Effects of anesthesia on conventional and speckle tracking echocardiographic parameters in a mouse model of pressure overload

Genetically‑modified mice are widely applied in cardiovascular studies as model organisms. Echocardiography is a key tool for evaluating cardiac and hemodynamic functions in mice. The present study aimed to examine the effects of isoflurane (ISF) on conventional and speckle tracking echocardiography (STE) parameters under healthy and pathological conditions using a murine model of pressure overload. In addition, the optimal dose of ISF in the process of echocardiographic measurement, with minimum cardiac contraction depression, was investigated. Conventional echocardiographic and STE examinations were performed on 38 adult C57BL/6 male mice. The mice were divided into the following three groups: The sham (n=15); mild thoracic aortic banding (TAB; n=15); and severe TAB (n=8) groups. ISF was administered under deep anesthesia (DA; 1‑2% ISF), light anesthesia (LA; 0.5‑1% ISF) and immediately prior to the mice waking up (awake; 0‑0.5% ISF). Conventional echocardiographic parameters were preserved within the sham and mild TAB groups (P>0.05 for each parameter) under LA and awake conditions. However, under DA conditions, the majority of these parameters were reduced compared with the LA and awake conditions (P<0.05). In the severe TAB group, conventional echocardiographic parameters remained constant under LA, DA and awake conditions. STE parameters in the groups remained similar between the LA and awake conditions, but were significantly reduced under DA conditions. Therefore, conventional echocardiography and STE may be performed using LA induced with low doses of ISF, under various pathological conditions without affecting cardiac function

Significance of myocardial tenascin-C expression in left ventricular remodelling and long-term outcome in patients with dilated cardiomyopathy

Aim Dilated cardiomyopathy (DCM) has a variety of causes, and no useful approach to predict left ventricular (LV) remodelling and long-term outcome has yet been established. Myocardial tenascin-C (TNC) is known to appear under pathological conditions, possibly to regulate cardiac remodelling. The aim of this study was to clarify the significance of myocardial TNC expression in LV remodelling and the long-term outcome in DCM. Methods and results One hundred and twenty-three consecutive DCM patients who underwent endomyocardial biopsy for initial diagnosis were studied. Expression of TNC in biopsy sections was analysed immunohistochemically to quantify the ratio of the TNC-positive area to the whole myocardial tissue area (TNC area). Clinical parameters associated with TNC area were investigated. The patients were divided into two groups based on receiver operating characteristic analysis of TNC area to predict death: high TNC group with TNC area ≥2.3% (22 patients) and low TNC group with TNC area <2.3% (101 patients). High TNC was associated with diabetes mellitus. Comparing echocardiographic findings between before and 9 months after endomyocardial biopsy, the low TNC group was associated with decreased LV end-diastolic diameter and increased LV ejection fraction, whereas the high TNC group was not. Survival analysis revealed a worse outcome in the high TNC group than in the low TNC group (P < 0.001). Multivariable Cox regression analysis revealed that TNC area was independently associated with poor outcome (HR = 1.347, P = 0.032). Conclusions Increased myocardial TNC expression was associated with worse LV remodeling and long-term outcome in DCM

Thyroid hormone levels within reference range are associated with heart rate, cardiac structure, and function in middle-aged men and women

