Surface Lin28A expression consistent with cellular stress parallels indicators of senescence.

AIMS: Declining cellular functional capacity resulting from stress or ageing is a primary contributor to impairment of myocardial performance. Molecular pathway regulation of biological processes in cardiac interstitial cells (CICs) is pivotal in stress and ageing responses. Altered localization of the RNA-binding protein Lin28A has been reported in response to environmental stress, but the role of Lin28A in response to stress in CICs has not been explored. Surface Lin28A redistribution is indicative of stress response in CIC associated with ageing and senescence. METHODS AND RESULTS: Localization of Lin28A was assessed by multiple experimental analyses and treatment conditions and correlated to oxidative stress, senescence, and ploidy in adult murine CICs. Surface Lin28A expression is present on 5% of fresh CICs and maintained through Passage 2, increasing to 21% in hyperoxic conditions but lowered to 14% in physiologic normoxia. Surface Lin28A is coincident with elevated senescence marker p16 and beta-galactosidase (ß-gal) expression in CICs expanded in hyperoxia, and also increases with polyploidization and binucleation of CICs regardless of oxygen culture. Transcriptional profiling of CICs using single-cell RNA-Seq reveals up-regulation of pathways associated with oxidative stress in CICs exhibiting surface Lin28A. Induction of surface Lin28A by oxidative stress is blunted by treatment of cells with the antioxidant Trolox in a dose-dependent manner, with 300 µM Trolox exposure maintaining characteristics of freshly isolated CICs possessing low expression of surface Lin28A and ß-gal with predominantly diploid content. CONCLUSION: Surface Lin28A is a marker of environmental oxidative stress in CICs and antioxidant treatment antagonizes this phenotype. The biological significance of Lin28 surface expression and consequences for myocardial responses may provide important insights regarding mitigation of cardiac stress and ageing.

'Youthful' phenotype of c-Kit+ cardiac fibroblasts.

Cardiac fibroblast (CF) population heterogeneity and plasticity present a challenge for categorization of biological and functional properties. Distinct molecular markers and associated signaling pathways provide valuable insight for CF biology and interventional strategies to influence injury response and aging-associated remodeling. Receptor tyrosine kinase c-Kit mediates cell survival, proliferation, migration, and is activated by pathological injury. However, the biological significance of c-Kit within CF population has not been addressed. An inducible reporter mouse detects c-Kit promoter activation with Enhanced Green Fluorescent Protein (EGFP) expression in cardiac cells. Coincidence of EGFP and c-Kit with the DDR2 fibroblast marker was confirmed using flow cytometry and immunohistochemistry. Subsequently, CFs expressing DDR2 with or without c-Kit was isolated and characterized. A subset of DDR2+ CFs also express c-Kit with coincidence in ~ 8% of total cardiac interstitial cells (CICs). Aging is associated with decreased number of c-Kit expressing DDR2+ CFs, whereas pathological injury induces c-Kit and DDR2 as well as the frequency of coincident expression in CICs. scRNA-Seq profiling reveals the transcriptome of c-Kit expressing CFs as cells with transitional phenotype. Cultured cardiac DDR2+ fibroblasts that are c-Kit+ exhibit morphological and functional characteristics consistent with youthful phenotypes compared to c-Kit- cells. Mechanistically, c-Kit expression correlates with signaling implicated in proliferation and cell migration, including phospho-ERK and pro-caspase 3. The phenotype of c-kit+ on DDR2+ CFs correlates with multiple characteristics of 'youthful' cells. To our knowledge, this represents the first evaluation of c-Kit biology within DDR2+ CF population and provides a fundamental basis for future studies to influence myocardial biology, response to pathological injury and physiological aging.

Fundamentals of vaping-associated pulmonary injury leading to severe respiratory distress.

Vaping of flavored liquids has been touted as safe alternative to traditional cigarette smoking with decreased health risks. The popularity of vaping has dramatically increased over the last decade, particularly among teenagers who incorporate vaping into their daily life as a social activity. Despite widespread and increasing adoption of vaping among young adults, there is little information on long-term consequences of vaping and potential health risks. This study demonstrates vaping-induced pulmonary injury using commercial JUUL pens with flavored vape juice using an inhalation exposure murine model. Profound pathological changes to upper airway, lung tissue architecture, and cellular structure are evident within 9 wk of exposure. Marked histologic changes include increased parenchyma tissue density, cellular infiltrates proximal to airway passages, alveolar rarefaction, increased collagen deposition, and bronchial thickening with elastin fiber disruption. Transcriptional reprogramming includes significant changes to gene families coding for xenobiotic response, glycerolipid metabolic processes, and oxidative stress. Cardiac systemic output is moderately but significantly impaired with pulmonary side ventricular chamber enlargement. This vaping-induced pulmonary injury model demonstrates mechanistic underpinnings of vaping-related pathologic injury.

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.

PIM1 Promotes Survival of Cardiomyocytes by Upregulating c-Kit Protein Expression.

