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Ribonucleoprotein (RNP) granules are biomolecular condensates—liquid–liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636Sallele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20mutation.

Chemical biology promises discovery of new and unexpected mechanistic pathways, protein functions and disease targets. Here, we probed the mechanism-of-action and protein targets of 3,5-disubstituted isoxazoles (Isx), cardiomyogenic small molecules that target Notch-activated epicardium-derived cells (NECs) in vivoand promote functional recovery after myocardial infarction (MI). Mechanistic studies in NECs led to an Isx-activated Gqprotein-coupled receptor (GqPCR) hypothesis tested in a cell-based functional target screen for GPCRs regulated by Isx. This screen identified one agonist hit, the extracellular proton/pH-sensing GPCR GPR68, confirmed through genetic gain- and loss-of-function. Overlooked until now, GPR68 expression and localization were highly regulated in early post-natal and adult post-infarct mouse heart, where GPR68-expressing cells accumulated subepicardially. Remarkably, GPR68-expressing cardiomyocytes established a proton-sensing cellular “buffer zone” surrounding the MI. Isx pharmacologically regulated gene expression (mRNAs and miRs) in this GPR68-enriched border zone, driving cardiomyogenic and pro-survival transcriptional programs in vivo. In conclusion, we tracked a (micromolar) bioactive small molecule’s mechanism-of-action to a candidate target protein, GPR68, and validated this target as a previously unrecognized regulator of myocardial cellular responses to tissue acidosis, setting the stage for future (nanomolar) target-based drug lead discovery.

Targeting native progenitors with small molecule pharmaceuticals that direct cell fate decisions is an attractive approach for regenerative medicine. Here, we show that 3,5-disubstituted isoxazoles (Isx), stem cell-modulator small molecules originally recovered in a P19 embryonal carcinoma cell-based screen, directed cardiac muscle gene expression in vivoin target tissues of adult transgenic reporter mice. Isx also stimulated adult mouse myocardial cell cycle activity. Narrowing our focus onto one target cardiac-resident progenitor population, Isx directed muscle transcriptional programs in vivoin multipotent Notch-activated epicardium-derived cells (NECs), generating Notch-activated adult cardiomyocyte-like precursors. Myocardial infarction (MI) preemptively differentiated NECs toward fibroblast lineages, overriding Isx’s cardiogenic influence in this cell population. Isx dysregulated gene expression in vivoin Notch-activated repair fibroblasts, driving distinctive (pro-angiogenesis) gene programs, but failed to mitigate fibrosis or avert ventricular functional decline after MI. In NECs in vitro, Isx directed partial muscle differentiation, which included biosynthesis and assembly of sarcomeric α-actinin premyofibrils, beaded structures pathognomonic of early developing cardiomyocytes. Thus, although Isx small molecules have promising in vivoefficacy at the level of cardiac muscle gene expression in native multipotent progenitors and are first in class in this regard, a greater understanding of the dynamic interplay between fibrosis and cardiogenic small molecule signals will be required to pharmacologically enable regenerative repair of the heart.

The regenerative capacity of skeletal muscle is due to the myogenic progenitor cell population that is resident in adult skeletal muscle. To enhance our understanding of this cell population, we examined the temporal–spatial expression pattern for filamin C during murine embryogenesis, adult muscle regeneration and in selected myopathic models of human disease. Using in situ hybridization, we observed filamin C to be restricted to mesodermal lineages including the developing heart and skeletal muscle during embryogenesis. Following cardiotoxin-induced muscle injury of adult skeletal muscle, filamin C expression was dynamically regulated in activated myogenic progenitor cells and newly regenerated myotubes. This expression pattern was further supported using RT-PCR analysis of filamin C expression in differentiating C2C12 myotubes. These results support the paradigm that the regulatory mechanisms of muscle regeneration largely recapitulate the fundamental events observed during muscle development and that filamin C may function in signal transduction or cellular migration of the myogenic progenitor cell population.

Muscle regeneration is a complex process requiring the coordinated interaction between the myogenic progenitor cells or satellite cells, growth factors, cytokines, inflammatory components, vascular components and the extracellular matrix (ECM). Previous studies have elegantly described the physiological modulation of the regenerative process in response to muscle injury, but the molecular response that characterizes stages of the repair process remains ill-defined. The recent completion of the Human and Mouse Genome Projects and the advent of technologies such as high-density oligonucleotide array analysis facilitate an expanded analysis of complex processes such as muscle regeneration. In the present study, we define cellular and molecular events that characterize stages of muscle injury and regeneration. Utilization of transcriptional profiling strategies revealed coordinated expression of growth factors [i.e., Tgfb1, Igf1, Egf, chemokine (C-C motif) ligand 6 and 7], the fetal myogenic program (Myod1, Myf5, Myf6), and the biomatrix (procollagen genes, Mmp3, Mmp9, biglycan, periostin) during muscle regeneration. Corroboration of the transcriptional profiling analysis included quantitative real-time RT-PCR and in situ hybridization analyses of selected candidate genes. In situ hybridization studies for periostin [osteoblast-specific factor 2 (fasciclin I-like)] and biglycan revealed that these genes are restricted to mesenchymal derivatives during embryogenesis and are significantly regulated during regeneration of the injured hindlimb skeletal muscle. We conclude that muscle regeneration is a complex process that requires the coordinated modulation of the inflammatory response, myogenic progenitor cells, growth factors, and ECM for complete restoration of muscle architecture.