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ISO acutely causes cardiomyocyte necrosis mainly targeting the sub-endocardial apex. a The fraction of necrotic cardiomyocytes (CM) was significantly increased in a dose-dependent manner at 1 day after ISO (200 and 400 mg/Kg) with higher frequency in the Apex sub-endocardium. n = 7 per group; *p < 0.05 vs. saline; #p < 0.05 vs. Epi; †p < 0.05 vs. Mid; ‡p < 0.05 vs. 200 mg/Kg (one-way ANOVA analysis with Tukey's multiple comparison test). b Representative images of necrotic CMs labelled in vivo with anti-myosin antibody (green) showing higher damage in the subendocardium when compared with myocardium and epicardium. c, d Representative images of necrotic CMs labelled in vivo with anti-myosin antibody (green) showing ISO dose-dependent higher damage in the apex when compared with mid and base in LV regions 1 day after ISO 200 (c) or 400 (d) mg/Kg, respectively. e Representative image of a non-necrotic anti-myosin antibody (green)-labelled normal cardiomyocyte. f Plasma cTnI was significantly elevated in a dose-dependent manner at 1 day after ISO. hs-TnT = high-sensitive cardiac Troponin T. n = 7 per group; *P < 0.05 vs. Saline; #p < 0.05 vs. 200 mg/Kg (one-way ANOVA analysis with Tukey's multiple comparison test). Scale bars = 50 µm except for b left panel = 300 µm. All data are mean ± SD
Source publication
An overdose of Isoproterenol (ISO) causes acute cardiomyocyte (CM) dropout and activates the resident cardiac c-kitpos stem/progenitor cells (CSCs) generating a burst of new CM formation that replaces those lost to ISO. Recently, unsuccessful attempts to reproduce these findings using c-kitCre knock-in (KI) mouse models were reported. We tested whe...
Contexts in source publication
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... ISO overdose causes CM necrosis and apoptosis in a dose-dependent manner in the LV sub-endocardial apex ISO at 200 mg/Kg 21 (n = 14) or at 400 mg/Kg (lethal dose in the study by Wallner et al. 22 ) (n = 14) caused diffuse CM necrosis in 12-week-old C57BL/6J male mice as revealed by in vivo myosin antibody labelling 27 1 day after ISO injection ( Fig. 1). Myosin-labelled CMs showed clear morphologic features of necrosis with loss of cell membrane integrity and architectural disarray (Fig. 1). The damage was distributed in an intensity/density progressive gradient from epicardium to sub-endocardium and from base to apex (Fig. 1a-c). CM death mostly concentrated to the sub-endocardial ...
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... 400 mg/Kg (lethal dose in the study by Wallner et al. 22 ) (n = 14) caused diffuse CM necrosis in 12-week-old C57BL/6J male mice as revealed by in vivo myosin antibody labelling 27 1 day after ISO injection ( Fig. 1). Myosin-labelled CMs showed clear morphologic features of necrosis with loss of cell membrane integrity and architectural disarray (Fig. 1). The damage was distributed in an intensity/density progressive gradient from epicardium to sub-endocardium and from base to apex (Fig. 1a-c). CM death mostly concentrated to the sub-endocardial apex where myosinlabelled necrotic CMs reached up to ~8% and ~12% after ISO 200 or 400 mg/Kg ISO, respectively (Fig. 1a-d). Only very rare ...
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... by in vivo myosin antibody labelling 27 1 day after ISO injection ( Fig. 1). Myosin-labelled CMs showed clear morphologic features of necrosis with loss of cell membrane integrity and architectural disarray (Fig. 1). The damage was distributed in an intensity/density progressive gradient from epicardium to sub-endocardium and from base to apex (Fig. 1a-c). CM death mostly concentrated to the sub-endocardial apex where myosinlabelled necrotic CMs reached up to ~8% and ~12% after ISO 200 or 400 mg/Kg ISO, respectively (Fig. 1a-d). Only very rare (~0,001% of total) myosin-labelled CMs were detected in the saline-injected mice; however, these antimyosin-labelled CMs were always normally ...
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... and architectural disarray (Fig. 1). The damage was distributed in an intensity/density progressive gradient from epicardium to sub-endocardium and from base to apex (Fig. 1a-c). CM death mostly concentrated to the sub-endocardial apex where myosinlabelled necrotic CMs reached up to ~8% and ~12% after ISO 200 or 400 mg/Kg ISO, respectively (Fig. 1a-d). Only very rare (~0,001% of total) myosin-labelled CMs were detected in the saline-injected mice; however, these antimyosin-labelled CMs were always normally shaped and without additional signs of necrosis (Fig. 1e). Increased cTnT blood levels independently confirmed CM necrosis directly proportional to the ISO dose (Fig. 1f). The ...
