Cultured Myoblasts Derived from Rat Soleus Muscle Show Altered Regulation of Proliferation and Myogenesis during the Course of Mechanical Unloading.

The structure and function of soleus muscle fibers undergo substantial remodeling under real or simulated microgravity conditions. However, unloading-induced changes in the functional activity of skeletal muscle primary myoblasts remain poorly studied. The purpose of our study was to investigate how short-term and long-term mechanical unloading would affect cultured myoblasts derived from rat soleus muscle. Mechanical unloading was simulated by rat hindlimb suspension model (HS). Myoblasts were purified from rat soleus at basal conditions and after 1, 3, 7, and 14 days of HS. Myoblasts were expanded in vitro, and the myogenic nature was confirmed by their ability to differentiate as well as by immunostaining/mRNA expression of myogenic markers. The proliferation activity at different time points after HS was analyzed, and transcriptome analysis was performed. We have shown that soleus-derived myoblasts differently respond to an early and later stage of HS. At the early stage of HS, the proliferative activity of myoblasts was slightly decreased, and processes related to myogenesis activation were downregulated. At the later stage of HS, we observed a decrease in myoblast proliferative potential and spontaneous upregulation of the pro-myogenic program.

Skeletal Muscle Mitochondria Dysfunction in Genetic Neuromuscular Disorders with Cardiac Phenotype.

Mitochondrial dysfunction is considered the major contributor to skeletal muscle wasting in different conditions. Genetically determined neuromuscular disorders occur as a result of mutations in the structural proteins of striated muscle cells and therefore are often combined with cardiac phenotype, which most often manifests as a cardiomyopathy. The specific roles played by mitochondria and mitochondrial energetic metabolism in skeletal muscle under muscle-wasting conditions in cardiomyopathies have not yet been investigated in detail, and this aspect of genetic muscle diseases remains poorly characterized. This review will highlight dysregulation of mitochondrial representation and bioenergetics in specific skeletal muscle disorders caused by mutations that disrupt the structural and functional integrity of muscle cells.

Clinical Response to Personalized Exercise Therapy in Heart Failure Patients with Reduced Ejection Fraction is Accompanied by Skeletal Muscle Histological Alterations.

Abstract: Heart failure (HF) is associated with skeletal muscle wasting and exercise intolerance. This study aimed to evaluate the exercise-induced clinical response and histological alterations. One hundred and forty-four HF patients were enrolled. The individual training program was determined as a workload at or close to the lactate threshold (LT1); clinical data were collected before and after 12 weeks/6 months of training. The muscle biopsies from eight patients were taken before and after 12 weeks of training: histology analysis was used to evaluate muscle morphology. Most of the patients demonstrated a positive response after 12 weeks of the physical rehabilitation program in one or several parameters tested, and 30% of those showed improvement in all four of the following parameters: oxygen uptake (VO2) peak, left ventricular ejection fraction (LVEF), exercise tolerance (ET), and quality of life (QOL); the walking speed at LT1 after six months of training showed a significant rise. Along with clinical response, the histological analysis detected a small but significant decrease in both fiber and endomysium thickness after the exercise training course indicating the stabilization of muscle mechanotransduction system. Together, our data show that the beneficial effect of personalized exercise therapy in HF patients depends, at least in part, on the improvement in skeletal muscle physiological and biochemical performance.

Association of tribbles homologue 1 gene expression in human umbilical vein endothelial cells with duration of intrauterine exposure to hyperglycaemia.

