Thomas G. Hampton

Mice mutant for Egfr and Shp2 have defective cardiac semilunar valvulogenesis.

Atrioventricular and semilunar valve abnormalities are common birth defects, but how cardiac valvulogenesis is directed remains largely unknown. During studies of genetic interaction between Egfr, encoding the epidermal growth factor receptor, and Ptpn11, encoding the protein-tyrosine-phosphatase Shp2, we discovered that Egfr is required for semilunar, but not atrioventricular, valve development. Although unnoticed in earlier studies, mice homozygous for the hypomorphic Egfr allele waved-2 (Egfrwa2/wa2) exhibit semilunar valve enlargement resulting from over-abundant mesenchymal cells. Egfr−/− mice (CD1 background) have similar defects. The penetrance and severity of the defects in Egfrwa2/wa2 mice are enhanced by heterozygosity for a targeted mutation of exon 2 of Ptpn11 (ref. 3). Compound (Egfrwa2/wa2:Ptpn11+/−) mutant mice also show premature lethality. Electrocardiography, echocardiography and haemodynamic analyses showed that affected mice develop aortic stenosis and regurgitation. Our results identify the Egfr and Shp2 as components of a growth-factor signalling pathway required specifically for semilunar valvulogenesis, support the hypothesis that Shp2 is required for Egfr signalling in vivo, and provide an animal model for aortic valve disease.

Abnormal cardiac function in the streptozotocin-induced, non–insulin-dependent diabetic rat: Noninvasive assessment with Doppler echocardiography and contribution of the nitric oxide pathway☆

OBJECTIVES We sought to evaluate in vivo and in vitro left ventricular (LV) geometry and function in streptozotocin-induced diabetic rats and the possible role of the nitric oxide (NO) pathway. BACKGROUND Diabetes results in cardiac dysfunction; however, the specific abnormalities are unknown. Because decreased NO contributes to abnormal vascular function in diabetics, we hypothesized that NO pathway abnormalities may contribute to diabetic cardiomyopathy. METHODS Control rats and those with non-insulin-dependent diabetes mellitus (NIDDM) underwent echocardiography, hemodynamic assessment, isolated heart perfusion and measurement of exhaled NO and LV endothelial constitutive nitric oxide synthase (ecNOS). RESULTS Diabetic rats had increased LV mass (3.3 +/- 0.6 vs. 2.6 +/- 0.3 g/g body weight [BW], p < 0.001) and cavity dimensions (diastolic 2.0 +/- 0.1 vs. 1.8 +/- 0.2 cm/cm tibial length [TL], p < 0.05). Diabetic rats had prolonged isovolumic relaxation time (IVRT) (40 +/- 8 vs. 26 +/- 6 ms, p < 0.0001), increased atrial contribution to diastolic filling (0.47 +/- 0.09 vs. 0.30 +/- 0.08 m/s, p < 0.0001), and elevated in vivo LV end-diastolic pressure (7 +/- 6 vs. 2 +/- 1 mm Hg, p = 0.04). Diabetic rats had increased chamber stiffness. Shortening was similar in both groups, despite reduced meridional wall stress in diabetics, suggesting impaired systolic contractility. Exhaled NO was lower in diabetic rats (1.8 +/- 0.2 vs. 3.3 +/- 0.3 parts per billion, p < 0.01) and correlated with Doppler LV filling. The ecNOS was similar between the groups. CONCLUSIONS Diabetic cardiomyopathy is characterized by LV systolic and diastolic dysfunction, the latter correlating with decreased exhaled NO. The NO pathway is intact, suggesting impaired availability of NO as contributor to cardiomyopathy.

Gait dynamics in mouse models of Parkinson's disease and Huntington's disease

BackgroundGait is impaired in patients with Parkinsons disease (PD) and Huntingtons disease (HD), but gait dynamics in mouse models of PD and HD have not been described. Here we quantified temporal and spatial indices of gait dynamics in a mouse model of PD and a mouse model of HD.MethodsGait indices were obtained in C57BL/6J mice treated with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg/day for 3 days) for PD, the mitochondrial toxin 3-nitropropionic acid (3NP, 75 mg/kg cumulative dose) for HD, or saline. We applied ventral plane videography to generate digital paw prints from which indices of gait and gait variability were determined. Mice walked on a transparent treadmill belt at a speed of 34 cm/s after treatments.ResultsStride length was significantly shorter in MPTP-treated mice (6.6 ± 0.1 cm vs. 7.1 ± 0.1 cm, P < 0.05) and stride frequency was significantly increased (5.4 ± 0.1 Hz vs. 5.0 ± 0.1 Hz, P < 0.05) after 3 administrations of MPTP, compared to saline-treated mice. The inability of some mice treated with 3NP to exhibit coordinated gait was due to hind limb failure while forelimb gait dynamics remained intact. Stride-to-stride variability was significantly increased in MPTP-treated and 3NP-treated mice compared to saline-treated mice. To determine if gait disturbances due to MPTP and 3NP, drugs affecting the basal ganglia, were comparable to gait disturbances associated with motor neuron diseases, we also studied gait dynamics in a mouse model of amyotrophic lateral sclerosis (ALS). Gait variability was not increased in the SOD1 G93A transgenic model of ALS compared to wild-type control mice.ConclusionThe distinct characteristics of gait and gait variability in the MPTP model of Parkinsons disease and the 3NP model of Huntingtons disease may reflect impairment of specific neural pathways involved.