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... the sub-endocardial apex where myosinlabelled necrotic CMs reached up to ~8% and ~12% after ISO 200 or 400 mg/Kg ISO, respectively (Fig. 1a-d). Only very rare (~0,001% of total) myosin-labelled CMs were detected in the saline-injected mice; however, these antimyosin-labelled CMs were always normally shaped and without additional signs of necrosis (Fig. 1e). Increased cTnT blood levels independently confirmed CM necrosis directly proportional to the ISO dose (Fig. 1f). The normal cTnT values baseline was established at <0.01 ng/ml in 15 consecutive control mice. Blood cTnT levels increased to 0.25 ± 0.14 and 0.58 ± 0.21 ng/ml 1 day after 200 or 400 mg/Kg ISO, respectively (Fig. ...
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... ISO, respectively (Fig. 1a-d). Only very rare (~0,001% of total) myosin-labelled CMs were detected in the saline-injected mice; however, these antimyosin-labelled CMs were always normally shaped and without additional signs of necrosis (Fig. 1e). Increased cTnT blood levels independently confirmed CM necrosis directly proportional to the ISO dose (Fig. 1f). The normal cTnT values baseline was established at <0.01 ng/ml in 15 consecutive control mice. Blood cTnT levels increased to 0.25 ± 0.14 and 0.58 ± 0.21 ng/ml 1 day after 200 or 400 mg/Kg ISO, respectively (Fig. ...
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... signs of necrosis (Fig. 1e). Increased cTnT blood levels independently confirmed CM necrosis directly proportional to the ISO dose (Fig. 1f). The normal cTnT values baseline was established at <0.01 ng/ml in 15 consecutive control mice. Blood cTnT levels increased to 0.25 ± 0.14 and 0.58 ± 0.21 ng/ml 1 day after 200 or 400 mg/Kg ISO, respectively (Fig. ...
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... assessment of EBD 28 -positive CMs at 1 day after ISO closely reproduced the CM necrosis data obtained with myosin antibody labelling ( Supplementary Fig. 1). In addition, CM necrotic death after ISO was also evident by H&E histochemistry (Supplementary Fig. 1B). ...
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... assessment of EBD 28 -positive CMs at 1 day after ISO closely reproduced the CM necrosis data obtained with myosin antibody labelling ( Supplementary Fig. 1). In addition, CM necrotic death after ISO was also evident by H&E histochemistry (Supplementary Fig. 1B). Finally, ISO exposure caused apoptotic CM death in a dose-dependent manner as identified by caspase-3 labelling (Supplementary Fig. 1C). ...
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... after ISO closely reproduced the CM necrosis data obtained with myosin antibody labelling ( Supplementary Fig. 1). In addition, CM necrotic death after ISO was also evident by H&E histochemistry (Supplementary Fig. 1B). Finally, ISO exposure caused apoptotic CM death in a dose-dependent manner as identified by caspase-3 labelling (Supplementary Fig. ...
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... apex (Fig. 3a). The fraction of BrdU pos and EdU pos CMs was ~7% and ~3% of the CMs in the subendocardial apical layer at 28 days, respectively, compared with ~0,1% in saline-treated mice. Newly formed CMs were significantly less abundant in the mid-and basal myocardial regions (Fig. 3b), in agreement with less CM damage in these areas (Fig. 1). Also, BrdU pos and EdU pos CMs were significantly less abundant with only 7 days compared with 28 days continuous thymidine analogues' administration after ISO (Fig. 3b). In all cases, however, EdU-labelled less (< 50%) newly formed CMs than BrdU either after 7-days or 28-days of continuous administration (Fig. ...
Citations
... Cardiomyocyte death and diminished pump function are difficult to reverse in adults. Scientists worldwide have focused on cardiac regeneration for the replacement of dead and damaged cardiomyocytes with new cells to improve heart function [3][4][5][6]. ...