Maternal gestational diabetes mellitus (GDM) is considered to be an important factor that epigenetically predisposes offspring to metabolic and cardiovascular diseases. However, the mechanisms of how intrauterine hyperglycaemia affects offspring have not been thoroughly studied. The mammalian tribbles homologue 1 (TRIB1) gene is associated with plasma lipid concentrations and coronary artery disease (CAD). Our aim was to study the effect of GDM and its treatment terms on the level of TRIB1 gene expression in human umbilical vein endothelial cells (HUVECs) of newborns from women with and without GDM. The study included 50 women with GDM and 25 women without GDM (control group). Women with GDM were divided into three groups according to their gestational age when the treatment of GDM started: 24-28 weeks (GDM1, N = 16), 29-32 weeks (GDM2, N = 25) and >34 weeks (GDM3, N = 9). The levels of TRIB1 gene expression in GDM3, GDM2, GDM1 and control groups were 2.8 ± 1.1, 4.2 ± 2.4, 6.0 ± 3.4 and 8.1 ± 6.1, respectively (p = 0.001). After comparison in pairs the difference was significant for the following pairs: GDM2-control (p = 0.004), GDM3-control (p = 0.002), GDM1-GDM3 (p = 0.012). Notably, if treatment had been started before the 28th week of gestation, the difference in TRIB1 gene expression in HUVECs was not significant (p = 0.320 for comparison between GDM1 and control groups). Our findings support the hypothesis that TRIB1 gene expression in HUVECs depends on the duration of intrauterine exposure to hyperglycaemia.

Various lamin A/C mutations alter expression profile of mesenchymal stem cells in mutation specific manner.

Various mutations in LMNA gene, encoding for nuclear lamin A/C protein, lead to laminopathies and contribute to over ten human disorders, mostly affecting tissues of mesenchymal origin such as fat tissue, muscle tissue, and bones. Recently it was demonstrated that lamins not only play a structural role providing communication between extra-nuclear structures and components of cell nucleus but also control cell fate and differentiation. In our study we assessed the effect of various LMNA mutations on the expression profile of mesenchymal multipotent stem cells (MMSC) during adipogenic and osteogenic differentiation. We used lentiviral approach to modify human MMSC with LMNA-constructs bearing mutations associated with different laminopathies--G465D, R482L, G232E, R527C, and R471C. The impact of various mutations on MMSC differentiation properties and expression profile was assessed by colony-forming unit analysis, histological staining, expression of the key differentiation markers promoting adipogenesis and osteogenesis followed by the analysis of the whole set of genes involved in lineage-specific differentiation using PCR expression arrays. We demonstrate that various LMNA mutations influence the differentiation efficacy of MMSC in mutation-specific manner. Each LMNA mutation promotes a unique expression pattern of genes involved in a lineage-specific differentiation and this pattern is shared by the phenotype-specific mutations.

The effect of bone marrow- and adipose tissue-derived mesenchymal stem cell transplantation on myocardial remodelling in the rat model of ischaemic heart failure.

This study aimed to investigate the effect of bone marrow- and adipose tissue-derived mesenchymal stem cell (BM-MSC and AD-MSC respectively) transplantation on left ventricular function and infarct area (IA) in the rat model of ischaemic heart failure. In anaesthetized Wistar rats, the left coronary artery (LCA) was occluded for 40 min with subsequent reperfusion for 7 days. Seven days following surgery, the animals with LCA occlusion/reperfusion were randomized into three groups: (i) Controls received intramyocardial injection of vehicle at three different locations within the peri-infarct zone, (ii) BM-MSC: cells were injected in the same way as in previous group (10(6) ), (iii) AD-MSC: using the same protocol as used in the BM-MSC group. In addition there was also a sham-treated group that had no injection. Two weeks following MSC transplantation, the hearts were isolated and perfused according to the Langendorff method followed by 30-min global ischaemia and 90-min reperfusion. After this IA was determined histologically. During Langendorff perfusion initial and postischaemic LV functions were the same in all groups although LV pressure at the 10th minute of reperfusion was higher in the AD-MSC group compared to controls. However, LV pressure during 30-min global ischaemia was significantly higher in BM-MSC as compared to controls and AD-MSC. The sham treated animals showed the same results as those seen with BM-MSC. Thus, BM-MSC transplantation, in contrast to transplantation of AD-MSC, resulted in better preservation of the LV ability to contract during ischaemia. Furthermore, IA was significantly smaller in BM-MSC group as compared to the controls and the AD-MSC groups. Thus this study has demonstrated that treatment with BM-MSC both ameliorates LV function and reduces histological scar size.