Gait dynamics in trisomic mice: quantitative neurological traits of Down syndrome

The segmentally trisomic mouse Ts65Dn is a model of Down syndrome (DS). Gait abnormalities are almost universal in persons with DS. We applied a noninvasive imaging method to quantitatively compare the gait dynamics of Ts65Dn mice (n=10) to their euploid littermates (controls) (n=10). The braking duration of the hind limbs in Ts65Dn mice was prolonged compared to that in control mice (60+/-3 ms vs. 49+/-2 ms, P<.05) at a slow walking speed (18 cm/s). Stride length and stride frequency of forelimbs and hind limbs were comparable between Ts65Dn mice and control mice. Stride dynamics were significantly different in Ts65Dn mice at a faster walking speed (36 cm/s). Stride length was shorter in Ts65Dn mice (5.9+/-0.1 vs. 6.3+/-0.3 cm, P<.05), and stride frequency was higher in Ts65Dn compared to control mice (5.9+/-0.1 vs. 5.3+/-0.1 strides/s, P<.05). Hind limb swing duration was prolonged in Ts65Dn mice compared to control mice (93+/-3 vs. 76+/-3 ms, P<.05). Propulsion of the forelimbs contributed to a significantly larger percentage of stride duration in Ts65Dn mice than in control mice at the faster walking speed. Indices of gait dynamics in Ts65Dn mice correspond to previously reported findings in children with DS. The methods used in the present study provide quantitative markers for genotype and phenotype relationship studies in DS. This technique may provide opportunities for testing the efficacy of therapies for motor dysfunction in persons with DS.

Method for non-invasively recording electrocardiograms in conscious mice

BackgroundThe rapid increase in the development of mouse models is resulting in a growing demand for non-invasive physiological monitoring of large quantities of mice. Accordingly, we developed a new system for recording electrocardiograms (ECGs) in conscious mice without anesthesia or implants, and created Internet-accessible software for analyzing murine ECG signals. The system includes paw-sized conductive electrodes embedded in a platform configured to record ECGs when 3 single electrodes contact 3 paws.ResultsWith this technique we demonstrated significantly reduced heart rate variability in neonates compared to adult mice. We also demonstrated that female mice exhibit significant ECG differences in comparison to age-matched males, both at baseline and in response to β-adrenergic stimulation.ConclusionsThe technology we developed enables non-invasive screening of large numbers of mice for ECG changes resulting from genetic, pharmacological, or pathophysiological alterations. Data we obtained non-invasively are not only consistent with what have been reported using invasive and expensive methods, but also demonstrate new findings regarding gender-dependent and age-dependent variations in ECGs in mice.

Neuregulin-1 attenuated doxorubicin-induced decrease in cardiac troponins

Neuregulin-1 (NRG1) is a potential therapeutic agent for the treatment of doxorubicin (Dox)-induced heart failure. NRG1, however, activates the erbB2 receptor, which is frequently overexpressed in breast cancers. It is, therefore, important to understand how NRG1, via erbB2, protects the heart against Dox cardiotoxicity. Here, we studied NRG1-erbB2 signaling in Dox-treated mice hearts and in isolated neonatal rat ventricular myocytes (NRVM). Male C57BL/6 mice were treated with recombinant NRG1 before and daily after a single dose of Dox. Cardiac function was determined by catheterization. Two-week survival was analyzed by the Kaplan-Meier method. Cardiac troponins [cardiac troponin I (cTnI) and cardiac troponin T (cTnT)] and phosphorylated Akt protein levels were determined in mice hearts and in NRVM by Western blot analysis. Activation of caspases and ubiquitinylation of troponins were determined in NRVM by caspase assay and immunoprecipitation. NRG1 significantly improved survival and cardiac function in Dox-treated mice. NRG1 reduced the decrease in cTnI, cTnT, and cardiac troponin C (cTnC) and maintained Akt phosphorylation in Dox-treated mice hearts. NRG1 reduced the decrease in cTnI and cTnT mRNA and proteins in Dox-treated NRVM. Inhibition of erbB2, phosphoinositide 3-kinase (PI3K), Akt, and mTOR blocked the protective effects of NRG1 on cTnI and cTnT in NRVM. NRG1 significantly reduced Dox-induced caspase activation, which degraded troponins, in NRVM. NRG1 reduced Dox-induced proteasome degradation of cTnI. NRG1 attenuates Dox-induced decrease in cardiac troponins by increasing transcription and translation and by inhibiting caspase activation and proteasome degradation of troponin proteins. NRG1 maintains cardiac troponins by the erbB2-PI3K pathway, which may lessen Dox-induced cardiac dysfunction.

