Mammalian heart renewal by pre-existing cardiomyocytes

Although recent studies have revealed that adult heart cells are generated after birth in mammals, the frequency and source of these new heart cells is unclear. Here we combined two different pulse-chase approaches--genetic fate-mapping and stable isotope labeling with Multi-isotope Imaging Mass Spectrometry (MIMS)--to determine the origin of cardiomyocytes in adult mice. MIMS allows quantitative imaging of stable isotope reporters in domains smaller than a micron cubed. The nuclear incorporation of a 15N stable isotope-enriched tracer here as 15N-thymidine is detectable with high precision by an increase in the 15N/14N ratio above natural abundance (0.37%) in a pattern resembling chromatin evident in cells having divided during a labeling period. We administered 15N-thymidine for 8 weeks to three age groups of C57BL6 mice starting at day-4 (neonate), at 10-weeks (young adult) and at 22-months (old adult) and extrapolated DNA synthesis yields a yearly rate of 5.5% in the young adult that declines further to 2.6% in advanced aged mice. Fig1a. To evaluate the origin of newly generated cardiomyocytes, we performed 15N-thymidine labeling of double-transgenic MerCreMer/ZEG mice, which we previously developed for genetic lineage mapping. Fig1b. After treatment with 4OH-tamoxifen, we show that DNA synthesis occurs at a low rate in predominantly pre-existing cardiomyocytes [77% expressed GFP, a frequency essentially identical to that of surrounding 15N- cardiomyocytes (15N, 77% vs 15N-, 84%; Fisher’s exact=n.s.)]. Fig1c. Cell cycle activity in cardiomyocytes led to polyploidy and multinucleation, and to new diploid, mononucleated cardiomyocytes [17% of total; predominantly GFP+ (83% vs 82%, p=n.s.)]. Fig1d. Our results support a low rate of cell division of cardiomyocytes as the origin of mammalian adult cardiogenesis and are inconsistent with a high rate of stem cell activity in the normal mammalian myocardium.

Multi-isotope imaging mass spectrometry (MIMS) allows high resolution quantitative imaging of protein and nucleic acid synthesis at the level of a single cell using stable isotope labels. We employed MIMS to determine the compartmental localization of selenoproteins tagged with stable isotope selenium compounds in human aortic endothelial cells (HAEC), and to compare the efficiency of labeling (to determine the ideal selenium source) from these compounds: [82Se]-selenite, [77Se]-seleno-methionine, and [76Se]-methyl-selenocysteine. We found that all three selenium sources appear to be localized in the nucleus as well as in the cytoplasm in HAEC. Seleno-methionine appears to be a better source for (seleno)protein synthesis. For MIMS detection, we compared freeze-drying to thin layer vs. thin sectioning for sample preparation. MIMS provides a unique and novel way to dissect selenoprotein synthesis in cells.

Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter1,2 but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with sub-micron resolution3,4. Here we apply MIMS to diverse organisms, including Drosophila, mice, and humans. We test the “immortal strand hypothesis,” which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labeling mice with 15N-thymidine from gestation through post-natal week 8, we find no 15N label retention by dividing small intestinal crypt cells after 4wk chase. In adult mice administered 15N-thymidine pulse-chase, we find that proliferating crypt cells dilute label consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human hematopoietic system. These studies show that MIMS provides high-resolution quantitation of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.

Quantitative imaging of subcellular metabolism with stable isotopes and multi-isotope imaging mass spectrometry

Design and rationale of a clinical trial to increase cardiomyocyte division in infants with tetralogy of Fallot

Multi-isotope imaging mass spectrometry (MIMS) allows the measurement of turnover of molecules within intracellular compartments with a spatial resolution down to 30 nm. We use molecules enriched in stable isotopes administered to animals by diet or injection, or to cells through the culture medium. The stable isotopes used are, in general, 15 N, 13 C, 18 O, and 2 H. For stem cell studies, we essentially use 15 N-thymidine, 13 C-thymidine, and 81 Br from BrdU. This protocol describes the practical use of MIMS with specific reference to applications in stem cell research. This includes choice and administration of stable isotope label(s), sample preparation, best practice for high-resolution imaging, secondary ion mass spectrometry using the Cameca NanoSIMS 50L, and methods for robust statistical analysis of label incorporation in regions of interest (ROI). © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Stable isotope labeling of DNA in cultured cells Basic Protocol 2: Stable isotope labeling of DNA in animals Basic Protocol 3: Preparation of Si chips, the general sample support for NanoSIMS analysis Basic Protocol 4: Stable isotope analysis of DNA replication in single nuclei in a population of cells with NanoSIMS Basic Protocol 5: Data reduction and processing.

