Reduced acute myocardial ischemia-reperfusion injury in IL-6-deficient mice employing a closed-chest model.

OBJECTIVE AND DESIGN: We examined the role of IL-6 in the temporal development of cardiac ischemia-reperfusion injury employing a closed-chest I/R model. MATERIALS/METHODS: Infarction, local and systemic inflammation, neutrophil infiltration, coagulation and ST elevation/resolution were compared between wild-type (WT) and IL-6-deficient (IL-6(-/-)) mice after 1 h ischemia and 0, ½, 3, and 24 h reperfusion. RESULTS: IL-6 deficiency reduced infarct size at 3 h reperfusion (28.8 ± 4.5 % WT vs 17.6 ± 2.5 % IL-6(-/-)), which reduction persisted and remained similar at 24 h reperfusion (25.1 ± 3.0 % WT vs 14.6 ± 4.4 % IL-6(-/-)). Serum Amyloid A was reduced at 24 h reperfusion only (57.5 ± 4.9 WT vs 24.8 ± 5.6 ug/ml IL-6(-/-) mice). Cardiac cytokines (IL-6, IL-1ß and TNFα) peaked at 3 h reperfusion, but IL-1ß and TNFα levels were unaffected by IL-6 deficiency. Significant neutrophil influx was only detected at 24 h reperfusion and was similar for WT and IL-6(-/-). Tissue factor peaked at 24 h reperfusion, whereas fibrin/fibrinogen peaked at 3 h reperfusion and was completely resolved at 24 h reperfusion; both coagulation factors were unaltered by IL-6 deficiency. Prolonged ST elevation was observed during ischemia that completely resolved for both genotypes at early reperfusion. CONCLUSIONS: The data suggest that, in the absence of major surgical intervention, IL-6 contributes to the development of infarct size in the early phase of reperfusion; this contribution did not depend on neutrophil influx, IL-1ß and TNFα, tissue factor and fibrin.

A transgenic mouse model for the simultaneous monitoring of ANF and BNP gene activity during heart development and disease.

AIM: The expression of Nppa (ANF) and Nppb (BNP) marks the chamber myocardium in the embryo, and both genes serve as early and accurate markers for hypertrophy and heart failure. Non-invasive visualization of Nppa-Nppb expression in living mice would enable to evaluate the disease state during the course of time in heart disease models. We sought to develop a method to assess the pattern and level of Nppa and Nppb expression within living mice. METHODS AND RESULTS: A modified bacterial artificial chromosome containing a genomic segment spanning the Nppa-Nppb locus was randomly integrated into the mouse genome. Firefly Luciferase was inserted into Nppa and the red fluorescent protein gene Katushka into Nppb. Both reporters precisely recapitulated the spatio-temporal patterns of Nppa and Nppb, respectively. In a hypertrophy model (transverse aortic constriction) and myocardial infarction model (left anterior descending coronary artery occlusion), the non-invasively measured bioluminescent signal from Luciferase correlated with Nppa expression, and the intensity of red fluorescence with levels of the expression of Katushka and Nppb. After myocardial infarction, the border zone of the infarct area was readily identified by an increased intensity of Katushka fluorescence. CONCLUSIONS: A genomic region sufficient to regulate the developmental pattern and stress response of Nppa and Nppb has been defined. The double reporter mice can be used for the functional imaging and investigation of cardiac hypertrophy and myocardial infarction in vivo.

Deletion of the innate immune NLRP3 receptor abolishes cardiac ischemic preconditioning and is associated with decreased Il-6/STAT3 signaling.

OBJECTIVE: Recent studies indicate that the innate immune system is not only triggered by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As triggers for the innate immune NLRP3 inflammasome protein complex appear to overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NLRP3 inflammasome is involved in IPC and acute I/R injury of the heart. PRINCIPAL FINDINGS: Baseline cardiac performance and acute I/R injury were investigated in isolated, Langendorff-perfused hearts from wild-type (WT), ASC(-/-) and NLRP3(-/-) mice. Deletion of NLRP3 inflammasome components ASC(-/-) or NLRP3(-/-) did not affect baseline performance. The deletions exacerbated I/R-induced mechanical dysfunction, but were without effect on I/R-induced cell death. When subjected to IPC, WT and ASC(-/-) hearts were protected against I/R injury (improved function and less cell death). However, IPC did not protect NLRP3(-/-) hearts against I/R injury. NLRP3(-/-) hearts had significantly decreased cardiac IL-6 levels with a trend towards lower IL-1ß levels at end reperfusion, suggesting abrogation of IPC through diminished IL-6 and/or IL-1ß signaling. Subsequent experiments showed that neutralising IL-6 using an antibody against IL-6 abrogated IPC in WT hearts. However, inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) did not abrogate IPC in WT hearts. Analysis of survival kinases after IPC demonstrated decreased STAT3 expression in NLRP3(-/-) hearts when compared to WT hearts. CONCLUSIONS: The data suggest that the innate immune NLRP3 protein, in an NLRP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6/STAT3 dependent mechanism.

