Metastatic gastric adenocarcinoma to the cutaneous neck and chest wall.

This case describes an atypical cutaneous presentation of metastatic gastric carcinoma in a patient initially presenting with dysphagia and a sclerotic red plaque overlying the anterior neck and chest. Skin biopsy revealed metastatic adenocarcinoma from the upper gastrointestinal tract. Esophagogastroduodenoscopy revealed stage IV metastatic gastric adenocarcinoma. Treatment with chemotherapy was initiated.

Timing of Regadenoson-induced Peak Hyperemia and the Effects on Coronary Flow Reserve.

Background: Regadenoson is used to induce hyperemia in cardiac imaging, facilitating diagnosis of ischemia and assessment of coronary flow reserve (CFR). While the regadenoson package insert recommends administration of radionuclide tracer 10-20 seconds after injection, peak hyperemia has been observed at approximately 100 seconds after injection in healthy volunteers undergoing cardiovascular magnetic resonance imaging (CMR). It is unclear when peak hyperemia occurs in a patient population. Objectives: The goal of this study was to determine time to peak hyperemia after regadenoson injection in healthy volunteers and patients, and whether the recommended image timing in the package insert underestimates CFR. Methods: Healthy volunteers (n=15) and patients (n=25) underwent stress CMR, including phase-contrast imaging of the coronary sinus at rest and multiple timepoints after 0.4 mg regadenoson injection. Coronary sinus flow (ml/min) was divided by resting values to yield CFR. Smoothed, time-resolved curves for CFR were generated with pointwise 95% confidence intervals. Results: CFR between 60 and 120 seconds was significantly higher than CFR at 30 seconds after regadenoson injection (p < 0.05) as shown by non-overlapping 95% confidence intervals for both healthy volunteers (30 s, [2.8, 3.4]; 60 s, [3.8, 4.4]; 90 s, [4.1, 4.7]; 120 s, [3.6, 4.3]) and patients (30 s, [2.1, 2.5]; 60 s, [2.6, 3.1]; 90 s, [2.7, 3.2]; 120 s, [2.5, 3.1]). Conclusion: Imaging at 90 seconds following regadenoson injection is the optimal approach to capture peak hyperemia. Imaging at 30 seconds, which is more aligned with the package insert recommendation, would yield an underestimate of CFR and confound assessment of microvascular dysfunction.

Brand ferumoxytol vs. generic ferumoxytol comparison across two dosing regimens: a cardiac MRI image quality study.

BACKGROUND: Ferumoxytol is becoming more widely used as an off-label blood-pool contrast agent for MR angiography (MRA) and four-dimensional (4D) flow imaging in pediatric cardiovascular disease. Brand and generic versions of ferumoxytol are available with no information on relative efficacy as a contrast agent and safety profiles. OBJECTIVE: This study evaluates patient safety and image quality of comparable dosages of generic ferumoxytol (GF) versus brand ferumoxytol (BF) with the following hypotheses: (1) Reducing the contrast dosage from 3 to 2 mg/kg will not affect imaging quality and diagnostic accuracy of MRA and four-dimensional 4D flow. (2) GF and BF have similar image quality. (3) GF and BF have similar patient safety profiles. MATERIALS AND METHODS: In an IRB-approved retrospective study, changes in vitals/clinical status between baseline, during infusion, and 30 min post-infusion were analyzed in 3 groups: group 1 (3 mg/kg BF, 216 patients, age: 19.29 ± 11.71 years ranging from 2 months to 62 years), group 2 (2 mg/kg BF, 47 patients, age: 15.35 ± 8.56 years ranging from 10 days to 41 years), and group 3 (2 mg/kg GF, 127 patients, age: 17.16 ± 12.18 years ranging from 6 days to 58 years). Both pediatric and adult patients with congenital heart disease (CHD) indications were included within the study. Adverse reactions were classified as mild, moderate, or severe. Quantitative analysis of MR image quality was performed with signal-to-noise ratio (SNR) on MRA and velocity-to-noise ratio (VNR) on 4D flow. Qualitative grading of imaging features was performed by 2 experienced observers. Two-way analysis of variance (ANOVA) and chi-square tests were used for comparison with a P value of ≤ 0.05 used for significance. RESULTS: No statistical difference was found in clinical status and vital signs (P>0.05). No severe reactions were reported. 7.9% of GF patients experienced an adverse reaction compared to 2.3% with 3 mg/kg BF and 8.4% with 2 mg/kg BF. There was no statistical difference in SNR between the 3 groups (P>0.05). For 4D flow, 2 mg/kg GF demonstrated an increase in VNR compared to 2 mg/kg BF (P = 0.005). The qualitative scores for MRA and 4D flow were high (≥ 3) across all 3 groups. CONCLUSIONS: No significant difference was identified between 2 mg/kg GF and BF in terms of safety profile and image quality. Given the small sample size of this study, further studies are required to confirm these results.

