Cell type-specific expression of endogenous cardiac Troponin I antisense RNA in the neonatal rat heart

Since the number of detected natural antisense RNA is growing, investigations upon the expression pattern of the antisense RNA become more important. As we focused our work on natural occurring antisense transcripts in human and rat heart tissues, we were interested in the question, whether the expression pattern of antisense and sense RNA can vary in different cell types of the same tissue. In our previous analysis of total neonatal rat heart tissue, we demonstrated the co-expression of both cTnI RNA species in this tissue. Now we investigated the expression of antisense and sense RNA quantitatively in neonatal cardiomyocytes (NCMs) and neonatal cardiac fibroblasts (NCFs). Performing northern blot as well as RT-PCR, we could detect natural antisense and sense RNA transcripts of cTnI in NCM and NCF implying that these transcripts are co-expressed in both cell types. The absolute amounts of the RNA transcripts were higher in NCM. Both RNA species showed identical sizes in the northern blot. Quantification by real-time PCR revealed a higher relative level of natural antisense RNA in NCF compared to NCM which points out to a cell type-specific expression of sense and antisense RNA. Our observations suggest that antisense RNA transcription may contribute to a cell type-specific regulation of the cTnI gen

Detection of a novel sense–antisense RNA-hybrid structure by RACE experiments on endogenous troponin I antisense RNA

Conformational changes in the troponin/tropomyosin complex significantly alter the mechanical properties of cardiac muscle. Phosphorylation of cardiac troponin I, part of the troponin/tropomyosin complex, reduces calcium affinity, which leads to increased relaxation of cardiac muscle. Because cardiac troponin I plays a central role in tuning the heart to different work demands, detailed knowledge of troponin I protein regulation is required. Our group previously detected naturally occurring antisense RNA for troponin I in human and rat hearts, and here, attempt to unravel the structure of rat cardiac troponin I antisense RNA. We performed rapid amplification of cDNA ends (RACE) experiments and discovered antisense sequences identical to a copy of the sense mRNA, which led us to conclude that the antisense RNA must be transcribed from troponin I mRNA in the cytoplasm. Moreover, we isolated RNA structures comprising sense and antisense sequences in one continuous molecule. As we found no homolog structures described in the literature, we called this “hybrid RNA.” Because a duplex formation was demonstrated previously we concluded that hybrid RNA is a consequence of a tight interaction between sense and antisense troponin I RNA in vivo, which we discuss in the article

Increased protocol adherence and safety during controlled normothermia as compared to hypothermia after cardiac arrest

Purpose: This study aims to compare protocol adherence, neurological outcome and adverse effects associated with a controlled hypothermia versus a controlled normothermia protocol in patients successfully resuscitated after cardiac arrest. Methods: In this retrospective single-center study in a university intensive care unit in Switzerland, post-cardiac arrest patients were compared before and after a protocol change from targeted temperature management at 33 °C (TTM-33) to 36 °C (TTM-36) using an intravascular cooling device. Protocol adherence was assessed as the primary outcome. Secondary outcomes were in-hospital mortality, neurological outcome and adverse effects. Results: 373 patients after cardiac arrest were screened, of whom a total of 133 patients were included. Protocol adherence was lower in the TTM-33 group (47% vs 87% of patients, p < 0.01). In-hospital mortality (59% vs 45%, p = 0.15) and neurological outcome (modified Rankin Score < 4 in 33% vs 39% and CPC-Score < 3 in 33% vs 39% of patients, p = 0.60 and 0.97) were similar. Overall incidence of adverse effects was comparable, with bradycardic arrhythmias occurring more frequently in the TTM-33 group. Conclusion: Protocol adherence was higher in the TTM-36 group. In-hospital mortality and neurological outcome were similar, while bradycardic arrhythmias were encountered more often in TTM-33

Inflammatory kidney and liver tissue response to different hydroxyethylstarch (HES) preparations in a rat model of early sepsis

