Postnatal Heart Development Research Articles

Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different age-dependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56–2.2 × 106 cells/heart) and viability (~70–100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell type-specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR < 0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rod-shaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

3D mitochondrial remodeling during postnatal heart development

The heart is featured by high mitochondrial volume, by which continuous contractile activity can be maintained. However, it is unclear how mitochondrial structures are matured during postnatal development. Using FIB‐SEM, we thus sought to identify 3D mitochondrial structural characteristics in the heart of mice at postnatal (P) day 1, 7, 14, and 42, respectively. Here, matured mitochondria (P14‐42) were shown to have larger and more spherical structures and were highly connected with adjacent mitochondria as compared to early postnatal days (P1‐7). Nevertheless, the immature mitochondria appeared to have more intense interaction with other subcellular organelles including lipid droplets and sarcotubular structures (i.e., SR/T‐tubules). Thus, our study suggests that initial mitochondrial development can be augmented by crosstalk with adjacent mitochondria as well as other subcellular components and that it would be important for the further mitochondrial maturation process in the heart muscle.

Proteogenomics Integrating Reveal a Complex Network, Alternative Splicing, Hub Genes Regulating Heart Maturation.

Heart maturation is an essentially biological process for neonatal heart transition to adult heart, thus illustrating the mechanism of heart maturation may be helpful to explore postnatal heart development and cardiac cardiomyopathy. This study combined proteomic analysis based on isobaric tags for relative and absolute quantitation (iTRAQ) and transcriptome analysis based on RNA sequencing to detect the proteins and genes associated with heart maturation in mice. The proteogenomics integrating analysis identified 254 genes/proteins as commonly differentially expressed between neonatal and adult hearts. Functional and pathway analysis demonstrated that these identified genes/proteins contribute to heart maturation mainly by regulating mRNA processing and energy metabolism. Genome-wide alternative splicing (AS) analysis showed that some important sarcomere and energy-associated genes undergo different AS events. Through the Cytoscape plug-in CytoHubba, a total of 23 hub genes were found and further confirmed by RT-qPCR. Next, we verified that the most up-regulated hub gene, Ogdhl, plays an essential role in heart maturation by detecting energy metabolism phenotype changes in the Ogdhl-interfering cardiomyocytes. Together, we revealed a complex gene network, AS genes and patterns, and candidate hub genes controlling heart maturation by proteome and transcriptome combination analysis.

Alternative splicing regulation of membrane trafficking genes during myogenesis.

Alternative splicing transitions occur during organ development, and, in numerous diseases, splicing programs revert to fetal isoform expression. We previously found that extensive splicing changes occur during postnatal mouse heart development in genes encoding proteins involved in vesicle-mediated trafficking. However, the regulatory mechanisms of this splicing-trafficking network are unknown. Here, we found that membrane trafficking genes are alternatively spliced in a tissue-specific manner, with striated muscles exhibiting the highest levels of alternative exon inclusion. Treatment of differentiated muscle cells with chromatin-modifying drugs altered exon inclusion in muscle cells. Examination of several RNA-binding proteins revealed that the poly-pyrimidine tract binding protein 1 (PTBP1) and quaking regulate splicing of trafficking genes during myogenesis, and that removal of PTBP1 motifs prevented PTBP1 from binding its RNA target. These findings enhance our understanding of developmental splicing regulation of membrane trafficking proteins which might have implications for muscle disease pathogenesis.

Epigenetic Regulator RNF20 Underlies Temporal Hierarchy of Gene Expression to Regulate Cardiomyocyte Polarization During Postnatal Heart Development

Epigenetic Regulator RNF20 Underlies Temporal Hierarchy of Gene Expression to Regulate Cardiomyocyte Polarization During Postnatal Heart Development

A roadmap for the characterization of energy metabolism in human cardiomyocytes derived from induced pluripotent stem cells

