Fibroblast transition to an endothelial “trans” state improves cell reprogramming efficiency

Fibroblast reprogramming offers the potential for myocardial regeneration via in situ cell transdifferentiation. We explored a novel strategy leveraging endothelial cell plasticity to enhance reprogramming efficiency. Rat cardiac endothelial cells and fibroblasts were treated with Gata4, Mef2c, and Tbx5 (GMT) to assess the cardio-differentiation potential of these cells. The endothelial cell transdifferentiation factor ETV2 was transiently over-expressed in fibroblasts followed by GMT treatment to assess “trans-endothelial” cardio-differentiation. Endothelial cells treated with GMT generated more cTnT+ cells than did cardiac fibroblasts (13% ± 2% vs 4% ± 0.5%, p < 0.01). Cardiac fibroblasts treated with ETV2 demonstrated increased endothelial cell markers, and when then treated with GMT yielded greater prevalence of cells expressing cardiomyocyte markers including cTnT than did fibroblasts treated with GMT or ETV2 (10.3% ± 0.2% vs 1.7% ± 0.06% and 0.6 ± 0.03, p < 0.01). Rat cardiac fibroblasts treated with GMT + ETV2 demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes, whereas cells treated with GMT or ETV2 alone failed to contract in co-culture experiments. Human cardiac fibroblasts treated with ETV2 and then GMT likewise demonstrated greater prevalence of cTnT expression than did cells treated with GMT alone (2.8-fold increase, p < 0.05). Cardiac fibroblast transitioning through a trans-endothelial state appears to enhance cardio-differentiation by enhancing fibroblast plasticity.

Diet-induced obese mice are resistant to improvements in cardiac function resulting from short-term adropin treatment

Previous studies have shown that treatment with recombinant adropin, a circulating peptide secreted by the liver and brain, restores glucose utilization in the hearts of diet-induced obese mice. This restoration of fuel substrate flexibility, which is lost in obese and diabetic animals, has the potential to improve contractile function in the diabetic heart. Using an ex vivo approach, we examined whether short-term adropin treatment could enhance cardiac function in a mouse model of diet-induced obesity. Our study showed that acute adropin treatment reduces inhibitory phosphorylation of pyruvate dehydrogenase in primary neonatal cardiomyocytes, and leads to moderate improvements in ex vivo cardiac function in mice fed a low fat diet. Conversely, short-term exposure to adropin led to a small decrease in cardiac function in mice fed a long-term high fat diet. Insulin treatment did not significantly alter cardiac function in adropin treated hearts from either low or high fat diet mice, however acute adropin treatment did moderately restore downstream insulin signaling in high fat diet fed mice. Overall, these data suggest that in an ex vivo setting, acute adropin treatment alone is not sufficient to promote improved cardiac function in obese animals.

Mapping the Proximity Interaction Network of STIM1 Reveals New Mechanisms of Cytoskeletal Regulation

Stromal interaction molecule 1 (STIM1) resides primarily in the sarco/endoplasmic reticulum, where it senses intraluminal Ca2+ levels and activates Orai channels on the plasma membrane to initiate Ca2+ influx. We have previously shown that STIM1 is involved in the dynamic remodeling of the actin cytoskeleton. However, the downstream effectors of STIM1 that lead to cytoskeletal remodeling are not known. The proximity-labeling technique (BioID) can capture weak and transient protein-protein interactions, including proteins that reside in the close vicinity of the bait, but that may not be direct binders. Hence, in the present study, we investigated the STIM1 interactome using the BioID technique. A promiscuous biotin ligase was fused to the cytoplasmic C-terminus of STIM1 and was stably expressed in a mouse embryonic fibroblast (MEF) cell line. Screening of biotinylated proteins identified several high confidence targets. Here, we report Gelsolin (GSN) as a new member of the STIM1 interactome. GSN is a Ca2+-dependent actin-severing protein that promotes actin filament assembly and disassembly. Results were validated using knockdown approaches and immunostaining. We tested our results in neonatal cardiomyocytes where STIM1 overexpression induced altered actin dynamics and cytoskeletal instability. This is the first time that BioID assay was used to investigate the STIM1 interactome. Our work highlights the role of STIM1/GSN in the structure and function of the cytoskeleton.

