Orlando Aristizábal

Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice

To elucidate the role of cardiac myosin-binding protein-C (MyBP-C) in myocardial structure and function, we have produced mice expressing altered forms of this sarcomere protein. The engineered mutations encode truncated forms of MyBP-C in which the cardiac myosin heavy chain-binding and titin-binding domain has been replaced with novel amino acid residues. Analogous heterozygous defects in humans cause hypertrophic cardiomyopathy. Mice that are homozygous for the mutated MyBP-C alleles express less than 10% of truncated protein in M-bands of otherwise normal sarcomeres. Homozygous mice bearing mutated MyBP-C alleles are viable but exhibit neonatal onset of a progressive dilated cardiomyopathy with prominent histopathology of myocyte hypertrophy, myofibrillar disarray, fibrosis, and dystrophic calcification. Echocardiography of homozygous mutant mice showed left ventricular dilation and reduced contractile function at birth; myocardial hypertrophy increased as the animals matured. Left-ventricular pressure-volume analyses in adult homozygous mutant mice demonstrated depressed systolic contractility with diastolic dysfunction. These data revise our understanding of the role that MyBP-C plays in myofibrillogenesis during cardiac development and indicate the importance of this protein for long-term sarcomere function and normal cardiac morphology. We also propose that mice bearing homozygous familial hypertrophic cardiomyopathy-causing mutations may provide useful tools for predicting the severity of disease that these mutations will cause in humans.

Design and fabrication of a 40-MHz annular array transducer

This paper investigates the feasibility of fabricating a five-ring, focused annular array transducer operating at 40 MHz. The active piezoelectric material of the transducer was a 9-/spl mu/m thick polyvinylidene fluoride (PVDF) film. One side of the PVDF was metallized with gold and forms the ground plane of the transducer. The array pattern of the transducer and electrical traces to each annulus were formed on a copper-clad polyimide film. The PVDF and polyimide were bonded with a thin layer of epoxy, pressed into a spherically curved shape, then back filled with epoxy. A five-ring transducer with equal area elements and 100 /spl mu/m kerfs between annuli was fabricated and tested. The transducer had a total aperture of 6 mm and a geometric focus of 12 mm. The pulse/echo response from a quartz plate located at the geometric focus, two-way insertion loss (IL), complex impedance, electrical crosstalk, and lateral beamwidth all were measured for each annulus. The complex impedance data from each element were used to perform electrical matching, and the measurements were repeated. After impedance matching, f/sub c/ /spl sim/ 36 MHz and -6-dB bandwidths ranged from 31 to 39%. The ILs for the matched annuli ranged from -28 to -38 dB.

Onset of Cardiac Function During Early Mouse Embryogenesis Coincides With Entry of Primitive Erythroblasts Into the Embryo Proper

When cardiac function and blood flow are first established are fundamental questions in mammalian embryogenesis. The earliest erythroblasts arise in yolk sac blood islands and subsequently enter the embryo proper to initiate circulation. Embryos staged 0 to 30 somites (S) were examined in utero with 40- to 50-MHz ultrasound biomicroscopy (UBM)-Doppler, to determine onset of embryonic heartbeat and blood flow and to characterize basic physiology of the very early mouse embryonic circulation. A heartbeat was first detected at 5 S, and blood vascular flow at 7 S. Heart rate, peak arterial velocity, and velocity-time integral showed progressive increases that indicated a dramatically increasing cardiac output from even the earliest stages. In situ hybridization revealed an onset of the heartbeat coincident with the appearance of yolk sac–derived erythroblasts in the embryo proper at 5 S. Early maturation of the circulation follows a tightly coordinated program.

Noninvasive, In Utero Imaging of Mouse Embryonic Heart Development With 40-MHz Echocardiography

BACKGROUND The increasing number of transgenic and targeted mutant mice with embryonic cardiac defects has resulted in the need for noninvasive techniques to examine cardiac structure and function in early mouse embryos. We report the first use of a novel 40-MHz ultrasound imaging system in the study of mouse cardiac development in utero. METHODS AND RESULTS Transabdominal scans of mouse embryos staged between 8.5 and 13.5 days of gestation (E8.5 to E13.5) were obtained in anesthetized mice. Atrial and ventricular contractions could be discerned from E9.5, and changes in cardiac morphology were observed from E9.5 to E13.5. Hyperechoic streaming patterns delineated flow through the umbilical, vitelline, and other major blood vessels. Diastolic and systolic ventricular areas were determined by planimetry of the epicardial borders, and fractional area change was measured as an index of contractile function. Significant increases in ventricular size were documented at each stage between E10.5 and E13.5, and the ability to perform serial imaging studies over 3 days of embryonic development is described. Finally, the detection of vascular cell adhesion molecule 1 (VCAM-1) homozygous null mutant embryos demonstrates the first example of noninvasive, in utero analysis of cardiac structure and function in a targeted mouse mutant. CONCLUSIONS We used 40-MHz echocardiography to identify key elements of the early mouse embryonic cardiovascular system and for noninvasive dimensional analysis of developing cardiac ventricles. The ability to perform serial measurements and to detect mutant embryos with cardiac defects highlights the usefulness of the technique for investigating normal and abnormal cardiovascular development.