Regulatory Action of Plasma from Patients with Obesity and Diabetes towards Muscle Cells Differentiation and Bioenergetics Revealed by the C2C12 Cell Model and MicroRNA Analysis.

Obesity and type 2 diabetes mellitus (T2DM) are often combined and pathologically affect many tissues due to changes in circulating bioactive molecules. In this work, we evaluated the effect of blood plasma from obese (OB) patients or from obese patients comorbid with diabetes (OBD) on skeletal muscle function and metabolic state. We employed the mouse myoblasts C2C12 differentiation model to test the regulatory effect of plasma exposure at several levels: (1) cell morphology; (2) functional activity of mitochondria; (3) expression levels of several mitochondria regulators, i.e., Atgl, Pgc1b, and miR-378a-3p. Existing databases were used to computationally predict and analyze mir-378a-3p potential targets. We show that short-term exposure to OB or OBD patients' plasma is sufficient to affect C2C12 properties. In fact, the expression of genes that regulate skeletal muscle differentiation and growth was downregulated in both OB- and OBD-treated cells, maximal mitochondrial respiration rate was downregulated in the OBD group, while in the OB group, a metabolic switch to glycolysis was detected. These alterations correlated with a decrease in ATGL and Pgc1b expression in the OB group and with an increase of miR-378a-3p levels in the OBD group.

LMNA Mutations G232E and R482L Cause Dysregulation of Skeletal Muscle Differentiation, Bioenergetics, and Metabolic Gene Expression Profile.

Laminopathies are a family of monogenic multi-system diseases resulting from mutations in the LMNA gene which include a wide range of neuromuscular disorders. Although lamins are expressed in most types of differentiated cells, LMNA mutations selectively affect only specific tissues by mechanisms that remain largely unknown. We have employed the combination of functional in vitro experiments and transcriptome analysis in order to determine how two LMNA mutations associated with different phenotypes affect skeletal muscle development and metabolism. We used a muscle differentiation model based on C2C12 mouse myoblasts genetically modified with lentivirus constructs bearing wild-type human LMNA (WT-LMNA) or R482L-LMNA/G232E-LMNA mutations, linked to familial partial lipodystrophy of the Dunnigan type and muscular dystrophy phenotype accordingly. We have shown that both G232E/R482L-LMNA mutations cause dysregulation in coordination of pathways that control cell cycle dynamics and muscle differentiation. We have also found that R482/G232E-LMNA mutations induce mitochondrial uncoupling and a decrease in glycolytic activity in differentiated myotubes. Both types of alterations may contribute to mutation-induced muscle tissue pathology.

Truncating Variant in Myof Gene Is Associated With Limb-Girdle Type Muscular Dystrophy and Cardiomyopathy.

Even though genetic studies of individuals with neuromuscular diseases have uncovered the molecular background of many cardiac disorders such as cardiomyopathies and inherited arrhythmic syndromes, the genetic cause of a proportion of cardiomyopathies associated with neuromuscular phenotype still remains unknown. Here, we present an individual with a combination of cardiomyopathy and limb-girdle type muscular dystrophy where whole exome sequencing identified myoferlin (MYOF)-a member of the Ferlin protein family and close homolog of DYSF-as the most likely candidate gene. The disease-causative role of the identified variant c.[2576delG; 2575G>C], p.G859QfsTer8 is supported by functional studies in vitro using the primary patient's skeletal muscle mesenchymal progenitor cells, including both RNA sequencing and morphological studies, as well as recapitulating the muscle phenotype in vivo in zebrafish. We provide the first evidence supporting a role of MYOF in human muscle disease.

Protein kinase M zeta regulation of Na/K ATPase: a persistent neuroprotective mechanism of ischemic preconditioning in hippocampal slice cultures.