Cutaneous adverse reactions resulting from targeted cancer therapies: histopathologic and clinical findings.

Targeted cancer treatments-designed to interfere with specific molecular signals responsible for tumor survival and progression-have shown benefit over conventional chemotherapies but may lead to diverse cutaneous adverse effects. This review highlights clinically significant dermatologic toxicities and their associated histopathologic findings, resulting from various targeted cancer drugs. Case reports and series, clinical trials, reviews, and meta-analyses are included for analysis and summarized herein. Cutaneous side effects resulting from targeted cancer therapies were reported with incidences as high as 90% for certain medications, and reactions are often predictable based on mechanism(s) of action of a given drug. Common and important reaction patterns included: acneiform eruptions, neutrophilic dermatoses, hand-foot skin reaction, secondary cutaneous malignancies, and alopecia. Clinical and histopathologic recognition of these toxicities remains impactful for patient care.

Cardioprotective Role for Paraoxonase-1 in Chronic Kidney Disease.

Paraoxonase-1 (PON-1) is a hydrolytic enzyme associated with HDL, contributing to its anti-inflammatory, antioxidant, and anti-atherogenic properties. Deficiencies in PON-1 activity result in oxidative stress and detrimental clinical outcomes in the context of chronic kidney disease (CKD). However, it is unclear if a decrease in PON-1 activity is mechanistically linked to adverse cardiovascular events in CKD. We investigated the hypothesis that PON-1 is cardioprotective in a Dahl salt-sensitive model of hypertensive renal disease. Experiments were performed on control Dahl salt-sensitive rats (SSMcwi, hereafter designated SS-WT rats) and mutant PON-1 rats (SS-Pon1em1Mcwi, hereafter designated SS-PON-1 KO rats) generated using CRISPR gene editing technology. Age-matched 10-week-old SS and SS-PON-1 KO male rats were maintained on high-salt diets (8% NaCl) for five weeks to induce hypertensive renal disease. Echocardiography showed that SS-PON-1 KO rats but not SS-WT rats developed compensated left ventricular hypertrophy after only 4 weeks on the high-salt diet. RT-PCR analysis demonstrated a significant increase in the expression of genes linked to cardiac hypertrophy, inflammation, and fibrosis, as well as a significant decrease in genes essential to left ventricular function in SS-PON-1 KO rats compared to SS-WT rats. A histological examination also revealed a significant increase in cardiac fibrosis and immune cell infiltration in SS-PON-1 KO rats, consistent with their cardiac hypertrophy phenotype. Our data suggest that a loss of PON-1 in the salt-sensitive hypertensive model of CKD leads to increased cardiac inflammation and fibrosis as well as a molecular and functional cardiac phenotype consistent with compensated left ventricular hypertrophy.

RNO3 QTL regulates vascular structure and arterial stiffness in the spontaneously hypertensive rat.

Increased arterial stiffness is an independent risk factor for hypertension, stroke, and cardiovascular morbidity. Thus, understanding the factors contributing to vascular stiffness is of critical importance. Here, we used a rat model containing a known quantitative trait locus (QTL) on chromosome 3 (RNO3) for vasoreactivity to assess potential genetic elements contributing to blood pressure, arterial stiffness, and their downstream effects on cardiac structure and function. Although no differences were found in blood pressure at any time point between parental spontaneously hypertensive rats (SHRs) and congenic SHR.BN3 rats, the SHRs showed a significant increase in arterial stiffness measured by pulse wave velocity. The degree of arterial stiffness increased with age in the SHRs and was associated with compensatory cardiac changes at 16 wk of age, and decompensatory changes at 32 wk, with no change in cardiac structure or function in the SHR.BN3 hearts at these time points. To evaluate the arterial wall structure, we used multiphoton microscopy to quantify cells and collagen content within the adventitia and media of SHR and SHR.BN3 arteries. No difference in cell numbers or proliferation rates was found, although phenotypic diversity was characterized in vascular smooth muscle cells. Herein, significant anatomical and physiological differences related to arterial structure and cardiovascular tone including collagen, pulse wave velocity (PWV), left ventricular (LV) geometry and function, and vascular smooth muscle cell (VSMC) contractile apparatus proteins were associated with the RNO3 QTL, thus providing a novel platform for studying arterial stiffness. Future studies delimiting the RNO3 QTL could aid in identifying genetic elements responsible for arterial structure and function.

