YTHDF1 is pivotal for maintenance of cardiac homeostasis.

The YTH-domain family (YTHDF) of RNA binding proteins can control gene expression at the post-transcriptional level by regulating mRNAs with N6-methyladenosine (m6A) modifications. Despite the established importance of m6A in the heart, the cardiac role of specific m6A-binding proteins remains unclear. Here, we characterized the function of YTHDF1 in cardiomyocytes using a newly generated cardiac-restricted mouse model. Deletion of YTHDF1 in adult cardiomyocytes led to hypertrophy, fibrosis, and dysfunction. Using mass spectrometry, we identified the necessity of YTHDF1 for the expression of cardiomyocyte membrane raft proteins. Specifically, YTHDF1 bound to m6A-modified Caveolin 1 (Cav1) mRNA and favored its translation. We further demonstrated that YTHDF1 regulates downstream ERK signaling. Altogether, our findings highlight a novel role for YTHDF1 as a post-transcriptional regulator of caveolar proteins which is necessary for the maintenance of cardiac function.

YTHDF2 governs muscle size through a targeted modulation of proteostasis.

The regulation of proteostasis is fundamental for maintenance of muscle mass and function. Activation of the TGF-ß pathway drives wasting and premature aging by favoring the proteasomal degradation of structural muscle proteins. Yet, how this critical post-translational mechanism is kept in check to preserve muscle health remains unclear. Here, we reveal the molecular link between the post-transcriptional regulation of m6A-modified mRNA and the modulation of SMAD-dependent TGF-ß signaling. We show that the m6A-binding protein YTHDF2 is essential to determining postnatal muscle size. Indeed, muscle-specific genetic deletion of YTHDF2 impairs skeletal muscle growth and abrogates the response to hypertrophic stimuli. We report that YTHDF2 controls the mRNA stability of the ubiquitin ligase ASB2 with consequences on anti-growth gene program activation through SMAD3. Our study identifies a post-transcriptional to post-translational mechanism for the coordination of gene expression in muscle.

In vitro inflammatory multi-cellular model of osteoarthritis.

Objective: Osteoarthritis (OA) is a chronic joint disease, with limited treatment options, characterized by inflammation and matrix degradation, and resulting in severe pain or disability. Progressive inflammatory interaction among key cell types, including chondrocytes and macrophages, leads to a cascade of intra- and inter-cellular events which culminate in OA induction. In order to investigate these interactions, we developed a multi-cellular in vitro OA model, to characterize OA progression, and identify and evaluate potential OA therapeutics in response to mediators representing graded levels of inflammatory severity. Methods: We compared macrophages, chondrocytes and their co-culture responses to "low" Interleukin-1 (IL-1) or "high" IL-1/tumor necrosis factor (IL-1/TNF) levels of inflammation. We also investigated response changes following the administration of dexamethasone (DEX) or mesenchymal stromal cell (MSC) treatment via a combination of gene expression and secretory changes, reflecting not only inflammation, but also chondrocyte function. Results: Inflamed chondrocytes presented an osteoarthritic-like phenotype characterized by high gene expression of pro-inflammatory cytokines and chemokines, up-regulation of ECM degrading proteases, and down-regulation of chondrogenic genes. Our results indicate that while MSC treatment attenuates macrophage inflammation directly, it does not reduce chondrocyte inflammatory responses, unless macrophages are present as well. DEX however, can directly attenuate chondrocyte inflammation. Conclusions: Our results highlight the importance of considering multi-cellular interactions when studying complex systems such as the articular joint. In addition, our approach, using a panel of both inflammatory and chondrocyte functional genes, provides a more comprehensive approach to investigate disease biomarkers, and responses to treatment.

Loss of YTHDF2 Alters the Expression of m6A-Modified Myzap and Causes Adverse Cardiac Remodeling.

How post-transcriptional regulation of gene expression, such as through N6-methyladenosine (m6A) messenger RNA methylation, impacts heart function is not well understood. We found that loss of the m6A binding protein YTHDF2 in cardiomyocytes of adult mice drove cardiac dysfunction. By proteomics, we found myocardial zonula adherens protein (MYZAP) within the top up-regulated proteins in knockout cardiomyocytes. We further demonstrated that YTHDF2 binds m6A-modified Myzap messenger RNA and controls its stability. Cardiac overexpression of MYZAP has been associated with cardiomyopathy. Thus, our findings provide an important new mechanism for the YTHDF2-dependent regulation of this target and therein its novel role in the maintenance of cardiac homeostasis.

Mineralocorticoid receptor antagonists and glucocorticoids differentially affect skeletal muscle inflammation and pathology in muscular dystrophy.

