Adult Cardiomyocyte Cell Cycle Detour: Offramp to Quiescent Destinations

Retrospective views on cardiomyocyte mitosis.

Considering the universally accepted conclusion that loss of cardiomyocytes is a major underlying cause of heart failure from acute pathologic injury or chronic stress, the answer of generating additional cardiomyocytes to restore structural and functional integrity of the heart seems a simple, clever, and achievable solution. However, inherent biological properties of the adult mammalian myocardium have rendered this overtly straightforward approach frustratingly difficult. Indeed, decades of research and thousands of publications have been dedicated to the singular goal of prompting adult mammalian cardiomyocytes to re-enter cell cycle and complete mitosis. The inescapable conclusion from collective efforts put forth is that adult mammalian cardiomyocytes are remarkably refractory to mitotic activity, unlike those found in either early postnatal mice or zebrafish. Nevertheless, new publications appear every year touting major advances in understanding and augmenting cardiomyocyte proliferation.[1-4] Therefore, it seems reasonable to briefly reflect upon where we are in this process, what factors are obstructing forward progress, and how the field could re-center with renewed focus and purpose to empower the ultimate goal of developing interventional approaches for therapeutic cardiomyogenesis.

Evolution of thinking on cardiomyocyte renewal.

Current literature is replete with masterful reviews on the topic of cardiomyogenesis that summarize the sophisticated and elegant studies carried out by hundreds of laboratories around the world.[5-10] The consensus opinion for many years remains that mammalian cardiomyocyte proliferation is readily observed in prenatal and early postnatal development.[11, 12] Furthering evidence for immature mammalian cardiomyocyte proliferative capacity, similar conclusions were reached observing cultured neonatal cardiomyocytes.[13-15] However, scant evidence exists to support adult mammalian cardiomyocyte division in vitro, but rather a “de-differentiation” process characterized by loss of myofibril organization, return to immature phenotypic properties, and expression of stem cell marker c-kit.[16-19] Even less encouraging, adult mammalian cardiomyocyte division in situ remained elusive, with reports of occasional mitotic figures without definitive proof of completed cytokinesis [20, 21], since labeling with proliferating cell nuclear antigen and bromodeoxyuridine (BrdU) are not definitive evidence of completed cell division.[22, 23] The new millennium witnessed a number of controversial turns in the search for evidence of adult mammalian cardiomyogenesis with the advent of cardiac stem cells[24, 25], carbon-14 estimates of turnover from nuclear bomb blasts[26, 27], as well as the rise and fall of related studies from the Anversa lab.[28] Retrospectively considering the arc of thinking on adult mammalian cardiomyocyte replacement, there is no disputing that researchers have failed to unlock regenerative potential of the adult mammalian myocardium sufficient to restore structure or function lost from pathologic damage, chronic stress or aging.[12] Acceptance of this humbling defeat stands in stark contrast to myocardial repair in lower vertebrates or neonatal mice where acute injury promotes cardiomyocyte replacement.[29, 30] Profound differences in reparative potential between mammalian neonatal versus adult hearts are intriguing, but the underlying explanation might simply be chalked up to neonatal mammalian cardiomyocytes having more in common with zebrafish than adult mammals.[31] So without a clear path forward, many have returned to re-examination of the adult cardiomyocyte armed with novel approaches and unflagging optimism, intent upon succeeding where so many others have failed before them.

Current renewed interest in cardiomyocyte proliferation.