Background: Triiodothyronine (T3) has many effects on the heart, and marked changes in cardiac function and structure occur in patients with (subclinical) thyroid disease. We investigated whether between-subject variation in thyroid hormone levels within the euthyroid range is also associated with heart rate and echocardiographic heart function and structure. Methods: Subjects were selected from the Asklepios study (n=2524), a population-representative random sample of patients aged between 35 and 55 years, free from overt cardiovascular disease at baseline. Analyses were restricted to 2078 subjects (1013 women and 1065 men), not using antihypertensive or thyroid medication nor having antithyroperoxidase antibody levels above clinical cut-off or thyrotropin (TSH) levels outside the reference range. All subjects were phenotyped in-depth and underwent comprehensive echocardiography, including diastolic evaluation. Thyroid function parameters were determined by automated electrochemiluminescence. Results: Heart rate was robustly positively associated with (quartiles of) free T3 (FT3) and T3, both in subjects with TSH levels within reference (0.27-4.2 μU/L) and in narrow TSH range (0.5-2.5 μU/L; p<0.0001). FT3 and T3 were negatively associated with left ventricular (LV) end-diastolic volume but positively associated with relative wall thickness. Total T3 (TT3) was associated with enhanced ventricular contraction (as assessed by tissue Doppler imaging). Free thyroxine, FT3, and TT3 were positively associated with late ventricular filling, and TT3 was associated with early ventricular filling. Conclusion: We have demonstrated a strong positive association between thyroid hormone levels within the euthyroid range and heart rate, and more subtle effects on cardiac function and structure. More specifically, we suggest a smaller LV cavity size (with increased relative wall thickness), an enhanced atrial and ventricular contraction, and LV relaxation with higher circulating thyroid hormones. These results illustrate that variation in thyroid hormone levels, even within the reference range, exerts effects on the heart

Inhibition of Autophagy Signaling via 3-methyladenine Rescued Nicotine-Mediated Cardiac Pathological Effects and Heart Dysfunctions.

Rationale: Cigarette smoking is a well-established risk factor for myocardial infarction and sudden cardiac death. The deleterious effects are mainly due to nicotine, but the mechanisms involved and theranostics remain unclear. Thus, we tested the hypothesis that nicotine exposure increases the heart sensitivity to ischemia/reperfusion injury and dysfunction, which can be rescued by autophagy inhibitor. Methods: Nicotine or saline was administered to adult rats via subcutaneous osmotic minipumps in the absence or presence of an autophagy inhibitor, 3-methyladenine (3-MA). After 30 days of nicotine treatment, the rats underwent the cardiac ischemia/reperfusion (I/R) procedure and echocardiography analysis, and the heart tissues were isolated for molecular biological studies. Results: Nicotine exposure increased I/R-induced cardiac injury and cardiac dysfunction as compared to the control. The levels of autophagy-related proteins including LC3 II, P62, Beclin1, and Atg5 were upregulated in the reperfused hearts isolated from nicotine-treated group. In addition, nicotine enhanced cardiac and plasma ROS production, and increased the phosphorylation of GSK3β (ser9) in the left ventricle tissues. Treatment with 3-MA abolished nicotine-mediated increase in the levels of autophagy-related proteins and phosphorylation of GSK3β, but had no effect on ROS production. Of importance, 3-MA ameliorated the augmented I/R-induced cardiac injury and dysfunction in the nicotine-treated group as compared to the control. Conclusion: Our results demonstrate that nicotine exposure enhances autophagy signaling pathway, resulting in development of ischemic-sensitive phenotype of heart. It suggests a potentially novel therapeutic strategy of autophagy inhibition for the treatment of ischemic heart disease

Losartan Treatment Attenuates Tumor-induced Myocardial Dysfunction

Fatigue and muscle wasting are common symptoms experienced by cancer patients. Data from animal models demonstrate that angiotensin is involved in tumor-induced muscle wasting, and that tumor growth can independently affect myocardial function, which could contribute to fatigue in cancer patients. In clinical studies, inhibitors of angiotensin converting enzyme (ACE) can prevent the development of chemotherapy-induced cardiovascular dysfunction, suggesting a mechanistic role for the renin–angiotensin–aldosterone system (RAAS). In the present study, we investigated whether an angiotensin (AT) 1-receptor antagonist could prevent the development of tumor-associated myocardial dysfunction. Methods and results: Colon26 adenocarcinoma (c26) cells were implanted into female CD2F1 mice at 8 weeks of age. Simultaneously, mice were administered Losartan (10 mg/kg) daily via their drinking water. In vivo echocardiography, blood pressure, in vitro cardiomyocyte function, cell proliferation assays, and measures of systemic inflammation and myocardial protein degradation were performed 19 days following tumor cell injection. Losartan treatment prevented tumor-induced loss of muscle mass and in vitro c26 cell proliferation, decreased tumor weight, and attenuated myocardial expression of interleukin-6. Furthermore, Losartan treatment mitigated tumor-associated alterations in calcium signaling in cardiomyocytes, which was associated with improved myocyte contraction velocity, systolic function, and blood pressures in the hearts of tumor-bearing mice. Conclusions: These data suggest that Losartan may mitigate tumor-induced myocardial dysfunction and inflammation

