We have previously reported that global ablation of NMHC II-B in mice resulted in hydrocephalus as early as E11.5 associated with defects in cell-cell adhesion of the neural epithelial cells lining the spinal canal and cerebral ventricles.9;15
mice, ablation of NMHC II-B was initiated at E10.5 controlled by the nestin promoter, which is consistent with the delayed onset of hydrocephalus. Of particular note despite the death of these mice between 12 to 22 days of age due most likely to severe hydrocephalus, there were no abnormalities found in the heart.
In addition to learning whether the defects we found in the B−/B− mouse hearts were related directly or indirectly to those found in the nervous system, we also wanted to study the role of NMII-B in the adult mouse heart. Most BαMHC/BαMHC mice manifested progressive cardiac abnormalities starting with myocyte hypertrophy, which was apparent as early as P0 and increased during postnatal development to 6 and 10 months of age. At 10 months there was also evidence for myocyte vacuolation and cell degeneration, interstitial fibrosis and an infiltration of the cardiac tissue with inflammatory cells. We hypothesize that the cardiac phenotype in the BαMHC/BαMHC mice is initiated by abnormalities specific to the cardiac myocytes, since NMII-B is ablated in these cells but not in the non-myocytes in these mice. This loss of NMII-B (and lack of compensation by NMII-A or II-C) results in a failure in cytokinesis as manifested by multinucleation and the bizarre nuclei found in these cells. It most likely contributes to abnormal enlargement of the cardiac myocytes as well as their decreased numbers at P0. We therefore reasoned that the interstitial fibrosis and infiltration of inflammatory cells are secondary to the primary abnormality in the cardiac myocytes, which is most likely myocyte degeneration.
The pathological changes in the hearts of cardiac-specific NMHC II-B knockout mice are in agreement with the echocardiographic and EKG studies. The marked decrease in the fractional shortening at 10 months is consistent with the compromised contractility of cardiac muscle. The abnormalities noted in EKGs (an abnormal electrical axis) could reflect the striking defects found in the IDs. Previous work has shown that in the adult heart NMII-B is localized to the Z-lines and IDs.18
The IDs are composed of adherans junctions, desmosomes and gap junctions that form cell-cell boundaries and connections between cardiac myocytes and allow the myocardium to function in synchrony. As noted above, work from a number of laboratories has shown that NMIIs play an important role in cell-cell adhesion7;15;20;22
and that abnormalities in a number of adhesion proteins result in either loss or structural changes in the cardiac IDs.20;23;24
provides evidence that loss of NMII-B primarily affects the adhesion junctions rather than the gap junctions or desmosomes. Moreover, BαMHC
hearts at 6 months show a milder defect in the adhesion junction of the IDs and no defects in the desmosomes and gap junctions (data not shown).
We have analyzed the expression of a number of ID proteins and found a significant decrease in the expression of mXinα in BαMHC
hearts compared to the wild type hearts. Mice ablated for mXinα also show abnormal IDs.19
Unlike the mXinα knockout hearts, NMII-B ablated hearts show no decrease in expression levels of N-cadherin orβ-catenin. Moreover there was no change in the distribution of β-catenin. We therefore attribute the primary cause of the disruption of the IDs to the loss of NMII-B. We speculate that the decrease of mXinα is secondary to the loss of NMII-B and the mechanism of this decrease is of ongoing interest. The decrease in both of these proteins, one of which (mXinα) has been demonstrated to also bind to β-catenin,21
would explain the marked disruption of the IDs.
The finding of a role for NMII-B in the cardiac ID is similar to the findings for NMII-B in the spinal canal. Our hypothesis is that NMII exerts tension and stabilizes actin filaments which in turn are required for maintenance of adhesion complexes between cells or in this case between the cardiac myocytes. The loss of NMII-B from the adhesion complex could therefore result in the gradual deterioration in the cardiac adhesion complex, including the loss of mXinα,
over a period of time and this would account for our failure to observe abnormal discs in B−
mice which died before birth. Interestingly, generation of mice in which NMII-A replaced NMII-B did not produce defects in the IDs.25
This is consistent with the hypothesis that in cases where NMIIs are apparently playing a structural rather than a motor role, one isoform is more likely to substitute for the other in vivo
as well as in cultured cells.6
When myosin is playing more of a motor role, for example in neural cell migration, because of significant differences in the kinetics of MgATPase hydrolysis and actin-binding properties between the myosin isoforms, successful substitution is much less likely, at least in vivo
These findings further support the idea that disruption of the IDs in BαMHC
mice is due to the loss of NM II and is secondary to the development of the cardiomyopathy.
These conditionally ablated mice demonstrate that the defects we observed in the hearts and brains of the B−/B− mice are independent of each other. The availability of NMHC II-B floxed mice will allow conditional ablation of NMHC II-B in a variety of tissues and cells and thus help to further define its role both in situ and in vivo.