Mineralocorticoid receptor antagonists (MRAs) slow cardiomyopathy in patients with Duchenne muscular dystrophy (DMD) and improve skeletal muscle pathology and function in dystrophic mice. However, glucocorticoids, known antiinflammatory drugs, remain a standard of care for DMD, despite substantial side effects. Exact mechanisms underlying mineralocorticoid receptor (MR) signaling contribution to dystrophy are unknown. Whether MRAs affect inflammation in dystrophic muscles and how they compare with glucocorticoids is unclear. The MRA spironolactone and glucocorticoid prednisolone were each administered for 1 week to dystrophic mdx mice during peak skeletal muscle necrosis to compare effects on inflammation. Both drugs reduced cytokine levels in mdx quadriceps, but prednisolone elevated diaphragm cytokines. Spironolactone did not alter myeloid populations in mdx quadriceps or diaphragms, but prednisolone increased F4/80hi macrophages. Both spironolactone and prednisolone reduced inflammatory gene expression in myeloid cells sorted from mdx quadriceps, while prednisolone additionally perturbed cell cycle genes. Spironolactone also repressed myeloid expression of the gene encoding fibronectin, while prednisolone increased its expression. Overall, spironolactone exhibits antiinflammatory properties without altering leukocyte distribution within skeletal muscles, while prednisolone suppresses quadriceps cytokines but increases diaphragm cytokines and pathology. Antiinflammatory properties of MRAs and different limb and respiratory muscle responses to glucocorticoids should be considered when optimizing treatments for patients with DMD.

Restoration of Cardiomyogenesis in Aged Mouse Hearts by Voluntary Exercise.

BACKGROUND: The human heart has limited capacity to generate new cardiomyocytes and this capacity declines with age. Because loss of cardiomyocytes may contribute to heart failure, it is crucial to explore stimuli of endogenous cardiac regeneration to favorably shift the balance between loss of cardiomyocytes and the birth of new cardiomyocytes in the aged heart. We have previously shown that cardiomyogenesis can be activated by exercise in the young adult mouse heart. Whether exercise also induces cardiomyogenesis in aged hearts, however, is still unknown. Here, we aim to investigate the effect of exercise on the generation of new cardiomyocytes in the aged heart. METHODS: Aged (20-month-old) mice were subjected to an 8-week voluntary running protocol, and age-matched sedentary animals served as controls. Cardiomyogenesis in aged hearts was assessed on the basis of 15N-thymidine incorporation and multi-isotope imaging mass spectrometry. We analyzed 1793 cardiomyocytes from 5 aged sedentary mice and compared these with 2002 cardiomyocytes from 5 aged exercised mice, followed by advanced histology and imaging to account for ploidy and nucleation status of the cell. RNA sequencing and subsequent bioinformatic analyses were performed to investigate transcriptional changes induced by exercise specifically in aged hearts in comparison with young hearts. RESULTS: Cardiomyogenesis was observed at a significantly higher frequency in exercised compared with sedentary aged hearts on the basis of the detection of mononucleated/diploid 15N-thymidine-labeled cardiomyocytes. No mononucleated/diploid 15N-thymidine-labeled cardiomyocyte was detected in sedentary aged mice. The annual rate of mononucleated/diploid 15N-thymidine-labeled cardiomyocytes in aged exercised mice was 2.3% per year. This compares with our previously reported annual rate of 7.5% in young exercised mice and 1.63% in young sedentary mice. Transcriptional profiling of young and aged exercised murine hearts and their sedentary controls revealed that exercise induces pathways related to circadian rhythm, irrespective of age. One known oscillating transcript, however, that was exclusively upregulated in aged exercised hearts, was isoform 1.4 of regulator of calcineurin, whose regulation and functional role were explored further. CONCLUSIONS: Our data demonstrate that voluntary running in part restores cardiomyogenesis in aged mice and suggest that pathways associated with circadian rhythm may play a role in physiologically stimulated cardiomyogenesis.

m6A RNA methylation: A dynamic regulator of cardiac muscle and extracellular matrix.

Post-transcriptional modifications encompass a large group of RNA alterations that control gene expression. Methylation of the N6-Adenosine (m6A) of mRNA is a prevalent modification which alters the life cycle of transcripts. The roles that m6A play in regulating cardiac homeostasis and injury response are an active area of investigation, but it is clear that this chemical modification is a critical controller of fibroblast to myofibroblast transition, cardiomyocyte hypertrophy and division, and the structure and function of the extracellular matrix. Here we discuss the latest findings of m6A in cardiac muscle and matrix.

CITED4 Protects Against Adverse Remodeling in Response to Physiological and Pathological Stress.