The role of cardiac troponin T quantity and function in cardiac development and dilated cardiomyopathy

Background: Hypertrophic (HCM) and dilated (DCM) cardiomyopathies results from sarcomeric protein mutations, including cardiac troponin T (cTnT, TNNT2). We determined whether TNNT2 mutations cause cardiomyopathies by altering cTnT function or quantity; whether the severity of DCM is related to the ratio of mutant to wildtype cTnT; whether Ca2+ desensitization occurs in DCM; and whether absence of cTnT impairs early embryonic cardiogenesis. Methods and Findings: We ablated Tnnt2 to produce heterozygous Tnnt2+/ mice, and crossbreeding produced homozygous null Tnnt2-/-embryos. We also generated transgenic mice overexpressing wildtype (TGWT) or DCM mutant (TGK210Δ) Tnnt2. Crossbreeding produced mice lacking one allele of Tnnt2, but carrying wildtype (Tnnt2+/-/TGWT) or mutant (Tnnt2+/-/TGK210Δ) transgenes. Tnnt2+/-mice relative to wildtype had significantly reduced transcript (0.82 ± 0.06 [SD] vs. 1.00 ± 0.12 arbitrary units; p = 0.025), but not protein (1.01 ± 0.20 vs. 1.00 ± 0.13 arbitrary units; p = 0.44). Tnnt2+/-mice had normal hearts (histology, mass, left ventricular end diastolic diameter [LVEDD], fractional shortening [FS]). Moreover, whereas Tnnt2+/-/ TGK210Δ mice had severe DCM, TGK210Δ mice had only mild DCM (FS 18 ± 4 vs. 29 ± 7%; p < 0.01). The difference in severity of DCM may be attributable to a greater ratio of mutant to wildtype Tnnt2 transcript in Tnnt2+/-/TGK210Δ relative to TGK210Δ mice (2.42±0.08, p = 0.03). Tnnt2+/-/TGK210Δ muscle showed Ca2+ desensitization (pCa50 = 5.34 ± 0.08 vs. 5.58 ± 0.03 at sarcomere length 1.9 μm. p<0.01), but no difference in maximum force generation. Day 9.5 Tnnt2-/-embryos had normally looped hearts, but thin ventricular walls, large pericardial effusions, noncontractile hearts, and severely disorganized sarcomeres. Conclusions: Absence of one Tnnt2 allele leads to a mild deficit in transcript but not protein, leading to a normal cardiac phenotype. DCM results from abnormal function of a mutant protein, which is associated with myocyte Ca2+ desensitization. The severity of DCM depends on the ratio of mutant to wildtype Tnnt2 transcript. cTnT is essential for sarcomere formation, but normal embryonic heart looping occurs without contractile activity. © 2008 Ahmad et al

Epigenetic Down-Regulation of Sirt 1 via DNA Methylation and Oxidative Stress Signaling Contributes to the Gestational Diabetes Mellitus-Induced Fetal Programming of Heart Ischemia-Sensitive Phenotype in Late Life.

