Kathirvel Gopalakrishnan

Targeted disruption of Adamts16 gene in a rat genetic model of hypertension

A disintegrin-like metalloproteinase with thrombospondin motifs–16 (Adamts16) is an important candidate gene for hypertension. The goal of the present study was to further assess the candidacy of Adamts16 by targeted disruption of this gene in a rat genetic model of hypertension. A rat model was generated by manipulating the genome of the Dahl Salt–sensitive (S) rat using zinc-finger nucleases, wherein the mutant rat had a 17 bp deletion in the first exon of Adamts16, introducing a stop codon in the transcript. Systolic blood pressure (BP) of the homozygous Adamts16mutant rats was lower by 36 mmHg compared with the BP of the S rats. The Adamts16mutant rats exhibited significantly lower aortic pulse wave velocity and vascular media thickness compared with S rats. Scanning electron and fluorescence microscopic studies indicated that the mechanosensory cilia of vascular endothelial cells from the Adamts16mutant rats were longer than that of the S rats. Furthermore, Adamts16mutant rats showed splitting and thickening of glomerular capillaries and had a longer survival rate, compared with the S rats. Taken together, these physiological observations functionally link Adamts16 to BP regulation and suggest the vasculature as the potential site of action of Adamts16 to lower BP.

Downregulation of SWI/SNF chromatin remodeling factor subunits modulates cisplatin cytotoxicity

Chromatin remodeling complex SWI/SNF plays important roles in many cellular processes including transcription, proliferation, differentiation and DNA repair. In this report, we investigated the role of SWI/SNF catalytic subunits Brg1 and Brm in the cellular response to cisplatin in lung cancer and head/neck cancer cells. Stable knockdown of Brg1 and Brm enhanced cellular sensitivity to cisplatin. Repair kinetics of cisplatin DNA adducts revealed that downregulation of Brg1 and Brm impeded the repair of both intrastrand adducts and interstrand crosslinks (ICLs). Cisplatin ICL-induced DNA double strand break repair was also decreased in Brg1 and Brm depleted cells. Altered checkpoint activation with enhanced apoptosis as well as impaired chromatin relaxation was observed in Brg1 and Brm deficient cells. Downregulation of Brg1 and Brm did not affect the recruitment of DNA damage recognition factor XPC to cisplatin DNA lesions, but affected ERCC1 recruitment, which is involved in the later stages of DNA repair. Based on these results, we propose that SWI/SNF chromatin remodeling complex modulates cisplatin cytotoxicity by facilitating efficient repair of the cisplatin DNA lesions.

Genome-Wide Identification of Long Noncoding RNAs in Rat Models of Cardiovascular and Renal Disease

Long noncoding RNAs (lncRNAs) are an emerging class of genomic regulatory molecules reported in various species. In the rat, which is one of the major mammalian model organisms, discovery of lncRNAs on a genome-wide scale is lagging. Renal lncRNA sequencing and lncRNA transcriptome analysis were conducted in 3 rat strains that are widely used in cardiovascular and renal research: the Dahl salt-sensitive rat, the spontaneously hypertensive rat, and the Dahl salt-resistant rat. Through the RNA sequencing approach, 3273 transcripts were identified as rat lncRNAs. A majority of lncRNAs were without predicted target genes. Differential expression of 273 and 749 lncRNAs was detected between Dahl salt-sensitive versus Dahl salt-resistant and Dahl salt-sensitive versus spontaneously hypertensive rat comparisons, respectively. To couple the observed differential expression of lncRNAs with the status of mRNAs, an mRNA transcriptome analysis was conducted. Several cis mRNA genes were coregulated with lncRNAs. Of these, the protein expression status of 4 target genes, Asb3, Chac2, Pex11b, and Sp5, were differentially expressed between the relevant strain comparisons, thereby suggesting that the differentially expressed lncRNAs associated with these genes are candidate genetic determinants of blood pressure. This study serves as a first-generation catalog of rat lncRNAs and illustrates the prioritization of lncRNAs as candidates for complex polygenic traits.

Augmented Rififylin Is a Risk Factor Linked to Aberrant Cardiomyocyte Function, Short-QT Interval and Hypertension

