Urszula Tyrankiewicz

Detection of mitochondrial dysfunction by EPR technique in mouse model of dilated cardiomyopathy

Tgalphaq44 mice with targeted overexpression of activated Galphaq protein in cardiomyocytes mimic many of the phenotypic characteristics of dilated cardiomyopathy in humans. However, it is not known whether the phenotype of Tgalphaq44 mice would also involve dysfunction of cardiac mitochondria. The aim of the present work was to examine changes in EPR signals of semiquinones and iron in Fe-S clusters, as compared to classical biochemical indices of mitochondrial function in hearts from Tgalphaq44 mice in relation to the progression of heart failure. Tgalphaq44 mice at the age of 14 months displayed pulmonary congestion, increased heart/body ratio and impairment of cardiac function as measured in vivo by MRI. However, in hearts from Tgalphaq44 mice already at the age of 10 months EPR signals of semiquinones, as well as cyt c oxidase activity were decreased, suggesting alterations in mitochondrial electron flow. Furthermore, in 14-months old Tgalphaq44 mice loss of iron in Fe-S clusters, impaired citrate synthase activity, and altered mitochondrial ultrastructure were observed, supporting mitochondrial dysfunction in Tgalphaq44 mice. In conclusion, the assessment of semiquinones content and Fe(III) analysis by EPR represents a rational approach to detect dysfunction of cardiac mitochondria. Decreased contents of semiquinones detected by EPR and a parallel decrease in cyt c oxidase activity occurs before hemodynamic decompensation of heart failure in Tgalphaq44 mice suggesting that alterations in function of cardiac mitochondria contribute to the development of the overt heart failure in this model.

Preserved cardiomyocyte function and altered desmin pattern in transgenic mouse model of dilated cardiomyopathy

Taking advantage of the unique model of slowly developing dilated cardiomyopathy in mice with cardiomyocyte-specific transgenic overexpression of activated Gαq protein (Tgαq*44 mice) we analyzed the contribution of the cardiomyocyte malfunction, fibrosis and cytoskeleton remodeling to the development of heart failure in this model. Left ventricular (LV) in vivo function, myocardial fibrosis, cytoskeletal proteins expression and distribution, Ca(2+) handling and contractile function of isolated cardiomyocytes were evaluated at the stages of the early, compensated, and late, decompensated heart failure in 4-, 12- and 14-month-old Tgαq*44 mice, respectively, and compared to age-matched wild-type FVB mice. In the 4-month-old Tgαq*44 mice significant myocardial fibrosis, moderate myocyte hypertrophy and increased expression of regularly arranged and homogenously distributed desmin accompanied by increased phosphorylation of desmin chaperone protein, αB-crystallin, were found. Cardiomyocyte shortening, Ca(2+) handling and LV function were not altered. At 12 and 14 months of age, Tgαq*44 mice displayed progressive deterioration of the LV function. The contractile performance of isolated myocytes was still preserved, and the amplitude of Ca(2+) transients was even increased probably due to impairment of Na(+)/Ca(2+) exchanger function, while fibrosis was more extensive than in younger mice. Moreover, substantial disarrangement of desmin distribution accompanied by decreasing phosphorylation of αB-crystallin appeared. In Tgαq*44 mice disarrangement of desmin, at least partly related to inadequate phosphorylation of αB-crystallin seems to be importantly involved in the progressive deterioration of contractile heart function.

Characterization of the cardiac response to a low and high dose of dobutamine in the mouse model of dilated cardiomyopathy by MRI in vivo

To assess the cardiac response to low (0.15–0.5 mg/kg i.p.) and high (1.5–20 mg/kg i.p.) doses of dobutamine in Tgαq*44 mice with dilated cardiomyopathy at the stage of advanced heart failure.

Retrospectively gated MRI for in vivo assessment of endothelium-dependent vasodilatation and endothelial permeability in murine models of endothelial dysfunction

Endothelial dysfunction is linked to impaired endothelial‐dependent vasodilatation and permeability changes. Here, we quantify both of these phenomena associated with endothelial dysfunction by MRI in vivo in mice.

