Prashanth K. Kandalla

Cardiac macrophages (cMΦ) are critical for early postnatal heart regeneration and fibrotic repair in the adult heart, but their origins and cellular dynamics during postnatal development have not been well characterized. Tissue macrophages can be derived from embryonic progenitors or from monocytes during inflammation. We report that within the first weeks after birth, the embryo-derived population of resident CX3CR1(+) cMΦ diversifies into MHCII(+) and MHCII(-) cells. Genetic fate mapping demonstrated that cMΦ derived from CX3CR1(+) embryonic progenitors persisted into adulthood but the initially high contribution to resident cMΦ declined after birth. Consistent with this, the early significant proliferation rate of resident cMΦ decreased with age upon diversification into subpopulations. Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative replacement of all cMΦ populations. Furthermore, parabiotic mice and BM chimeras of nonirradiated recipient mice revealed a slow but significant donor contribution to cMΦ. Together, our observations indicate that in the heart, embryo-derived cMΦ show declining self-renewal with age and are progressively substituted by monocyte-derived macrophages, even in the absence of inflammation.

BACKGROUND: Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. METHODS: Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. RESULTS: The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. CONCLUSIONS: Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.

Most cells in adult tissues are nondividing. In skeletal muscle, differentiated myofibers have exited the cell cycle permanently, whereas satellite stem cells withdraw transiently, returning to active proliferation to repair damaged myofibers. We have examined the epigenetic mechanisms operating in conditional quiescence by analyzing the function of a predicted chromatin regulator mixed lineage leukemia 5 (MLL5) in a culture model of reversible arrest. MLL5 is induced in quiescent myoblasts and regulates both the cell cycle and differentiation via a hierarchy of chromatin and transcriptional regulators. Knocking down MLL5 delays entry of quiescent myoblasts into S phase, but hastens S-phase completion. Cyclin A2 (CycA) mRNA is no longer restricted to S phase, but is induced throughout G(0)/G(1), with activation of the cell cycle regulated element (CCRE) in the CycA promoter. Overexpressed MLL5 physically associates with the CCRE and impairs its activity. MLL5 also regulates CycA indirectly: Cux, an activator of CycA promoter and S phase is induced in RNAi cells, and Brm/Brg1, CCRE-binding repressors that promote differentiation are repressed. In knockdown cells, H3K4 methylation at the CCRE is reduced, reflecting quantitative global changes in methylation. MLL5 appears to lack intrinsic histone methyl transferase activity, but regulates expression of histone-modifying enzymes LSD1 and SET7/9, suggesting an indirect mechanism. Finally, expression of muscle regulators Pax7, Myf5, and myogenin is impaired in MLL5 knockdown cells, which are profoundly differentiation defective. Collectively, our results suggest that MLL5 plays an integral role in novel chromatin regulatory mechanisms that suppress inappropriate expression of S-phase-promoting genes and maintain expression of determination genes in quiescent cells.