Using congenic strains of the Dahl salt-sensitive (S) rat introgressed with genomic segments from the normotensive Lewis rat, a blood pressure quantitative trait locus was previously mapped within 104 kb on chromosome 10. The goal of the current study was to conduct extensive phenotypic studies and to further fine-map this locus. At 14 weeks of age, the blood pressure of the congenic rats fed a low-salt diet was significantly higher by 47 mm Hg (P<0.001) compared with that of the S rat. A time-course study showed that the blood pressure effect was significant from very young ages of 50 to 52 days (13 mm Hg; P<0.01). The congenic strain implanted with electrocardiography transmitters demonstrated shorter-QT intervals and increased heart rate compared with S rats (P<0.01). The average survival of the congenic strain was shorter (134 days) compared with the S rat (175 days; P<0.0007). The critical region was narrowed to <42.5 kb containing 171 variants and a single gene, rififylin. Both the mRNA and protein levels of rififylin were significantly higher in the hearts of the congenic strain. Overexpression of rififylin is known to delay endocytic recycling. Endocytic recycling of fluorescently labeled holotransferrin from cardiomyocytes of the congenic strain was slower than that of S rats (P<0.01). Frequency of cardiomyocyte beats in the congenic strain (62±9 bpm) was significantly higher than that of the S rat (24±6 bpm; P<0.001). Taken together, our study provides evidence to suggest that early perturbations in endocytic recycling caused by the overexpression of Rffl is a novel physiological mechanism potentially underlying the development of hypertension.

Refined mapping of blood pressure quantitative trait loci using congenic strains developed from two genetically hypertensive rat models

Previously linkage and substitution mapping were conducted between the Dahl Salt-sensitive (S) rat and the Spontaneously Hypertensive Rat (SHR) to address the hypothesis that genetic contributions to blood pressure (BP) in two genetically hypertensive rat strains are different. Among the BP quantitative trait loci (QTLs) detected, two are located on chromosome 9 within large genomic segments. The goal of the current study was to develop new iterations of congenic substrains, to further resolve both of these BP QTLs on chromosome 9 as independent congenic segments. A total of 10 new congenic substrains were developed and characterized. The newly developed congenic substrains S.SHR(9)x8Ax11A and S.SHR(9)x10Ax1, with introgressed segments of 2.05 and 6.14 Mb, represented the shortest genomic segments. Both of these congenic substrains, S.SHR(9)x8Ax11A and S.SHR(9)x10Ax1 lowered BP of the S rat by 56 mm Hg (P<0.001) and 15 mm Hg (P<0.039), respectively. The BP measurements were corroborated by radiotelemetry. Urinary protein excretion was significantly lowered by SHR alleles within S.SHR(9)x10Ax1 but not by S.SHR(9)x8Ax11A. The shorter of the two congenic segments, 2.05 Mb was further characterized and found to contain a single differentially expressed protein-coding gene, Tomoregulin-2 (Tmeff2). The protein expression of Tmeff2 was higher in the S rat compared with S.SHR(9)x8Ax11A, which also had lower cardiac hypertrophy as measured by echocardiography. Tmeff2 is known to be upregulated in patients from multiple cohorts with cardiac hypertrophy. Taken together, Tmeff2 can be prioritized as a candidate gene for hypertension and associated cardiac hypertrophy in both rats and in humans.

Construction of two novel reciprocal conplastic rat strains and characterization of cardiac mitochondria.

Because of the lack of appropriate animal models, the potentially causal contributions of inherited mitochondrial genomic factors to complex traits are less well studied compared with inherited nuclear genomic factors. We previously detected variations between the mitochondrial DNA (mtDNA) of the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR). Specifically, multiple variations were detected in mitochondrial genes coding for subunits of proteins essential for electron transport, in mitochondrial reactive oxygen species production, and within the D-loop region. To evaluate the effects of these mtDNA variations in the absence of the corresponding nuclear genomic factors as confounding variables, novel reciprocal strains of S and SHR were constructed and characterized. When compared with that of the S rat, the heart tissue from the S.SHR(mt) conplastic strain wherein the mtDNA of the S rat was substituted with that of the SHR had a significant increase in mtDNA copy number and decrease in mitochondrial reactive oxygen species production. A corresponding increase in aerobic treadmill running capacity and a significant increase in survival that was not related to changes in blood pressure were observed in the S.SHR(mt) rats compared with the S rat. The reciprocal SHR.S(mt) rats did not differ from the SHR in any phenotype tested, suggesting lower penetrance of the S mtDNA on the nuclear genomic background of the SHR. These novel conplastic strains serve as invaluable tools to further dissect the relationship between heart function, aerobic fitness, cardiovascular disease progression, and mortality.

Epistatic genetic determinants of blood pressure and mortality in a salt-sensitive hypertension model.