Yap Promotes Noncanonical Wnt Signals From Cardiomyocytes for Heart Regeneration

Rationale: During neonatal heart regeneration, the fibrotic response, which is required to prevent cardiac rupture, resolves via poorly understood mechanisms. Deletion of the Hippo pathway gene Sav in adult cardiomyocytes increases Yap activity and promotes cardiac regeneration, partly by inducing fibrosis resolution. Deletion of Yap in neonatal cardiomyocytes leads to increased fibrosis and loss of neonatal heart regeneration, suggesting that Yap inhibits fibrosis by regulating intercellular signaling from cardiomyocytes to cardiac fibroblasts (CFs). Objective: We investigated the role of Wntless ( Wls ), which is a direct target gene of Yap, in communication between cardiomyocytes and CFs during neonatal heart regeneration. Methods and Results: We generated 2 mouse models to delete Wls specifically in cardiomyocytes ( Myh6-Cas9 combined with AAV9-Wls-g RNAs, and Myh6 cre-ERT2/+ ; Wls flox/flox ). Reanalysis of single-cell RNA sequencing data revealed that Wnt ligands are expressed in cardiomyocytes, whereas Wnt receptors are expressed in CFs, suggesting that Wnt signaling is directional from cardiomyocytes to CFs during neonatal heart regeneration. Wls deletion in neonatal cardiomyocytes disrupted Wnt signaling, revealed by reduced noncanonical Wnt signaling in non-cardiomyocytes. Four weeks after neonatal heart myocardial infarction, heart function was measured by echocardiography. Wls deletion in neonatal cardiomyocytes after myocardial infarction impairs neonatal heart regeneration, marked by decreased contractile function and increased fibrosis. Wls mutant hearts display CF activation, characterized by increased extracellular matrix secretion, inflammation, and CF proliferation. Conclusions: These data indicate that during neonatal heart regeneration, intercellular signaling from cardiomyocytes to CFs occurs via noncanonical Wnt signaling to rebuild cardiac architecture after myocardial infarction.

A standardized method to purify cardiomyocytes from individual mouse hearts of any age

Rationale: Primary cardiomyocytes are invaluable for understanding postnatal heart development and elucidating disease mechanisms in genetic and pharmacological models, however, a method to obtain freshly purified cardiomyocytes at any postnatal age, without using different age-dependent isolation procedures and cell culture, is lacking. Objective: To develop a standardized method that allows rapid isolation and purification of cardiomyocytes in high yield and viability from individual neonatal, infant, and adult mice. Methods and Results: Hearts of C57BL/6J mice were cannulated 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 were then subjected to Langendorff retrograde perfusion and enzymatic digestion. Cardiomyocytes were isolated after subsequent tissue disaggregation and filtration, in high yield (1.56-2.2x106 cardiomyocytes/heart) and viability (~70-100%). The larger size of infant (P10 and P13) and adult (P70), but not neonatal, cardiomyocytes relative to non-myocytes, allowed enrichment by differential centrifugation. Cardiomyocytes from all ages were further purified by immunomagnetic bead-based depletion of non-myocytes. Together, these procedures resulted in the isolation of highly purified cardiomyocytes (~94%) within 1 hour, enabling experiments using individual replicates. For example, RNA-sequencing of cardiomyocytes purified from one P2 male and female heart per litter (n=4 litters) showed distinct clustering by litters and sex differences for nine differentially expressed genes (FDR<0.005). In situ fixation via coronary perfusion, performed immediately after tissue digestion, preserved the cytoarchitecture of isolated cardiomyocytes (yielding ~94% rod-shaped cardiomyocytes at all ages), allowing capture of spindle-shaped neonatal cells undergoing mitosis, as well as enabling accurate quantitation of cardiomyocyte area and nucleation state. Conclusion: The procedures developed here provide a universal protocol for the rapid isolation and purification of high-quality cardiomyocytes from hearts of any postnatal age, even those of neonates, thereby enabling direct comparisons between individual hearts.

