Developmental changes in lung function of mice are independent of sex as a biological variable.

Pulmonary function testing (PFT) in mice includes biomechanical assessment of lung function relevant to physiology in health and its alteration in disease, hence, it is frequently used in preclinical modeling of human lung pathologies. Despite numerous reports of PFT in mice of various ages, there is a lack of reference data for developing mice collected using consistent methods. Therefore, we profiled PFTs in male and female C57BL/6J mice from 2 to 23 wk of age, providing reference values for age- and sex-dependent changes in mouse lung biomechanics during development and young adulthood. Although males and females have similar weights at birth, females weigh significantly less than males after 5 wk of age (P < 0.001) with largest weight gain observed between 3 and 8 wk in females and 3 and 13 wk in males, after which weight continued to increase more slowly up to 23 wk of age. Lung function parameters including static compliance and inspiratory capacity also increased rapidly between 3 and 8 wk in female and male mice, with male mice having significantly greater static compliance and inspiratory capacity than female mice (P < 0.001). Although these parameters appear higher in males at a given age, allometric scaling showed that static compliance and inspiratory compliance were comparable between the two sexes. This suggests that differences in measurements of lung function are likely body weight-based rather than sex-based. We expect these data to facilitate future lung disease research by filling a critical knowledge gap in our field.NEW & NOTEWORTHY This study provides reference values for changes in mouse lung biomechanics from 2 to 23 wk of age. There are rapid developmental changes in lung structure and function of male and female mice between the ages of 3 and 8 wk. Male mice become noticeably heavier than female mice at or about 5 wk of age. We identified that differences in normal lung function measurements are likely weight-based, not sex-based.

Regional myocardial strain analysis via 2D speckle tracking echocardiography: validation with sonomicrometry and correlation with regional blood flow in the presence of graded coronary stenoses and dobutamine stress.

BACKGROUND: Quantitative regional strain analysis by speckle tracking echocardiography (STE) may be particularly useful in the assessment of myocardial ischemia and viability, although reliable measurement of regional strain remains challenging, especially in the circumferential and radial directions. We present an acute canine model that integrates a complex sonomicrometer array with microsphere blood flow measurements to evaluate regional myocardial strain and flow in the setting of graded coronary stenoses and dobutamine stress. We apply this unique model to rigorously evaluate a commercial 2D STE software package and explore fundamental regional myocardial flow-function relationships. METHODS: Sonomicrometers (16 crystals) were implanted in epicardial and endocardial pairs across the anterior myocardium of anesthetized open chest dogs (n = 7) to form three adjacent cubes representing the ischemic, border, and remote regions, as defined by their relative locations to a hydraulic occluder on the mid-left anterior descending coronary artery (LAD). Additional cardiac (n = 3) and extra-cardiac (n = 3) reference crystals were placed to define the cardiac axes and aid image registration. 2D short axis echocardiograms, sonometric data, and microsphere blood flow data were acquired at baseline and in the presence of mild and moderate LAD stenoses, both before and during low-dose dobutamine stress (5 µg/kg/min). Regional end-systolic 2D STE radial and circumferential strains were calculated with commercial software (EchoInsight) and compared to those determined by sonomicrometry and to microsphere blood flow measurements. Post-systolic indices (PSIs) were also calculated for radial and circumferential strains. RESULTS: Low-dose dobutamine augmented both strain and flow in the presence of mild and moderate stenoses. Regional 2D STE strains correlated moderately with strains assessed by sonomicrometry (Rradial = 0.56, p < 0.0001; Rcirc = 0.55, p < 0.0001) and with regional flow quantities (Rradial = 0.61, Rcirc = 0.63). Overall, correspondence between 2D STE and sonomicrometry was better in the circumferential direction (Bias ± 1.96 SD: - 1.0 ± 8.2% strain, p = 0.06) than the radial direction (5.7 ± 18.3%, p < 0.0001). Mean PSI values were greatest in low flow conditions and normalized with low-dose dobutamine. CONCLUSIONS: 2D STE identifies changes in regional end-systolic circumferential and radial strain produced by mild and moderate coronary stenoses and low-dose dobutamine stress. Regional 2D STE end-systolic strain measurements correlate modestly with regional sonomicrometer strain and microsphere flow measurements.

Multimodality Imaging of Aortic Valve Calcification and Function in a Murine Model of Calcific Aortic Valve Disease and Bicuspid Aortic Valve.