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are an increasingly employed model in cardiac research and drug discovery. As cellular metabolism plays an integral role in determining phenotype, the characterization of the metabolic profile of hiPSC-CM during maturation is crucial for their translational application. In this study we employ a combination of methods including extracellular flux, 13C-glucose enrichment and targeted metabolomics to characterize the metabolic profile of hiPSC-CM during their maturation in culture from 6 weeks, up to 12 weeks. Results show a progressive remodeling of pathways involved in energy metabolism and substrate utilization along with an increase in sarcomere regularity. The oxidative capacity of hiPSC-CM and particularly their ability to utilize fatty acids increased with time. In parallel, relative glucose oxidation was reduced while glutamine oxidation was maintained at similar levels. There was also evidence of increased coupling of glycolysis to mitochondrial respiration, and away from glycolytic branch pathways at later stages of maturation. The rate of glycolysis as assessed by lactate production was maintained at both stages but with significant alterations in proximal glycolytic enzymes such as hexokinase and phosphofructokinase. We observed a progressive maturation of mitochondrial oxidative capacity at comparable levels of mitochondrial content between these time-points with enhancement of mitochondrial network structure. These results show that the metabolic profile of hiPSC-CM is progressively restructured, recapitulating aspects of early post-natal heart development. This would be particularly important to consider when employing these cell model in studies where metabolism plays an important role.

Abstract 11948: A Standardized Method to Isolate and Purify Cardiomyocytes from Individual Mouse Hearts Irrespective of Postnatal Age

Rational: Primary cardiomyocytes (CMs) are invaluable for understanding postnatal heart development and elucidating disease mechanisms in genetic and pharmacological models, however, a method to obtain freshly purified CMs at any postnatal age, without the need for different age-dependent isolation procedures and cell culture, is lacking. Objective: To develop a standardized method that allows rapid isolation and purification of CMs in high yield and viability from individual neonatal, infant, and adult mice. Methods and Results: Using a novel in situ aortic cannulation procedure optimized to allow cannulation of even the very small vessel of neonates [postnatal day 0-2 (P0-2)], hearts of C57BL/6J mice were subjected to Langendorff retrograde perfusion and enzymatic digestion. After subsequent tissue disaggregation and filtration, CMs were isolated in high yield (1.55-2.2 x 10 6 CMs/heart) and viability (~78%). The larger size of infant (P10 and P13) and adult (P70), but not neonatal CMs, compared to non-myocytes, allowed their further enrichment by differential centrifugation, before purification at the bench by bead-based immunomagnetic cell separation. This involved depletion of endothelial cells (for infant and adult preparations) or non-myocytes (neonatal preparations). Together, these procedures resulted in highly purified CMs (~95%) from hearts of all ages within 1 hour. Moreover, in situ fixation immediately after tissue digestion via coronary perfusion, preserved the cytoarchitecture of isolated CMs (~94% rod-shaped CMs at all postnatal ages), allowing capture of spindle-shaped neonatal cells undergoing mitosis, as well as enabling accurate quantitation of CM area and nucleation state. RNA-sequencing of CMs purified from one P2 male and female heart per litter (n=4 litters) showed distinct clustering by litters rather than by sexes; a finding consistent with cardiac size and shape being indistinguishable between P2 male (n=6) and female (n=6) hearts as determined by micro-computed tomography (X-ray 3D imaging). Conclusion: The procedures developed here provide a universal protocol for the rapid purification of high-quality CMs from individual hearts at any postnatal age, even those of neonates.

P38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1.

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

Inhibition of SENP2-mediated Akt deSUMOylation promotes cardiac regeneration via activating Akt pathway.

Post-translational modification (PTM) by small ubiquitin-like modifier (SUMO) is a key regulator of cell proliferation and can be readily reversed by a family of SUMO-specific proteases (SENPs), making SUMOylation an ideal regulatory mechanism for developing novel therapeutic strategies for promoting a cardiac regenerative response. However, the role of SUMOylation in cardiac regeneration remains unknown. In the present study, we assessed whether targeting protein kinase B (Akt) SUMOylation can promote cardiac regeneration. Quantitative PCR and Western blotting results showed that small ubiquitin-like modifier-specific protease 2 (SENP2) is up-regulated during postnatal heart development. SENP2 deficiency promoted P7 and adult cardiomyocyte (CM) dedifferentiation and proliferation both in vitro and in vivo. Mice with SENP2 deficiency exhibited improved cardiac function after MI due to CM proliferation and angiogenesis. Mechanistically, the loss of SENP2 up-regulated Akt SUMOylation levels and increased Akt kinase activity, leading to a decrease in GSK3β levels and subsequently promoting CM proliferation and angiogenesis. In summary, inhibition of SENP2-mediated Akt deSUMOylation promotes CM differentiation and proliferation by activating the Akt pathway. Our results provide new insights into the role of SUMOylation in cardiac regeneration.

Progressive stretch enhances growth and maturation of 3D stem-cell-derived myocardium.