Although genetic determinants protecting against the development of elevated blood pressure (BP) are well investigated, less is known regarding their impact on longevity. We concomitantly assessed genomic regions of rat chromosomes 3 and 7 (RNO3 and RNO7) carrying genetic determinants of BP without known epistasis, for their independent and combinatorial effects on BP and the presence of genetic determinants of survival using Dahl salt-sensitive (S) strains carrying congenic segments from Dahl salt-resistant (R) rats. Although congenic and bicongenic S.R strains carried independent BP quantitative trait loci within the RNO3 and RNO7 congenic regions, only the RNO3 allele(s) independently affected survival. The bicongenic S.R strain showed epistasis between R-rat RNO3 and RNO7 alleles for BP under salt-loading conditions, with less-than-additive effects observed on a 2% NaCl diet and greater-than-additive effects observed after prolonged feeding on a 4% NaCl diet. These RNO3 and RNO7 congenic region alleles had more-than-additive effects on survival. Increased survival of bicongenic compared with RNO3 congenic rats was attributable, in part, to maintaining lower BP despite chronic exposure to an increased dietary salt (4% NaCl) intake, with both strains showing delays in reaching highest BP. R-rat RNO3 alleles were also associated with superior systolic function, with the S.R bicongenic strain showing epistasis between R-rat RNO3 and RNO7 alleles leading to compensatory hypertrophy. Whether these alleles affect survival by additional actions within other BP-regulating tissues/organs remains unexplored. This is the first report of simultaneous detection of independent and epistatic loci dictating, in part, longevity in a hypertensive rat strain.

Defining a rat blood pressure quantitative trait locus to a <81.8 kb congenic segment: comprehensive sequencing and renal transcriptome analysis

Evidence from multiple linkage and genome-wide association studies suggest that human chromosome 2 (HSA2) contains alleles that influence blood pressure (BP). Homologous to a large segment of HSA2 is rat chromosome 9 (RNO9), to which a BP quantitative trait locus (QTL) was previously mapped. The objective of the current study was to further resolve this BP QTL. Eleven congenic strains with introgressed segments spanning <81.8 kb to <1.33 Mb were developed by introgressing genomic segments of RNO9 from the Dahl salt-resistant (R) rat onto the genome of the Dahl salt-sensitive (S) rat and tested for BP. The congenic strain with the shortest introgressed segment spanning <81.8 kb significantly lowered BP of the hypertensive S rat by 25 mmHg and significantly increased its mean survival by 45 days. In contrast, two other congenic strains had increased BP compared with the S. We focused on the <81.8 kb congenic strain, which represents the shortest genomic segment to which a BP QTL has been mapped to date in any species. Sequencing of this entire region in both S and R rats detected 563 variants. The region did not contain any known or predicted rat protein coding genes. Furthermore, a whole genome renal transcriptome analysis between S and the <81.8 kb S.R congenic strain revealed alterations in several critical genes implicated in renal homeostasis. Taken together, our results provide the basis for future studies to examine the relationship between the candidate variants within the QTL region and the renal differentially expressed genes as potential causal mechanisms for BP regulation.

Mitochondrial polymorphisms in rat genetic models of hypertension

Hypertension is a complex trait that has been studied extensively for genetic contributions of the nuclear genome. We examined mitochondrial genomes of the hypertensive strains: the Dahl Salt-Sensitive (S) rat, the Spontaneously Hypertensive Rat (SHR), and the Albino Surgery (AS) rat, and the relatively normotensive strains: the Dahl Salt-Resistant (R) rat, the Milan Normotensive Strain (MNS), and the Lewis rat (LEW). These strains were used previously for linkage analysis for blood pressure (BP) in our laboratory. The results provide evidence to suggest that variations in the mitochondrial genome do not account for observed differences in blood pressure between the S and R rats. However, variants were detected among the mitochondrial genomes of the various hypertensive strains, S, SHR, and AS, and also among the normotensive strains R, MNS, and LEW. A total of 115, 114, 106, 106, and 16 variations in mtDNA were observed between the comparisons S versus LEW, S versus MNS, S versus SHR, S versus AS, and SHR versus AS, respectively. Among the 13 genes coding for proteins of the electron transport chain, 8 genes had nonsynonymous variations between S, LEW, MNS, SHR, and AS. The lack of any sequence variants between the mitochondrial genomes of S and R rats provides conclusive evidence that divergence in blood pressure between these two inbred strains is exclusively programmed through their nuclear genomes. The variations detected among the various hypertensive strains provides the basis to construct conplastic strains and further evaluate the effects of these variants on hypertension and associated phenotypes.

Mutation within the hinge region of the transcription factor Nr2f2 attenuates salt-sensitive hypertension

Genome-wide association studies (GWAS) have prioritized a transcription factor, Nuclear Receptor 2 Family 2 (NR2F2), as being associated with essential hypertension in humans. Here we provide evidence that validates this association and indicates that Nr2f2 is a genetic determinant of blood pressure (BP). Using the zinc-finger nuclease technology, the generation of a targeted Nr2f2-edited rat model is reported. The resulting gene-edited rats have a 15bp deletion in exon 2 leading to a 5 amino acid deletion in the hinge region of the mutant Nr2f2 protein. Both systolic and diastolic blood pressures of the Nr2f2mutant rats are significantly lower than controls. Because the hinge region of Nr2f2 is required for interaction with Friend of Gata2 (Fog2), protein-protein interaction is examined. Interaction of Nr2f2mutant protein with Fog2 is greater than that with the wild type Nr2f2 indicating that the extent of interaction between these two transcription factors critically influences BP.