Rationale: The incidence of gestational diabetes mellitus (GDM) is increasing worldwide. However, whether and how GDM exposure induces fetal programming of adult cardiac dysfunctional phenotype, especially the underlying epigenetic molecular mechanisms and theranostics remain unclear. To address this problem, we developed a late GDM rat model. Methods: Pregnant rats were made diabetic on day 12 of gestation by streptozotocin (STZ). Experiments were conducted in 6 weeks old offspring. Results: There were significant increases in ischemia-induced cardiac infarction and gender-dependent left ventricular (LV) dysfunction in male offspring in GDM group as compared to controls. Exposure to GDM enhanced ROS level and caused a global DNA methylation in offspring cardiomyocytes. GDM attenuated cardiac Sirt 1 protein and p-Akt/Akt levels, but enhanced autophagy-related proteins expression (Atg 5 and LC3 II/LC3 I) as compared to controls. Ex-vivo treatment of DNA methylation inhibitor, 5-Aza directly inhibited Dnmt3A and enhanced Sirt 1 protein expression in fetal hearts. Furthermore, treatment with antioxidant, N-acetyl-cysteine (NAC) in offspring reversed GDM-mediated DNA hypermethylation, Sirt1 repression and autophagy-related gene protein overexpression in the hearts, and rescued GDM-induced deterioration in heart ischemic injury and LV dysfunction. Conclusion: Our data indicated that exposure to GDM induced offspring cardiac oxidative stress and DNA hypermethylation, resulting in an epigenetic down-regulation of Sirt1 gene and aberrant development of heart ischemia-sensitive phenotype, which suggests that Sirt 1-mediated signaling is the potential therapeutic target for the heart ischemic disease in offspring

Sarcospan Regulates Cardiac Isoproterenol Response and Prevents Duchenne Muscular Dystrophy-Associated Cardiomyopathy.

BackgroundDuchenne muscular dystrophy is a fatal cardiac and skeletal muscle disease resulting from mutations in the dystrophin gene. We have previously demonstrated that a dystrophin-associated protein, sarcospan (SSPN), ameliorated Duchenne muscular dystrophy skeletal muscle degeneration by activating compensatory pathways that regulate muscle cell adhesion (laminin-binding) to the extracellular matrix. Conversely, loss of SSPN destabilized skeletal muscle adhesion, hampered muscle regeneration, and reduced force properties. Given the importance of SSPN to skeletal muscle, we investigated the consequences of SSPN ablation in cardiac muscle and determined whether overexpression of SSPN into mdx mice ameliorates cardiac disease symptoms associated with Duchenne muscular dystrophy cardiomyopathy.Methods and resultsSSPN-null mice exhibited cardiac enlargement, exacerbated cardiomyocyte hypertrophy, and increased fibrosis in response to β-adrenergic challenge (isoproterenol; 0.8 mg/day per 2 weeks). Biochemical analysis of SSPN-null cardiac muscle revealed reduced sarcolemma localization of many proteins with a known role in cardiomyopathy pathogenesis: dystrophin, the sarcoglycans (α-, δ-, and γ-subunits), and β1D integrin. Transgenic overexpression of SSPN in Duchenne muscular dystrophy mice (mdx(TG)) improved cardiomyofiber cell adhesion, sarcolemma integrity, cardiac functional parameters, as well as increased expression of compensatory transmembrane proteins that mediate attachment to the extracellular matrix.ConclusionsSSPN regulates sarcolemmal expression of laminin-binding complexes that are critical to cardiac muscle function and protects against transient and chronic injury, including inherited cardiomyopathy

Inflammatory Serine Proteases Play a Critical Role in the Early Pathogenesis of Diabetic Cardiomyopathy.