Several excellent reviews have previously covered many aspects of current knowledge and obstacles in the pursuit of cardiomyogenesis for adult mammalian hearts.[23, 29, 32, 33] Highlighting the distinction of adult mammalian hearts is important, as substantial time and effort has been expended defining the indisputable cardiomyogenic activity inherent to postnatal mouse myocardium as well as zebrafish hearts. Yet there is abundant evidence that the inherent biological milieu of hearts from postnatal mice or zebrafish is profoundly distinct from adult mammalian myocardium, leaving translatability of such research unresolved. Clearly, it stands to reason that cardiomyogenic testing for adult mammalian hearts is best tested in the setting of an in vivo adult mammal model to achieve the most dependable and reliable results. And yet, even in the setting of adult murine models there has been lack of consensus on cardiomyogenic cell sources, proliferative activity, and quantitation of mitotic activity. For example, a rigorous study of cardiomyogenesis in mice during postnatal development concluded that a very brief period of cardiomyogenic potential exists after birth that disappears in the adult heart,[34] consistent with more recent re-visitation of this topic using the apical resection model[35, 36] as well as neonatal pigs.[37, 38] None of these studies address potential induction on cardiomyogenesis following pathologic damage in an adult setting, but a recent consensus statement from the American Heart Association focused upon endogenous cardiomyogenesis (rather than cell-based therapeutic approaches) concluded “1) Cardiomyocyte renewal rates may be higher after injury than under normal conditions, and 2) The experimental determination of cardiomyocyte turnover after cardiac injury can be challenging owing to inflammation, proliferation of stromal and vascular cells, and scar formation.”[39] After decades of unrelenting investigation, the consensus is that answers related to cardiomyocyte turnover in the pathological setting remain unresolved. Clearly, new approaches and additional knowledge are required. Selected primary considerations that have hampered the field are presented in the next few paragraphs, highlighting longstanding limitations as well as the way forward proposed in this review.

To avoid detours and stay on the main path toward mitosis, we need to understand where the offramps are and bypass them.

Paradoxically, a primary issue hampering studies of adult mammalian cardiomyogenesis has been the difficulty of determining cardiomyocyte proliferation using markers of cell cycling. While such demonstrations are readily reproduced in neonatal mice or zebrafish, the biological responses of adult cardiomyocytes to mitotic stimuli render typical measures of cell division irrelevant. For example, multiple markers of cell cycle have been developed for investigations of non- myocardial cell biology and co-opted to assess cardiomyocyte proliferation (Fig. 1). Each of these markers has been used to infer mitotic activity, yet none of them alone are truly definitive indicators of authentic cell division when working with cardiomyocytes. Specifically, these markers indicate progression through cell cycle including mitosis. However, in the context of cardiomyocytes, many of these markers are present at multiple stages of cell cycle and it is impossible to distinguish cells that are progressing through mitosis from those that arrest at various mitotic checkpoints.

Numerous approaches for assessing mitosis – most inauthentic.

All sorts of results have been reported in the adult mammalian context with widely varying observations of cardiomyocyte “proliferation” using a plethora of markers and metrics to assess de novo cardiomyogenesis.[34, 40, 41] Lack of standardization, varied experimental approaches, and underappreciation for distinctive cell cycle regulation of cardiomyocytes has led to substantial confusion and, in some cases, hyperbolic claims of translational potential that have not as yet been borne out through the passage of time and practical experience.

Warnings from published articles on flawed methodology.

Limitations of using these markers to document cardiomyocyte proliferation have been highlighted in previous publications,[40, 42] but despite these admonitions the presentation of these labels as evidence of cardiomyocyte mitotic activity continues. This serious problem for the field is indicative of disconnects in recognizing the atypical mitotic resistance of cardiomyocytes relative to other cell types where such labels could be accurate and appropriate. A recent study pointed out these limitations and offered a way forward using two novel proteins (RhoA and IQGAP3)[43] or midbody positioning[44] as definitive markers of cardiomyocyte division, but unfortunately use of these markers also rests upon a tour-de-force confocal analysis of intracellular localization at a critical transient moment in the penultimate steps of mitosis. Demonstration of cardiomyocyte mitosis using individual proteins or structures will require further development of tools to monitor these proteins in real-time to follow intracellular localization that is beyond the capabilities of current typical investigations of cardiomyocyte analyses.

Complexity of demonstrating mitosis in vivo.

