the ras association domain family ( rassf ) consists of 10 members : rassf1 - 10 .
importantly , all isoforms contain a ras association ( ra ) domain either in their c - terminal ( rassf1 - 6 ) or n - terminal ( rassf7 - 10 ) regions . to date , no known catalytic activity has been described for this family , and the general consensus supposes that rassf proteins function as scaffolds to localize signaling in the cell .
rassf1 isoform a ( rassf1a ) is the most characterized member of the rassf family .
the rassf1 gene encodes multiple splice variants , including the two predominant isoforms , rassf1a and c. the rassf1a isoform is the longest variant of the rassf1 gene .
structurally , rassf1a is a product of exons 1 , 2/ , 3 , 4 , 5 , and 6 , while rassf1c consists of exons 2 , 3 , 4 , 5 , and 6 . both isoforms contain a c - terminal ra domain ; however , rassf1a has an additional c1 domain that is not present in rassf1c .
this short arm of chromosome 3 is known to exhibit loss of heterozygosity in many tumor models and is thought to harbor tumor suppressor genes . as the literature has shown , rassf1a fits this description .
the rassf1a promoter contains a cpg island that shows a high frequency of hypermethylation in tumors , thereby silencing rassf1a expression in many human cancers including lung , breast , ovarian , renal , and bladder [ 47 ] .
rassf1a expression is also lost in numerous cancer cell lines , while rassf1c expression is seemingly unaffected .
interestingly , recent work suggests that rassf1c may actually promote tumor progression [ 8 , 9 ] , further distinguishing these two splice variants .
all rassf proteins have an ra domain , which is thought to necessitate their binding to activated , gtp - bound ras proteins . while rassf5 ( nore1 ) is thought to bind ras directly , whether rassf1a is able to associate with ras is less clear .
it has been shown that rassf1a binds k - ras in vitro , and an interaction between ectopically expressed rassf1a and activated k - ras has been observed in hek293 cells [ 11 , 12 ] .
however , other work has found that this interaction only occurs in the presence of nore1 , arguing for an indirect association .
importantly , to our knowledge , there are no reports demonstrating the interaction of endogenous rassf1a and ras proteins .  
it has been implicated in the negative regulation of cell cycle progression , cell proliferation , and cell survival .
rassf1a has been shown to localize to microtubules of proliferating cells , increasing microtubule stability and inhibiting cell division [ 14 , 15 ] .
this may be mediated through direct binding or though interaction with microtubule - associated proteins such as c19orf5 .
rassf1a has also been shown to inhibit proliferation by inhibiting the accumulation of cyclin d1 and arresting cell division [ 17 , 18 ] .  
rassf1a also promotes apoptosis , which can reportedly occur through multiple mechanisms and is likely cell - type dependent .
one mechanism that mediates the apoptotic function of rassf1a involves protein interaction with modulator of apoptosis-1 ( moap-1 or map-1 ) .
moap-1 is normally sequestered in an inactive form in healthy cells . upon death receptor stimulation ,
rassf1a binds moap-1 , causing its activation and subsequent association with bax , which leads to apoptosis .
previous work has also demonstrated enhancement of rassf1a / mst - mediated cell death by the scaffold cnk1 .
rassf1a can also elicit inhibitory effects on growth and survival through engagement of the hippo pathway .
the hippo signaling pathway is a highly conserved kinase cascade that was originally discovered in drosophila and has been shown to be a critical regulator of cell proliferation , survival , and organ growth .
three members of this pathway , drassf , salvador and hippo , contain the sarah ( salvador - rassf - hippo ) domain , which is conserved in its mammalian counterparts rassf1 - 6 , ww45 , and mst1/2 , respectively .
while the drosophila ortholog drassf is known to antagonize hippo activation in the fly , it has been demonstrated that rassf1a promotes phosphorylation and activation of mst 1/2 by inhibiting the phosphatase pp2a in mammalian systems [ 29 , 30 ] .
the biological relevance of rassf1a - mediated activation of hippo signaling has also been investigated .
reported a rassf1a - mst2-yap - p73-puma signaling axis that promotes apoptosis in mammalian cells .
hippo signaling is also important for maintaining intestinal homeostasis and tissue regeneration in response to injury .
mouse models with conditional disruption of either mst1/2 or sav1 in the intestinal epithelium displayed hyperactivation of yes - associated protein ( yap ) , increased intestinal stem cell ( isc ) proliferation , and increased polyp formation following dextran sodium sulfate ( dss ) treatment [ 32 , 33 ] .
similarly , loss - of - function mutations of hippo components in the fly midgut caused increased isc proliferation .
these findings suggest that perhaps hippo signaling serves a more global role in regulating organ integrity , structure , and response to injury , and that perturbation of this pathway can lead to aberrant growth and dysfunction .
in 2005 , two independent groups generated and published findings regarding the systemic deletion of the rassf1a gene variant in mice [ 35 , 36 ] .