RATIONALE: Cardiac CITED4 (CBP/p300-interacting transactivators with E [glutamic acid]/D [aspartic acid]-rich-carboxylterminal domain4) is induced by exercise and is sufficient to cause physiological hypertrophy and mitigate adverse ventricular remodeling after ischemic injury. However, the role of endogenous CITED4 in response to physiological or pathological stress is unknown. OBJECTIVE: To investigate the role of CITED4 in murine models of exercise and pressure overload. METHODS AND RESULTS: We generated cardiomyocyte-specific CITED4 knockout mice (C4KO) and subjected them to an intensive swim exercise protocol as well as transverse aortic constriction (TAC). Echocardiography, Western blotting, qPCR, immunohistochemistry, immunofluorescence, and transcriptional profiling for mRNA and miRNA (microRNA) expression were performed. Cellular crosstalk was investigated in vitro. CITED4 deletion in cardiomyocytes did not affect baseline cardiac size or function in young adult mice. C4KO mice developed modest cardiac dysfunction and dilation in response to exercise. After TAC, C4KOs developed severe heart failure with left ventricular dilation, impaired cardiomyocyte growth accompanied by reduced mTOR (mammalian target of rapamycin) activity and maladaptive cardiac remodeling with increased apoptosis, autophagy, and impaired mitochondrial signaling. Interstitial fibrosis was markedly increased in C4KO hearts after TAC. RNAseq revealed induction of a profibrotic miRNA network. miR30d was decreased in C4KO hearts after TAC and mediated crosstalk between cardiomyocytes and fibroblasts to modulate fibrosis. miR30d inhibition was sufficient to increase cardiac dysfunction and fibrosis after TAC. CONCLUSIONS: CITED4 protects against pathological cardiac remodeling by regulating mTOR activity and a network of miRNAs mediating cardiomyocyte to fibroblast crosstalk. Our findings highlight the importance of CITED4 in response to both physiological and pathological stimuli.

Alginate encapsulation for bupivacaine delivery and mesenchymal stromal cell immunomodulatory cotherapy.

PURPOSE: Mesenchymal stromal cells (MSCs) are used to treat various inflammatory conditions. In parallel, to mitigate pain associated with inflammation, analgesics or opioids are prescribed, often with significant side effects. Local anesthetics (LAs) offer a promising alternative to these medications. However, their short duration and negative effects on anti-inflammatory MSCs have limited their therapeutic effectiveness. To mitigate these negative effects and to move toward developing a cotherapy, we engineered a sustained release bupivacaine alginate-liposomal construct that enables up to 4 days of LA release. By encapsulating MSC in alginate (eMSC), we demonstrate that we can further increase drug concentration to clinically relevant levels, without compromising eMSC viability or anti-inflammatory function. MATERIALS AND METHODS: MSCs were freely cultured or encapsulated in alginate microspheres ± TNFα/IFN-γ and were left untreated or dosed with bolus, liposomal, or construct bupivacaine. After 24, 48, and 96 hours, the profiles were assessed to quantify secretory function associated with LA-MSC interactions. To approximate LA exposure over time, a MATLAB model was generated. RESULTS: eMSCs secrete similar levels of IL-6 and prostaglandin E2 (PGE2) regardless of LA modality, whereas free MSCs secrete larger amounts of IL-6 and lower amounts of anti-inflammatory PGE2. Modeling the system indicated that higher doses of LA can be used in conjunction with eMSC while retaining eMSC viability and function. In general, eMSC treated with higher doses of LA secreted similar or higher levels of immunomodulatory cytokines. CONCLUSION: eMSCs, but not free MSC, are protected from LA, regardless of LA modality. Increasing the LA concentration may promote longer and stronger pain mitigation while the protected eMSCs secrete similar, if not higher, immunomodulatory cytokine levels. Therefore, we have developed an approach, using eMSC and the LA construct that can potentially be used to reduce pain as well as improve MSC anti-inflammatory function.

Exercise induces new cardiomyocyte generation in the adult mammalian heart.

Loss of cardiomyocytes is a major cause of heart failure, and while the adult heart has a limited capacity for cardiomyogenesis, little is known about what regulates this ability or whether it can be effectively harnessed. Here we show that 8 weeks of running exercise increase birth of new cardiomyocytes in adult mice (~4.6-fold). New cardiomyocytes are identified based on incorporation of 15N-thymidine by multi-isotope imaging mass spectrometry (MIMS) and on being mononucleate/diploid. Furthermore, we demonstrate that exercise after myocardial infarction induces a robust cardiomyogenic response in an extended border zone of the infarcted area. Inhibition of miR-222, a microRNA increased by exercise in both animal models and humans, completely blocks the cardiomyogenic exercise response. These findings demonstrate that cardiomyogenesis can be activated by exercise in the normal and injured adult mouse heart and suggest that stimulation of endogenous cardiomyocyte generation could contribute to the benefits of exercise.