Bio-engineered myocardium has great potential to substitute damaged myocardium and for studies of myocardial physiology and disease, but structural and functional immaturity still implies limitations. Current protocols of engineered heart tissue (EHT) generation fall short of simulating the conditions of postnatal myocardial growth, which are characterized by tissue expansion and increased mechanical load. To investigate whether these two parameters can improve EHT maturation, we developed a new approach for the generation of cardiac tissues based on biomimetic stimulation under application of continuously increasing stretch.Methods: EHTs were generated by assembling cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) at high cell density in a low collagen hydrogel. Maturation and growth of the EHTs were induced in a custom-made biomimetic tissue culture system that provided continuous electrical stimulation and medium agitation along with progressive stretch at four different increments. Tissues were characterized after a three week conditioning period.Results: The highest rate of stretch (S3 = 0.32 mm/day) increased force development by 5.1-fold compared to tissue with a fixed length, reaching contractility of 11.28 mN/mm². Importantly, intensely stretched EHTs developed physiological length-dependencies of active and passive forces (systolic/diastolic ratio = 9.47 ± 0.84), and a positive force-frequency relationship (1.25-fold contractility at 180 min-1). Functional markers of stretch-dependent maturation included enhanced and more rapid Ca2+ transients, higher amplitude and upstroke velocity of action potentials, and pronounced adrenergic responses. Stretch conditioned hiPSC-CMs displayed structural improvements in cellular volume, linear alignment, and sarcomere length (2.19 ± 0.1 µm), and an overall upregulation of genes that are specifically expressed in adult cardiomyocytes.Conclusions: With the intention to simulate postnatal heart development, we have established techniques of tissue assembly and biomimetic culture that avoid tissue shrinkage and yield muscle fibers with contractility and compliance approaching the properties of adult myocardium. This study demonstrates that cultivation under progressive stretch is a feasible way to induce growth and maturation of stem cell-derived myocardium. The novel tissue-engineering approach fulfills important requirements of disease modelling and therapeutic tissue replacement.

Adult-Onset Myopathy with Constitutive Activation of Akt following the Loss of hnRNP-U.

SummarySkeletal muscle has the remarkable ability to modulate its mass in response to changes in nutritional input, functional utilization, systemic disease, and age. This is achieved by the coordination of transcriptional and post-transcriptional networks and the signaling cascades balancing anabolic and catabolic processes with energy and nutrient availability. The extent to which alternative splicing regulates these signaling networks is uncertain. Here we investigate the role of the RNA-binding protein hnRNP-U on the expression and splicing of genes and the signaling processes regulating skeletal muscle hypertrophic growth. Muscle-specific Hnrnpu knockout (mKO) mice develop an adult-onset myopathy characterized by the selective atrophy of glycolytic muscle, the constitutive activation of Akt, increases in cellular and metabolic stress gene expression, and changes in the expression and splicing of metabolic and signal transduction genes. These findings link Hnrnpu with the balance between anabolic signaling, cellular and metabolic stress, and physiological growth.

Single-cell analysis of murine fibroblasts identifies neonatal to adult switching that regulates cardiomyocyte maturation

Cardiac maturation lays the foundation for postnatal heart development and disease, yet little is known about the contributions of the microenvironment to cardiomyocyte maturation. By integrating single-cell RNA-sequencing data of mouse hearts at multiple postnatal stages, we construct cellular interactomes and regulatory signaling networks. Here we report switching of fibroblast subtypes from a neonatal to adult state and this drives cardiomyocyte maturation. Molecular and functional maturation of neonatal mouse cardiomyocytes and human embryonic stem cell-derived cardiomyocytes are considerably enhanced upon co-culture with corresponding adult cardiac fibroblasts. Further, single-cell analysis of in vivo and in vitro cardiomyocyte maturation trajectories identify highly conserved signaling pathways, pharmacological targeting of which substantially delays cardiomyocyte maturation in postnatal hearts, and markedly enhances cardiomyocyte proliferation and improves cardiac function in infarcted hearts. Together, we identify cardiac fibroblasts as a key constituent in the microenvironment promoting cardiomyocyte maturation, providing insights into how the manipulation of cardiomyocyte maturity may impact on disease development and regeneration.

Gene-environment regulatory circuits of right ventricular pathology in tetralogy of fallot.