both described similar phenotypes involving the spontaneous generation of tumors , particularly in aged mice , thus further supporting the notion that rassf1a is a bona fide tumor suppressor [ 35 , 36 ] .
not surprisingly , nearly all studies involving rassf1a to date are related to cancer biology with few reports related to the cardiovascular field .
rassf1a is ubiquitously expressed and has been detected in heart tissue [ 3 , 37 , 38 ] .
initial investigation into the role of rassf1 gene products in a cardiac context came from the neyses laboratory .
their findings demonstrated that both rassf1a and rassf1c could associate with the sarcolemmal calcium pump , pmca4b , in neonatal rat cardiac myocytes .
this interaction was shown to mediate the inhibition of erk , and subsequent elk transcription and suggested the possibility that rassf1a could modulate cardiac myocyte growth .  
five years later , the same group demonstrated that rassf1a does in fact negatively regulate cardiac hypertrophy in vivo using rassf1a mice .
although these mice have increased susceptibility to spontaneous tumorigenesis , no apparent cardiovascular phenotype was observed under basal conditions , that is , no differences in heart size , morphology , or function compared to wt .
however , when rassf1a mice were challenged with pressure overload , they responded with an exaggerated hypertrophic response , evidenced by significantly greater increases in heart weight / body weight and hypertrophic gene expression ( anp , bnp , -mhc ) .
cardiac myocytes of rassf1a mice were significantly larger , which explains the augmented heart growth .
chamber dilation of rassf1a mouse hearts was observed by echocardiography , consistent with eccentric hypertrophic remodeling .
hemodynamic analysis of wt and rassf1a mice showed a rightward shift in pv loops following pressure overload in rassf1a hearts , yet dp / dtmax , dp / dtmin , and fractional shortening were not altered in rassf1a mice compared to wt .   to examine rassf1a function in cardiac myocytes , oceandy et al .
utilized a neonatal rat cardiac myocyte ( nrcm ) culture and the forced expression of rassf1a through adenoviral gene transfer .
increased rassf1a expression inhibited phenylephrine-(pe- ) induced cardiac myocyte growth and suppressed raf-1 and erk1/2 activation by pe treatment .
conversely , both raf-1 and erk1/2 phosphorylation were increased in  rassf1a hearts following pressure overload , suggesting negative regulation of mapk signaling by rassf1a .
deletion mutants of rassf1a revealed an important function of the n - terminus of rassf1a that disrupts the binding of active ras and raf-1 , thus preventing erk activation and cardiac myocyte growth .   to better understand the function of rassf1a in cardiac myocytes in vivo , we crossed genetically altered mice harboring a floxed rassf1a allele   with mice harboring the cre recombinase transgene driven by the -mhc promoter .
this strategy disrupted endogenous rassf1a gene expression and ensured cardiac myocyte specificity [ 38 , 40 ] .
similar to the rassf1a  mice , rassf1a - cre mice had no obvious baseline cardiac phenotype .
although we also found exaggerated heart growth in the rassf1a mice in response to pressure overload , the rassf1a - cre mice unexpectedly had attenuated hypertrophy , that is , smaller hearts and cardiac myocytes , compared to rassf1a and -mhc - cre controls .
furthermore , rassf1a - cre mice had significantly less fibrosis and myocyte apoptosis , and better cardiac function following pressure overload .
this was in stark contrast to the rassf1a mice , which presented significantly more fibrosis and a decline in cardiac function comparable to the levels found in wt mice .  
as an alternative approach we also generated two different cardiac - specific transgenic mouse lines : the first expressing wild - type rassf1a and the second expressing a rassf1a sarah domain point mutant ( l308p ) that renders it unable to bind mst1 .
interestingly , we found that increased rassf1a expression in the heart caused increases in mst1 activation , cardiac myocyte apoptosis , and fibrosis , and led to worsened function following pressure overload .
conversely , rassf1a l308p tg mice had significant reductions in mst1 activation , apoptosis and fibrosis , while cardiac function was preserved after stress .
these opposing phenotypes strongly implicate mst1 as a critical effector of rassf1a - mediated myocardial dysfunction .  
in cultured nrcms , increased rassf1a expression elicited activation of mst1 and caused mst1-mediated apoptosis .
however , in primary rat cardiac fibroblasts , rassf1a had a more pronounced effect on inhibition of cell proliferation rather than survival .
additionally , rassf1a depletion led to an upregulation of nf-b - dependent tnf- expression and secretion in cardiac fibroblasts , while no change in il-1 , il-6 , or tgf-1 was observed . through conditioned medium transfer experiments , we demonstrated that tnf- secretion from fibroblasts promotes cardiac myocyte growth .
furthermore , treatment of rassf1a mice with a neutralizing antibody against tnf- was able to rescue the augmented heart growth and fibrosis observed following pressure overload .
these data strongly implicated tnf- as a critical paracrine factor influencing the cardiac myocyte growth response to stress in vivo .
this work also demonstrated the cell - type specificity of rassf1a signaling in the heart and highlighted a novel signaling pathway downstream of rassf1a / mst1 that mediates a paracrine effect in vivo ( see figure 1 ) .