In ischemic preconditioning, a sublethal ischemic insult protects neurons from subsequent ischemia. In organotypic hippocampal slice cultures a sublethal 5-minute hypoxia-hypoglycemia treatment prevented neuronal loss after a 10-minute experimental ischemic (EI) treatment of hypoxia-hypoglycemia. Whereas preconditioning protected against EI given 24 h later, it did not protect when EI was given 2 h later, suggesting a slow development of neuroprotection. This model identified two regulators of ischemic preconditioning: the atypical protein kinase M zeta (PKMzeta), and the Na/K ATPase. Two hours following preconditioning, when there was no neuroprotection, Na/K ATPase activity was unchanged. In contrast, Na/K ATPase activity significantly increased 24 h after the preconditioning treatment. Elevated Na/K ATPase activity was accompanied by increased surface expression of the alpha1 and alpha2 isoforms of the Na/K ATPase. Similarly, active PKMzeta levels were increased at 24 h, but not 2 h, after preconditioning. PKMzeta overexpression by sindbis virus vectors also increased Na/K ATPase activity. To examine PKMzeta regulation of Na/K ATPase, occlusion experiments were performed using marinobufagenin to inhibit alpha1, dihydroouabain to inhibit alpha2/3 and a zeta-pseudosubstrate peptide to inhibit PKMzeta. These experiments showed that PKMzeta regulated both the activity and surface expression of the alpha1 isoform of the Na/K ATPase. Marinobufagenin, dihydroouabain, and zeta-pseudosubstrate peptide were used to determine if PKMzeta or the alpha1 and alpha2 Na/K ATPase isoforms protected neurons. All three compounds blocked neuroprotection following ischemic preconditioning. PKMzeta levels were elevated 3 days after ischemic preconditioning. These data indicate key roles of PKMzeta and Na/K ATPase in ischemic preconditioning.

New Approach to Cryopreservation of Primary Noncultivated Human Umbilical Vein Endothelium in Biobanking.

It is widely accepted that endothelial dysfunction (ED) is a common feature and a risk factor for cardiovascular diseases and metabolic disorders. Cultures of human umbilical vein endothelial cells (HUVECs) are routinely used in cell-based models to study in vitro molecular and cellular mechanisms of development of different aspects of ED. The methods of the HUVEC extraction and expansion are well developed and standardized. However, when large collections of samples are needed for certain projects, or when samples from a rare population of patients should be collected for future experimental use, HUVEC samples should be transferred to a biobank to be saved in liquid nitrogen for a long period of time until the required collection is completed. This scenario is not always convenient since it requires a lot of effort, a large quantity of expensive culture reagents with limited expiration periods, and sometimes special facilities and well-trained cell biologists among the biobank staff. In this project, we evaluated a method of HUVEC cryopreservation, where the stage of cell culturing and expansion before the transfer of samples to the biobank is eliminated. A total of 55 samples of umbilical cord (UC) were obtained from women immediately after delivery. A primary endothelium pellet derived from 17 UC samples was isolated, frozen, and placed in long-term storage in a liquid nitrogen freezer. Other samples were used to obtain HUVEC cultures. We have demonstrated that cryopreservation of primary endothelium pellets from UC veins without culturing and expansion steps does not affect the physiological features of HUVECs. This new approach would improve the efficiency of biobanking logistics, especially in the case of banking of large collections of endothelial samples.

Genetic Control of Serum Marinobufagenin in the Spontaneously Hypertensive Rat and the Relationship to Blood Pressure.

BACKGROUND: We have investigated serum levels of immunoreactive marinobufagenin (MBG) in 16- to 20-week-old spontaneously hypertensive rats (SHRs)-A3 and in the normotensive Wistar-Kyoto (WKY) rat strain in the absence of salt loading, and we have investigated the genetic control of serum MBG. METHODS AND RESULTS: We genotyped the F2 progeny of an SHR-A3×WKY intercross using a genome-wide panel of 253 single-nucleotide polymorphism markers that were dimorphic between SHR-A3 and WKY and measured serum MBG by ELISA. Serum MBG levels were lower in SHR-A3 than WKY rats (0.39±0.07 and 1.27±0.40 nmol/L, respectively), suggesting that MBG may not play a role in the markedly divergent blood pressure measured by telemetry in rats of these 2 strains (SHR-A3 and WKY, 198.3±4.43 and 116.8±1.51 mm Hg, respectively). The strain difference in serum MBG was investigated to determine whether genomic regions influencing MBG might be identified by genetic mapping. Quantitative trait locus mapping indicated a single locus influencing serum MBG in the region of chromosome 6q12. Homozygosity of WKY alleles at this locus was associated with increased serum MBG levels. We surveyed whole genome sequences from our SHR-A3 and WKY lines, seeking coding sequence variation between SHR-A3 and WKY within the mapped locus that might explain the inherited strain difference in serum MBG. CONCLUSIONS: We identified amino acid substitution in the sterol transport protein Abcg5, present in SHR-A3, but absent in WKY, that is a potential mechanism influencing MBG levels.

