Pim1 maintains telomere length in mouse cardiomyocytes by inhibiting TGFß signalling.

AIMS: Telomere attrition in cardiomyocytes is associated with decreased contractility, cellular senescence, and up-regulation of proapoptotic transcription factors. Pim1 is a cardioprotective kinase that antagonizes the aging phenotype of cardiomyocytes and delays cellular senescence by maintaining telomere length, but the mechanism remains unknown. Another pathway responsible for regulating telomere length is the transforming growth factor beta (TGFß) signalling pathway where inhibiting TGFß signalling maintains telomere length. The relationship between Pim1 and TGFß has not been explored. This study delineates the mechanism of telomere length regulation by the interplay between Pim1 and components of TGFß signalling pathways in proliferating A549 cells and post-mitotic cardiomyocytes. METHODS AND RESULTS: Telomere length was maintained by lentiviral-mediated overexpression of PIM1 and inhibition of TGFß signalling in A549 cells. Telomere length maintenance was further demonstrated in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1 and by pharmacological inhibition of TGFß signalling. Mechanistically, Pim1 inhibited phosphorylation of Smad2, preventing its translocation into the nucleus and repressing expression of TGFß pathway genes. CONCLUSION: Pim1 maintains telomere lengths in cardiomyocytes by inhibiting phosphorylation of the TGFß pathway downstream effectors Smad2 and Smad3, which prevents repression of telomerase reverse transcriptase. Findings from this study demonstrate a novel mechanism of telomere length maintenance and provide a potential target for preserving cardiac function.

Cardiac tissue engineering therapeutic products to enhance myocardial contractility.

Researchers continue to develop therapeutic products for the repair and replacement of myocardial tissue that demonstrates contractility equivalent to normal physiologic states. As clinical trials focused on pure adult stem cell populations undergo meta-analysis for preclinical through clinical design, the field of tissue engineering is emerging as a new clinical frontier to repair the myocardium and improve cardiac output. This review will first discuss the three primary tissue engineering product themes that are advancing in preclinical to clinical models: (1) cell-free scaffolds, (2) scaffold-free cellular, and (3) hybrid cell and scaffold products. The review will then focus on the products that have advanced from preclinical models to clinical trials. In advancing the cardiac regenerative medicine field, long-term gains towards discovering an optimal product to generate functional myocardial tissue and eliminate heart failure may be achieved.

Adult human cardiac stem cell supplementation effectively increases contractile function and maturation in human engineered cardiac tissues.

BACKGROUND: Delivery of stem cells to the failing heart is a promising therapeutic strategy. However, the improvement in cardiac function in animal studies has not fully translated to humans. To help bridge the gap between species, we investigated the effects of adult human cardiac stem cells (hCSCs) on contractile function of human engineered cardiac tissues (hECTs) as a species-specific model of the human myocardium. METHODS: Human induced pluripotent stem cell-derived cardiomyoctes (hCMs) were mixed with Collagen/Matrigel to fabricate control hECTs, with an experimental group of hCSC-supplemented hECT fabricated using a 9:1 ratio of hCM to hCSC. Functional testing was performed starting on culture day 6, under spontaneous conditions and also during electrical pacing from 0.25 to 1.0 Hz, measurements repeated at days 8 and 10. hECTs were then frozen and processed for gene analysis using a Nanostring assay with a cardiac targeted custom panel. RESULTS: The hCSC-supplemented hECTs displayed a twofold higher developed force vs. hCM-only controls by day 6, with approximately threefold higher developed stress and maximum rates of contraction and relaxation during pacing at 0.75 Hz. The spontaneous beat rate characteristics were similar between groups, and hCSC supplementation did not adversely impact beat rate variability. The increased contractility persisted through days 8 and 10, albeit with some decrease in the magnitude of the difference of the force by day 10, but with developed stress still significantly higher in hCSC-supplemented hECT; these findings were confirmed with multiple hCSC and hCM cell lines. The force-frequency relationship, while negative for both, control (- 0.687 Hz- 1; p = 0.013 vs. zero) and hCSC-supplemented (- 0.233 Hz- 1;p = 0.067 vs. zero) hECTs, showed a significant rectification in the regression slope in hCSC-supplemented hECT (p = 0.011 vs. control). Targeted gene exploration (59 genes) identified a total of 14 differentially expressed genes, with increases in the ratios of MYH7/MHY6, MYL2/MYL7, and TNNI3/TNNI1 in hCSC-supplemented hECT versus controls. CONCLUSIONS: For the first time, hCSC supplementation was shown to significantly improve human cardiac tissue contractility in vitro, without evidence of proarrhythmic effects, and was associated with increased expression of markers of cardiac maturation. These findings provide new insights about adult cardiac stem cells as contributors to functional improvement of human myocardium.

