The data presented in our study suggest that a BP QTL is further fine-mapped within <42.5kb containing a single candidate protein coding gene, Rififylin and 171 candidate quantitative trait nucleotides. The congenic strain containing LEW alleles at this QTL region has elevated BP, develops cardiac hypertrophy and has a shorter QT interval. These phenotypes are associated with an elevated mRNA and protein expression of rififylin for unknown reasons. There are two known functions of overexpression of both the transcript and protein product of the gene, Rififylin: 1) delayed recycling of endosomes [2
] and 2) increased polyubiquitination of proteins [3
]. Both of these functions were differential between the congenic strain and S. The delay in recycling of endosomes was demonstrated in neonatal cardiomyocytes of the congenic strain. These physiological differences were associated with alterations in beats/min of neonatal cardiomyocytes from the congenic animals which also had shorter QT intervals and increased heart rates. These early changes in cardiac function are known predictors of hypertension [16
] and observed to occur before significant differences in blood pressure are detected between the congenic strain and S. Taken together, the data suggests that overexpression of Rffl
represents one of the mechanistic factors likely to be contributing to the observed increase in blood pressure of the congenic strain. Cardiac hypertrophy observed later in the study is likely influenced by the higher blood pressure, but the concentric nature of the hypertrophy remains intriguing.
The contractile function of cardiomyocytes is primarily dependent on the surface expression of ion channels [18
]. While mutations in many ion channels per se
are discovered to cause alterations in myocardial repolarization [19
], it is possible that functional alterations in proteins involved in the intracellular transport of the channels to and from the plasma membrane could also affect membrane repolarization by regulating the dynamic control of their endocytosis and exocytosis from intracellular storage compartments. One such storage compartment is the endocytic recycling compartment (ERC), a peri-nuclear collection of tubular organelles that mature from sorting endosomes [22
]. An independent study has demonstrated that the recycling of endosomes is delayed in the presence of an excess of rififylin [2
]. We have demonstrated that this function of rififylin is recapitulated in cardiomyocytes from the congenic strain which overexpress rififylin. This observation lead us to suspect alterations in myocardial repolarization, which was further supported by the shorter QT-intervals observed in the congenic strain because QT-intervals reflect myocardial repolarization as measured by the electrocardiogram[23
Rififylin is also reported to be an endosome-associated ubiquitin ligase 3 [3
]. Therefore we assessed the polyubiquitination status of cardiac proteins. Higher levels of polyubiquitinated proteins observed in the congenic strain compared with the S is suggestive of increased availability of endosome-associated ubiquitin ligase 3 activity as a result of increased expression of Rffl
. Because the ubiquitin-proteasome system influences transcription [13
], we reasoned that the observed downregulation of Mbd2
is perhaps a consequence of the increased polyubiquitination of cardiac proteins in the congenic strain compared with S.
An obvious question that arises from our current study is, ‘why is Rffl overexpressed in the congenic strain compared with S?’ The various possibilities include the following:(1) exonic or intronic variants within the Rffl gene, (2) variants within the promoter of Rffl, (3) variants upstream and downstream of Rffl that are binding sites for transcription enhancers/suppressors, (4) variants in non-coding RNAs, (5) variants within yet undefined regulatory elements of Rffl and (6) Trans-elements on the remainder of the S rat genome that interact with LEW alleles within the <42.5kb QTL region to regulate the expression of Rffl. Sequencing has revealed that while there are no exonic variants of Rffl, there are 171 candidate variants between S and LEW within the <42.5kb region. To begin addressing the question of why Rffl is overexpressed in the congenic strain, a detailed follow-up study of each of these candidate variants is required, but this is beyond the scope of the current study which was primarily focused on extended phenotyping. Nevertheless, the importance of the current study is that it provides evidence for aberrant endocytic recycling in cardiomyocytes to be further considered as a novel physiological mechanism potentially underlying blood pressure regulation.
Several mapping studies point to candidate genes on human chromosome 17 as plausible for blood pressure control in humans [24
]. Two independent genome-wide association studies (GWAS) [10
] also point to single nucleotide polymorphisms (SNPs) on human chromosome 17 that are associated with systolic BP. Further, one of these groups conducted a large meta-analysis of three GWAS in 13,685 individuals of European ancestry from the Framingham Heart Study, the Rotterdam Study and the Cardiovascular Health Study, as part of the QTGEN consortium and identified association of multiple minor alleles near the RFFL
locus in humans with shorter QT-intervals [10
]. This association was also confirmed in a second study [11
]. Our finding of a genomic segment containing rififylin similarly linked to QT-intervals in rats serves as a functional validation of these GWAS [10
]. Further, our observation in rats that early changes in QT-interval contributed to the development of hypertension suggests that these individuals could be at risk for developing hypertension. Interestingly, over 29% of the cohorts screened by GWAS [10
] were hypertensive, thus lending support to our interpretation. Overall, our studies provide the impetus to target the endosomal recycling machinery via Rffl
as a novel mechanism for prevention of aberrant heart function leading to hypertension in individuals with abnormal heart rhythms.