BACKGROUND/AIMS: Diabetic cardiomyopathy (DCM) is characterized by structural and functional alterations that can lead to heart failure. Several mechanisms are known to be involved in the pathogenesis of DCM, however, the molecular mechanism that links inflammation to DCM is incompletely understood. To learn about this mechanism, we investigated the role of inflammatory serine proteases (ISPs) during the development of DCM. METHODS: Eight weeks old mice with deletion of dipeptidyl peptidase I (DPPI), an enzyme involved in the maturation of major ISPs, and wild type (WT) mice controls were injected with streptozotocin (50 mg/kg for 5 days intraperitoneally) and studied after 4, 8, 16, and 20 week after induction of type 1 diabetes mellitus (T1DM). Induction of diabetes was followed by echocardiographic measurements, glycemic and hemoglobulin A1c profiling, immunoblot, qPCR, enzyme activity assays, and immunohistochemistry (IHC) analysis of DPPI, ISPs, and inflammatory markers. Fibrosis was determined from left ventricular heart by Serius Red staining and qPCR. Apoptosis was determined by TUNEL assay and immunoblot analysis. RESULTS: In the diabetic WT mice, DPPI expression increased along with ISP activation, and DPPI accumulated abundantly in the left ventricle mainly from infiltrating neutrophils. In diabetic DPPI-knockout (DPPI-KO) mice, significantly decreased activation of ISPs, myocyte apoptosis, fibrosis, and cardiac function was improved compared to diabetic WT mice. In addition, DPPI-KO mice showed a decrease in overall inflammatory status mediated by diabetes induction which was manifested by decreased production of pro-inflammatory cytokines like TNF-α, IL-1β and IL-6. CONCLUSION: This study elucidates a novel role of ISPs in potentiating the immunological responses that lead to the pathogenesis of DCM in T1DM. To the best of our knowledge, this is the first study to report that DPPI expression and activation promotes the inflammation that enhances myocyte apoptosis and contributes to the adverse cardiac remodeling that subsequently leads to DCM

Alterations in protein P53 expression during the development of pressure overload-induced left ventricular hypertrophy in rats

The relation between myocardial structural and functional changes and p53 proteins during the development of left ventricular hypertrophy (LVH) has not yet been reported. The purpose of this study was to determine whether p53 protein expression is altered during development of LVH, induced by pressure overload. LVH was induced in 80 male Wistar adult rats by abdominal aortic banding (AAB) and was monitored at the 10th, 15th, 20th, 25th, 35th and 45th post-operation days by echocardiography and validated by postmortem examination. Sham operated (SO) rats (n=60) went the same operation without banding. The expression of p53 protein in the nuclear and cytoplasmic extracts from left ventricular tissue from AAB and SO rats were analyzed by immunoblotting at each of the pointed days. Relative to SO, echo-left ventricular mass-to-tibia length (LVM/TL) ratio in AAB increased progressively on the 10th and the 15th day. On the 20th day we observed a short lasting regression of LVH, followed by a new marked increase on the 25th and 35th day; then there was presented a plateau. Relative to SO the changes in LV%FS (77% ± 2.2 %) in AAB rats remained at the same level throughout the studied period without significant differences in body weight or tibia length (TL). Results from echocardiography were validated by necropsy. Echo LVM/TL correlated significantly with actual heart weight-to-tibia length ratio (r=0.69, P&lt;0.0001, n=140). The level of p53 protein expression in the nuclear extracts from LV tissues obtained from AAB rats increased significantly by 77%, 78%, 35% and 63% at day 10th, 20th, 35th and 45th, respectively compared with SO, and returned to SO levels by day 15th and 25th post-operation. The highest level of p53 protein in the nuclear extracts from LV tissues obtained from AAB rats was determined on 20th day concomitant with significant reduction of the actual heart mass in the group with AAB rats, compared with the same groups on the other days. In the cytozol the expression of p53 was always higher in the SO, compared with AAB groups. Furthermore, the level of p53 protein in the nuclear extracts from AAB rats significantly correlated with the actual heart weight during the studied period (r= -0.59, P&lt;0.0001, n=60). Following the development of LVH over a relatively long period of time and providing the changes in short intervals, it was found a short lasting regression during ongoing pressure overload concomitant with significantly increased expression of p53 protein in the nuclear extracts from AAB rats. This study adds new important concepts for the time dependency of LVH and suggests that p53 protein may modulate the adaptive growth of pressure overload induced LVH