Prevalence of comorbidities in heart failure patients and those treated with cellular therapeutics.

Introduction: Changes in our daily living, particularly in work routines, diet, and physical exercise, have influenced a worldwide crisis for life-threatening comorbidities and the likelihood of cardiovascular disease diagnosis. Cardiovascular regenerative medicine researchers continue to investigate new therapeutic approaches and reexamine completed clinical trials to design more effective future studies. As the frequency of cardiovascular disease diagnosis continues to rise, investigations of how to repair and regenerate the failing myocardium remains an essential mission for human health. Areas covered: This review first examines the aging process, the rising rate of comorbidities, and the likelihood of developing heart disease. In the ongoing efforts to recapitulate human health needs in clinical trials, a review of clinical trials involving cellular therapeutics for cardiac repair, with a focus on the patient population and patients' complex medical records, is presented. Expert opinion: The expert opinion first draws attention to the changing demographics of patients diagnosed with diseases that lead to heart failure and focusing on obesity as a primary driver for increased cardiovascular disease. The opinion focuses on the importance of designing preclinical models and experimentation that better mimic the patient population and clinical situations to evaluate the effectiveness of potential future therapeutic interventions.

Adult Cardiomyocyte Cell Cycle Detour: Off-ramp to Quiescent Destinations.

Ability to promote completion of mitotic cycling of adult mammalian cardiomyocytes remains an intractable and vexing challenge, despite being one of the most sought after 'holy grails' of cardiovascular research. While some of the struggle is attributable to adult cardiomyocytes themselves that are notoriously post-mitotic, another contributory factor rests with difficulty in definitive tracking of adult cardiomyocyte cell cycle and lack of rigorous measures to track proliferation in situ. This review summarizes past, present, and future directions to promote adult mammalian cardiomyocyte cell cycle progression, proliferation, and renewal. Establishing relationship(s) between cardiomyocyte cell cycle progression and cellular biological properties is sorely needed to understand the mechanistic basis for cardiomyocyte cell cycle withdrawal to enhance cardiomyocyte cell cycle progression and mitosis.

Cardiac interstitial tetraploid cells can escape replicative senescence in rodents but not large mammals.

Cardiomyocyte ploidy has been described but remains obscure in cardiac interstitial cells. Ploidy of c-kit+ cardiac interstitial cells was assessed using confocal, karyotypic, and flow cytometric technique. Notable differences were found between rodent (rat, mouse) c-kit+ cardiac interstitial cells possessing mononuclear tetraploid (4n) content, compared to large mammals (human, swine) with mononuclear diploid (2n) content. In-situ analysis, confirmed with fresh isolates, revealed diploid content in human c-kit+ cardiac interstitial cells and a mixture of diploid and tetraploid content in mouse. Downregulation of the p53 signaling pathway provides evidence why rodent, but not human, c-kit+ cardiac interstitial cells escape replicative senescence. Single cell transcriptional profiling reveals distinctions between diploid versus tetraploid populations in mouse c-kit+ cardiac interstitial cells, alluding to functional divergences. Collectively, these data reveal notable species-specific biological differences in c-kit+ cardiac interstitial cells, which could account for challenges in extrapolation of myocardial from preclinical studies to clinical trials.

Cardiomyocyte cell cycle dynamics and proliferation revealed through cardiac-specific transgenesis of fluorescent ubiquitinated cell cycle indicator (FUCCI).