Cumulative background information presented thus far in this section certainly is sufficiently disconcerting to prompt skepticism and reservations related to recent publications of enhanced cardiomyocyte proliferation. Of note, one recent publication from 2017 asserts that frequency of mononuclear diploid cardiomyocytes correlates with increased cardiac regenerative potential[45] with an associated editorial,[46] yet this study did not rigorously discriminate between ploidy levels resulting from endomitosis, endoreplication, or cellular division. In a different publication from 2017, administration of miRNA mimics was touted to induce cardiomyocyte passage through mitosis, yet these conclusions were based upon Aurora B and phospho-histone 3 immunolocalization[3] spawning an editorial comment in the same issue.[47] A third high profile study based upon a defined set of four cyclin-related factors (4F) concludes adult cardiomyocyte proliferation was evident based upon EdU and phospho-histone 3 as well as histologic assessment of Mosaic Analysis with Double Markers (MADM) transgenic mice.[2] Cardiac-specific mouse models for clonal analysis including MADM have been comprehensively covered in an excellent review by Leone et al.[23] MADM has been used to assess mitosis in postnatal cardiomyocytes or adult cardiomyocytes with relatively low labeling efficiency of 0.78% or 0.9%, respectively.[48] Low efficiency cardiomyocyte labeling in MADM is likely due to the requirement for cell division coupled with Cre-mediated recombination in G2 phase to allow for recombinant alleles to segregate into separate daughter cells representative of mitosis.[49] [50] Catching mitosis and Cre-recombination simultaneously given the rarity of cardiomyocyte division in an adult heart is clearly challenging, and the requirement for three separate alleles (Cre as well as two MADM) into a single mouse for MADM presents a daunting prospect for mouse breeding schemes to introduce additional genetic modifications.[51] MADM is much more amenable to use with delivery of inductive agents to adult mice, as in a recent study touting unprecedentedly high adult cardiomyocyte mitotic activity following combined adenoviral delivery of four cell-cycle regulators.[2] Lastly, yet another publication in 2018 shows “birth of new cardiomyocytes in adult mice” following 8 weeks of running exercise identified based on incorporation of ¹⁵N-thymidine by multi-isotope imaging mass spectrometry (MIMS) and on being mononucleate/diploid.[1] MIMS also relies upon quantitation from a very limited number of ¹⁵N labeled diploid mononuclear cardiomyocytes (0.14% - 0.09% in non-injured hearts in one study; 0.25% in sedentary mice in a second study) leaving the technique susceptible to substantial influence from finding small numbers of additional labeled cells.[1, 52] MIMS technology, while impressive, is certainly not a widely adopted technique for assessing cardiomyocyte proliferation and validation using more broadly available techniques is warranted to substantiate the conclusion that exercise prompts a ~4.6 fold increase in new cardiomyocytes.[1] Whether such profound elevation of adult mammalian cardiomyocyte mitosis in these studies can be authenticated by other laboratories remains to be seen, as previous controversial claims for a postnatal burst of cardiomyocyte proliferation[53] were subsequently challenged by multiple laboratories unable to replicate these results.[54-56]

Considerable resources, time, and effort have been poured into studies of cardiomyogenesis in experimental models characterized by repair after acute injury, most notably in neonatal mice and zebrafish. The excitement and enthusiasm with which these models have been pursued is indisputable, but translating findings from these models to promote productive adult mammalian cardiomyocyte cell cycle progression and mitosis remains unfulfilled.[29] Aside from controversies of cardiomyogenesis versus ‘regeneration’ in the neonatal mouse apical resection model[30, 31, 36, 57-59] and the role of stem cells[60-62], shared reparative capabilities of neonatal mice and zebrafish appear to rest with the immature phenotype of the tissues relative to the adult mammalian myocardium.[31] The proteomic analysis concluded “the profound differences in structural gene expression place the (regenerative) zebrafish heart rather in the vicinity of the (proliferative) neonatal, but not the adult mouse hearts… It is therefore questionable if promitotic stimuli that drive cardiac regeneration in zebrafish may be capable of inducing cardiac regeneration in adult mammalian cardiomyocytes.” [29] Additional significant differences includes presence of intact centrosomes in cardiomyocytes of adult zebrafish or neonatal mammalian rodents versus absence in adult mammalian hearts.[63] Narrowing to the nexus of this challenge leads to defining causes and consequences for mammalian adult cardiomyocytes to withdraw from and their intractability to re-enter cell cycle.