Intrinsic Exercise Capacity and Mitochondrial DNA Lead to Opposing Vascular-Associated Risks.

Exercise capacity is a strong predictor of all-cause morbidity and mortality in humans. However, the associated hemodynamic traits that link this valuable indicator to its subsequent disease risks are numerable. Additionally, exercise capacity has a substantial heritable component and genome-wide screening indicates a vast amount of nuclear and mitochondrial DNA (mtDNA) markers are significantly associated with traits of physical performance. A long-term selection experiment in rats confirms a divide for cardiovascular risks between low- and high-capacity runners (LCR and HCR, respectively), equipping us with a preclinical animal model to uncover new mechanisms. Here, we evaluated the LCR and HCR rat model system for differences in vascular function at the arterial resistance level. Consistent with the known divide between health and disease, we observed that LCR rats present with resistance artery and perivascular adipose tissue dysfunction compared to HCR rats that mimic qualities important for health, including improved vascular relaxation. Uniquely, we show by generating conplastic strains, which LCR males with mtDNA of female HCR (LCR-mtHCR/Tol) present with improved vascular function. Conversely, HCR-mtLCR/Tol rats displayed indices for cardiac dysfunction. The outcome of this study suggests that the interplay between the nuclear genome and the maternally inherited mitochondrial genome with high intrinsic exercise capacity is a significant factor for improved vascular physiology, and animal models developed on an interaction between nuclear and mtDNA are valuable new tools for probing vascular risk factors in the offspring.

Vertical selection for nuclear and mitochondrial genomes shapes gut microbiota and modifies risks for complex diseases.

Here we postulate that the heritability of complex disease traits previously ascribed solely to the inheritance of the nuclear and mitochondrial genomes is broadened to encompass a third component of the holobiome, the microbiome. To test this, we expanded on the selectively bred low capacity runner/high capacity runner (LCR/HCR) rat exercise model system into four distinct rat holobiont model frameworks including matched and mismatched host nuclear and mitochondrial genomes. Vertical selection of varying nuclear and mitochondrial genomes resulted in differential acquisition of the microbiome within each of these holobiont models. Polygenic disease risk of these novel models were assessed and subsequently correlated with patterns of acquisition and contributions of their microbiomes in controlled laboratory settings. Nuclear-mitochondrial-microbiotal interactions were not for exercise as a reporter of health, but significantly noted for increased adiposity, increased blood pressure, compromised cardiac function, and loss of long-term memory as reporters of disease susceptibility. These findings provide evidence for coselection of the microbiome with nuclear and mitochondrial genomes as an important feature impacting the heritability of complex diseases.

Liver-specific rescuing of CEACAM1 reverses endothelial and cardiovascular abnormalities in male mice with null deletion of Ceacam1 gene.

OBJECTIVE: Mice with global null mutation of Ceacam1 (Cc1-/-), display impairment of insulin clearance that causes hyperinsulinemia followed by insulin resistance, elevated hepatic de novo lipogenesis, and visceral obesity. In addition, they manifest abnormal vascular permeability and elevated blood pressure. Liver-specific rescuing of Ceacam1 reversed all of the metabolic abnormalities in Cc1-/-liver+ mice. The current study examined whether Cc1-/- male mice develop endothelial and cardiac dysfunction and whether this relates to the metabolic abnormalities caused by defective insulin extraction. METHODS AND RESULTS: Myography studies showed reduction of agonist-stimulated nitric oxide production in resistance arterioles in Cc1-/-, but not Cc1-/-liver+ mice. Liver-based rescuing of CEACAM1 also attenuated the abnormal endothelial adhesiveness to circulating leukocytes in parallel to reducing plasma endothelin-1 and recovering plasma nitric oxide levels. Echocardiography studies revealed increased septal wall thickness, cardiac hypertrophy and reduced cardiac performance in Cc1-/-, but not Cc1-/-xliver+ mice. Insulin signaling experiments indicated compromised IRS1/Akt/eNOS pathway leading to lower nitric oxide level, and activated Shc/MAPK pathway leading to more endothelin-1 production in the aortae and hearts of Cc1-/-, but not Cc1-/-xliver+ mice. The increase in the ratio of endothelin-1 receptor A/B indicated an imbalance in the vasomotor activity of Cc1-/- mice, which was normalized in Cc1-/-xliver+ mice. CONCLUSIONS: The data underscore a critical role for impaired CEACAM1-dependent hepatic insulin clearance pathways and resulting hyperinsulinemia and lipid accumulation in aortae and heart in regulating the cardiovascular function.

Preconditioning and Postconditioning by Cardiac Glycosides in the Mouse Heart.

Ouabain preconditioning (OPC) initiated by low concentrations of the cardiac glycoside (CG) ouabain binding to Na/K-ATPase is relayed by a unique intracellular signaling and protects cardiac myocytes against ischemia/reperfusion injury. To explore more clinically applicable protocols based on CG properties, we tested whether the FDA-approved CG digoxin could trigger cardioprotective effects comparable with those of ouabain using PC, preconditioning and PostC, postconditioning protocols in the Langendorff-perfused mouse heart subjected to global ischemia and reperfusion. Ouabain or digoxin at 10 µmol/L inhibited Na/K-ATPase activity by approximately 30% and activated PKCε translocation by approximately 50%. Digoxin-induced PC (DigPC), initiated by a transient exposure before 40 minutes of ischemia, was as effective as OPC as suggested by the recovery of left ventricular developed pressure, end-diastolic pressure, and cardiac Na/K-ATPase activity after 30 minutes of reperfusion. DigPC also significantly decreased lactate dehydrogenase release and reduced infarct size, comparable with OPC. PostC protocols consisting of a single bolus injection of 100 nmoles of ouabain or digoxin in the coronary tree at the beginning of reperfusion both improved significantly the recovery of left ventricular developed pressure and decreased lactate dehydrogenase release, demonstrating a functional and structural protection comparable with the one provided by OPC. Given the unique signaling triggered by OPC, these results suggest that DigPostC could be considered for patients with risk factors and/or concurrent treatments that may limit effectiveness of ischemic PostC.

Positional cloning of quantitative trait nucleotides for blood pressure and cardiac QT-interval by targeted CRISPR/Cas9 editing of a novel long non-coding RNA.

Multiple GWAS studies have reported strong association of cardiac QT-interval to a region on HSA17. Interestingly, a rat locus homologous to this region is also linked to QT-intervals. The high resolution positional mapping study located the rat QT-interval locus to a <42.5kb region on RNO10. This region contained no variants in protein-coding sequences, but a prominent contiguous 19bp indel polymorphism was noted within a novel predicted long non-coding RNA (lncRNA), which we named as Rffl-lnc1. To assess the candidacy of this novel lncRNA on QT-interval, targeted CRISPR/Cas9 based genome-engineering approaches were applied on the rat strains used to map this locus. Targeted disruption of the rat Rffl-lnc1 locus caused aberrant, short QT-intervals and elevated blood pressure. Further, to specifically examine the significance of the 19bp polymorphism within the Rffl-lnc1 locus, a CRISPR/Cas9 based targeted knock-in rescue model was constructed by inserting the 19bp into the strain which contained the deletion polymorphism. The knock-in alleles successfully rescued the aberrant QT-interval and blood pressure phenotypes. Further studies revealed that the 19bp polymorphism was necessary and sufficient to recapitulate the phenotypic effect of the previously mapped <42.5kb rat locus. To our knowledge, this study is the first demonstration of a combination of both CRISPR/Cas9 based targeted disruption as well as CRISPR/Cas9 based targeted knock-in rescue approaches applied for a mammalian positional cloning study, which defines the quantitative trait nucleotides (QTNs) within a rat long non-coding RNA as being important for the pleiotropic regulation of both cardiac QT-intervals and blood pressure.

Differential roles of caveolin-1 in ouabain-induced Na+/K+-ATPase cardiac signaling and contractility.

Binding of ouabain to cardiac Na+/K+-ATPase initiates cell signaling and causes contractility in cardiomyocytes. It is widely accepted that caveolins, structural proteins of caveolae, have been implicated in signal transduction. It is known that caveolae play a role in Na+/K+-ATPase functions. Regulation of caveolin-1 in ouabain-mediated cardiac signaling and contractility has never been reported. The aim of this study is to compare ouabain-induced cardiac signaling and contractility in wild-type (WT) and caveolin-1 knockout (cav-1 KO) mice. In contrast with WT cardiomyocytes, ouabain-induced signaling e.g., activation of phosphoinositide 3-kinase-α/Akt and extracellular signal-regulated kinases (ERK)1/2, and hypertrophic growth were significantly reduced in cav-1 KO cardiomyocytes. Interactions of the Na+/K+-ATPase α1-subunit with caveolin-3 and the Na+/K+-ATPase α1-subunit with PI3K-α were also decreased in cav-1 KO cardiomyocytes. The results from cav-1 KO mouse embryonic fibroblasts also proved that cav-1 significantly attenuated ouabain-induced ERK1/2 activation without alteration in protein and cholesterol distribution in caveolae/lipid rafts. Intriguingly, the effect of ouabain induced positive inotropy in vivo (via transient infusion of ouabain, 0.48 nmol/g body wt) was not attenuated in cav-1 KO mice. Furthermore, ouabain (1-100 µM) induced dose-dependent contractility in isolated working hearts from WT and cav-1 KO mice. The effects of ouabain on contractility between WT and cav-1 KO mice were not significantly different. These results demonstrated differential roles of cav-1 in the regulation of ouabain signaling and contractility. Signaling by ouabain, in contrast to contractility, may be a redundant property of Na+/K+-ATPase.

In vivo assessment of arterial stiffness in the isoflurane anesthetized spontaneously hypertensive rat.

BACKGROUND: Rodent models are increasingly used to study the development and progression of arterial stiffness. Both the non-invasive Doppler derived Pulse Wave Velocity (PWV) and the invasively determined arterial elastance index (EaI) have been used to assess arterial stiffness in rats and mice, but the need for anesthetic agents to make these in vivo estimates may limit their utility. Thus, we sought to determine: 1) if known differences in arterial stiffness in spontaneously hypertensive rats (SHR) are detectable by PWV and EaI measurements when made under isoflurane anesthesia, and 2) if these two uniquely acquired assessments of arterial elasticity correlate. METHODS: We obtained PWV and EaI measurements in isoflurane anesthetized young and old SHRs, which are known to have significant differences in arterial stiffness. Doppler pulse waves were recorded from carotid and iliac arteries and the distance (D) between probe applantation sites was recorded. Simultaneously, an EKG was obtained, and the time intervals between the R-wave of the EKG to the foot of the Doppler waveforms were measured and averaged over three cardiac cycles. Pulse-transit time (T) of the carotid to iliac artery was determined, and PWV was calculated as Distance (D)/Time (T), where D = the distance from the carotid to the iliac notch and T = (R to iliac foot) - (R to carotid foot). EaI was subsequently determined from pressure volumes loops obtained via left ventricle catheterization. RESULTS: PWV and EaI were found to be significantly faster in the older rats (13.2 ± 2.0 vs. 8.0 ± 0.8 m/sec, p < 0.001; 120 ± 20 vs. 97 ± 16 mmHg/µl/g, p <0.05). Bland-Altman analyses of intra- and inter-observer measures demonstrate a statistically significant relationship between readings (p < 0.0001). PWV and EaI measurements were found to be significantly and positively correlated with a correlation coefficient of 0.53 (p < 0.05). CONCLUSION: Our study suggests that isoflurane administration does not limit Doppler PWV or EaI measures in their ability to provide accurate, in vivo assessments of relative arterial stiffness in isoflurane anesthetised SHR rats. Furthermore, PWV data obtained in these rats correlate well with invasively determined EaI.

Different roles of the cardiac Na+/Ca2+-exchanger in ouabain-induced inotropy, cell signaling, and hypertrophy.

Previous studies have shown that digitalis drugs, acting as specific inhibitors of cardiac Na(+)/K(+)-ATPase, not only cause positive inotropic effects, but also activate cell signaling pathways that lead to cardiac myocyte hypertrophy. A major aim of this work was to assess the role of Na(+)/Ca(2+)-exchanger, NCX1, in the above two seemingly related drug effects. Using a mouse with ventricular-specific knockout (KO) of NCX1, ouabain-induced positive inotropy that was evident in isolated wild-type (Wt) hearts was clearly reduced in KO hearts. Ouabain also increased Ca(2+) transient amplitudes in Wt myocytes, but not in KO myocytes. Ouabain-induced activations of ERK 1/2 were noted in Wt myocytes, but not in KO myocytes; however, ouabain activated PI3K1A and Akt in both Wt and KO myocytes. Protein synthesis rate, as a measure of hypertrophy, was increased by ouabain in Wt and KO myocytes; these drug effects were prevented by a PI3K inhibitor but not by a MEK/ERK inhibitor. Hypertrophy caused by ET-1, but not that induced by ouabain, was accompanied by upregulation of BNP gene in Wt and KO myocytes. The findings indicate 1) the necessity of NCX1 for positive inotropic action of ouabain; 2) the irrelevance of NCX1 and ERK 1/2 activation to ouabain-induced hypertrophy; and 3) that hypertrophy caused by ouabain through the activation of PI3K1A/Akt pathway is likely to be beneficial to the heart.

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 Adamts16(mutant) rats was lower by 36 mmHg compared with the BP of the S rats. The Adamts16(mutant) 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 Adamts16(mutant) rats were longer than that of the S rats. Furthermore, Adamts16(mutant) 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.

Sarin causes autonomic imbalance and cardiomyopathy: an important issue for military and civilian health.

Sarin, a lethal chemical nerve agent, may be a causative factor in multifactorial syndrome implicated in the Gulf War and Tokyo terrorist attacks. Although a high dose results in seizure and death, low-dose exposure may lead to autonomic imbalance and chronic cardiac pathologies. In this study, echocardiography and electrocardiography were used to examine the late-onset effects of a low-dose sarin on cardiac structure and function in mice. Adrenal corticosterone and tyrosine hydroxylase mRNA levels were measured. Stress responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis was also tested. Findings demonstrate changes consistent with a dilated cardiomyopathy, including left ventricular dilatation, reduced contractility, and altered electrophysiological and inotropic responses to ß-adrenergic stimulation. Results also indicate reduced adrenal tyrosine hydroxylase mRNA, corticosterone and altered stress responsiveness of HPA indicating autonomic imbalance. The role of low-dose sarin/organophosphate exposure needs to be considered in the military and civilian populations that suffer from autonomic imbalance and/or cardiomyopathies of indeterminate origin.

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.

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.

Preconditioning by subinotropic doses of ouabain in the Langendorff perfused rabbit heart.

Short exposure to low concentrations of digitalis drugs like ouabain protects the rat heart against ischemia/reperfusion injury through the activation of the Na/K-adenosine triphosphatase (ATPase)/Src receptor complex and subsequent stimulation of key intracellular cardioprotective signals. Rat Na/K-ATPase, however, is relatively insensitive to digitalis, and it is not known if similar results could be obtained in species with higher sensitivity. Thus, to determine whether ouabain pretreatment protects against ischemic injury and activates the Na/K-ATPase signaling cascade in a species with cardiac glycoside sensitivity comparable to humans, the present study was conducted in the rabbit model. In Langendorff perfused rabbit hearts, 20-minute exposure to 500-nM ouabain resulted in positive inotropy as evidenced by a significant increase in +dP/dt, and this increase was accompanied by the activation of several well-characterized downstream mediators of the cardiac Na/K-ATPase receptor pathway, including Src, Akt, ERK1/2, and protein kinase Cepsilon. A short (4 minutes) administration of a subinotropic dose of ouabain (100 nM) followed by an 8-minute washout before 30 minutes of global ischemia and 120 minutes of reperfusion resulted in protection against cell death, as evidenced by a significant decrease in infarct size. These data indicate that ouabain administration activates the Na/K-ATPase signaling cascade and protects against ischemic injury in a species with high cardiac Na/K-ATPase sensitivity.