RATIONALE: Understanding and manipulating the cardiomyocyte cell cycle has been the focus of decades of research, however the ultimate goal of activating mitotic activity in adult mammalian cardiomyocytes remains elusive and controversial. The relentless pursuit of controlling cardiomyocyte mitosis has been complicated and obfuscated by a multitude of indices used as evidence of cardiomyocyte cell cycle activity that lack clear identification of cardiomyocyte "proliferation" versus cell cycle progression, endoreplication, endomitosis, and even DNA damage. Unambiguous appreciation of the complexity of cardiomyocyte replication that avoids oversimplification and misinterpretation is desperately needed. OBJECTIVE: Track cardiomyocyte cell cycle activity and authenticate fidelity of proliferation markers as indicators of de novo cardiomyogenesis in post-mitotic cardiomyocytes. METHODS AND RESULTS: Cardiomyocytes expressing the FUCCI construct driven by the α-myosin heavy chain promoter were readily and uniformly detected through the myocardium of transgenic mice. Cardiomyocyte cell cycle activity peaks at postnatal day 2 and rapidly declines thereafter with almost all cardiomyocytes arrested at the G1/S cell cycle transition. Myocardial infarction injury in adult hearts prompts transient small increases in myocytes progressing through cell cycle without concurrent mitotic activity, indicating lack of cardiomyogenesis. In comparison, cardiomyogenic activity during early postnatal development correlated with coincidence of FUCCI and cKit+ cells that were undetectable in the adult myocardium. CONCLUSIONS: Cardiomyocyte-specific expression of Fluorescence Ubiquitination-based Cell Cycle Indicators (FUCCI) reveals previously unappreciated aspects of cardiomyocyte cell cycle arrest and biological activity in postnatal development and in response to pathologic damage. Compared to many other methods and model systems, the FUCCI transgenic (FUCCI-Tg) mouse represents a valuable tool to unambiguously track cell cycle and proliferation of the entire cardiomyocyte population in the adult murine heart. FUCCI-Tg provides a desperately needed novel approach in the armamentarium of tools to validate cardiomyocyte proliferative activity that will reveal cell cycle progression, discriminate between cycle progression, DNA replication, and proliferation, and provide important insight for enhancing cardiomyocyte proliferation in the context of adult myocardial tissue.

In situ transcriptome characteristics are lost following culture adaptation of adult cardiac stem cells.

Regenerative therapeutic approaches for myocardial diseases often involve delivery of stem cells expanded ex vivo. Prior studies indicate that cell culture conditions affect functional and phenotypic characteristics, but relationship(s) of cultured cells derived from freshly isolated populations and the heterogeneity of the cultured population remain poorly defined. Functional and phenotypic characteristics of ex vivo expanded cells will determine outcomes of interventional treatment for disease, necessitating characterization of the impact that ex vivo expansion has upon isolated stem cell populations. Single-cell RNA-Seq profiling (scRNA-Seq) was performed to determine consequences of culture expansion upon adult cardiac progenitor cells (CPCs) as well as relationships with other cell populations. Bioinformatic analyses demonstrate that identity marker genes expressed in freshly isolated cells become undetectable in cultured CPCs while low level expression emerges for thousands of other genes. Transcriptional profile of CPCs exhibited greater degree of similarity throughout the cultured population relative to freshly isolated cells. Findings were validated by comparative analyses using scRNA-Seq datasets of various cell types generated by multiple scRNA-Seq technology. Increased transcriptome diversity and decreased population heterogeneity in the cultured cell population may help account for reported outcomes associated with experimental and clinical use of CPCs for treatment of myocardial injury.

Enhancement Strategies for Cardiac Regenerative Cell Therapy: Focus on Adult Stem Cells.

The idiom heart of the matter refers to the focal point within a topic and, with regard to health and longevity, the heart is truly pivotal for quality of life. Societal trends worldwide continue toward increased percent body fat and decreased physical activity with coincident increases in chronic diseases including cardiovascular disease as the top global cause of death along with insulin resistance, accelerated aging, cancer. Although long-term survival rates for cardiovascular disease patients are grim, intense research efforts continue to improve both prevention and treatment options. Pharmacological interventions remain the predominant interventional strategy for mitigating progression and managing symptoms, but cellular therapies have the potential to cure or even mediate remission of cardiovascular disease. Adult stem cells are the most studied cellular therapy in both preclinical and clinical investigation. This review will focus on the advanced therapeutic strategies to augment products and methods of delivery, which many think heralds the future of clinical investigations. Advanced preclinical strategies using adult stem cells are examined to promote synergism between preclinical and clinical research, streamline implementation, and improve this imminent matter of the heart.

Cardiac ageing: extrinsic and intrinsic factors in cellular renewal and senescence.

Cardiac ageing manifests as a decline in function leading to heart failure. At the cellular level, ageing entails decreased replicative capacity and dysregulation of cellular processes in myocardial and nonmyocyte cells. Various extrinsic parameters, such as lifestyle and environment, integrate important signalling pathways, such as those involving inflammation and oxidative stress, with intrinsic molecular mechanisms underlying resistance versus progression to cellular senescence. Mitigation of cardiac functional decline in an ageing organism requires the activation of enhanced maintenance and reparative capacity, thereby overcoming inherent endogenous limitations to retaining a youthful phenotype. Deciphering the molecular mechanisms underlying dysregulation of cellular function and renewal reveals potential interventional targets to attenuate degenerative processes at the cellular and systemic levels to improve quality of life for our ageing population. In this Review, we discuss the roles of extrinsic and intrinsic factors in cardiac ageing. Animal models of cardiac ageing are summarized, followed by an overview of the current and possible future treatments to mitigate the deleterious effects of cardiac ageing.