Quantification of stable isotope tracers after metabolic labeling provides a snapshot of the dynamic state of living cells and tissue. A form of imaging mass spectrometry quantifies isotope ratios with a lateral resolution <50 nm, using a methodology that we refer to as multi-isotope imaging mass spectrometry (MIMS). Despite lateral resolution exceeding diffraction-limited light microscopy, lack of contrast has largely limited use of MIMS to large or specialized subcellular structures, such as the nucleus and stereocilia. In this study, we repurpose the engineered peroxidase APEX2 as the first genetically encoded marker for MIMS. Coupling APEX2 labeling of lysosomes and metabolic labeling of protein, we identify that individual lysosomes exhibit substantial heterogeneity in protein age, which is lost in iPSC-derived neurons lacking the lysosomal protein progranulin. This study expands the practical use of MIMS for cell biology by enabling measurements of metabolic function from stable isotope labeling within individual organelles in situ.

Background: Cardiomyocyte proliferation, the fundamental mechanism of heart development and regeneration, is active in infants. We investigated cardiomyocyte proliferation in infants with heart disease. Objective/Hypothesis: Infants with tetralogy of Fallot (ToF, the most common type of cyanotic congenital heart disease) or heart failure (HF) exhibit altered cardiomyocyte proliferation. Methods: The DNA replication marker, 15 N-thymidine, was administered to infants with unrepaired ToF (n = 13) and to infants with heart failure waiting for transplantation (n = 5). One of the infants with ToF was also treated with the beta-blocker, propranolol. Myocardial samples were ascertained at the time of ToF surgery, placement or removal of left ventricular assist devices (LVAD), and heart transplantation. Retention of 15 N-thymidine in cells that had undergone DNA replication was assessed using Multi-Isotope Imaging Mass Spectrometry (MIMS). Polyploidy was quantified to identify post-mitotic, non-dividing cardiomyocytes. Results: Within the first 4 months after birth, 0.74 billion new cardiomyocytes were generated—3.4-fold higher than after the age of 4 months (P = 0.0417). Average per nucleus DNA content in 15 N-labeled cardiomyocytes in ToF infants (n = 11) and HF infants (n = 3) was significantly greater (1.38-fold) than that in individuals without heart disease (n = 11, P = 0.0358), indicating increased formation of post-mitotic cardiomyocytes in diseased hearts. An infant with HF received 15 N-thymidine while on LVAD. Despite clinical improvement and LVAD removal, cardiomyocyte generation remained below age-appropriate levels. In contrast, a ToF infant treated with propranolol, showed a reduced incidence of polyploid nuclei, suggesting enhanced cardiomyocyte division in response to β-blockade. Conclusion: Infants with heart disease generate new cardiomyocytes, with the majority of this occurring in the first four months after birth, which identifies a window for regenerative therapies. Beta-blockade may promote cardiomyocyte division, highlighting a direction for regenerative therapy.

The quantification of cell cycle activity is a prerequisite to defining the dynamics and scope of organ development or regeneration. Multi-isotope imaging mass spectrometry (MIMS) merges stable isotope tracers with an imaging mass spectrometry platform called NanoSIMS, which can quantitatively measure the incorporation of stable isotope tracers with high precision in suborganelle domains. MIMS has been applied to quantify the dynamics of postnatal cardiogenesis and mammalian cardiomyocyte regeneration during aging or in response to injury. Here, we present an approach to the conduct of MIMS experiments, with an emphasis on the application to the field of cardiac regeneration; however, the approach is also applicable, with, at most, minor modifications to broader biological questions.

Multi-isotope imaging mass spectrometry (MIMS) combines stable isotope tracers with the quantitative imaging of NanoSIMS ion microscopy. With extensive safety precedent, use of stable isotopes in MIMS applications opens the possibility of studying a wide array of biological questions in humans[1]. Here we describe a series of approaches to increase the effective analytical throughput for detecting rare nuclear labeling events with MIMS. At the level of sample preparation, cells in suspension were either smeared at high density or pelleted cells were embedded and sectioned to reach nuclear depth. Presputtering conditions were optimized for each cell type to ensure the reproducible sampling of nuclei. Adipose tissue posed a different challenge as the large volume of adipocytes results in an obligatorily low density of nuclei in any given plane. Before introducing samples to the NanoSIMS instrument, all nuclei were fluorescently stained, imaged, and their coordinates recorded, allowing automated analysis of fields that contained at least one nucleus and therefore minimizing analysis of dead space. These data emphasize unique challenges posed by human studies, where both ethical and practical issues may limit the administration of stable isotope labels for prolonged periods of time as may be necessary to achieve high labeling frequencies in cells that divide infrequently.