Adaptive stress response in segmental progeria resembles long-lived dwarfism and calorie restriction in mice.

How congenital defects causing genome instability can result in the pleiotropic symptoms reminiscent of aging but in a segmental and accelerated fashion remains largely unknown. Most segmental progerias are associated with accelerated fibroblast senescence, suggesting that cellular senescence is a likely contributing mechanism. Contrary to expectations, neither accelerated senescence nor acute oxidative stress hypersensitivity was detected in primary fibroblast or erythroblast cultures from multiple progeroid mouse models for defects in the nucleotide excision DNA repair pathway, which share premature aging features including postnatal growth retardation, cerebellar ataxia, and death before weaning. Instead, we report a prominent phenotypic overlap with long-lived dwarfism and calorie restriction during postnatal development (2 wk of age), including reduced size, reduced body temperature, hypoglycemia, and perturbation of the growth hormone/insulin-like growth factor 1 neuroendocrine axis. These symptoms were also present at 2 wk of age in a novel progeroid nucleotide excision repair-deficient mouse model (XPD(G602D/R722W)/XPA(-/-)) that survived weaning with high penetrance. However, despite persistent cachectic dwarfism, blood glucose and serum insulin-like growth factor 1 levels returned to normal by 10 wk, with hypoglycemia reappearing near premature death at 5 mo of age. These data strongly suggest changes in energy metabolism as part of an adaptive response during the stressful period of postnatal growth. Interestingly, a similar perturbation of the postnatal growth axis was not detected in another progeroid mouse model, the double-strand DNA break repair deficient Ku80(-/-) mouse. Specific (but not all) types of genome instability may thus engage a conserved response to stress that evolved to cope with environmental pressures such as food shortage.

Modified two-step model for studying the inflammatory response during myocardial ischemia and reperfusion in mice.

Studies of myocardial ischemia-reperfusion (MI-R) in the mouse can be accomplished by use of reversible ligation of the left interventricular branch artery (LIB). To study interactions of coagulation, inflammation, and reperfusion injury, the model should not be influenced by effects of the surgery. In existing closed-thorax mouse models, the release of inflammatory factors attributable to surgical intervention could be separated from the release resulting from induction of MI-R. In these models, the final myocardial injury was induced by reversible closure of the LIB several days after preparative surgery that included median thoracotomy. In an attempt to develop a less invasive procedure to approach the LIB, we replaced median thoracotomy with lateral thoracotomy. After this procedure, body weight was regained within four days, and on days 9 to 11 after the preparative surgery, cytokine values were back to baseline. During one hour of ischemia, mean arterial pressure (MAP) remained at 78 +/- 2 mmHg. After induction of reperfusion, MAP was 67 +/- 4 mmHg, indicating better perfusion pressure of myocardial tissue at the microcirculatory level than that in simple open-thorax models. Electrocardiographic recording revealed transient ST elevation indicating reversible transmural ischemia and reperfusion. Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining visualized the extent of area of infarction (AOI) and area at risk (AAR). The procedure-related mortality was 13%, which compared well with published data from median thoracotomy studies. We conclude that our new model provides stable near-physiologic hemodynamics and allows study of the inflammatory response resulting from MI-R, with low procedure-related mortality.

Fentanyl-fluanisone-midazolam combination results in more stable hemodynamics than does urethane alpha-chloralose and 2,2,2-tribromoethanol in mice.

Near-physiologic hemodynamic conditions for several hours were needed to study cardiovascular physiology in a murine model. We compared two commonly used anesthetic treatments, urethane alpha-chloralose (U-alphaCh; 968 mg U and 65 mg alphaCh/kg) and 2,2,2-tribromoethanol (TBE; 435 mg/kg) and fentanyl fluanisone midazolam (FFM; 3.313 mg fentanyl, 104.8 mg fluanisone, and 52.42 mg midazolam/kg) with respect to mean arterial blood pressure (MAP) and heart rate (HR) for 100 min at similar levels of surgical anesthesia. Assessed every 10 to 15 min, the U-alphaCh+TBE group maintained a significantly (P < 0.001) lower mean MAP (49 4 mmHg) than did the FFM group (78 5 mmHg). Mean HR in the U-alphaCh+TBE group significantly (P < 0.001) increased from 308 34 bpm at the beginning to 477 43 bpm at the end of the experiment. In comparison, the FFM group showed a stable HR of 431 37 bpm. The MAP and HR of the U-alphaCh+TBE group were extremely unstable, with sudden and unpredictable changes in MAP when examined at 1-min intervals. The results of our study show that U-alphaCh+TBE anesthesia should not be used in murine models in which stable, near-physiologic hemodynamics are needed for cardiovascular studies.