this mechanism involving multiple cell types , and paracrine signaling among them is rather unique and contrasts with more established signaling paradigms of cardiac hypertrophy including calcineurin / nfat , hdac / mef2 and mek / erk pathways , which have been elucidated in the cardiac myocyte .  
using genetically altered mouse models we showed that increased expression of mst1 , and subsequent activation of the hippo pathway , caused increased apoptosis , dilated cardiomyopathy , and premature death .
interestingly , expression of mst1 also attenuated cardiac myocyte hypertrophy thereby impairing the heart 's ability to appropriately respond to stress .
in contrast , expression of a kinase - inactive mst1 mutant ( dn - mst1 ) prevented cell death and protected the heart from insult .
lats1/2 kinases ( mammalian homologs of warts ) are targets of mst1/2 that can phosphorylate and inactivate yap , thereby inhibiting yap - mediated gene transcription .
similar to our findings related to mst1 , we demonstrated that transgenic expression of lats2 in the heart led to inhibited growth and worsened function .
conversely , kinase - inactive lats2 ( dn - lats2 ) transgenic mice had larger hearts both at baseline and following pressure overload and displayed attenuated cardiac myocyte apoptosis in response to stress
. taken together , these results provide further evidence that activation of hippo signaling , via increased mst1 or lats2 expression , inhibits cardiac myocyte growth and promotes apoptosis in the adult heart
. furthermore , selective inhibition of hippo signaling in the cardiac myocyte ( dn - mst1 or dn - lats2 tg ) confers protection against insult , similar to what we observed in the cardiac myocyte - specific rassf1a deleted mice .
however , the hypertrophic response in these two models was opposite , which may result from a hippo - independent pathway(s ) downstream of rassf1a .
it should be pointed out that studies of adult mouse models using cardiac myocyte - restricted deletion of mst1/2 , lats1/2 or yap have not been published .
findings from these models should be helpful in further elucidating the role of hippo signaling components in the adult murine heart .
recent work from the martin laboratory demonstrated the importance of mammalian hippo signaling during cardiac development and cardiac myocyte proliferation .
conditional deletion of salvador ( sav1 ) in the embryonic heart , driven by nkx2.5-cre expression , caused increased myocyte proliferation and cardiac enlargement and was mediated by hyperactivation of yap and subsequent wnt/-catenin - regulated gene expression . in a similar vein , direct targeting of yap expression in the developing mouse heart further demonstrated its role in governing both myocyte proliferation and heart growth .
interestingly , both reports described an interaction between yap and wnt signaling , highlighting additional hippo signaling crosstalk in the heart .
  fueled by the initial reports described herein , investigation into the role of rassf1a in cardiovascular biology has begun to accelerate . yet many questions remain outstanding . among them
, what are the upstream inputs that regulate rassf1a function ? what is the mechanism responsible for rassf1a cell - type - specific
what are the molecular constituents of the rassf1a complex ? does rassf1a have additional mst1-independent functions in the heart , as has been demonstrated in tumor cell lines ?
recent work identified activated k - ras as a promoter of rassf1a signaling in colorectal cancer cells .
this finding begs the question of whether k - ras or additional ras isoforms regulate rassf1a in other systems and cell types . based on our findings in rassf1a - deleted mice
, we speculate that the difference in proliferative capacity between cardiac myocytes and fibroblasts may explain the distinct effects of rassf1a signaling in the heart . there may also be differences in the expression or localization of signaling components , thereby modulating their ability to effectively signal in certain cell types .
exposure to diverse signals and cues in the extracellular milieu may also contribute to varied outcomes downstream rassf1a . as we continue to elucidate the role of rassf1a and hippo signaling in the heart
our work has shed light on the importance of cell type specificity rassf1a in determining pathological outcomes .
we also defined a paracrine mechanism functioning downstream of rassf1a in response to cardiac stress .
it is likely that additional complexities remain to be uncovered and will ultimately influence possible interventions to manipulate rassf1a and treat heart disease .
rassf1a signaling is diverse and our knowledge regarding rassf1a function is rapidly expanding . given that a bridge from cancer to cardiovascular biology is in place , it is likely that as additional rassf1a mechanisms of action are discovered , its impact on cardiac biology will continue to grow .
 the rassf proteins are a family of polypeptides , each containing a conserved ras association domain , suggesting that these scaffold proteins may be effectors of activated ras or ras - related small gtpases . 
 rassf proteins are characterized by their ability to inhibit cell growth and proliferation while promoting cell death . 
 rassf1 isoform a is an established tumor suppressor and is frequently silenced in a variety of tumors and human cancer cell lines . 
 however , our understanding of its function in terminally differentiated cell types , such as cardiac myocytes , is relatively nascent 
 . herein , we review the role of rassf1a in cardiac physiology and disease and highlight signaling pathways that mediate its function . 
1. Introduction
2. RASSF1A
3. Cardiovascular Function of RASSF1A
4. Conclusion