Electrocardiographic findings in mdx mice: A cardiac phenotype of Duchenne muscular dystrophy

The mdx mouse is a model of Duchenne muscular dystrophy (DMD). As many DMD patients die of cardiac failure, we investigated whether mdx mice exhibited clinically relevant cardiac phenotypes. We applied a recently developed method for noninvasively recording electrocardiograms (ECGs) to study male mdx mice (n = 15) and control mice (n = 15). The mdx mice had significant tachycardia and decreased heart rate variability, consistent with observations in DMD patients. Heart rate was nearly 15% faster in mdx mice than control mice (P < 0.05). The rate‐corrected QT interval duration and PR interval were shorter in mdx compared to control mice (P < 0.05). The muscarinic antagonist atropine significantly increased heart rate and decreased PR interval in C57 mice. In contrast, atropine significantly decreased heart rate and increased PR interval in all mdx mice. Pharmacological autonomic blockade and baroreflex sensitivity testing demonstrated an imbalance in autonomic nervous system modulation of heart rate, with decreased parasympathetic activity and increased sympathetic activity in mdx mice. Baseline ECGs and contrary responses to muscarinic blockade by atropine in mice deficient in neuronal nitric oxide synthase (nNOS) suggest that the autonomic dysfunction in mdx mice may be independent of decreased myocardial nNOS. These electrocardiographic findings in dystrophin‐deficient mice may provide new bases for diagnosing, understanding, and treating DMD patients.

Ethanol's effects on gait dynamics in mice investigated by ventral plane videography.

BACKGROUND Performance of mice in motor function tests for ethanol sensitivity is often task dependent, not reflective of coordinated movement, and reported qualitatively. Therefore, we applied a new imaging technique to record and quantify coordinated gait dynamics in mice in response to ethanol. METHODS We applied ventral plane videography to record and report gait indices in mice walking on a transparent treadmill belt. We examined the effects of ethanol on gait in C57BL/6J (B6) and DBA/2J (D2) mice walking at a speed of 25 cm/sec. B6 and D2 are two inbred strains that are widely used to study the genetic influences of ethanol on motor function. RESULTS Gait posture in D2 mice was less stable than in B6 mice. B6 mice always showed an alternate step sequence, whereas D2 mice sometimes showed cruciate and rotary step sequences. Ethanol in increasing doses increased stride frequency, decreased stride length, and increased stride length variability in D2 mice but not in B6 mice. The forelimb braking duration was significantly shortened and the hind limb propulsion duration was significantly prolonged with a high dose of ethanol in D2 mice but not in B6 mice. Differences in gait indices between the two strains of mice were more pronounced of the forelimbs with the highest dose of ethanol (2.75 g/kg). CONCLUSION Our data suggest that the higher susceptibility of D2 compared with B6 mice to the effects of ethanol on motor function may be attributed to less stable basal gait characteristics that are perturbed by ethanol. The ability of this method to quantify step sequence patterns and gait indices of forelimb and hind limbs could provide new data regarding ethanol-induced motor incoordination.

Intracellular calcium dynamics in mouse model of myocardial stunning.