The phenotypic spectrum of congenital heart defects (CHDs) is contributed by both genetic and environmental factors. Their interactions are profoundly heterogeneous but may operate on common pathways as in the case of hypoxia signaling during postnatal heart development in the context of CHDs. Tetralogy of Fallot (TOF) is the most common cyanotic (hypoxemic) CHD. However, how the hypoxic environment contributes to TOF pathogenesis after birth is poorly understood. We performed Genome-wide transcriptomeanalysis on right ventricle outflow tract (RVOT) specimens from cyanotic and noncyanotic TOF. Co-expression network analysis identified gene modules specifically associated with clinical diagnosis and hypoxemia status in the TOF hearts. In particular, hypoxia-dependent induction of myocyte proliferation is associated with E2F1-mediated cell cycle regulation and repression of the WNT11-RB1 axis. Genes enriched in epithelial mesenchymal transition (EMT), fibrosis, and sarcomere were also repressed in cyanotic TOF patients. Importantly, transcription factor analysis of the hypoxia-regulated modules suggested CREB1 as a putative regulator of hypoxia/WNT11-RB1 circuit. The study providesa high-resolution landscape of transcriptome programming associated with TOF phenotypes and unveiled hypoxia-induced regulatory circuit in cyanotic TOF. Hypoxia-induced cardiomyocyte proliferation involves negative modulation of CREB1 activity upstream of the WNT11-RB1 axis. KEY MESSAGES: Genetic and environmental factors contribute to congenital heart defects (CHDs). How hypoxia contributes to Tetralogyof Fallot (TOF) pathogenesis after birth is unclear. Systems biology-based analysis revealed distinct molecular signature in CHDs. Gene expression modules specifically associated with cyanotic TOF were uncovered. Key regulatory circuits induced by hypoxia in TOF pathogenesis after birth were unveiled.

De novo dNTP production is essential for normal postnatal murine heart development

The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in WT postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in WT hearts.

Abstract 521: Cardiac Fibroblasts are Activated During Postnatal Extracellular Matrix Remodeling

Objectives: During the postnatal period, P0 to P30 in the mouse, the myocardial extracellular matrix (ECM) transitions to a mature profile necessary for increased cardiac output. We hypothesize that, during the postnatal period, cardiac fibroblasts (CF) become activated, as indicated by Periostin (Postn) expression, and remodel the ECM, followed by quiescence in the mature heart. Our goal is to study CF activation and its role in postnatal heart development. Methods: CF activation was monitored by Postn MerCreMer(MCM) ;Rosa GFP lineage analysis and the resident CF were studied using a TCF21 MCM ;R26 GFP model. Tamoxifen induction of Cre MCM was initiated in the postnatal period and lineage-specific CF were quantified. Cardiac ECM maturation and CF proliferation, together with bone marrow-derived and endothelial cells, were assessed by Immunofluorescence (IF). TCF21 , Postn , Col1a1 , TnnI and CD31 transcripts were examined by RNAscope, and Fibronectin (FN) compartmentalization, an indicator of ECM maturation, also was examined. Results: FN, Postn, and Postn MCM R26 GFP lineage cells were more prominent at P0-7 than at P30. Postn -expressing cells also co-express Tcf21 and Col1a1 , but not TnnI (myocytes) or PECAM (endothelial cells), as determined by RNAscope. In agreement, Postn MCM R26 GFP CF do not express CD31, CD45, or the myofibroblast marker alphaSMA at P7. At P30, Postn, FN, and Col1a1 expression is reduced in CF, but TCF21 MCM ;R26 GFP is maintained, suggesting a transient period of CF activation followed by quiescence. Similarly, proliferation rates of the Postn MCM R26 GFP cells are higher than TCF21 MCM ;R26 GFP CF at P7, followed by decreased proliferation at P30. Conclusions: Activation and proliferation of CFs and ECM gene expression is increased in the week after birth relative to quiescent CFs at P30. Postn+ cells also peak at P7 but are not detected in appreciable numbers at P30, suggesting a critical role in ECM remodeling and myocardial maturation in the postnatal period. Studies are ongoing to determine the codependence of postnatal activated CF, ECM maturation and myocardial regeneration.