The ability of the adult mammalian heart to regenerate can save the cardiac muscle from a loss of function caused by injury. Cardiomyocyte regeneration is a key aspect of research for the treatment of cardiovascular diseases. The mouse heart shows temporary regeneration in the first week after birth; thus, the newborn mouse heart is an ideal model to study heart muscle regeneration. In this study, proteomic analysis was used to investigate the differences in protein expression in the hearts of neonatal mice at days 1 (P1 group), 4 (P4 group), and 7 (P7 group). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed changes in several groups of proteins, including the protein kinase A (PKA) signaling pathway. Moreover, it was found that PKA inhibitors and agonists regulated cardiomyocyte replication in neonatal mouse hearts. These findings suggest that PKA may be a target for the regulation of the cardiomyocyte cell cycle.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12953-023-00219-4.
... To assess the phenotypic and transcriptional changes occurring during CSC myogenic specification, cardiomyocyte-depleted cardiac cell preparations were obtained from wild-type C57BL/6J mice, as well as from the Cre reporter ROSA26 mT/mG and ROSA26 floxed-stop/dTomato mice (both on a C57BL/6J background), through mechanical (gentleMAC Dissociator) dissociation in collagenase type-II-based digestion buffer, followed by gravity separation [23,24]. These three mouse strains were chosen to eventually obtain CSCs and CSC-derived CSs useful for cell tracking in vivo upon transplantation [23,35,36]. Cardiomyocyte-depleted cardiac cells were efficiently depleted of CD45-and CD31-expressing cells by magneticactivated cell sorting (MACS) [23,24] and were then FACS-sorted to obtain a pure (over 95%) population of CD45 neg /CD31 neg /c-kit pos cardiac cells [23,24], which we shown to be enriched for multipotent CSCs [23,24]. ...
Background:
Three-dimensional cell culture systems hold great promise for bridging the gap between in vitro cell-based model systems and small animal models to study tissue biology and disease. Among 3D cell culture systems, stem-cell-derived spheroids have attracted significant interest as a strategy to better mimic in vivo conditions. Cardiac stem cell/progenitor (CSC)-derived spheroids (CSs) provide a relevant platform for cardiac regeneration.
Methods:
We compared three different cell culture scaffold-free systems, (i) ultra-low attachment plates, (ii) hanging drops (both requiring a 2D/3D switch), and (iii) agarose micro-molds (entirely 3D), for CSC-derived CS formation and their cardiomyocyte commitment in vitro.
Results:
The switch from a 2D to a 3D culture microenvironment per se guides cell plasticity and myogenic differentiation within CS and is necessary for robust cardiomyocyte differentiation. On the contrary, 2D monolayer CSC cultures show a significant reduced cardiomyocyte differentiation potential compared to 3D CS culture. Forced aggregation into spheroids using hanging drop improves CS myogenic differentiation when compared to ultra-low attachment plates. Performing CS formation and myogenic differentiation exclusively in 3D culture using agarose micro-molds maximizes the cardiomyocyte yield.
Conclusions:
A 3D culture system instructs CS myogenic differentiation, thus representing a valid model that can be used to study adult cardiac regenerative biology.
... In addition, it still remains unclear if M1 macrophages can switch to an M2 type, representing a mixed phenotype, or if these two subsets of macrophages necessarily originate from completely different sources [22]. Nevertheless, it has been established that the cellular response to heart damage can be divided into three distinct phases: the inflammatory, proliferative, and resolutive phases ( Figure 2) [23]. ...
... However, it is also w accepted that the M1/M2 paradigm is just a simplification, and the exact source phenotypes of macrophages are yet to be fully clarified. In addition, it still remains un if M1 macrophages can switch to an M2 type, representing a mixed phenotype, or if two subsets of macrophages necessarily originate from completely different sources Nevertheless, it has been established that the cellular response to heart damage c divided into three distinct phases: the inflammatory, proliferative, and resolutive p ( Figure 2) [23]. ...
... However, the experimental design employed by Vagnozzi et al. did not allow for the detection of new cardiomyocyte formation [94]. However, other studies have demonstrated the generation of new cardiomyocytes following injury [23,95]. For instance, an acute cardiomyocyte loss caused by an isoproterenol overdose (ISO) activates the resident cardiac c-kit + stem/progenitor cells (CSCs), leading to the generation of new cardiomyocytes that replace those lost after ISO-induced damage [23]. ...