Quantification of cellular proliferation in humans is important for understanding biology and responses to injury and disease. However, existing methods require administration of tracers that cannot be ethically administered in humans. We present a protocol for the direct quantification of cellular proliferation in human hearts. The protocol involves administration of non-radioactive, non-toxic stable isotope 15Nitrogen-enriched thymidine (15N-thymidine), which is incorporated into DNA during S-phase, in infants with tetralogy of Fallot, a common form of congenital heart disease. Infants with tetralogy of Fallot undergo surgical repair, which requires the removal of pieces of myocardium that would otherwise be discarded. This protocol allows for the quantification of cardiomyocyte proliferation in this discarded tissue. We quantitatively analyzed the incorporation of 15N-thymidine with multi-isotope imaging spectrometry (MIMS) at a sub-nuclear resolution, which we combined with correlative confocal microscopy to quantify formation of binucleated cardiomyocytes and cardiomyocytes with polyploid nuclei. The entire protocol spans 3-8 months, which is dependent on the timing of surgical repair, and 3-4.5 researcher days. This protocol could be adapted to study cellular proliferation in a variety of human tissues.

Purpose of ReviewThe typical remodeling process after cardiac injury is scarring and compensatory hypertrophy. The limited regeneration potential of the adult heart is thought to be due to the post-mitotic status of postnatal cardiomyocytes, which are mostly binucleated and/or polyploid. Nevertheless, there is evidence for cardiomyocyte turnover in the adult heart. The purpose of this review is to describe the recent findings regarding the proliferative potential of mononuclear cardiomyocytes and to evaluate their function in cardiac turnover and disease.Recent FindingsThere is overwhelming evidence from carbon-dating in humans and multi-isotope imaging mass spectrometry in mice that there is a very low but detectable level of turnover of cardiomyocytes in the heart. The source of this renewal is not clear, but recent evidence points to a population of mononuclear, diploid cardiomyocytes that are still capable of authentic cell division. Controversy arises when their role in cardiac repair is considered, as some studies claim that they contribute to repair by cell division while other studies do not find evidence for hyperplasia but hypertrophy. Stimulation of the mononuclear cardiomyocyte population has been proposed as a therapeutic strategy in cardiac disease.SummaryThe studies reviewed here agree on the existence of a low annual cardiomyocyte turnover rate which can be attributed to the proliferation of mononuclear cardiomyocytes. Potential roles of mononucleated cardiomyocytes in cardiac repair after injury are discussed.

To measure single-cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labelling of microbial populations with heavy water (a passive tracer) and (15) N ammonium in combination with multi-isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single-cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single-cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (∼0.1 atom % (2) H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (∼6 h, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium versus amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments.

Recent studies report that postnatal mammalian hearts undergo cardiomyocyte refreshment. While the exact origin of the cells involved in postnatal cardiomyogenesis remains unclear. Here, we identified a pool of Nkx2.5 enhancer expressing cells in the postnatal mouse heart with cardiomyogenic differentiation potential in vitro. We tracked the expression of a cardiac-specific enhancer of Nkx2.5 using inducible Nkx2.5 enhancer-Cre mice from embryonic development to adulthood and post-myocardial infarction (MI) and documented the Nkx2.5 enhancer expressing cells directly contribute to postnatal cardiomyogenesis in vivo. Upon genetic ablation of these activated progenitors after myocardial injury, the cardiac function deteriorated. Transcriptomic analysis of Nkx2.5 enhancer expressing cells showed high expression of heart development genes. To trace the developmental origin of the activated Nkx2.5 cardiomyogenic progenitor cells, we created different lineage-Cre/Nkx2.5 enh-eGFP/ROSA26 reporter triple transgenic mice. Post-MI Nkx2.5 cardiomyogenic progenitor cells originated from the embryonic epicardial cells, not from the pre-existing cardiomyocytes, endothelial cells, cardiac neural crest cells, or perinatal/postnatal epicardial cells. Together, this study confirmed that cardiac lineage-specific progenitor cells, which originate from embryonic epicardium-derived cells, contribute to postnatal mammalian cardiomyogenesis.

![Figure][1] J. Patrick Kampf Torrey Pines Institute for Molecular Studies, San Diego, California ![Figure][1] Alan M. Kleinfeld Torrey Pines Institute for Molecular Studies, San Diego, California akleinfeld{at}tpims.org The mechanism of free fatty acid (FFA)