Ouabain- and marinobufagenin-induced proliferation of human umbilical vein smooth muscle cells and a rat vascular smooth muscle cell line, A7r5.

BACKGROUND: We studied the growth-promoting effects of 2 sodium pump-selective cardiotonic steroids, ouabain and marinobufagenin, on cultured cells from vascular smooth muscle (VSMCs) from human umbilical vein and a rat VSMC line, A7r5. METHODS AND RESULTS: Both ouabain and marinobufagenin activated proliferation of these cells in a concentration-dependent manner, reflecting the cardiotonic steroid sensitivity of the specific alpha1 subunit contained within each cell source. The observed effective concentration ranges of both compounds was below that necessary to induce cytoplasmic ion alterations by sodium pump inhibition. CONCLUSIONS: These data indicate that the ouabain-activated proliferative effect previously observed in canine VSMCs occurs in other VSMC sources. This growth effect seems to be initiated by drug interaction with the sodium pump, reflected by the affinity of the steroid for the pump, and is independent of altered transmembrane ionic gradients.

Regulation of adrenocortical cardiotonic steroid production by dopamine and PKA signaling.

There is growing evidence that the adrenal cortex is the source of cardiotonic steroid (CS) regulators of sodium, potassium-ATPase (NKA). The control of adrenocortical production CS may play a critical role in mediating renal and vascular responses involved in arterial hypertension. Dopamine (DA) controls renal NKA by direct regulatory phosphorylation and indirectly by modification of aldosterone release. In the present studies, Y-1 adrenocortical cell cultures which have been shown to produce a cardiotonic steroid indistinguishable from the known vertebrate steroid, marinobufagenin (MBG), were treated with various agents to stimulate or antagonize dopamine signaling pathways. We demonstrate that Y-1 cells express both pharmacological types of dopamine receptor (DA1 and DA2). Treatment of Y-1 cells with DA stimulated MBG production in a dose range similar to that shown to inhibit aldosterone production by the adrenal cortex. Experiments with specific DA1 and DA2 receptor agonists and antagonists were performed and allowed us to attribute the DA stimulatory effect to a DA1 type receptor. The DA stimulatory effect on MBG depended on protein kinase A (PKA) and could be blocked by Rp-cAMPS. Although both basal and forskolin-stimulated progesterone production by Y-1 cells were profoundly inhibited in Y-1 cell lines expressing the dominant negative type I regulatory subunit of PKA, both basal and forskolin-stimulated MBG production were demonstrated in these lines. This evidence suggests a possible role of DA1 signaling through camp-mediated activation of the type II PKA holoenzyme in the adrenal cortex.

Functional properties of bone marrow derived multipotent mesenchymal stromal cells are altered in heart failure patients, and could be corrected by adjustment of expansion strategies.

BACKGROUND: Bone marrow multipotent mesenchymal stromal cells (BM-MMSC) considered as a prospective substrate for cell therapy applications, however adult stem cells could be affected by donor-specific factors: age, gender, medical history. Our aim was to investigate how HF affects the functional properties of BM-MMSC. MATERIALS AND METHODS: BM-MMSC from 10 healthy donors (HD), and 16 donors with chronic HF were evaluated for proliferative activity, ability to differentiate, replicative senescence, expression of genes that affect regeneration and fibrosis. The effect of culturing conditions on efficiency of BM-MMSC expansion was determined. RESULTS: HF-derived BM-MMSC demonstrated early decrease of proliferative activity and upregulation of genes that control both, regeneration and fibrosis: Tgf-ß pathway, synthesis of ECM, remodeling enzymes, adhesion molecules. We assume that these effects were related to increase of frequency of myofibroblast-like CD146+/SMAα+ CFU-F in HF samples; (ii) low seeding density and hypoxia resulted in predominant purification and expansion of CD146+/SMAα- CFU-Fs. (iii) the activity of NPs system was downregulated in HF BM-MMSC; CONCLUSIONS: downregulation of NP signaling in combination with upregulation of Tgf-ß pathway in BM-MMSC would result in pro-fibrotic phenotype and make these cells non-effective for therapeutic applications; the corrections in culturing strategy resulted in 2(3)-2(7) increase of expansion efficiency.