In vitro CSC-derived cardiomyocytes exhibit the typical microRNA-mRNA blueprint of endogenous cardiomyocytes

AbstractmiRNAs modulate cardiomyocyte specification by targeting mRNAs of cell cycle regulators and acting in cardiac muscle lineage gene regulatory loops. It is unknown if or to-what-extent these miRNA/mRNA networks are operative during cardiomyocyte differentiation of adult cardiac stem/progenitor cells (CSCs). Clonally-derived mouse CSCs differentiated into contracting cardiomyocytes in vitro (iCMs). Comparison of “CSCs vs. iCMs” mRNome and microRNome showed a balanced up-regulation of CM-related mRNAs together with a down-regulation of cell cycle and DNA replication mRNAs. The down-regulation of cell cycle genes and the up-regulation of the mature myofilament genes in iCMs reached intermediate levels between those of fetal and neonatal cardiomyocytes. Cardiomyo-miRs were up-regulated in iCMs. The specific networks of miRNA/mRNAs operative in iCMs closely resembled those of adult CMs (aCMs). miR-1 and miR-499 enhanced myogenic commitment toward terminal differentiation of iCMs. In conclusions, CSC specification/differentiation into contracting iCMs follows known cardiomyo-MiR-dependent developmental cardiomyocyte differentiation trajectories and iCMs transcriptome/miRNome resembles that of CMs.

Samm50 Promotes Hypertrophy by Regulating Pink1-Dependent Mitophagy Signaling in Neonatal Cardiomyocytes

Pathological cardiac hypertrophy, the adaptive response of the myocardium to various pathological stimuli, is one of the primary predictors and predisposing factors of heart failure. However, its molecular mechanisms underlying pathogenesis remain poorly understood. Here, we studied the function of Samm50 in mitophagy during Ang II-induced cardiomyocyte hypertrophy via lentiviruses mediated knockdown and overexpression of Samm50 protein. We first found that Samm50 is a key positive regulator of cardiac hypertrophy, for western blot and real-time quantitative PCR detection revealed Samm50 was downregulated both in pressure-overload-induced hypertrophic hearts and Ang II-induced cardiomyocyte hypertrophy. Then, Samm50 overexpression exhibits enhanced induction of cardiac hypertrophy marker genes and cell enlargement in primary mouse cardiomyocytes by qPCR and immunofluorescence analysis, respectively. Meanwhile, Samm50 remarkably reduced Ang II-induced autophagy as indicated by decreased mitophagy protein levels and autophagic flux, whereas the opposite phenotype was observed in Samm50 knockdown cardiomyocytes. However, the protective role of Samm50 deficiency against cardiac hypertrophy was abolished by inhibiting mitophagy through Vps34 inhibitor or Pink1 knockdown. Moreover, we further demonstrated that Samm50 interacted with Pink1 and stimulated the accumulation of Parkin on mitochondria to initiate mitophagy by co-immunoprecipitation analysis and immunofluorescence. Thus, these results suggest that Samm50 regulates Pink1-Parkin-mediated mitophagy to promote cardiac hypertrophy, and targeting mitophagy may provide new insights into the treatment of cardiac hypertrophy.

Abstract 103: Mechanistic Interplay Between Cardioprotection And Heart Regeneration Mediated By The ER-bound Transcription Factor Nrf1

Cardiomyocyte loss is the underlying basis for a majority of heart diseases. Preventing cardiomyocytes from death (cardioprotection) and replenishing the lost myocardium (regeneration) are the central goals for heart repair. Although cardioprotection and heart regeneration have been traditionally thought to involve separate mechanisms, protection of cardiomyocytes from injury or disease stimuli is a prerequisite to any meaningful regenerative response. In our study, we sought to understand how neonatal cardiomyocytes cope with injury-induced stress to regenerate damaged myocardium and whether the underlying mechanisms could be leveraged to promote heart regeneration and repair in adults. Using spatial transcriptomic profiling, we visualized regenerative cardiomyocytes reconstituting damaged myocardium after ischemia, and found that they are marked by expression of Nrf1, an ER-bound stress responsive transcription factor. Single-nucleus RNA sequencing revealed that genetic deletion of Nrf1 prevented neonatal cardiomyocytes from activating a transcriptional program required for heart regeneration. Conversely, overexpression of Nrf1 protected the adult mouse heart from ischemia/reperfusion injury. Nrf1 also protected human induced pluripotent stem cell-derived cardiomyocytes from cardiotoxicity induced by the chemotherapeutic drug doxorubicin. The cardioprotective function of Nrf1 is mediated by a dual stress response mechanism involving activation of the proteasome and maintenance of redox balance. Taken together, our study uncovers a unique adaptive mechanism activated in response to injury that maintains the tissue homeostatic balance required for heart regeneration. Reactivating these mechanisms in the adult heart represents a potential therapeutic approach for cardiac repair.