardiac performance. The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time. Injury to a target organ is sensed by distant stem cells, which migrate to the site of damage and undergo alternate stem cell differentiation; these events promote structural and functional repair. This high degree of stem cell plasticity prompted us to test whether dead myocardium could be restored by transplanting bone marrow cells in infarcted mice. We sorted lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein by fluorescence-activated cell sorting on the basis of c-kit expression. Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct. Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells. The developing tissue comprised proliferating myocytes and vascular structures. Our studies indicate that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease.
Three lines of investigation have suggested that interactions between Survivin and the chromosomal passenger proteins INCENP and Aurora-B kinase may be important for mitotic progression. First, interference with the function of Survivin/BIR1, INCENP, or Aurora-B kinase leads to similar defects in mitosis and cytokinesis [1-7] (see [8] for review). Second, INCENP and Aurora-B exist in a complex in Xenopus eggs [9] and in mammalian cultured cells [7]. Third, interference with Survivin or INCENP function causes Aurora-B kinase to be mislocalized in mitosis in both C. elegans and vertebrates [5, 7, 9]. Here, we provide evidence that Survivin, Aurora-B, and INCENP interact physically and functionally. Direct visualization of Survivin-GFP in mitotic cells reveals that it localizes identically to INCENP and Aurora-B. Survivin binds directly to both Aurora-B and INCENP in yeast two-hybrid and in vitro pull-down assays. The in vitro interaction between Survivin and Aurora-B is extraordinarily stable in that it resists 3 M NaCl. Finally, Survivin and INCENP interact functionally in vivo; in cells in which INCENP localization is disrupted, Survivin adheres to the chromosomes and no longer concentrates at the centromeres or transfers to the anaphase spindle midzone. Our data provide the first biochemical evidence that Survivin can interact directly with members of the chromosomal passenger complex.
c-Myc, a protooncogene, mediates both proliferative and cellular growth in many cell types. Although not expressed in the adult heart under normal physiological conditions, Myc expression is rapidly upregulated in response to hypertrophic stimuli. Although Myc is capable of sustaining hyperplastic growth in fetal myocytes, the effects of its re-expression in adult postmitotic myocardium and its role in mediating cardiac hypertrophy are unknown. To determine the effects of de novo Myc activity in adult postmitotic myocardium in vivo, we created a novel transgenic model in which Myc is expressed and inducibly activated specifically in cardiac myocytes. Activation of Myc in adult myocardium was sufficient to reproduce the characteristic changes in myocyte size, protein synthesis, and cardiac-specific gene expression seen in cardiac hypertrophy. Despite the increased cardiac mass, left ventricular function remained normal. Activation of Myc also provoked cell cycle reentry in postmitotic myocytes, which led to increased nuclei per myocyte and DNA content per nuclei.
Ki67 protein: the immaculate deception?
This article updates our previous review of Ki67 published in Histopathology 10 years ago. In this period the numbers of papers published featuring this antibody has increased 10-fold from 338 to 3489 indicating the considerable enthusiasm with which this antibody has been studied. This review attempts to provide an update on the characterization of the Ki67 protein, its function and its use as a prognostic or diagnostic tool.