Calcific aortic valve disease (CAVD) is a prevailing disease with increasing occurrence and no known medical therapy. Dcbld2-/- mice have a high prevalence of bicuspid aortic valve (BAV), spontaneous aortic valve calcification, and aortic stenosis (AS). 18F-NaF PET/CT can detect the aortic valve calcification process in humans. However, its feasibility in preclinical models of CAVD remains to be determined. Here, we sought to validate 18F-NaF PET/CT for tracking murine aortic valve calcification and leveraged it to examine the development of calcification with aging and its interdependence with BAV and AS in Dcbld2-/- mice. Methods: Dcbld2-/- mice at 3-4 mo, 10-16 mo, and 18-24 mo underwent echocardiography, 18F-NaF PET/CT (n = 34, or autoradiography (n = 45)), and tissue analysis. A subset of mice underwent both PET/CT and autoradiography (n = 12). The aortic valve signal was quantified as SUVmax on PET/CT and as percentage injected dose per square centimeter on autoradiography. The valve tissue sections were analyzed by microscopy to identify tricuspid and bicuspid aortic valves. Results: The aortic valve 18F-NaF signal on PET/CT was significantly higher at 18-24 mo (P < 0.0001) and 10-16 mo (P < 0.05) than at 3-4 mo. Additionally, at 18-24 mo BAV had a higher 18F-NaF signal than tricuspid aortic valves (P < 0.05). These findings were confirmed by autoradiography, with BAV having significantly higher 18F-NaF uptake in each age group. A significant correlation between PET and autoradiography data (Pearson r = 0.79, P < 0.01) established the accuracy of PET quantification. The rate of calcification with aging was significantly faster for BAV (P < 0.05). Transaortic valve flow velocity was significantly higher in animals with BAV at all ages. Finally, there was a significant correlation between transaortic valve flow velocity and aortic valve calcification by both PET/CT (r = 0.55, P < 0.001) and autoradiography (r = 0.45, P < 0.01). Conclusion: 18F-NaF PET/CT links valvular calcification to BAV and aging in Dcbld2-/- mice and suggests that AS may promote calcification. In addition to addressing the pathobiology of valvular calcification, 18F-NaF PET/CT may be a valuable tool for evaluation of emerging therapeutic interventions in CAVD.

Atrial AMP-activated protein kinase is critical for prevention of dysregulation of electrical excitability and atrial fibrillation.

Metabolic stress is an important cause of pathological atrial remodeling and atrial fibrillation. AMPK is a ubiquitous master metabolic regulator, yet its biological function in the atria is poorly understood in both health and disease. We investigated the impact of atrium-selective cardiac AMPK deletion on electrophysiological and structural remodeling in mice. Loss of atrial AMPK expression caused atrial changes in electrophysiological properties and atrial ectopic activity prior to the onset of spontaneous atrial fibrillation. Concomitant transcriptional downregulation of connexins and atrial ion channel subunits manifested with delayed left atrial activation and repolarization. The early molecular and electrophysiological abnormalities preceded left atrial structural remodeling and interstitial fibrosis. AMPK inactivation induced downregulation of transcription factors (Mef2c and Pitx2c) linked to connexin and ion channel transcriptional reprogramming. Thus, AMPK plays an essential homeostatic role in atria, protecting against adverse remodeling potentially by regulating key transcription factors that control the expression of atrial ion channels and gap junction proteins.

Arteriovenous Fistula-induced Cardiac Remodeling Shows Cardioprotective Features in Mice.

OBJECTIVE---: Arteriovenous fistulae (AVF) placed for hemodialysis have high flow rates that can stimulate left ventricular (LV) hypertrophy. LV hypertrophy generally portends poor cardiac outcomes, yet clinical studies point to superior cardiac-specific outcomes for patients with AVF when compared to other dialysis modalities. We hypothesize that AVF induce physiologic cardiac hypertrophy with cardioprotective features. METHODS---: 9-11 week C57Bl/6 male and female mice were treated with sham laparotomy or an aortocaval fistula via a 25Ga needle. Cardiac chamber size and function were assessed with serial echocardiography, and cardiac CTA. Hearts were harvested at 5 weeks post-operatively, and collagen content assessed with Masson's trichrome. Bulk mRNA sequencing was performed from LV of sham and AVF mice at 10 days. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis (Qiagen) to identify affected pathways and predict downstream biological effects. RESULTS---: Mice with AVF had similar body weight and wet lung mass, but increased cardiac mass compared to sham-operated mice. AVF increased cardiac output while preserving LV systolic and diastolic function, as well as indices of right heart function; all 4 cardiac chambers were enlarged, with slight decrement in relative LV wall thickness. Histology showed preserved collagen density within each of the 4 chambers without areas of fibrosis. RNA sequencing captured 19,384 genes, of which 857 were significantly differentially expressed, including transcripts from extracellular matrix-related genes, ion channels, metabolism, and cardiac fetal genes. Top upstream regulatory molecules predicted include activation of angiogenic (Vegf, Akt1), pro-cardiomyocyte survival (Hgf, Foxm1, Erbb2, Lin9, Areg), and inflammation-related (CSF2, Tgfb1, TNF, Ifng, Ccr2, IL6) genes, as well as the inactivation of cardiomyocyte antiproliferative factors (Cdkn1a, FoxO3, α-catenin). Predicted downstream effects include reduction to heart damage, and increased arrhythmia, angiogenesis, and cardiogenesis. There were no significant sex-dependent differences in the AVF-stimulated cardiac adaptation. CONCLUSIONS---: AVF stimulate adaptive cardiac hypertrophy in wild-type mice without heart failure or pathological fibrosis. Transcriptional correlates suggest AVF-induced cardiac remodeling has some cardioprotective, although also arrhythmogenic features.