BACKGROUND Tissue hypoperfusion and inflammation in sepsis can lead to organ failure including kidney and liver. In sepsis, mortality of acute kidney injury increases by more than 50%. Which type of volume replacement should be used is still an ongoing debate. We investigated the effect of different volume strategies on inflammatory mediators in kidney and liver in an early sepsis model. MATERIAL AND METHODS Adult male Wistar rats were subjected to sepsis by cecal ligation and puncture (CLP) and assigned to three fluid replenishment groups. Animals received 30mL/kg of Ringer's lactate (RL) for 2h, thereafter RL (75mL/kg), hydroxyethyl starch (HES) balanced (25mL/kg), containing malate and acetate, or HES saline (25mL/kg) for another 2h. Kidney and liver tissue was assessed for inflammation. In vitro rat endothelial cells were exposed to RL, HES balanced or HES saline for 2h, followed by stimulation with tumor necrosis factor-α (TNF-α) for another 4h. Alternatively, cells were exposed to malate, acetate or a mixture of malate and acetate, reflecting the according concentration of these substances in HES balanced. Pro-inflammatory cytokines were determined in cell supernatants. RESULTS Cytokine mRNA in kidney and liver was increased in CLP animals treated with HES balanced compared to RL, but not after application of HES saline. MCP-1 was 3.5fold (95% CI: 1.3, 5.6) (p<0.01) and TNF-α 2.3fold (95% CI: 1.2, 3.3) (p<0.001) upregulated in the kidney. Corresponding results were seen in liver tissue. TNF-α-stimulated endothelial cells co-exposed to RL expressed 3529±1040pg/mL MCP-1 and 59±23pg/mL CINC-1 protein. These cytokines increased by 2358pg/mL (95% CI: 1511, 3204) (p<0.001) and 29pg/ml (95% CI: 14, 45) (p<0.01) respectively when exposed to HES balanced instead. However, no further upregulation was observed with HES saline. PBS supplemented with acetate increased MCP-1 by 1325pg/mL (95% CI: 741, 1909) (p<0.001) and CINC-1 by 24pg/mL (95% CI: 9, 38) (p<0.01) compared to RL. Malate as well as HES saline did not affect cytokine expression. CONCLUSION We identified HES balanced and specifically its component acetate as pro-inflammatory factor. How important this additional inflammatory burden on kidney and liver function is contributing to the sepsis-associated inflammatory burden in early sepsis needs further evaluation

Modulation of Early Inflammatory Response by Different Balanced and Non-Balanced Colloids and Crystalloids in a Rodent Model of Endotoxemia

<div><p>The use of hydroxyethyl starch (HES) in sepsis has been shown to increase mortality and acute kidney injury. However, the knowledge of the exact mechanism by which several fluids, especially starch preparations may impair end-organ function particularly in the kidney, is still missing. The aim of this study was to measure the influence of different crystalloid and colloid fluid compositions on the inflammatory response in the kidney, the liver and the lung using a rodent model of acute endotoxemia. Rats were anesthetized and mechanically ventilated. Lipopolysaccharide (5 mg/kg) was administered intravenously. After one hour crystalloids [lactate-buffered (RLac) or acetate-buffered (RAc)] were infused i.v. (30 ml/kg) in all groups. At 2 hours rats either received different crystalloids (75 ml/kg of RLac or RAc) or colloids (25 ml/kg of HES in saline or HES in RAc or gelatin in saline). Expression of messenger RNA for cytokine-induced neutrophil chemoattractant-1 (CINC-1), monocyte chemotactic protein-1 (MCP-1), necrosis factor α (TNFα) and intercellular adhesion molecule 1 (ICAM-1) was assessed in kidney, liver and lung tissue by real-time PCR after 4 hours. The use of acetate-buffered solutions was associated with a significantly higher expression of CINC-1 and TNFα mRNA in the liver, in the kidney and in the lung. Only marginal effects of gelatin and hydroxyethyl starch on mRNA expression of inflammatory mediators were observed. The study provides evidence that the type of buffering agent of different colloidal and crystalloid solutions might be a crucial factor determining the extent of early end-organ inflammatory response in sepsis.</p></div

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<p>Influence of CLP and different fluid resuscitation procedures on inflammatory mediator expression (mRNA) in kidneys.</p

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<p>The table shows B coefficients (95% confidence intervals) of the linear regression. Animals which received solely Ringers' lactate were used as reference group. mRNA expression is expressed as a fold-difference relative to the reference group. The different fluid ingredients (HES, gelatin, and acetate buffer) were entered as binary independent predictors in the regression model. Significance: ^ap≤0.001, ^bp≤0.01, ^cp<0.05; CINC-1  =  cytokine-induced neutrophil chemoattractant-1; MCP-1  =  monocyte chemoattractant protein-1; TNFα  =  tumor necrosis factor α; ICAM-1  =  intercellular adhesion molecule-1.</p

Composition of fluids.

<p>Composition of fluids.</p

Changes in inflammatory mediator expression of the kidneys.

<p>The table shows B coefficients (95% confidence intervals) of the linear regression. Animals which received solely Ringers' lactate were used as reference group. mRNA expression is expressed as a fold-difference relative to the reference group. The different fluid ingredients (HES, gelatin, and acetate buffer) were entered as binary independent predictors in the regression model. Significance: ^ap≤0.001, ^bp≤0.01, ^cp<0.05; CINC-1  =  cytokine-induced neutrophil chemoattractant-1; MCP-1  =  monocyte chemoattractant protein-1; TNFα  =  tumor necrosis factor α; ICAM-1  =  intercellular adhesion molecule-1.</p