Intracellular calcium ([Formula: see text]) and left ventricular (LV) function were determined in the coronary-perfused mouse heart to study[Formula: see text]-related mechanisms of injury from myocardial ischemia and reperfusion. Specifics for loading of the photoprotein aequorin into isovolumically contracting mouse hearts under constant-flow conditions are provided. The method allows detection of changes in [Formula: see text] on a beat-to-beat basis in a model of myocardial stunning and permits correlation of interventions that regulate Ca2+ exchange with functional alterations. Twenty-three coronary-perfused mouse hearts were subjected to 15 min of ischemia followed by 20 min of reperfusion. In 13 hearts, the perfusate included the calmodulin antagonist W7 (10 μM) to inhibit Ca2+-calmodulin-regulated mechanisms. Peak [Formula: see text] was 0.77 ± 0.03 μM in the control group and was unaffected by W7 at baseline. Ischemia was characterized by a rapid decline in LV function, followed by ischemic contracture, accompanied by a gradual rise in[Formula: see text]. Reperfusion was characterized by an initial burst of [Formula: see text] and a gradual recovery to nearly normal systolic[Formula: see text] while LV pressure recovered to 55% after 20 min of reperfusion (stunned myocardium). These results in the mouse heart confirm that stunning does not result from deficiency of [Formula: see text] but rather from a decreased myofilament responsiveness to [Formula: see text] due to changes in the myofilaments themselves. In hearts perfused with W7, the rise in [Formula: see text] during ischemia was significantly attenuated, as was the magnitude of mean[Formula: see text] during early reflow. Ischemic contracture was abolished or delayed. Hearts perfused with W7 showed significantly improved recovery of LV pressure, rate of contraction, and rate of relaxation. Diastolic [Formula: see text]was increased in control hearts during stunning but returned to baseline in hearts perfused with W7. Simultaneous assessment of[Formula: see text] and LV function demonstrates that calmodulin-regulated mechanisms may contribute to the pathogenesis of myocardial stunning in the mouse heart.Intracellular calcium (Cai2+) and left ventricular (LV) function were determined in the coronary-perfused mouse heart to study Cai2+-related mechanisms of injury from myocardial ischemia and reperfusion. Specifics for loading of the photoprotein aequorin into isovolumically contracting mouse hearts under constant-flow conditions are provided. The method allows detection of changes in Cai2+ on a beat-to-beat basis in a model of myocardial stunning and permits correlation of interventions that regulate Ca2+ exchange with functional alterations. Twenty-three coronary-perfused mouse hearts were subjected to 15 min of ischemia followed by 20 min of reperfusion. In 13 hearts, the perfusate included the calmodulin antagonist W7 (10 microM) to inhibit Ca(2+)-calmodulin-regulated mechanisms. Peak Cai2+ was 0.77 +/- 0.03 microM in the control group and was unaffected by W7 at baseline. Ischemia was characterized by a rapid decline in LV function, followed by ischemic contracture, accompanied by a gradual rise in Cai2+. Reperfusion was characterized by an initial burst of Cai2+ and a gradual recovery to nearly normal systolic Cai2+ while LV pressure recovered to 55% after 20 min of reperfusion (stunned myocardium). These results in the mouse heart confirm that stunning does not result from deficiency of Cai2+ but rather from a decreased myofilament responsiveness to Cai2+ due to changes in the myofilaments themselves. In hearts perfused with W7, the rise in Cai2+ during ischemia was significantly attenuated, as was the magnitude of mean Cai2+ during early reflow. Ischemic contracture was abolished or delayed. Hearts perfused with W7 showed significantly improved recovery of LV pressure, rate of contraction, and rate of relaxation. Diastolic Cai2+ was increased in control hearts during stunning but returned to baseline in hearts perfused with W7. Simultaneous assessment of Cai2+ and LV function demonstrates that calmodulin-regulated mechanisms may contribute to the pathogenesis of myocardial stunning in the mouse heart.

Cardiovascular Abnormalities in Transgenic Mice With Reduced Brown Fat An Animal Model of Human Obesity

BACKGROUND A new model of murine obesity has recently been developed through transgenic ablation of brown adipose tissue that manifests typical metabolic complications of obesity, including insulin resistance and non-insulin-dependent diabetes mellitus. The cardiovascular phenotype has not been defined. METHODS AND RESULTS Transthoracic echocardiography, aortic catheterization, isolated whole-heart studies, and morphometric histology defined cardiac structure and function in 30 transgenic mice with reduced brown fat and 30 matched wild-type controls. Obesity was indicated by a 77% increase in body weight and was accompanied by elevated systemic pressures (mean aortic blood pressure 85+/-1 versus 66+/-2 mm Hg; P<0.01), left ventricular dilation and hypertrophy (mass/body weight 4.0+/-0.2 versus 2.7+/-0.3 mg/g; P<0.01), and high cardiac output (cardiac index 3.2+/-0.4 versus 2.4+/-0.1 mL x kg(-1) x min(-1); P<0.01). Baseline functional parameters assessed in vitro were not different, but after imposition of zero-flow ischemia, significant relaxation impairment developed in obese mice. Although morphometrically determined myocyte diameters were similar, the percentage of interstitial fibrosis was significantly increased in transgenic mice compared with wild-type controls (7.5+/-2% versus 4. 2+/-0.2%; P<0.01). CONCLUSIONS Transgenic ablation of brown adipose tissue is associated not only with obesity but also with systemic hypertension, left ventricular hypertrophy with eccentric remodeling and fibrosis, and high cardiac output, a unique constellation of findings strikingly similar to that seen in human obesity. Mice with reduced brown fat may serve as a new model for the cardiovascular morbid complications associated with obesity in humans.