Rac1-mediated cardiac damage causes diastolic dysfunction in a mouse model of subacute doxorubicin-induced cardiotoxicity.

The anticancer efficacy of anthracyclines is limited by congestive heart failure. Clinically established markers of early onset of cardiotoxicity following anthracycline treatment and preventive measures are missing. Although statins are reported to alleviate anthracycline-induced cardiotoxicity in vivo, the molecular mechanisms involved remain elusive. In vitro data point to Rac1 as major target of the cytoprotective statin effects. Here we investigated whether specific inhibition of Rac1 by NSC23766 is as effective as lovastatin in preventing subacute cardiotoxicity following doxorubicin treatment. C57BL/6 mice were treated over 3 weeks with multiple low doses of doxorubicin (6 × 3 mg/kg BW, i.p.) and the level of DNA damage, apoptosis and regenerative proliferation as well as pro-inflammatory, pro-fibrotic and oxidative stress responses were investigated. Moreover, heart function was monitored by echocardiography. Doxorubicin induced subacute cardiotoxicity which was reflected on the level of residual DNA damage, frequency of apoptotic and mitotic cells as well as elevated mRNA expression of markers of heart failure, remodeling and mitochondrial biogenesis. These molecular markers of cardiotoxicity were mitigated to a similar extent by co-treatment with either lovastatin (10 mg/kg BW, p.o.) or NSC23766 (5 mg/kg BW, i.p.) three times a week. Moreover, doxorubicin caused diastolic dysfunction as reflected by increased E-wave acceleration time (EAT), which again was prevented by pharmacological inhibition of Rac1. Inhibition of Rac1 signaling is of major relevance for the cardioprotective effects of lovastatin in the context of anthracycline-induced cardiotoxicity. Moreover, EAT is a useful marker of subacute cardiotoxicity caused by persisting harmful effects of doxorubicin.

Endothelial NOS (NOS3) impairs myocardial function in developing sepsis.

Endothelial nitric oxide synthase (NOS)3-derived nitric oxide (NO) modulates inotropic response and diastolic interval for optimal cardiac performance under non-inflammatory conditions. In sepsis, excessive NO production plays a key role in severe hypotension and myocardial dysfunction. We aimed to determine the role of NOS3 on myocardial performance, NO production, and time course of sepsis development. NOS3(-/-) and C57BL/6 wildtype mice were rendered septic by cecum ligation and puncture (CLP). Cardiac function was analyzed by serial echocardiography, in vivo pressure and isolated heart measurements. Cardiac output (CO) increased to 160 % of baseline at 10 h after sepsis induction followed by a decline to 63 % of baseline after 18 h in wildtype mice. CO was unaltered in septic NOS3(-/-) mice. Despite the hyperdynamic state, cardiac function and mean arterial pressure were impaired in septic wildtype as early as 6 h post CLP. At 12 h, cardiac function in septic wildtype was refractory to catecholamines in vivo and respective isolated hearts showed impaired pressure development and limited coronary flow reserve. Hemodynamics remained stable in NOS3(-/-) mice leading to significant survival benefit. Unselective NOS inhibition in septic NOS3(-/-) mice diminished this survival benefit. Plasma NO( x )- and local myocardial NO( x )- and NO levels (via NO spin trapping) demonstrated enhanced NO( x )- and bioactive NO levels in septic wildtype as compared to NOS3(-/-) mice. Significant contribution by inducible NOS (NOS2) during this early phase of sepsis was excluded. Our data suggest that NOS3 relevantly contributes to bioactive NO pool in developing sepsis resulting in impaired cardiac contractility.

Non- invasive in vivo analysis of a murine aortic graft using high resolution ultrasound microimaging.

INTRODUCTION: As yet, murine aortic grafts have merely been monitored histopathologically. The aim of our study was to examine how these grafts can be monitored in vivo and non-invasively by using high-resolution ultrasound microimaging to evaluate function and morphology. A further aim was to prove if this in vivo monitoring can be correlated to immunohistological data that indicates graft integrity. METHODS: Murine infrarenal aortic isografts were orthotopically transplanted into 14 female mice (C57BL/6-Background) whereas a group of sham-operated animals (n = 10) served as controls. To assess the graft morphology and hemodynamics, we examined the mice over a post-operative period of 8 weeks with a sophisticated ultrasound system (Vevo 770, Visual Sonics). RESULTS: The non-invasive graft monitoring was feasible in all transplanted mice. We could demonstrate a regular post-transplant graft function and morphology, such as anterior/posterior wall displacement and wall thickness. Mild alterations of anterior wall motion dynamics could only be observed at the site of distal graft anastomosis (8 weeks after grafting (transplant vs. sham mice: 0.02 mm ± 0.01 vs. 0.03 mm ± 0.01, p<0.05). However, the integrity of the entire graft wall could be confirmed by histopathological evaluation of the grafts. CONCLUSIONS: With regard to graft patency, function and morphology, high resolution ultrasound microimaging has proven to be a valuable tool for longitudinal, non-invasive, in vivo graft monitoring in this murine aortic transplantation model. Consequently, this experimental animal model provides an excellent basis for molecular and pharmacological studies using genetically engineered mice.

Double-edged role of the CXCL12/CXCR4 axis in experimental myocardial infarction.

OBJECTIVES: Here we assess the intrinsic functions of the chemokine receptor CXCR4 in remodeling after myocardial infarction (MI) using Cxcr4 heterozygous (Cxcr4(+/-)) mice. BACKGROUND: Myocardial necrosis triggers complex remodeling and inflammatory changes. The chemokine CXCL12 has been implicated in protection and therapeutic regeneration after MI through recruiting angiogenic outgrowth cells, improving neovascularization and cardiac function, but the endogenous role of its receptor CXCR4 is unknown. METHODS: MI was induced by ligation of the left descending artery. Langendoff perfusion, echocardiography, quantitative immunohistochemistry, flow cytometry, angiogenesis assays, and cardiomyocyte analysis were performed. RESULTS: After 4 weeks, infarct size was reduced in Cxcr4(+/-) mice compared with wild-type mice and in respective bone marrow chimeras compared with controls. This was associated with altered inflammatory cell recruitment, decreased neutrophil content, delayed monocyte infiltration, and a predominance of Gr1(low) over classic Gr1(high) monocytes. Basal coronary flow and its recovery after MI were impaired in Cxcr4(+/-)mice, paralleled by reduced angiogenesis, myocardial vessel density, and endothelial cell count. Notably, no differences in cardiac function were seen in Cxcr4(+/-)mice compared with wild-type mice. Despite defective angiogenesis, Cxcr4(+/-) mouse hearts showed no difference in CXCL12, vascular endothelial growth factor or apoptosis-related gene expression. Electron microscopy revealed lipofuscin-like lipid accumulation in Cxcr4(+/-) mouse hearts and analysis of lipid extracts detected high levels of phosphatidylserine, which protect cardiomyocytes from hypoxic stress in vitro. CONCLUSIONS: CXCR4 plays a crucial role in endogenous remodeling processes after MI, contributing to inflammatory/progenitor cell recruitment and neovascularization, whereas its deficiency limits infarct size and causes adaptation to hypoxic stress. This should be carefully scrutinized when devising therapeutic strategies involving the CXCL12/CXCR4 axis.