Enhancing cardiomyocyte survival is crucial to blunt deterioration of myocardial structure and function following pathological damage. PIM1 (Proviral Insertion site in Murine leukemia virus (PIM) kinase 1) is a cardioprotective serine threonine kinase that promotes cardiomyocyte survival and antagonizes senescence through multiple concurrent molecular signaling cascades. In hematopoietic stem cells, PIM1 interacts with the receptor tyrosine kinase c-Kit upstream of the ERK (Extracellular signal-Regulated Kinase) and Akt signaling pathways involved in cell proliferation and survival. The relationship between PIM1 and c-Kit activity has not been explored in the myocardial context. This study delineates the interaction between PIM1 and c-Kit leading to enhanced protection of cardiomyocytes from stress. Elevated c-Kit expression is induced in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1. Co-immunoprecipitation and proximity ligation assay reveal protein-protein interaction between PIM1 and c-Kit. Following treatment with Stem Cell Factor, PIM1-overexpressing cardiomyocytes display elevated ERK activity consistent with c-Kit receptor activation. Functionally, elevated c-Kit expression confers enhanced protection against oxidative stress in vitro. This study identifies the mechanistic relationship between PIM1 and c-Kit in cardiomyocytes, demonstrating another facet of cardioprotection regulated by PIM1 kinase.

Human CardioChimeras: Creation of a Novel "Next-Generation" Cardiac Cell.

Background CardioChimeras produced by fusion of murine c-kit+ cardiac interstitial cells with mesenchymal stem cells promote superior structural and functional recovery in a mouse model of myocardial infarction compared with either precursor cell alone or in combination. Creation of human CardioChimeras (hCCs) represents the next step in translational development of this novel cell type, but new challenges arise when working with c-kit+ cardiac interstitial cells isolated and expanded from human heart tissue samples. The objective of the study was to establish a reliable cell fusion protocol for consistent optimized creation of hCCs and characterize fundamental hCC properties. Methods and Results Cell fusion was induced by incubating human c-kit+ cardiac interstitial cells and mesenchymal stem cells at a 2:1 ratio with inactivated Sendai virus. Hybrid cells were sorted into 96-well microplates for clonal expansion to derive unique cloned hCCs, which were then characterized for various cellular and molecular properties. hCCs exhibited enhanced survival relative to the parent cells and promoted cardiomyocyte survival in response to serum deprivation in vitro. Conclusions The generation of hCC is demonstrated and validated in this study, representing the next step toward implementation of a novel cell product for therapeutic development. Feasibility of creating human hybrid cells prompts consideration of multiple possibilities to create novel chimeric cells derived from cells with desirable traits to promote healing in pathologically damaged myocardium.

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.

Hypoxia Prevents Mitochondrial Dysfunction and Senescence in Human c-Kit+ Cardiac Progenitor Cells.

Senescence-associated dysfunction deleteriously affects biological activities of human c-Kit+ cardiac progenitor cells (hCPCs), particularly under conditions of in vitro culture. In comparison, preservation of self-renewal and decreases in mitochondrial reactive oxygen species (ROS) are characteristics of murine CPCs in vivo that reside within hypoxic niches. Recapitulating hypoxic niche oxygen tension conditions of ∼1% O2 in vitro for expansion of hCPCs rather than typical normoxic cell culture conditions (21% O2 ) could provide significant improvement of functional and biological activities of hCPCs. hCPCs were isolated and expanded under permanent hypoxic (hCPC-1%) or normoxic (hCPC-21%) conditions from left ventricular tissue explants collected during left ventricular assist device implantation. hCPC-1% exhibit increased self-renewal and suppression of senescence characteristics relative to hCPC-21%. Oxidative stress contributed to higher susceptibility to apoptosis, as well as decreased mitochondrial function in hCPC-21%. Hypoxia prevented accumulation of dysfunctional mitochondria, supporting higher oxygen consumption rates and mitochondrial membrane potential. Mitochondrial ROS was an upstream mediator of senescence since treatment of hCPC-1% with mitochondrial inhibitor antimycin A recapitulated mitochondrial dysfunction and senescence observed in hCPC-21%. NAD+ /NADH ratio and autophagic flux, which are key factors for mitochondrial function, were higher in hCPC-1%, but hCPC-21% were highly dependent on BNIP3/NIX-mediated mitophagy to maintain mitochondrial function. Overall, results demonstrate that supraphysiological oxygen tension during in vitro expansion initiates a downward spiral of oxidative stress, mitochondrial dysfunction, and cellular energy imbalance culminating in early proliferation arrest of hCPCs. Senescence is inhibited by preventing ROS through hypoxic culture of hCPCs. Stem Cells 2019;37:555-567.

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.

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.

Effects of electromagnetic fields on reelin and Dab1 expression in the developing cerebral cortex.

Many studies describe the biological effects of electromagnetic fields (EMF) including brain damages, neuronal migration and neurogenesis within the central nervous system. Neuronal cell produced in the neuroepithelium migrates along radial glial fibers into the cortical plate. Reelin, which is produced by Cajal-Retzius cells directs neuronal migration. It was shown that Disabled 1 (Dab1) functions downstream of reelin signal transduction pathway that directs the correct cytoarchitecture of the developing cortex. In this study, the EMF effects on total protein concentration (TPC), reelin and Dab1 expression in the developing cortex was studied. 30 pregnant Balb/c mice were separated into three groups: control (n = 10), EMF (n = 10) and SHAM groups (n = 10). The 15-day pregnant mice were placed inside the solenoid for a daily EMF exposure of 5 h for 3 consecutive days (15-17). The SHAM group was also located in the same coil with no exposure. Mice were sacrificed 24 h after the final exposure session. TPC, reelin and Dab1 expression were studied by Bio-Rad protein assay and western blot. No significant change in the TPC was seen in the EMF-treated cerebral cortex samples compared with those from the SHAM and control groups. It was also shown that the reelin and Dab1 expression increases in the EMF-treated cerebral cortex extracts as compared to controls and SHAM group. It is concluded that EMF may play important role in the neural cell migration by increasing reelin and Dab1 expression in the developing cortex.