WISP1 is a novel adipokine linked to inflammation in obesity.

WISP1 (Wnt1-inducible signaling pathway protein-1, also known as CCN4) is a member of the secreted extracellular matrix-associated proteins of the CCN family and a target gene of the Wingless-type (WNT) signaling pathway. Growing evidence links the WNT signaling pathway to the regulation of adipogenesis and low-grade inflammation in obesity. We aimed to validate WISP1 as a novel adipokine. Human adipocyte differentiation was associated with increased WISP1 expression and secretion. Stimulation of human macrophages with WISP1 led to a proinflammatory response. Circulating WISP1 and WISP1 subcutaneous adipose tissue expression were regulated by weight changes in humans and mice. WISP1 expression in visceral and subcutaneous fat tissue was associated with markers of insulin resistance and inflammation in glucose-tolerant subjects. In patients with nonalcoholic fatty liver disease, we found no correlation among disease activity score, liver fat content, and WISP1 expression. Insulin regulated WISP1 expression in adipocytes in vitro but had no acute effect on WISP1 gene expression in subcutaneous fat tissue in overweight subjects who had undergone hyperinsulinemic clamp experiments. The data suggest that WISP1 may play a role in linking obesity to inflammation and insulin resistance and could be a novel therapeutic target for obesity.

Cardiotonic steroids: potential endogenous sodium pump ligands with diverse function.

The highly conserved cardiotonic steroid (CS) binding site present on the ubiquitous membrane sodium pump, sodium, potassium-ATPase, appears to have been conserved by no force other than its capacity to bind CS: a family that includes plant-derived cardiac glycosides and putative endogenous vertebrate counterparts. Binding of ligand is inhibited by increased extracellular potassium. This implies functional coordination because inhibition of the sodium pump would be counterproductive when extracellular potassium is elevated. The interesting biology of the CS binding site continues to stimulate investigations into the identity of endogenous ligands, their role as pump regulators at the cellular level, and as mediators of body fluid balance and blood pressure regulation. In addition to inhibition of sodium and potassium transport, there is considerable recent evidence suggesting that the sodium pump may act as a cell signaling receptor activated by CS binding and responding by coordination of intracellular signaling pathways that can be dependent on and also independent of the reduction in transmembrane ion flux resulting directly from pump inhibition. This signaling may influence cell survival, growth, and differentiation. Recent insight into the biology of pump regulation by CS is reviewed.

Bone marrow- and subcutaneous adipose tissue-derived mesenchymal stem cells: differences and similarities.

Bone marrow (BM) and subcutaneous adipose tissue (Ad) are both considered being prospective sources of MSC for therapeutic applications. However, functional properties and therapeutic efficacy of MSC derived from different tissues of the same patient are still poorly investigated. In our study, BM-MSC and F-MSC cultures from 43 adult donors were evaluated in successive passages for immunophenotype, secretion of VEGF, SDF1, MCP1, IL6 and TGFß1, frequency of colony-forming units (CFU-F), frequency of adipo- and osteo-progenitors (CFU-Ad, CFU-Ost), and for onset of in vitro replicative senescence. We have demonstrated that at early passages (P2-P4 or up to 14-15 in vitro population doublings) BM- and Ad- derived MSC cultures are comparable in such important characteristics as proliferation rate (population doubling time: 3.4±0.2% in BM-MSC, 3±0.3% in F-MSC), clonogenity (CFU-F frequency: 32±5% in BM-MSC, 31±5% in F-MSC), differentiation potential (CFU-Ad frequency: 10.4±2% in BM-MSC, 13±3% in F-MSC; CFU-Ost frequency: 18.5±5.5% in BM-MSC, 18±5% in F-MSC), but differ significantly in abundance of CD146⁺ fraction within the sample (25±5% in BM-MSC, 7±3% in F-MSC) and in a level of VEGF, SDF-1, MCP1 and TGFß1 secretion. We have also demonstrated that BM-MSC enter senescence after P3-4 while most of F-MSC did not show senescence features up to P6-8. Together, these data demonstrate that specific properties of MSC from different sources should be always taken into account, when developing and optimizing the specific protocols for MSC expansion and evaluation for each particular clinical application.