There is an increasing interest in understanding the connection between the immune and cardiovascular systems, which are highly integrated and communicate through finely regulated cross-talking mechanisms. Recent evidence has demonstrated that the immune system does indeed have a key role in the response to cardiac injury and in cardiac regeneration. Among the immune cells, macrophages appear to have a prominent role in this context, with different subtypes described so far that each have a specific influence on cardiac remodeling and repair. Similarly, there are significant differences in how the innate and adaptive immune systems affect the response to cardiac damage. Understanding all these mechanisms may have relevant clinical implications. Several studies have already demonstrated that stem cell-based therapies support myocardial repair. However, the exact role that cardiac macrophages and their modulation may have in this setting is still unclear. The current need to decipher the dual role of immunity in boosting both heart injury and repair is due, at least for a significant part, to unresolved questions related to the complexity of cardiac macrophage phenotypes. The aim of this review is to provide an overview on the role of the immune system, and of macrophages in particular, in the response to cardiac injury and to outline, through the modulation of the immune response, potential novel therapeutic strategies for cardiac regeneration.
... Isoproterenol (ISO, Sigma-Aldrich #I6504; St. Louis, MO, USA) was prepared by dissolving a desired amount of powder in NaCl 0.9% [8][9][10]. The solution obtained was protected from the light and kept on ice until the injections. ...
... Before any ISO/Saline injection, the body weight of animals was determined, mice were anesthetized using isoflurane, and baseline echocardiography was obtained. Then, mice were randomly divided in the different groups and, on awakening, prepared to receive saline or ISO at dose of 200 mg/Kg [9,10]. The solutions were injected subcutaneously under the inter-scapular skin. ...
... All ISO injections were administered to male 12/14-weeks-old mice. After 72 h of receiving ISO at dose of 200 mg/Kg, mice received systemic administration of 5-FU (15 mg/Kg/day) for 25 days through subcutaneously implantation of mini-osmotic pumps [9,10]. Before pumps implantation, the mice were anesthetized using isoflurane. ...
Appropriate dilated cardiomyopathy (DCM) animal models are highly desirable considering the pathophysiological and clinical heterogeneity of DCM. Genetically modified mice are the most widely and intensively utilized research animals for DCM. However, to translate discoveries from basic science into new and personalized medical applications, research in non-genetically based DCM models remains a key issue. Here, we characterized a mouse model of non-ischemic DCM induced by a stepwise pharmacologic regime of Isoproterenol (ISO) high dose bolus followed by a low dose systemic injection of the chemotherapy agent, 5-Fluorouracil (5-FU). C57BL/6J mice were injected with ISO and, 3 days after, were randomly assigned to saline or 5-FU. Echocardiography and a strain analysis show that ISO + 5FU in mice induces progressive left ventricular (LV) dilation and reduced systolic function, along with diastolic dysfunction and a persistent global cardiac contractility depression through 56 days. While mice treated with ISO alone recover anatomically and functionally, ISO + 5-FU causes persistent cardiomyocyte death, ensuing in cardiomyocyte hypertrophy through 56 days. ISO + 5-FU-dependent damage was accompanied by significant myocardial disarray and fibrosis along with exaggerated oxidative stress, tissue inflammation and premature cell senescence accumulation. In conclusions, a combination of ISO + 5FU produces anatomical, histological and functional cardiac alterations typical of DCM, representing a widely available, affordable, and reproducible mouse model of this cardiomyopathy.
... The adult mammalian heart contains a large amount of endogenous CSCs, which is clonogenic, self-renewing and pluripotent. CSCs were involved in the response to cardiac injury and physiological CMs transition during the life cycle, and have a significant capacity for cardiac tissue regeneration [100]. Reactivation of developmental signaling factors in the heart leads to metabolic reprogramming of CMs, which favors increased cell-cycle activity and myocardial repair after injury. ...
Cardiac aging is a natural process accompanied by cardiomyocyte hypertrophy and dysfunction. These changes can lead to adverse organ remodeling and ultimately lead to the development of heart failure. The study of cardiac aging is helpful to explore the mechanism of senescence and is of great significance for preventing cardiac aging. Cardiac aging is accompanied by changes in various metabolic functions. In this process, due to the change of metabolic substrates and enzyme activities, oxidative stress response increases, and reactive oxygen species (ROS) increases, accompanied by mitochondrial dysfunction and gene expression changes, so related protein metabolism also changes. Hormone metabolism and autophagy are also involved in the process of cardiac aging. Based on these findings, changes in diet, caloric restriction, improvement of mitochondrial function and promotion of autophagy have been proven to have positive effects in delaying cardiac aging. This article reviews the metabolic changes involved in the process of cardiac aging from different aspects, and briefly reviews the measures to improve cardiac aging.
