Human ear grown on petri-dish (above). Salamander limb regeneration (below).
Regeneration has been a fascination to humans since the dawn of modern biological science. What gives starfish and salamanders the ability to regenerate lost limbs and why don’t humans have that ability? One of the first organisms studied for regenerative abilities is the Hydra, a freshwater polyp, which has the ability to regenerate a clone of itself from even the smallest tissue fragment (Lenhoff and Lenhoff, 1986; Noda, 1971; Gierer et al., 1972). But humans are vastly different from the simple Hydra, so we need another model organism on which we conduct research. Mus musculus, or the common house mouse, is a perfect model organism for research that will lead to human applications due to various important reasons: their genome is 95% identical to humans, they are cost effective, have short generation times and the ease of genome manipulation. This page outlines a study on mice hearts that offers insight into organ regeneration that will hopefully result in medical applications for humans.
But first, let’s begin with the heart. Cardiac tissue is composed of two types of muscle cells: cardiomyocytes and cardiac pacemaker cells. Lower vertebrates have the ability to completely regenerate cardiac tissue, but the phenomenon is significantly decreased in mammals. Mammals are born with a set number of cardiomyocytes that only increase in size over the course of development and aging. However, new research suggests these cells actually turnover during the lifespan of the organism, but that will not be covered here. Not surprisingly, the neonatal heart shows a regenerative ability similar to that of lower vertebrates. What if cardiomyocytes had the ability to replicate like normal somatic cells?
Currently, the only method we have to treat heart failure or other cardiac injuries is total organ transplantation. The procedure may result in organ rejection and requires a lifetime of medication. Research in the field of cardiac tissue regeneration will have resounding benefits in the medical field. Study of these pre-birth cardiocyocytes will change modern medicine by giving us the ability to simply re-grow organs using a patient’s own tissue samples. This negates the risk of organ rejection by providing a heart that functions the same, if not better than the original.
Findings of Mahmoud et. al. 2013
Previous research suggests that a family of homeodomain transcription factors that follow the TALE (three amino acid loop extension) structure, Meis, is responsible for cardiac differentiation during embryonic development.
Initial experiments such as global knockout of Meis1 show that the gene is essential for normal embryonic haematopeiesis and heart development, but the exact nature of the function of this gene is unknown and the main question that Mahmoud and his team are trying to answer.
Initially, the expression pattern was analyzed during day 1 and day 7 after birth (Figure 1a). Meis1 activity increases after birth, suggesting a cell cycle inhibiting function. Meis1 is known to interact with mammalian Hox genes, so a qRT-PCR on the entire Hox family was done. It showed 5 Hox proteins known to interact with Meis1 by stabilizing Meis1 DNA binding to enhance transcriptional activity.
In order to figure out the role of Meis1 in cardiomyocyte proliferation, in vitro siRNA knockdown of Meis1 showed a 3-fold increase in cardiomyocyte replication (Fig 1e). To determine if Meis1 affected other normal heart development, cardiomyocyte specific KO mice were generated and compared to normal control mice to show gene expression was consistent with Meis1 deletion . Phenotypically, the KO mice had smaller cardiomyocytes, but heart function and heart size were unaffected. Another assay was run to determine if these KO cardiomyocutes were actively proliferating since heart size was unaffected. Using a mitosis marker pH3 and a cytokinesis marker Aurora B kinase, a >9-fold increase in proliferation was determined (Fig 2g). After it was shown that the cardiomyocytes were actively proliferating, Mahmoud and his team asked if the deletion increased the total number of cells.
Along with an increase in total number of cardiomyocytes, an increase in mononucleated cells and decrease in binucleated cells was noticed. This suggests the Meis1 deletion affects cell cycle activity and postnatal nucleation.
Next they examined how Meis1 deletion affect the adult heart. With global deletion of Meis1 during late gestation, no change in the adult heart size was noted. In addition, cardiomyocytes continued to show mitosis into adulthood.
Only a few adult cells can reenter proliferation phase (mononuclear cells). Inducible knockout mice created by corssing Meis1 f/f mice with alpha-MHC-MerCreMer mice (Meis1 iKO) and used to confirm that Meis1 deletion did not affect morphology or fibrosis (Fig. 3c), but 6 weeks later an increase in heart to body weight ratio was noted. Along with a few more experiments not noted here, cell cycle reentry was induced by Meis1 deletion.
The opposite was attempted by trying to overexpress Meis1 to inhibit neonatal proliferation. Cardiac-specific Meis1 overexpressing mice were created by crossing two different genotypes. The mice were allowed to express Meis1 from birth until the absence of tetracyclin. No significant gain in heart to body weight ratio was noted in the overexpressing mice (Fig 4c, d), but cardiomycoyte size was increased. Since the ratio remained unchanged while cell size increases, we can conclude that cell count decreased. The mice also exhibited an upregulation of CDK inhibitors (p21), which as we know inhibits mitosis. The results conclude cell cycle arrest in neonatal myocytes.
Suppl. Figure 4a (above), Suppl. Figure 3a,b.
Finally we come to the question: How does Meis1 regulate proliferation? A cell cycle PCR array and qRT-PCR assay (Suppl. Fig 4a) both confirmed that Meis1 deleted downregulated CDK-inhibitors (p16, p15, p19, p21 and p57) and upregulated positive cell cycle regulators (Suppl. Fig 3a, b).
Studies over the course of two decades have suggested cell cycle regulators play an important part in cardiomycoyte cell cycle arrest. Exit is associated with downregulation of positive cell cycle regulators and induction of cell cycle inhibitors. But, the mechanism of these direct cell cycle effoctors in cardiomyocytes are unclear.
Meis1 deletion results in upregulation of positive cell cycle regulators and downregulation of other negative regualtiors. The mechanism behind the activation of Meis1 is not fully understood, but Mahmoud et al’s results show the gene as an important transcriptional regulator of cardiomyocyte replication and its expression results in an upregulation of 3 of the 5 known Hox genes that interact with Meis1.
The paper is very detailed in the methods and results. The hypothesis and direction of the paper was stated clearly in the abstract and introduction. The figures support the results very clearly.
The results of the paper define many of the functions and interactions of Meis1. The author outlines several possible studies that will further define the role of Meis1 on cardiomyocyte proliferation after birth. One of such future directions include defining the transcriptional network involved in mediating the effect of Meis1 on postnatal cardiomyocytes since it is known to upregulate 3 of the 5 Hox genes that interact with Meis1.
Gierer, A., S. Berking, H. Bode, C.N. David, K. Flick, G. Hansmann, H. Schaller, E. Trenkner. 1972. Regeneration of hydra from reaggregated cells. Nat New Biol. 239:98–101.doi:10.1038/239098a0
Lenhoff, S.G., H.M. Lenhoff. 1986. Hydra and the Birth of Experimental Biology: Abraham Trembley’s Memoirs Concerning the Natural History of a Type of Freshwater Polyp with Arms Shaped like Horns. The Boxwood Press, Pacific Grove, CA. 192 pp.
Mahmoud, Ahmed I., et al. “Meis1 regulates postnatal cardiomyocyte cell cycle arrest.” Nature 497.7448 (2013): 249-253.
Noda, K. 1971. Reconstitution of dissociated cells of Hydra. Zool Mag. 80:27–31.