Computed Tomographic Angiography Assessment of Epicardial Coronary Vasoreactivity for Early Detection of Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: The vascular endothelium is a novel target for the detection, management, and prevention of doxorubicin (DOX)-induced cardiotoxicity. OBJECTIVES: The study aimed to: 1) develop a methodology by computed tomography angiography (CTA) to evaluate stress-induced changes in epicardial coronary diameter; and 2) apply this to a chronic canine model of DOX-induced cardiotoxicity to assess vascular toxicity. METHODS: To develop and validate quantitative methods, sequential retrospectively gated coronary CTAs were performed in 16 canines. Coronary diameters were measured at prespecified distances during rest, adenosine (ADE) (280 µg/kg/min), rest 30 min post-ADE, and dobutamine (DOB) (5 µg/kg/min). A subgroup of 8 canines received weekly intravenous DOX (1 mg/kg) for 12 to 15 weeks, followed by rest-stress CTA at cumulative doses of ∼4-mg/kg (3 to 5 mg/kg), ∼8-mg/kg (7 to 9 mg/kg), and ∼12-mg/kg (12 to 15 mg/kg) of DOX. Echocardiograms were performed at these timepoints to assess left ventricular ejection fraction and global longitudinal strain. RESULTS: Under normal conditions, epicardial coronary arteries reproducibly dilated in response to ADE (left anterior descending coronary artery [LAD]: 12 ± 2%, left circumflex coronary artery [LCx]: 13 ± 2%, right coronary artery [RCA]: 14 ± 2%) and DOB (LAD: 17 ± 3%, LCx: 18 ± 2%, RCA: 15 ± 3%). With DOX, ADE vasodilator responses were impaired after ∼4-mg/kg (LAD: -3 ± 1%, LCx: 0 ± 2%, RCA: -5 ± 2%) and ∼8-mg/kg (LAD: -3 ± 1%, LCx: 0 ± 1%, RCA: -2 ± 2%). The DOB dilation response was preserved at ∼4-mg/kg of DOX (LAD: 18 ± 4%, LCx: 11 ± 3%, RCA: 11 ± 2%) but tended to decrease at ∼8-mg/kg of DOX (LAD: 4 ± 2%, LCx: 8 ± 3%, RCA: 3 ± 2%). A significant left ventricular ejection fraction reduction was observed only at 12 to 15 mg/kg DOX (baseline: 63 ± 2%, 12-mg/kg: 45 ± 3%). Global longitudinal strain was abnormal at ∼4-mg/kg of DOX (p = 0.011). CONCLUSIONS: CTA can reliably assess epicardial coronary diameter in response to pharmacological stressors, providing a noninvasive functional index of coronary vasoreactivity. Impaired epicardial vasodilation occurs early in DOX-induced cardiotoxicity.

Dual Angiotensin Receptor-Neprilysin Inhibition With Sacubitril/Valsartan Attenuates Systolic Dysfunction in Experimental Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: Doxorubicin (DOX) induces cardiotoxicity in part by activation of matrix metalloproteinases (MMPs). Sacubitril/valsartan (Sac/Val) exerts additive cardioprotective actions over renin-angiotensin-aldosterone inhibitors in preclinical models of myocardial infarction and in heart failure patients. We hypothesized that Sac/Val would be more cardioprotective than Val in a rodent model of progressive DOX-induced cardiotoxicity, and this benefit would be associated with modulation of MMP activation. OBJECTIVES: We sought to investigate the efficacy of Sac/Val for the treatment of anthracycline-induced cardiotoxicity. METHODS: Male Wistar rats received DOX intraperitoneally (15 mg/kg cumulative) or saline over 3 weeks. Following the first treatment, control animals were gavaged daily with water (n = 25), while DOX-treated animals were gavaged daily with water (n = 25), Val (31 mg/kg; n = 25) or Sac/Val (68 mg/kg; n = 25) for either 4 or 6 weeks. Echocardiography was performed at baseline, and 4 and 6 weeks after DOX initiation. In addition, myocardial MMP activity was assessed with 99mTc-RP805, and cardiotoxicity severity was assessed by histology at these time points in a subgroup of animals. RESULTS: Left ventricular ejection fraction decreased by 10% at 6 weeks in DOX and DOX + Val rats (both p < 0.05), while this reduction was attenuated in DOX + Sac/Val rats. MMP activity was increased at 6 weeks by 76% in DOX-alone rats, and tended to increase in DOX + Val rats (36%; p = 0.051) but was similar in DOX + Sac/Val rats as compared with time-matched control animals. Both therapies attenuated histological evidence of cellular toxicity and fibrosis (p < 0.05). CONCLUSIONS: Sac/Val offers greater protection against left ventricular remodeling and dysfunction compared with standard angiotensin receptor blocker therapy in a rodent model of progressive DOX-induced cardiotoxicity.