Narrow time window of metabolic changes associated with transition to overt heart failure in Tgaq*44 mice

BACKGROUND The timing and consequences of alternations in substrate utilization in heart failure (HF) and their relationship with structural changes remain unclear. This study aimed to analyze metabolic changes associated with transition to overt heart failure in transgenic mouse model of HF resulting from cardiac-specific overexpression of constitutively active Gαq*. METHODS Structural changes quantified by morphometry, relative cardiac mRNA and protein expression of PPARα, FAT/CD36, CPT-1, GLUT-4 and glycolytic efficiency following administration of 1-(13)C glucose were investigated in 4-14-month-old Tgαq*44 mice (TG), compared with age-matched FVB wild type mice (WT). RESULTS Initial hypertrophy in TG (4-10-month of age) was featured by an accelerated glycolytic pathway that was not accompanied by structural changes in cardiomyocytes. In 10-month-old TG, cardiomyocyte elongation and hypertrophic remodeling and increased glycolytic flux was accompanied by relatively low expression of FAT/CD36, CPT-1 and PPARα. During the transition phase (12-month-old TG), a pronounced increase in PPARα with an increase in relative fatty acid (FA) flux was associated with anomalies of cardiomyocytes with accumulation of lipid droplets and glycogen as well as cell death. At the stage of overt heart failure (14-month-old TG), an accelerated glycolytic pathway with a decline in FA oxidation was accompanied by further structural changes. CONCLUSION Tgαq*44 mice display three distinct phases of metabolic/structural changes during hypertrophy and progression to HF, with relatively short period of increase in FA metabolism, highlighting a narrow metabolic changes associated with transition to overt heart failure in Tgaq*44 mice that have therapeutic significance.

The effect of the renin–angiotensin–aldosterone system inhibition on myocardial function in early and late phases of dilated cardiomyopathy in Tgaq*44 mice

BACKGROUND The renin-angiotensin-aldosterone system (RAAS) determines progression of heart failure (HF) in humans, and RAAS inhibition is a major therapeutic strategy in HF. AIM To assess the effect of angiotensin-converting enzyme inhibitor (ACE-I) and aldosterone receptor antagonist (ARA) therapy on the development of HF at its early and late stage in a murine model of dilated cardiomyopathy (Tgaq*44 mice). METHODS Tgaq*44 mice at the early or advanced stage of HF received combined therapy including ACE-I (perindopril 2 mg/kg) and ARA (canrenone 20 mg/kg). Cardiac function was assessed by magnetic resonance imaging before and after 2 months of treatment. RESULTS Combined therapy with perindopril and canrenone resulted in preserved systolic function at the early stage and reduced chamber dilatation at the advanced stage of HF in Tgaq*44 mice. CONCLUSIONS Activation of the RAAS is involved in progression of HF in Tgaq*44 mice with dilated cardiomyopathy. Therapeutic efficacy of ACE-I and ARA to inhibit systolic dysfunction and cardiac chamber dilation depends on the stage of HF development.

Functional and Biochemical Endothelial Profiling In Vivo in a Murine Model of Endothelial Dysfunction; Comparison of Effects of 1-Methylnicotinamide and Angiotensin-converting Enzyme Inhibitor

Although it is known that 1-methylnicotinamide (MNA) displays vasoprotective activity in mice, as yet the effect of MNA on endothelial function has not been demonstrated in vivo. Here, using magnetic resonance imaging (MRI) we profile the effects of MNA on endothelial phenotype in mice with atherosclerosis (ApoE/LDLR-/-) in vivo, in comparison to angiotensin (Ang) -converting enzyme (ACE) inhibitor (perindopril), with known vasoprotective activity. On a biochemical level, we analyzed whether MNA- or perindopril-induced improvement in endothelial function results in changes in ACE/Ang II-ACE2/Ang-(1–7) balance, and L-arginine/asymmetric dimethylarginine (ADMA) ratio. Endothelial function and permeability were evaluated in the brachiocephalic artery (BCA) in 4-month-old ApoE/LDLR-/- mice that were non-treated or treated for 1 month or 2 months with either MNA (100 mg/kg/day) or perindopril (10 mg/kg/day). The 3D IntraGate®FLASH sequence was used for evaluation of BCA volume changes following acetylcholine (Ach) administration, and for relaxation time (T1) mapping around BCA to assess endothelial permeability using an intravascular contrast agent. Activity of ACE/Ang II and ACE2/Ang-(1–7) pathways as well as metabolites of L-arginine/ADMA pathway were measured using liquid chromatography/mass spectrometry-based methods. In non-treated 6-month-old ApoE/LDLR-/- mice, Ach induced a vasoconstriction in BCA that amounted to –7.2%. 2-month treatment with either MNA or perindopril resulted in the reversal of impaired Ach-induced response to vasodilatation (4.5 and 5.5%, respectively) and a decrease in endothelial permeability (by about 60% for MNA-, as well as perindopril-treated mice). Improvement of endothelial function by MNA and perindopril was in both cases associated with the activation of ACE2/Ang-(1–7) and the inhibition of ACE/Ang II axes as evidenced by an approximately twofold increase in Ang-(1–9) and Ang-(1–7) and a proportional decrease in Ang II and its active metabolites. Finally, MNA and perindopril treatment resulted in an increase in L-arginine/ADMA ratio by 107% (MNA) and 140% (perindopril), as compared to non-treated mice. Functional and biochemical endothelial profiling in ApoE/LDLR-/- mice in vivo revealed that 2-month treatment with MNA (100 mg/kg/day) displayed a similar profile of vasoprotective effect as 2-month treatment with perindopril (10 mg/kg/day): i.e., the improvement in endothelial function that was associated with the beneficial changes in ACE/Ang II-ACE2/Ang (1–7) balance and in L-arginine/ADMA ratio in plasma.