Fluorescent Ubiquitin Cell Cycle Indicator (FUCCI) to study cardiomyocyte cell cycle.

Adult mammalian cardiomyocytes are notoriously indifferent to stimuli well known to drive mitosis in other cell types such as serum stimulation, oncogenic stimuli, or forced cell cycle reentry, yet it is clear that they do respond in alternative ways. These longstanding ambiguities have rendered claims of induced cardiomyocyte cell cycling open to debate and skepticism, although pervasive doubts are sharply contrasted against the abundance of publications in support of induced cardiomyocyte mitotic activity. Our group assessed cardiomyocyte regulation from a new perspective using FUCCI reporters [67] as a tool to dissect cell cycle progression (Fig. 1). Implementation of FUCCI labeling has yielded advances in biological systems ranging from cell culture to zebrafish, flies, mice, and embryonic stem cells.[67] The novel transgenic mouse is based upon well documented and proven FUCCI technology adapted to in vivo cell cycle monitoring via cardiomyocyte-specific transgenesis (FUCCI-Tg).[22] Although the FUCCI system has previously been studied in the cardiovascular context,[68-71] none of these prior studies used cardiomyocyte-specific expression and none were concerned with demonstration of enhanced adult cardiomyogenesis. Cardiomyocyte division is not the derived readout of the FUCCI-Tg, but rather the study of cell cycle progression. Indeed, to address the eventual outcome of cell cycle progression to discriminate between endoreplication, endomitosis, or mitosis, additional readouts are required for incorporation with imaging to determine ploidy state of nuclei as well as nuclearity of cardiomyocytes. The FUCCI-Tg is particularly valuable as a novel tool to assess cardiomyocyte proliferation because: 1) every cardiomyocyte in the heart is visualized for cell cycle status, not just “cycling myocytes”, 2) four distinct stages of cell cycle progression are revealed with inherent implications for cardiomyocyte mitosis, 3) quantitation of cell cycle status for collective myocyte populations is possible, 4) the system can be used in combination with DNA labeling to correlate cell cycle progression with DNA synthesis versus DNA damage, and most importantly 5) in vivo labeling is assessed in the adult mammalian heart – the ultimate testing milieu for authentic proliferative activity (to validate observations from in vitro or postnatal environments). The oscillation of FUCCI signal occurs in real time unlike long term tracking or genetic tracing approaches, so several time points need to be analyzed to find optimal timing for detection, for example after treatment, when cardiomyocyte proliferation occurs. Functionality and utility of the FUCCI-Tg documented in our publication[22] not only reinforced many prior studies of cardiomyocyte biological properties, but also revealed a previously unappreciated aspect of withdrawal from cell cycle: the canonical restriction point (R-point) first described by Pardee in 1974.[72]

R-point.

R-point cell cycle withdrawal could be mistaken for another cell cycle checkpoint, but the processes involved differ substantially. Whereas a checkpoint involves primarily intracellular sensors of metabolic state, genome integrity, and sequential execution of prior cell cycle steps, the R-point transition rests upon cellular integration of signals received from the environment over an extended period of time to determine whether growth is warranted.[73] Without permission to proceed past R-point, the cell withdraws from cycling and enters an arrested state. The R-point has been narrowed to a mid-to-late G1 stage known as the G1/S boundary when cellular resources are focused upon maintenance and preservation of ongoing processes rather than proliferative growth. Cardiomyocyte arrest at the R-point is intuitively attractive given the high metabolic demands of contractile function and the need to maintain structural integrity for normal function. The R-point integrates a multifaceted “knot of mitogen and inhibitory signaling” intrinsically dedicated to preventing cell cycle progression[74], which likely accounts for both the extended lifespan of cardiomyocytes as well as their notorious reluctance to undergo mitotic activity.