Cardiac c-Kit Biology Revealed by Inducible Transgenesis.

RATIONALE: Biological significance of c-Kit as a cardiac stem cell marker and role(s) of c-Kit+ cells in myocardial development or response to pathological injury remain unresolved because of varied and discrepant findings. Alternative experimental models are required to contextualize and reconcile discordant published observations of cardiac c-Kit myocardial biology and provide meaningful insights regarding clinical relevance of c-Kit signaling for translational cell therapy. OBJECTIVE: The main objectives of this study are as follows: demonstrating c-Kit myocardial biology through combined studies of both human and murine cardiac cells; advancing understanding of c-Kit myocardial biology through creation and characterization of a novel, inducible transgenic c-Kit reporter mouse model that overcomes limitations inherent to knock-in reporter models; and providing perspective to reconcile disparate viewpoints on c-Kit biology in the myocardium. METHODS AND RESULTS: In vitro studies confirm a critical role for c-Kit signaling in both cardiomyocytes and cardiac stem cells. Activation of c-Kit receptor promotes cell survival and proliferation in stem cells and cardiomyocytes of either human or murine origin. For creation of the mouse model, the cloned mouse c-Kit promoter drives Histone2B-EGFP (enhanced green fluorescent protein; H2BEGFP) expression in a doxycycline-inducible transgenic reporter line. The combination of c-Kit transgenesis coupled to H2BEGFP readout provides sensitive, specific, inducible, and persistent tracking of c-Kit promoter activation. Tagging efficiency for EGFP+/c-Kit+ cells is similar between our transgenic versus a c-Kit knock-in mouse line, but frequency of c-Kit+ cells in cardiac tissue from the knock-in model is 55% lower than that from our transgenic line. The c-Kit transgenic reporter model reveals intimate association of c-Kit expression with adult myocardial biology. Both cardiac stem cells and a subpopulation of cardiomyocytes express c-Kit in uninjured adult heart, upregulating c-Kit expression in response to pathological stress. CONCLUSIONS: c-Kit myocardial biology is more complex and varied than previously appreciated or documented, demonstrating validity in multiple points of coexisting yet heretofore seemingly irreconcilable published findings.

Mechanisms of Cardiac Repair and Regeneration.

Cardiovascular regenerative therapies are pursued on both basic and translational levels. Although efficacy and value of cell therapy for myocardial regeneration can be debated, there is a consensus that profound deficits in mechanistic understanding limit advances, optimization, and implementation. In collaboration with the TACTICS (Transnational Alliance for Regenerative Therapies in Cardiovascular Syndromes), this review overviews several pivotal aspects of biological processes impinging on cardiac maintenance, repair, and regeneration. The goal of summarizing current mechanistic understanding is to prompt innovative directions for fundamental studies delineating cellular reparative and regenerative processes. Empowering myocardial regenerative interventions, whether dependent on endogenous processes or exogenously delivered repair agents, ultimately depends on mastering mechanisms and novel strategies that take advantage of rather than being limited by inherent myocardial biology.

PIM1-minicircle as a therapeutic treatment for myocardial infarction.

PIM1, a pro-survival gene encoding a serine/ threonine kinase, influences cell proliferation and survival. Modification of cardiac progenitor cells (CPCs) or cardiomyocytes with PIM1 using a lentivirus-based delivery method showed long-term improved cardiac function after myocardial infarction (MI). However, lentivirus based delivery methods have stringent FDA regulation with respect to clinical trials. To provide an alternative and low risk PIM1 delivery method, this study examined the use of a non-viral modified plasmid-minicircle (MC) as a vehicle to deliver PIM1 into mouse CPCs (mCPCs) in vitro and the myocardium in vivo. MC containing a turbo gfp reporter gene (gfp-MC) was used as a transfection and injection control. PIM1 was subcloned into gfp-MC (PIM1-MC) and then transfected into mCPCs at an efficiency of 29.4±3.7%. PIM1-MC engineered mCPCs (PIM1-mCPCs) exhibit significantly (P<0.05) better survival rate under oxidative treatment. PIM1-mCPCs also exhibit 1.9±0.1 and 2.2±0.2 fold higher cell proliferation at 3 and 5 days post plating, respectively, as compared to gfp-MC transfected mCPCs control. PIM1-MC was injected directly into ten-week old adult FVB female mice hearts in the border zone immediately after MI. Delivery of PIM1 into myocardium was confirmed by GFP+ cardiomyocytes. Mice with PIM1-MC injection showed increased protection compared to gfp-MC injection groups measured by ejection fraction at 3 and 7 days post injury (P = 0.0379 and P = 0.0262 by t-test, respectively). Success of PIM1 delivery and integration into mCPCs in vitro and cardiomyocytes in vivo by MC highlights the possibility of a non-cell based therapeutic approach for treatment of ischemic heart disease and MI.