... To track myocardial cell regeneration, four weeks after STZ injection, the T1DM, T2DM and CTRL mice were implanted subcutaneously (between the two scapulae) with miniosmotic pumps to systemically release BrdU (Bromodeoxyuridine/5-bromo-2 -deoxyuridine, 50 mg/Kg/Day both) for 28 days. BrdU is an analogue of the nucleoside thymidine, whose cell incorporation in vivo is widely used to identify proliferating cells and, when administered continuously, as in this study, the detection of which provides the number of cumulative newly formed cells [27,28]. Cardiac sections from the T1DM and T2DM mice displayed a significantly lower percentage of BrdU-positive CMs when compared to the CTRL mice: 0.009 ± 0.004% vs. 0.05 ± 0.003% vs. 0.12 ± 0.02%, respectively ( Figure 4A). ...
... Mice that underwent echocardiographic evaluation were prepared as previously described in detail [22,23,27]. All echo images and videos were obtained from the mice at heart rates > 400 b.p.m. ...
... All echo images and videos were obtained from the mice at heart rates > 400 b.p.m. Echocardiographic images and videos were obtained with a Vevo 3100 system (Visualsonics, Inc., Toronto, Canada) equipped with a MX550D ultrahigh-frequency linear-array transducer [22,23,27]. B-mode, M-mode and speckle-tracking images were analyzed through Vevo LAB analysis software Version 3.2.0 ...
The main cause of morbidity and mortality in diabetes mellitus (DM) is cardiovascular complications. Diabetic cardiomyopathy (DCM) remains incompletely understood. Animal models have been crucial in exploring DCM pathophysiology while identifying potential therapeutic targets. Streptozotocin (STZ) has been widely used to produce experimental models of both type 1 and type 2 DM (T1DM and T2DM). Here, we compared these two models for their effects on cardiac structure, function and transcriptome. Different doses of STZ and diet chows were used to generate T1DM and T2DM in C57BL/6J mice. Normal euglycemic and nonobese sex- and age-matched mice served as controls (CTRL). Immunohistochemistry, RT-PCR and RNA-seq were employed to compare hearts from the three animal groups. STZ-induced T1DM and T2DM affected left ventricular function and myocardial performance differently. T1DM displayed exaggerated apoptotic cardiomyocyte (CM) death and reactive hypertrophy and fibrosis, along with increased cardiac oxidative stress, CM DNA damage and senescence, when compared to T2DM in mice. T1DM and T2DM affected the whole cardiac transcriptome differently. In conclusion, the STZ-induced T1DM and T2DM mouse models showed significant differences in cardiac remodeling, function and the whole transcriptome. These differences could be of key relevance when choosing an animal model to study specific features of DCM.
... To date, numerous different types of stem/progenitor cells expressing c-kit have been reported as a source of new CMs in the adult heart: cardiac stem cells, epicardium-derived cells, endothelial progenitor cells, Isl1 positive cardiac progenitor cell and CardioSphere-derived cells are examples of source of cells evaluated for their capacity to replace lost CMs and recover the myocardial tissue by transplantation into the post-infarcted myocardium [70][71][72][73][120][121][122][123][124]. It has been extensively demonstrated that c-Kit is a relatively ubiquitous marker (monocytes within the tissue, endothelial or hematopoietic cells express c-kit), and that its expression alone is insufficient to define a specific cardiac progenitor c-Kit positive cell population [72]. ...
... Therefore, better cell surface marker signatures are still needed to resolve c-kit positive cardiac cell heterogeneity [71,72]. Adult endogenous cardiac stem/progenitor cells (CSCs) have been shown to contribute to the cardiomyogenic cell lineage both in vitro and in vivo when delivered after injury; they also can be reactivated in situ to induce cardiac regeneration following cardiac injury [70][71][72]123]. Through gainand loss-of-function experiments we have shown the involvement of Wnt/β-catenin and TGF-β/SMAD signaling in growth promotion and cardiomyogenic specification of the CSCs [71,72]. ...