Abstract 424: Postnatal Resetting of Cardiomyocytes Toward Ground State as a Principal Step in Heart Maturation

The mammalian postnatal heart undergoes orchestrated and systematic changes to culminate in functional maturation, yet little is known about the cellular and molecular foundations underlying these processes. Here, we reported that cardiomyocytes (CMs) epigenetically and transcriptionally shut down cardiac gene expression immediately after birth, and reset into a ground state with potential enhancers for further programming CMs into distinct subtypes during heart maturation. Large-scale single-cell RNA-sequencing and genetic lineage tracing confirmed the presence of a postnatal ground state of CMs (pngCMs) that were in highest abundance around postnatal day 7 (P7), and recapitulated the developmental route of postnatal CM development. Importantly, this fate resetting mechanism used by CMs was conserved in neonatal heart regeneration upon surgical resection. Furthermore, P7-pngCMs exhibited remarkable regenerative capacity in infarcted hearts. Collectively, our data uncovered CM fate resetting as a pivotal mechanism in postnatal heart development and regeneration, and supported a new model to understand how organs adapt to abrupt environmental changes to gain maturity upon birth.

Abstract 106: Cardiac Fibroblast as a Critical Constituent of the Cellular Niche Driving Cardiac Maturation

Rationale: Cardiac maturation lays the foundation of postnatal heart development and disease, yet little is known about the contributions of the microenvironment to cardiomyocyte maturation. Objective: To identify non-cardiomyocyte cell types and important signaling pathways central to cardiomyocyte maturation. Methods and Results: Through building cell-cell interactomes and signaling regulatory networks from single-cell RNA-sequencing data of mouse hearts at various postnatal stages, we identified switching of fibroblast subclusters state as a key driving force behind cardiac maturation. Molecular and functional maturation of immature (neonatal) mouse cardiomyocytes or human embryonic stem cell-derived cardiomyocytes were considerably enhanced when co-cultured with their corresponding adult cardiac fibroblasts in vitro . Further, single-cell analysis of the cardiac maturation trajectory revealed highly conserved signaling pathways and ligands in fibroblast-induced cardiomyocyte maturation in vivo and in vitro . Targeting these signaling pathways using inhibitors significantly delayed cardiac maturation in postnatal hearts, and markedly induced cardiomyocyte proliferation in infarcted hearts. Conclusions: Our study unveiled cardiac fibroblasts as a major constituent in the microenvironment driving cardiac maturation. Targeting signaling pathways and ligand-receptor interactions involved in their crosstalk with cardiomyocytes may yield useful strategies to modulate cardiomyocyte maturation state in cardiac diseases and for regeneration purposes. Keywords: Cardiac Maturation, Fibroblast, Single-Cell RNA-Sequencing

Mitochondrial fusion is required for regulation of mitochondrial DNA replication.

Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA.

Cardiac fibroblast lineages and extracellular matrix maturation in postnatal heart development

During postnatal heart development in mice, the ventricular myocardium undergoes extensive remodeling with increased organization and maturation of cardiomyocytes, fibroblasts and endothelial cells. In addition, the extracellular matrix (ECM), consisting mostly of mature fibrillar collagen is produced, providing necessary mechanical support for increased cardiac output. In adult hearts, the matricellular protein periostin (postn) is induced during cardiac injury and is required for collagen deposition in scar formation and pathologic fibrosis. During postnatal heart remodeling, postn also is expressed in cardiac fibroblasts coincident with collagen 1a1 gene expression. At the same time, fibronectin expression indicative of immature ECM permissive for cardiomyocyte proliferation is decreased. By postnatal day 30, postn expression is decreased and the collagen‐rich ECM surrounding individual cardiomyocytes is apparent. The expression of postn during postnatal heart remodeling and ECM maturation supports shared molecular regulation of cardiac fibroblasts and collagen fibrillogenesis in development and disease. However, the specific contributions of postn‐expressing cardiac fibroblasts to the initial production of the collagen‐rich ECM of the heart or to maturation and hypertrophy of adjacent cardiomyocytes are not yet known.Support or Funding InformationThis work was supported by grants from the NIH/NHLBI (P01HL069779 and R01HL142217)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of mitochondrial fusion in cardiac energy metabolism

Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. The goal of our work was to determine the importance of mitochondrial fusion on heart energy homeostasis. To decipher the link between mitochondrial dynamics and mtDNA maintenance and heart energy metabolism, we studied heart conditional knockout mouse and mouse embryonic fibroblasts (MEFs) with disruption of mitochondrial fusion. Super-resolution microscopy analyses revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to clustering and loss of nucleoids. Remarkably, fluorescence in situ hybridization (FISH) in MEFs, bromouridine labeling of MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. The profound mtDNA depletion in hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but, instead, we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA.