Pharmacological inhibition of DNA methylation attenuates pressure overload-induced cardiac hypertrophy in rats.

BACKGROUND: Heart failure is associated with altered gene expression and DNA methylation. De novo DNA methylation is associated with gene silencing, but its role in cardiac pathology remains incompletely understood. We hypothesized that inhibition of DNA methyltransferases (DNMT) might prevent the deregulation of gene expression and the deterioration of cardiac function under pressure overload (PO). To test this hypothesis, we evaluated a DNMT inhibitor in PO in rats and analysed DNA methylation in cardiomyocytes. METHODS AND RESULTS: Young male Wistar rats were subjected to PO by transverse aortic constriction (TAC) or to sham surgery. Rats from both groups received solvent or 12.5 mg/kg body weight of the non-nucleosidic DNMT inhibitor RG108, initiated on the day of the intervention. After 4 weeks, we analysed cardiac function by MRI, fibrosis with Sirius Red staining, gene expression by RNA sequencing and qPCR, and DNA methylation by reduced representation bisulphite sequencing (RRBS). RG108 attenuated the ~70% increase in heart weight/body weight ratio of TAC over sham to 47% over sham, partially rescued reduced contractility, diminished the fibrotic response and the downregulation of a set of genes including Atp2a2 (SERCA2a) and Adrb1 (beta1-adrenoceptor). RG108 was associated with significantly lower global DNA methylation in cardiomyocytes by ~2%. The differentially methylated pathways were "cardiac hypertrophy", "cell death" and "xenobiotic metabolism signalling". Among these, "cardiac hypertrophy" was associated with significant methylation differences in the group comparison sham vs. TAC, but not significant between sham+RG108 and TAC+RG108 treatment, suggesting that RG108 partially prevented differential methylation. However, when comparing TAC and TAC+RG108, the pathway cardiac hypertrophy was not significantly differentially methylated. CONCLUSIONS: DNMT inhibitor treatment is associated with attenuation of cardiac hypertrophy and moderate changes in cardiomyocyte DNA methylation. The potential mechanistic link between these two effects and the role of non-myocytes need further clarification.

An alternative renin isoform is cardioprotective by modulating mitochondrial metabolism.

The renin-angiotensin system promotes oxidative stress, apoptosis, necrosis, fibrosis, and thus heart failure. Secretory renin plays a central role in these processes, initiating the generation of angiotensins. Nevertheless, alternative renin transcripts exist, which code for a cytosolically localized renin isoform (cyto-renin) that is cardioprotective. We tested the hypothesis that the protective effects are associated with a beneficial switch of metabolic and mitochondrial functions. To assess H9c2 cell mitochondrial parameters, we used the Seahorse XF analyser. Cardiac H9c2 cells overexpressing cyto-renin exhibited enhanced nonmitochondrial oxygen consumption, lactate accumulation, and LDH activity, reflecting a switch to more aerobic glycolysis known as Warburg effect. Additionally, mitochondrial spare capacity and cell respiratory control ratio were enhanced, indicating an increased potential to tolerate stress conditions. Renin knockdown induced opposite effects on mitochondrial functions without influencing metabolic parameters. Thus, the protective effects of cyto-renin are associated with an altered bioenergetic profile and an enhanced stress tolerance, which are favourable under ischaemic conditions. Therefore, cyto-renin is a promising new target for the prevention of ischaemia-induced myocardial damage.

Evaluation of a Modified Pamidronate Protocol for the Treatment of Osteogenesis Imperfecta.

Intravenous pamidronate is widely used to treat children with osteogenesis imperfecta (OI). In a well-studied protocol ('standard protocol'), pamidronate is given at a daily dose of 1 mg per kg body weight over 4 h on 3 successive days; infusion cycles are repeated every 4 months. Here, we evaluated renal safety of a simpler protocol for intravenous pamidronate infusions (2 mg per kg body weight given in a single infusion over 2 h, repeated every 4 months; 'modified protocol'). Results of 18 patients with OI types I, III, or IV treated with the modified protocol for 12 months were compared to 18 historic controls, treated with standard protocol. In the modified protocol, mild transient post-infusion increases in serum creatinine were found during each infusion but after 12 months serum creatinine remained similar from baseline [0.40 mg/dl (SD: 0.13)] to the end of the study [0.41 mg/dl (SD: 0.11)] (P = 0.79). The two protocols led to similar changes in serum creatinine during the first pamidronate infusion [modified protocol: +2% (SD: 21%); standard protocol: -3% (SD: 8%); P = 0.32]. Areal lumbar spine bone mineral density Z-scores increased from -2.7 (SD: 1.5) to -1.8 (SD: 1.4) with the modified protocol, and from -4.1 (SD: 1.4) to -3.1 (SD: 1.1) with standard protocol (P = 0.68 for group differences in bone density Z-score changes). The modified pamidronate protocol is safe and may have similar effects on bone density as the standard pamidronate protocol. More studies are needed with longer follow-up to prove anti-fracture efficacy.

Regression of cardiac hypertrophy in cyp1a1ren-2 transgenic rats.

PURPOSE: To evaluate the usefulness of the cyp1a1ren-2 transgenic rat model of inducible hypertension for studies of the development and regression of cardiac hypertrophy. MATERIALS AND METHODS: Cyp1a1ren-2 rats received a diet containing 0% or 0.167% indole-3-carbinonl (I3C) for 4 weeks to induce hypertension. Cardiac magnetic resonance imaging (MRI) at 7 T was performed every second week for 10 weeks to measure left ventricular mass and the ejection fraction. Concomitantly, in six cyp1a1ren-2 rats blood pressure was recorded telemetrically. RESULTS: Plasma prorenin concentrations rose from 138 ± 38 to 15,490 ± 3990 ng/angiotensin I/mL/h (P < 0.001) in I3C-treated transgenic rats and returned to basal levels after cessation of I3C. Mean blood pressure increased to a plateau of 169 ± 11 mmHg by the second week of induction. After cessation of I3C (day 28), arterial pressure dropped to values slightly below those prior to induction within 4 days (basal: 106 ± 7 mmHg, day 32: 103 ± 21 mmHg; NS). At day 28, left ventricular mass was increased by 39% vs. 4% in controls (P < 0.001) without changes of the ejection fraction. Cardiac hypertrophy was completely reversed at day 70, as evaluated by MRI. CONCLUSION: The cyp1a1ren-2 transgenic rat is a useful model to study reversal and healing in the absence of surgical interventions.

The renin-angiotensin system as a primary cause of polyarteritis nodosa in rats.

Polyarteritis nodosa is a necrotizing vasculitis of medium-sized arteries of unknown origin. Hypertension is present in 30% of patients with polyarteritis nodosa. In those cases, high renin levels are thought to be secondary to renal involvement. The present study was performed to identify causal factors of polyarteritis nodosa. In cyp1a1ren-2 transgenic rats, vasculitis of medium-sized arteries resembling classical polyarteritis nodosa can be induced. In this model, oral administration of indole-3-carbinol (I3C) activates the liver-specific cyp1a1 promoter, leading to prorenin expression in a dose-dependent manner. After the first 6 weeks of chronic induction with 0.125% I3C, the mean arterial pressure reached a plateau of about 170 mmHg. Ten out of 11 I3C-treated rats, which were chronically instrumented with a telemetric device to measure blood pressure, developed polyarteritis nodosa within 10 weeks of I3C treatment. I3C alone or instrumentation alone did not cause polyarteritis nodosa. The angiotensin-converting enzyme inhibitor captopril completely prevented the development of polyarteritis nodosa, indicating that local angiotensin II generation is a pathogenetic factor in this model. The renin-angiotensin system can play a primary role in the development of polyarteritis nodosa in rats.

Correlations among vitamin K intake, body fat, lipid profile and glucose homeostasis in adults and the elderly.

Objective Recent research has investigated the possible inverse relationship between vitamin K intake and body fat. In addition, an increasing number of studies are supporting a key role for this vitamin in improving lipid profile and insulin sensitivity and reducing the risk of type 2 diabetes mellitus, but little is known about what mechanisms would be involved. Thus, the objective of this study was to investigate the relationship between vitamin K intake (in the form of phylloquinone - PK), body fat, lipid profile and markers of glucose homeostasis in adults and the elderly. Subjects and methods A cross-sectional study with 298 participants (46% men) in the São Paulo Health Survey 2014-2015. Spearman correlations were performed to evaluate the associations between vitamin K intake and the biochemical and body composition measures. Results Among normal-weight male adults (n = 15), PK intake presented a positive correlation with the quantitative insulin sensitivity check index (QUICKI) (r = 0.525; p = 0.045). Among men with high fat mass index (FMI) (n = 101), PK intake had a negative correlation with homeostasis model assessment estimate for ß-cell function (HOMA-ß) (r = -0.227; p = 0.022). In women with high FMI (n = 122), PK intake had a negative correlation with HOMA-ß (r = -0.199, p = 0.032) and insulin (r = -0.207, p = 0.026). No correlations were found between PK intake and lipid profile. Conclusions Our findings support a potential relationship among PK intake, body fat and markers of glucose homeostasis in adults and the elderly.

Cytosolic renin is targeted to mitochondria and induces apoptosis in H9c2 rat cardiomyoblasts.

One important goal in cardiology is to prevent necrotic cell death in the heart. Necrotic cell death attracts neutrophils and monocytes into the injured myocardium. The consequences are fibrosis, remodelling and cardiac failure. The renin-angiotensin system promotes the development of cardiac failure. Recently, alternative renin transcripts have been identified lacking the signal sequence for a cotranslational transport to the endoplasmatic reticulum. These transcripts encode for a cytosolic renin with unknown functions. The expression of this alternative transcript increases after myocardial infarction. We hypothesized that cytosolic renin plays a role in survival and death of cardiomyocytes. To test this hypothesis, we overexpressed secretory or cytosolic renin in H9c2 cardiomyblasts and determined the rate of proliferation, necrosis and apoptosis. Proliferation rate, as indicated by BrdU incorporation into DNA, was reduced by secretory and cytosolic renin (cells transfected with control vector: 0.33 +/- 0.06; secretory renin: 0.12 +/- 0.02; P < 0.05; cytosolic renin: 0.15 +/- 0.03; P < 0.05). Necrosis was increased by secretory renin but decreased by cytosolic renin (LDH release after 10 days from cells transfected with control vector: 68.5 +/- 14.9; secretory renin: 100.0 +/- 0; cytosolic renin: 25.5 +/- 5.3% of content, each P < 0.05). Mitochondrial apoptosis, as indicated by phosphatidylserin translocation to the outer membrane, was unaffected by secretory renin but increased by cytosolic renin (controls: 23.8 +/- 3.9%; secretory renin: 22.1 +/- 4.7%; cytoplasmatic renin: 41.2 +/- 3.8%; P < 0.05). The data demonstrate that a cytosolic renin exists in cardiomyocytes, which in contradiction to secretory renin protects from necrosis but increases apoptosis. Non-secretory cytosolic renin can be considered as a new target for cardiac failure.

Lack of cardiac fibrosis in a new model of high prorenin hyperaldosteronism.

The aim of the present study was to test the hypothesis that elevation of prorenin in plasma is sufficient to induce cardiac fibrosis. Normotensive cyp1a1ren-2 transgenic rats with normal plasma prorenin and aldosterone levels were given 0.125% indole-3-carbinol (I3C) orally for a period of 12 wk. Plasma prorenin and aldosterone levels were determined in 4-wk intervals, and cardiac marker enzymes for hypertrophy, fibrosis, and oxidative stress as well as cardiac pathology were investigated. In I3C-treated cyp1a1 ren-2 transgenic rats, plasma prorenin concentrations were >100-fold elevated (> or = 7.1 + or - 2.6 microg ANG I.ml(-1).h(-1) vs. < or = 0.07 + or - 0.1; P < 0.001), whereas active renin levels were suppressed (0.09 + or - 0.02 vs. 0.2 + or - 0.1; P < 0.05). Aldosterone concentrations were elevated three- to fourfold for a period of >4 wk (574 + or - 51 vs. 160 + or - 68 pg/ml; P < 0.01). After 12 wk of I3C, rats exhibited moderate cardiac hypertrophy (heart weight/body weight 2.5 + or - 0.04 vs. 3.1 + or - 0.1 mg/g; P < 0.01). There was a slight increase in mRNA contents of endothelin 1 (1.21 + or - 0.08 vs. 0.75 + or - 0.007; P < 0.001), NADP oxidase-2 (1.03 + or - 0.006 vs. 0.76 + or - 0.04; P < 0.001), transforming growth factor-beta (0.99 + or - 0.06 vs. 0.84 + or - 0.04; P < 0.05), collagen type I (1.32 + or - 0.32 vs. 0.94 + or - 0.18; P < 0.05), and intercellular adhesion molecule-1 (1.12 + or - 0.12 vs. 0.84 + or - 0.08; P < 0.05). These genes are known to be stimulated by the renin-angiotensin system. There were no histological signs of fibrosis in the heart. We found that prorenin and aldosterone alone are not sufficient to induce considerable cardiac fibrosis in the absence of sodium load.

Folate, vitamin B6 and vitamin B12 in adolescence: serum concentrations, prevalence of inadequate intakes and sources in food.

OBJECTIVE: To investigate serum concentrations and the prevalence of inadequate folate intake and also vitamin B6 and vitamin B12 intakes and to identify those foods that make a major contribution to intake levels of these nutrients. METHODS: This was a cross-sectional, observational study of adolescents of both sexes aged 16 to 19 years from the town of Indaiatuba, SP, Brazil. Data collection was by non-consecutive 3-day dietary record. The samples' habitual diet was estimated by removing intraindividual variability, and the prevalence rates of inadequate intakes were calculated using the estimated average requirement as cutoff points. Biochemical assays for folate, vitamin B6 and vitamin B12 were conducted in accordance with the methods accepted in the literature. RESULTS: The study sample comprised 99 adolescents, the majority of whom were female (58.6%), with a mean age of 17.6 [standard deviation, (SD) 0.9]. Mean serum concentrations for folate, vitamin B6 and vitamin B12 were 9.2 (SD 3.4) ng/mL, 18.7 (SD 5.1) nmol/L and 397.5 (SD 188.4) pg/mL, respectively; and the prevalence rates of inadequate intake for these vitamins were 15.2, 10.2 and < 1%, respectively. The foods that made a major contribution to vitamin intakes were French bread, pasta and beans for folate; white rice, chicken and beef for vitamin B6; and lean beef, whole milk and fatty beef for vitamin B12. CONCLUSIONS: The prevalence rates of inadequate folate, vitamin B6 and vitamin B12 intakes were low, which is possibly the result of improved access to and availability of foods that are dietary sources of these vitamins. Beans, which are a part of the traditional Brazilian diet, remain one of the primary food items that contribute to folate intake, even after mandatory fortification with folic acid in Brazil.

Folato, B6 e B12 na adolescência: níveis séricos, prevalência de inadequação de ingestão e alimentos contribuintes / Folate, vitamin B6 and vitamin B12 in adolescence: serum concentrations, prevalence of inadequate intakes and sources in food

OBJETIVO: Investigar os níveis séricos e a prevalência de inadequação da ingestão dietética de folato e das vitaminas B6 e B12, identificando os alimentos contribuintes para a ingestão desses nutrientes. MÉTODOS: Estudo observacional, transversal, em adolescentes de 16 a 19 anos, de ambos os sexos, conduzido em Indaiatuba (SP). Coletou-se o registro alimentar de 3 dias não consecutivos. A dieta habitual foi estimada pela remoção da variabilidade intrapessoal, e a prevalência de inadequação da ingestão, pelo método da estimated average requirement como ponto de corte. As análises bioquímicas de folato, B6 e B12 foram conduzidas de acordo com os métodos aceitos na literatura. RESULTADOS: O estudo foi conduzido com 99 adolescentes, a maioria do sexo feminino (58,6 por cento), com média de idade de 17,6 (desvio padrão, DP 0,9) anos. As médias da concentração sérica de folato, B6 e B12 foram de 9,2 (DP 3,4) ng/mL, 18,7 (DP 5,1) nmol/L e 397,5 (DP 188,4) pg/mL, respectivamente; e a prevalência de inadequação da ingestão das vitaminas foi de 15,2, 10,2 e < 1 por cento, respectivamente. Os alimentos que mais contribuíram para a ingestão dos nutrientes foram, para folato: pão francês, macarrão e feijões; para B6: arroz branco, carne de frango e carne bovina; e para B12: carne bovina magra, leite integral e carne bovina gorda. CONCLUSÕES: As prevalências de inadequação de folato, B6 e B12 mostraram-se baixas, possivelmente em decorrência da melhoria do acesso e da disponibilidade de alimentos, fontes dietéticas das vitaminas. Os feijões, presentes na dieta tradicional brasileira, ainda estão entre os principais alimentos que contribuíram para a ingestão de folato, mesmo após a fortificação mandatória com ácido fólico no Brasil.

OBJECTIVE: To investigate serum concentrations and the prevalence of inadequate folate intake and also vitamin B6 and vitamin B12 intakes and to identify those foods that make a major contribution to intake levels of these nutrients. METHODS: This was a cross-sectional, observational study of adolescents of both sexes aged 16 to 19 years from the town of Indaiatuba, SP, Brazil. Data collection was by non-consecutive 3-day dietary record. The samples’ habitual diet was estimated by removing intraindividual variability, and the prevalence rates of inadequate intakes were calculated using the estimated average requirement as cutoff points. Biochemical assays for folate, vitamin B6 and vitamin B12 were conducted in accordance with the methods accepted in the literature. RESULTS: The study sample comprised 99 adolescents, the majority of whom were female (58.6 percent), with a mean age of 17.6 [standard deviation, (SD) 0.9]. Mean serum concentrations for folate, vitamin B6 and vitamin B12 were 9.2 (SD 3.4) ng/mL, 18.7 (SD 5.1) nmol/L and 397.5 (SD 188.4) pg/mL, respectively; and the prevalence rates of inadequate intake for these vitamins were 15.2, 10.2 and < 1 percent, respectively. The foods that made a major contribution to vitamin intakes were French bread, pasta and beans for folate; white rice, chicken and beef for vitamin B6; and lean beef, whole milk and fatty beef for vitamin B12. CONCLUSIONS: The prevalence rates of inadequate folate, vitamin B6 and vitamin B12 intakes were low, which is possibly the result of improved access to and availability of foods that are dietary sources of these vitamins. Beans, which are a part of the traditional Brazilian diet, remain one of the primary food items that contribute to folate intake, even after mandatory fortification with folic acid in Brazil.