Cardiac muscle damage-induced loss of cardiomyocytes (CMs) and dysfunction of the remaining ones leads to heart failure, which nowadays is the number one killer worldwide. Therapies fostering effective cardiac regeneration are the holy grail of cardiovascular research to stop the heart failure epidemic. The main goal of most myocardial regeneration protocols is the generation of new functional CMs through the differentiation of endogenous or exogenous cardiomyogenic cells. Understanding the cellular and molecular basis of cardiomyocyte commitment, specification, differentiation and maturation is needed to devise innovative approaches to replace the CMs lost after injury in the adult heart. The transcriptional regulation of CM differentiation is a highly conserved process that require sequential activation and/or repression of different genetic programs. Therefore, CM differentiation and specification have been depicted as a step-wise specific chemical and mechanical stimuli inducing complete myogenic commitment and cell-cycle exit. Yet, the demonstration that some microRNAs are sufficient to direct ESC differentiation into CMs and that four specific miRNAs reprogram fibroblasts into CMs show that CM differentiation must also involve negative regulatory instructions. Here, we review the mechanisms of CM differentiation during development and from regenerative stem cells with a focus on the involvement of microRNAs in the process, putting in perspective their negative gene regulation as a main modifier of effective CM regeneration in the adult heart.
... This experimental approach was challed into question later, showing that the used c-kit + /Cre system does not reliably track a Lin − c-kit low cardiac stem cell population [32]. Moreover, it has been shown that the knock-in of the Cre recombinase to the c-kit locus generates c-kit haploinsufficient mice with an impaired endogenous cardiac repair after injury compared to c-kit diploid mice [33]. However, more detailed experiments showed that additional depletion of CD45 + cells resulted in a c-kit + CD45 − subpopulation that is capable of supporting functional myocardial regeneration in a mouse model of myocardial injury [34]. ...
Tissue regeneration substantially relies on the functionality of tissue-resident endogenous adult stem cell populations. However, during aging, a progressive decline in organ function and regenerative capacities impedes endogenous repair processes. Especially the adult human heart is considered as an organ with generally low regenerative capacities. Interestingly, beneficial effects of systemic factors carried by young blood have been described in diverse organs including the heart, brain and skeletal muscle of the murine system. Thus, the interest in young blood or blood components as potential therapeutic agents to target age-associated malignancies led to a wide range of preclinical and clinical research. However, the translation of promising results from the murine to the human system remains difficult. Likewise, the establishment of adequate cellular models could help to study the effects of human blood plasma on the regeneration of human tissues and particularly the heart. Facing this challenge, this review describes the current knowledge of blood plasma-mediated protection and regeneration of aging tissues. The current status of preclinical and clinical research examining blood borne factors that act in stem cell-based tissue maintenance and regeneration is summarized. Further, examples of cellular model systems for a more detailed examination of selected regulatory pathways are presented.
... Similarly, myocardial damage following infarction prompted c-Kit + and DDR2 + and coinciding with a peak at 7 day post-MI, consistent with a protective role for cardiac cells expressing c-Kit and DDR2 alone or in combination (Fig. 2c, d). This observation is consistent with the prior reports using transgenic mice demonstrating c-Kit haploinsufficiency impairs structural and functional recovery of the damaged heart and aggravates fibrotic remodeling following myocardial injury [60,61]. ...
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.
... Consequently, c-kit + /Lin − cells resident in the heart have become one of the most promising targets for cell therapy [31,[76][77][78][79]. The regenerative activities of these cardiac stem cells have been demonstrated under ischemic injury [80,81], pressure overload [81], and overdose of isoproterenol [82]. In contrast, some studies have reported that cardiac stem cells minimally contribute to cardiomyocytes in the adult heart or have more assertively reported that the adult heart lacks an endogenous functional pool of myogenic precursor cells [83][84][85][86]. ...
In the adult mammalian heart, no data have yet shown the existence of cardiomyocyte-differentiable stem cells that can be used to practically repair the injured myocardium. Atypically shaped cardiomyocytes (ACMs) are found in cultures of the cardiomyocyte-removed fraction obtained from cardiac ventricles from neonatal to aged mice. ACMs are thought to be a subpopulation of cardiomyocytes or immature cardiomyocytes, most closely resembling cardiomyocytes due to their spontaneous beating, well-organized sarcomere and the expression of cardiac-specific proteins, including some fetal cardiac gene proteins. In this review, we focus on the characteristics of ACMs compared with ventricular myocytes and discuss whether these cells can be substitutes for damaged cardiomyocytes. ACMs reside in the interstitial spaces among ventricular myocytes and survive under severely hypoxic conditions fatal to ventricular myocytes. ACMs have not been observed to divide or proliferate, similar to cardiomyocytes, but they maintain their ability to fuse with each other. Thus, it is worthwhile to understand the role of ACMs and especially how these cells perform cell fusion or function independently in vivo. It may aid in the development of new approaches to cell therapy to protect the injured heart or the clarification of the pathogenesis underlying arrhythmia in the injured heart.