Myocardial Regeneration for Humans　- Modifying Biology and Manipulating Evolution.

Cardiovascular disease remains the leading cause of death worldwide and developing novel therapies to treat and cure the disease remains a high priority in the healthcare research community. Adult stem cells were successful in entering numerous clinical trials over the past 15 years in attempts to regenerate the heart. First-generation adult stem cell therapies for myocardial regeneration were highly promising in small animal models but realized benefits in humans were far more modest. Consequently, second-generation therapeutic approaches in early implementation phases have focused on enhancing cellular properties with higher survival and regenerative potential. Genetic programming dictates cellular fate, so understanding genetic composition and responses at the gene level to influence the outcome of the cell is essential for successful outcomes in regenerative medicine. Genetic editing is at the forefront of scientific innovation and as basic scientific research continues to expand upon understanding eukaryotic regenerative themes, a clearer vision of the possible future therapeutic approaches can be realized. Ultimately, enhancing biology and manipulating evolutional selection of cellular properties will be critical to achieving clinically relevant and biologically meaningful cardiac regeneration.

Empowering Adult Stem Cells for Myocardial Regeneration V2.0: Success in Small Steps.

Much has changed since our survey of the landscape for myocardial regeneration powered by adult stem cells 4 years ago.(1) The intervening years since that first review has witnessed an explosive expansion of studies that advance both understanding and implementation of adult stem cells in promoting myocardial repair. Painstaking research from innumerable laboratories throughout the world is prying open doors that may lead to restoration of myocardial structure and function in the wake of pathological injury. This global effort has produced deeper mechanistic comprehension coupled with an evolving appreciation for the complexity of myocardial regeneration in the adult context. Undaunted by both known and (as yet) unknown challenges, pursuit of myocardial regenerative medicine mediated by adult stem cell therapy has gathered momentum fueled by tantalizing clues and visionary goals. This concise review takes a somewhat different perspective than our initial treatise, taking stock of the business sector that has become an integral part of the field while concurrently updating state of affairs in cutting edge research. Looking retrospectively at advancement over the years as all reviews eventually must, the fundamental lesson to be learned is best explained by Jonatan Mårtensson: "Success will never be a big step in the future. Success is a small step taken just now."

Cardiomyocyte subdomain contractility arising from microenvironmental stiffness and topography.

Cellular structure and function are interdependent. To understand this relationship in beating heart cells, individual neonatal rat ventricular myocytes (NRVMs) were analyzed one and 3 days after plating when cultured on different stiffness (100, 400 kPa) and surface structures (flat or [Formula: see text] high, [Formula: see text] diameter, microposts spaced [Formula: see text] apart) manufactured from polydimethylsiloxane. Myofibril structure seen by immunohistochemistry was organized in three dimensions when NRVMs were attached to microposts. On day three, paxillin distribution near the post serving as cellular anchorage was quantified on both soft posts (12.04 % of total voxel count) and stiff posts (8.16 %). Living NRVMs were analyzed using line scans for sarcomeric shortening and shortening velocity, and traction force microscopy for surface stress and surface tension. One day after plating, NRVMs shortened more on soft posts ([Formula: see text] at [Formula: see text]) compared to either soft flat ([Formula: see text] at [Formula: see text]), stiff posts ([Formula: see text] at [Formula: see text]) or stiff flat ([Formula: see text] at [Formula: see text]). NRVMs have decreased shortening and shortening velocity on soft posts ([Formula: see text] at [Formula: see text]) compared to soft flat ([Formula: see text] at [Formula: see text]) substrates. The surface stress and surface tension increased over time for both soft post ([Formula: see text] and [Formula: see text] to [Formula: see text] and [Formula: see text]) and flat ([Formula: see text] and [Formula: see text] to [Formula: see text] and [Formula: see text]) substrates. Paxillin displacement during contraction on day three was significantly greater in NRVMs attached to soft posts [Formula: see text] compared to flat [Formula: see text] substrates. The volume and time creating four-dimensional data, interpreted by structural engineering theory, demonstrate subdomain structure is maintained by the counterbalance between the external load acting upon and the internal forces generated by the cardiomyocyte. These findings provide further insight into localized regulation of cellular mechanical function.