Comprehensive MRI for the detection of subtle alterations in diastolic cardiac function in apoE/LDLR(-/-) mice with advanced atherosclerosis.

ApoE/LDLR–/– mice represent a reliable model of atherosclerosis. However, it is not clear whether cardiac performance is impaired in this murine model of atherosclerosis. Here, we used MRI to characterize cardiac performance in vivo in apoE/LDLR–/– mice with advanced atherosclerosis. Six‐month‐old apoE/LDLR–/– mice and age‐matched C57BL/6J mice (control) were examined using highly time‐resolved cine‐MRI [whole‐chamber left ventricle (LV) imaging] and MR tagging (three slices: basal, mid‐cavity and apical). Global and regional measures of cardiac function included LV volumes, kinetics, time‐dependent parameters, strains and rotations. Histological analysis was performed using OMSB (orceine with Martius, Scarlet and Blue) and ORO (oil red‐O) staining to demonstrate the presence of advanced coronary atherosclerosis. MR‐tagging‐based strain analysis in apoE/LDLR–/– mice revealed an increased frequency of radial and circumferential systolic stretch (25% and 50% of segments, respectively, p ≤ 0.012), increased radial post‐systolic strain index (45% of segments, p = 0.009) and decreased LV untwisting rate (−30.3° (11.6°)/cycle, p = 0.004) when compared with control mice. Maximal strains and LV twist were unchanged. Most of the cine‐MRI‐based LV functional and anatomical parameters also remained unchanged in apoE/LDLR–/–mice, with only a lower filling rate, longer filling time, shorter isovolumetric contraction time and slower heart rate observed in comparison with control mice. The coronary arteries displayed severe atherosclerosis, as evidenced by histological analysis. Using comprehensive MRI methods, we have demonstrated that, despite severe coronary atherosclerosis in six‐month‐old apoE/LDLR–/– mice, cardiac performance including global parameters, twist and strains, was well preserved. Only subtle diastolic alterations, possibly of ischemic background, were uncovered. Copyright

Activation pattern of ACE2/Ang-(1-7) and ACE/Ang II pathway in course of heart failure assessed by multiparametric MRI in vivo in Tgαq*44 mice

Here, we analyzed systemic (plasma) and local (heart/aorta) changes in ACE/ACE-2 balance in Tgαq*44 mice in course of heart failure (HF). Tgαq*44 mice with cardiomyocyte-specific Gαq overexpression and late onset of HF were analyzed at different age for angiotensin pattern in plasma, heart, and aorta using liquid chromatography/mass spectrometry, for progression of HF by in vivo magnetic resonance imaging under isoflurane anesthesia, and for physical activity by voluntary wheel running. Six-month-old Tgαq*44 mice displayed decreased ventricle radial strains and impaired left atrial function. At 8-10 mo, Tgαq*44 mice showed impaired systolic performance and reduced voluntary wheel running but exhibited preserved inotropic reserve. At 12 mo, Tgαq*44 mice demonstrated a severe impairment of basal cardiac performance and modestly compromised inotropic reserve with reduced voluntary wheel running. Angiotensin analysis in plasma revealed an increase in concentration of angiotensin-(1-7) in 6- to 10-mo-old Tgαq*44 mice. However, in 12- to 14-mo-old Tgαq*44 mice, increased angiotensin II was noted with a concomitant increase in Ang III, Ang IV, angiotensin A, and angiotensin-(1-10). The pattern of changes in the heart and aorta was also compatible with activation of ACE2, followed by activation of the ACE pathway. In conclusion, mice with cardiomyocyte Gαq protein overexpression develop HF that is associated with activation of the systemic and the local ACE/Ang II pathway. However, it is counterbalanced by a prominent ACE2/Ang-(1-7) activation, possibly allowing to delay decompensation. NEW & NOTEWORTHY Changes in ACE/ACE-2 balance were analyzed based on measurements of a panel of nine angiotensins in plasma, heart, and aorta of Tgαq*44 mice in relation to progression of heart failure (HF) characterized by multiparametric MRI and exercise performance. The early stage of HF was associated with upregulation of the ACE2/angiotensin-(1-7) pathway, whereas the end-stage HF was associated with downregulation of ACE2/angiotensin-(1-7) and upregulation of the ACE/Ang II pathway. ACE/ACE-2 balance seems to determine the decompensation of HF in this model.