Genome-wide identification of allelic expression in hypertensive rats.

BACKGROUND: Identification of genes involved in complex cardiovascular disease traits has proven challenging. Inbred animal models can facilitate genetic studies of disease traits. The spontaneously hypertensive rat (SHR) is an inbred model of hypertension that exists in several closely related but genetically distinct lines. METHODS AND RESULTS: We used renal gene-expression profiling across 3 distinct SHR lines to identify genes that show different expression in SHR than in the genetically related normotensive control strain, Wistar-Kyoto. To ensure robust discovery of genes showing SHR-specific expression differences, we considered only those genes in which differential expression is replicated in multiple animals of each of multiple hypertensive rat lines at multiple time points during the ontogeny of hypertension. Mutation analysis was performed on the identified genes to uncover allelic variation. We identified those genes in which all SHR lines share a single allele of the gene when normotensive controls (Wistar-Kyoto) have fixed the alternative allele. We then identified which of the differentially expressed genes show expression that is controlled by the alleleic variation present in and around the gene. Allelic expression was demonstrated by observing the effect on gene expression of alleles inherited in the freely segregating F(2) progeny of a cross between SHR and Wistar-Kyoto animals. CONCLUSIONS: The result of these studies is the identification of several genes (Ptprj, Ela1, Dapk-2, and Gstt2) in which each of 4 SHR lines examined have fixed the same allele and in which each of 2 Wistar-Kyoto lines have a contrasting allele for which the inherited allele influences the level of gene expression. We further show that alleles of these genes lie in extensive haplotype blocks that have been inherited identical by descent in the hypertensive lines.

Hepatocyte nuclear factor 1 and hypertensive nephropathy.

Hypertension in spontaneously hypertensive rat (SHR) is associated with renal redox stress, and we hypothesized that nephropathy arises in SHR-A3 from altered capacity to mitigate redox stress compared with nephropathy-resistant SHR lines. We measured renal expression of redox genes in distinct lines of the spontaneously hypertensive rat (SHR-A3, SHR-B2, SHR-C) and the normotensive Wistar-Kyoto (WKY) strain. The SHR lines differ in either resisting (SHR-B2, SHR-C) or experiencing hypertensive nephropathy (SHR-A3). Immediately before the emergence of hypertensive renal injury expression of redox genes in SHR-A3 was profoundly altered compared with the injury-resistant SHR lines and WKY. This change appeared to arise in antioxidant genes where 16 of 28 were expressed at 34.3% of the level in the reference strain (WKY). No such change was observed in the injury-resistant SHR lines. We analyzed occurrence of transcription factor matrices in the promoters of the downregulated antioxidant genes. In these genes, the hepatocyte nuclear factor 1 (HNF1) transcription factor matrix was found to be nearly twice as likely to be present and the overall frequency of HNF1 sites was nearly 5 times higher, compared with HNF1 transcription factor matrices in antioxidant genes that were not downregulated. We identified 35 other (nonredox) renal genes regulated by HNF1. These were also significantly downregulated in SHR-A3, but not in SHR-B2 or SHR-C. Finally, expression of genes that comprise HNF1 (Tcf1, Tcf2, and Dcoh) was also downregulated in SHR-A3. The present experiments uncover a major change in transcriptional control by HNF1 that affects redox and other genes and precedes emergence of hypertensive renal injury.