Polyploidy.

Contributing to the confusion, biological phenomena of endomitosis, endoreplication, and DNA damage are often underappreciated or unaccounted for in assessments of cardiomyocyte proliferation.[75] Consistent with their atypical nature and resistance to cell division, cardiomyocytes enter mitosis and exit without generating daughter cells but rather by mere duplication of DNA without new nucleation or by adding additional nuclei.[45, 76-81] Although the terms ‘endoreplication’, ‘endomitosis’, and ‘endoreduplication’ are often used interchangeably,[82] for this proposal endoreplication refers to DNA duplication without karyokinesis, whereas endomitosis refers to karyokinesis without cytokinesis. These two processes involve progression through cell cycle that presents as DNA synthesis often misrepresented as mitotic activity (Fig. 2). Cardiomyocytes exhibit increased levels of ploidy within single nuclei well as by accumulating multiple nuclei, even as a normal process of aging.[34, 80, 83] So too, cardiomyocytes incorporate DNA labeling agents consequential to attempting DNA repair response following environmental challenge such as oxidative stress.[84, 85] Failure to appreciate these normal aspects of cardiomyocyte cell biology leads to controversial claims of proliferation rates and potentially erroneous claims of regeneration.[86-88]

We are only as strong as our weakest links – factoring in the entire organism.

Given everything written up to this point certainly is sufficient to give one pause regarding prospects for restoration of myocardial function through promotion of adult cardiomyocyte cell cycle. In keeping with allusions to the cell cycle highway, staying on track for adult mammalian cardiomyocyte may be facilitated by shifting gears rather than hitting the accelerator. Namely, focusing upon cell biology rather than narrow heavy-handed molecular interventions, recognizing that changing fundamental phenotype of cardiomyocyte to a more pliable and accommodating condition is inextricably linked to changing the potential for cell cycle progression, and taking cues from other adult organs and cells such as the liver.[93, 94] Lastly, although this review has centered upon adult mammalian cardiomyocytes, the involvement of the cardiac interstitial cell population should not be discounted or overlooked since those support cells regulate the surrounding environment.[102-106] And in the final analysis, a complex web of intrinsic and extrinsic factors all provide signposts in a medley that influences receptivity of the tissue to reparative action.[107, 108]

Realistic expectations, believable outcomes, and achievable destinations.

The quest for cardiomyogenic approaches in the adult mammalian heart remains a top priority for cardiovascular research and therapeutic interventional strategies to treat heart failure, even after decades of frustration and what can be characterized, at best, as modest outcomes. As previously observed, some of the impasse is attributable to the plethora of approaches and interpretations used in prior published studies. Even today, new tools allowing for increased understanding and improved accuracy for assessments are desperately needed. Application of rigorous and consistent measures to determine induction of cardiomyocyte cell cycle progression in the adult mammalian myocardium is essential to validate and compare the ever-expanding series of methods and practices developed throughout the world. Inconsistent measures, inappropriately applied measures, and overinterpretation of findings have been and continue to be problematic for achieving resolution in advancing mechanistic understanding of cardiomyocyte cell cycle regulation. Paradoxically, while substantial information has been gathered on the unique characteristics of the cardiomyocyte cell cycle relative to other cell types, assessments of outcomes often fail to fully and faithfully encompass the spectrum of possibilities with high rigor and reproducibility. The research community should coalesce around a commonly shared set of principles used to guide measurement of cardiomyocyte cell cycle and allow all researchers to benefit by comparative measures with standardized references such as the FUCCI-Tg serving as a platform for adult mammalian myocardial cell-cycle analysis.[22] Accomplishing the goal of unraveling cardiomyocyte cell cycle control will provide a path forward to reconcile disparate observations, thereby improving the accuracy and reproducibility of interventions intended to enhance adult mammalian cardiomyocyte cell cycle progression.