Members of the Smyd protein family have been shown to be involved in the regulation of cellular differentiation processes 
. It has become increasingly apparent that the functional role of Smyd proteins is of particular importance for the differentiation of muscle tissue 
. Targeted gene disruption revealed Smyd1 to be essential for early cardiac development 
by acting as a downstream effector of the cardiac transcription factor, MEF2C, in the developing heart 
. However, functional characterization of other Smyd-family members in the heart has not been performed. Since we have recently identified Smyd2 as a distinct Smyd -family member that is most highly expressed in heart and brain 
, we performed a study aimed at expanding the understanding of Smyd proteins in the heart with specific focus on Smyd2.
Our results reveal that Smyd2 is differentially expressed during cardiac development, displaying highest expression levels around birth in rats and mice. In contrast to Smyd1 deficiency, loss of Smyd2 does not result in embryonic lethality, consistent with implications from expression data that Smyd2 functions later in development. Smyd2 cKO animals are viable and are born in normal Mendelian ratios with no obvious changes in heart morphology or function. Thus, Smyd2 does not appear to be essential for early heart formation.
Given a peak expression of Smyd2 in the first week of postnatal life, one might anticipate that Smyd2 is important for the biological processes occurring during this time period, namely the irreversible exit from cell cycle 
as well as the change from mainly lactate and glucose catabolism to mitochondrial fatty acid oxidation 
. If Smyd2 was essential for these processes, we would have expected deregulation of genes associated with either cell cycle control (cyclins, CDKs, cell cycle inhibitors) or key regulatory enzymes for cardiac energy metabolism, such as carnitine palmitoyl transferase-I
or medium-chain acyl-CoA dehydrogenase 
. However, microarray analyses did not reveal significant changes of such marker genes in P5 mouse heart ventricles, nor did adult Smyd2
cKO hearts exhibit differences in size or weight as would have been expected if the proliferation of cardiomyocytes was affected 
. Surprisingly, we found that the majority of genes affected by cardiac Smyd2 deletion are functionally associated with translation. Interestingly, a number of the down regulated genes (eg, Mrpl45, Mrps18a and Mrpl3) belong to the nuclear encoded repertoire of mitochondrial ribosomal subunits 
. To our knowledge there are no previous data showing a transcriptional increase in components of the translational machinery occurring after birth. Nonetheless, our results suggest that the hypertrophic growth of the heart just after birth might be facilitated by a temporary increase in protein translation. Such a phenomenon is consistent with previous results which demonstrated increased ribosome expression during pathologic hypertrophy of cardiomyocytes (for review see Hannan et al. 
). The fact that we do not observe hypertrophy suggests that Smyd2 is not a key regulator of normal growth. It will, however, be of interest to test how Smyd2
cKO mice react to stress.
We and others have previously characterized Smyd2 as a histone methyltransferase with capacity to methylate H3K36 
as well as H3K4 
. As these findings were based on in vitro
as well as cell culture studies, our current study provided the opportunity to test whether corresponding effects could be observed in vivo
. The observed absence of any detectable changes in global H3K36 or H3K4 methylation, while unexpected, indicates that redundant HMTases might compensate in the developing heart. In particular, Smyd1, also has H3K4 methyltransferase activity 
. Since Smyd1 expression is slightly (but statistically significantly) elevated upon Smyd2 deletion, it is possible that this function of Smyd2 might be partially compensated by Smyd1. An alternative and trivial explanation might be that Smyd2 is predominantly expressed in cardiomyocytes which make up only 56% of all cell types in the murine heart 
. Assuming that Smyd2 might be involved in the transcriptional regulation of a subset of target genes in cardiomyocytes, one might not expect to detect global changes in histone methylation using crude heart tissue by western blot techniques. Therefore, a more detailed analysis of histone methylation status on isolated murine cardiomyocytes at confirmed target site promoters will be conducted in future experiments.
The finding that most of the deregulated genes in Smyd2 cKO hearts were repressed indicate a role for Smyd2 as an activator in the developing heart. This is consistent with other data from overexpression studies, showing that Smyd2 gain of function predominantly results in the up-regulation of genes 
. The finding that Smyd2 is capable of interacting with RNA Polymerase II as well as the RNA helicase, HELZ, suggests that Smyd2 might share functional similarities with Smyd3 
. Although we do not provide evidence for a functional consequence of the interaction between Smyd2 and RNAPolII or HELZ regarding the regulation of transcription, one might speculate that Smyd2 might also facilitate target gene expression via the elongation of transcription.
In addition to its molecular function as a histone methyltransferase, Huang et al. recently proposed a distinct role for Smyd2 as a putative oncogene by methylating p53 and thereby repressing its tumor suppressive function 
. Although we did not specifically address the functional consequence of Smyd2 deficiency for p53 activity in vivo
, one might have expected a pronounced phenotype, at least in adult Smyd2 cKO animals. This seemed reasonable, as it has been shown that cardiac deletion of Mdm4, another inhibitor of p53 functional activity, results in p53-dependent dilated cardiomyopathy 
. However, functional misregulation of p53 by Smyd2 in vivo
seems unlikely for the heart, as Smyd2 cKO hearts showed no noticeable change in the levels of apoptosis or necrosis, nor transcriptional changes in the p53 target genes Mdm2 and p21 (Figure S1A
). Additionally we did not observe any differences in p53 protein stability (Figure S1B
/C). Given the importance of understanding the precise mechanisms of p53 regulation in vivo
, our Smyd2 cKO mice will provide a useful tool for gathering such information in the heart as well as other organs. The relevance of Smyd2 in the heart will be particularly interesting in regard to stress models (myocardial infarction, hypoxia), as functional misregulation of p53 and other stress sensors might be masked under physiologic conditions, becoming apparent only when an acute need is present.
In summary, our data reveal that Smyd2 is dispensable for cardiac development and maturation in the mouse under normal physiologic conditions. They further suggest that Smyd2 might be involved in the transcriptional regulation of genes associated with protein translation.