Neonatal cardiomyopathy in mice homozygous for the Arg403Gln mutation in the α cardiac myosin heavy chain gene

Heterozygous mice bearing an Arg403Gln missense mutation in the alpha cardiac myosin heavy chain gene (alpha-MHC403/+) exhibit the histopathologic features of human familial hypertrophic cardiomyopathy. Surprisingly, homozygous alpha-MHC403/403 mice die by postnatal day 8. Here we report that neonatal lethality is caused by a fulminant dilated cardiomyopathy characterized by myocyte dysfunction and loss. Heart tissues from neonatal wild-type and alpha-MHC403/403 mice demonstrate equivalent switching of MHC isoforms; alpha isoforms in each increase from 30% at birth to 70% by day 6. Cardiac dimensions and function, studied for the first time in neonatal mice by high frequency (45 MHz) echocardiography, were normal at birth. Between days 4 and 6, alpha-MHC403/403 mice developed a rapidly progressive cardiomyopathy with left ventricular dilation, wall thinning, and reduced systolic contraction. Histopathology revealed myocardial necrosis with dystrophic calcification. Electron microscopy showed normal architecture intermixed with focal myofibrillar disarray. We conclude that 45-MHz echocardiography is an excellent tool for assessing cardiac physiology in neonatal mice and that the concentration of Gln403 alpha cardiac MHC in myocytes influences both cell function and cell viability. We speculate that variable incorporation of mutant and normal MHC into sarcomeres of heterozygotes may account for focal myocyte death in familial hypertrophic cardiomyopathy.

40 MHz Doppler characterization of umbilical and dorsal aortic blood flow in the early mouse embryo.

Physiological study of the developing mouse circulation has lagged behind advances in molecular cardiology. Using an innovative high-frequency Doppler system, we noninvasively characterized circulatory hemodynamics in early mouse embryos. We used image-guided 43 MHz pulsed-wave (PW) Doppler ultrasound to study the umbilical artery and vein, or dorsal aorta in 109 embryos. Studies were conducted on embryonic days (E) 9.5-14.5. Heart rate, peak blood flow velocities, and velocity time integrals in all vessels increased from E9.5-14.5, indicating increasing stroke volume and cardiac output. Heart rate, ranging from 192 bpm (E9.5) to 261 bpm (E14.5), was higher than previously reported. Placental impedance, assessed by the time delay between the peaks of the umbilical arterial and venous waveforms and by venous pulsatility, decreased with gestation. Acceleration time, a load-independent Doppler index of cardiac contractility, remained constant but seemed sensitive to heart rate. High-frequency PW Doppler is a powerful tool for the quantitative, noninvasive investigation of early mouse circulatory development.

40-MHz echocardiography scanner for cardiovascular assessment of mouse embryos

Congenital heart disease results from genetic defects that are manifested at early stages of embryogenesis. The mouse is the preferred animal model for studies of mammalian embryonic development and for an increasing number of human disease models. A number of genes identified in the mouse are critical for normal cardiovascular development, but an understanding of the underlying mechanisms regulating heart development is still incomplete, in part because of the lack of methods to measure hemodynamics in live mouse embryos. We describe the development of a 40-MHz ultrasound scanner, which allows image-guided continuous-wave and pulsed Doppler blood flow measurements in mouse embryos, in utero, at the critical early developmental stages. Doppler waveforms acquired from mouse embryonic umbilical vessels, descending aorta, and cardiac ventricles are presented to demonstrate the utility of the method. By combining image-guided ultrasound Doppler with the many available mouse mutants, this approach should lead to new insights into embryonic cardiovascular structure-function relationships.

Embryonic Heart Failure in NFATc1-/- Mice. Novel Mechanistic Insights From In Utero Ultrasound Biomicroscopy

Gene targeting in the mouse has become a standard approach, yielding important new insights into the genetic factors underlying cardiovascular development and disease. However, we still have very limited understanding of how mutations affect developing cardiovascular function, and few studies have been performed to measure altered physiological parameters in mouse mutant embryos. Indeed, although in utero lethality due to embryonic heart failure is one of the most common results of gene targeting experiments in the mouse, the underlying physiological mechanisms responsible for embryonic demise remain elusive. Using in utero ultrasound biomicroscopy (UBM), we studied embryonic day (E) 10.5 to 14.5 NFATc1−/− embryos and control littermates. NFATc1−/−mice, which lack outflow valves, die at mid-late gestation from presumed defects in forward blood flow with resultant heart failure. UBM showed increasing abnormal regurgitant flow in the aorta and extending into the embryonal–placental circulation, which was evident after E12.5 when outflow valves normally first develop. Reduced NFATc1−/− net volume flow and diastolic dysfunction contributed to heart failure, but contractile function remained unexpectedly normal. Among 107 NFATc1−/− embryos imaged, only 2 were observed to be in acute decline with progressive bradyarrhythmia, indicating that heart failure occurs rapidly in individual NFATc1−/− embryos. This study is among the first linking a specific physiological phenotype with a defined genotype, and demonstrates that NFATc1−/−embryonic heart failure is a complex phenomenon not simply attributable to contractile dysfunction.

Large-scale reorganization of the tonotopic map in mouse auditory midbrain revealed by MRI

The cortex is thought to be the primary site of sensory plasticity, particularly during development. Here, we report that large-scale reorganization of the mouse auditory midbrain tonotopic map is induced by a specific sound-rearing environment consisting of paired low- (16 kHz) and high-frequency (40 kHz) tones. To determine the potential for plasticity in the mouse auditory midbrain, we used manganese-enhanced MRI to analyze the midbrain tonotopic maps of control mice during normal development and mice reared in the two-tone (16 + 40 kHz) environment. We found that the tonotopic map emerged during the third postnatal week in normal mice. Before 3 weeks, a larger percentage of auditory midbrain responded to each of the suprathreshold test frequencies, despite the fact that the primary afferent projections are in place even before hearing onset. By 3 weeks, the midbrain tonotopic map of control mice was established, and manganese-enhanced MRI showed a clear separation between the 16- and 40-kHz responses. Two-tone rearing dramatically altered the appearance of these discrete frequency-specific responses. A significant volume of the auditory midbrain became responsive to both rearing frequencies, resulting in a large-scale reorganization of the tonotopic map. These results indicate that developmental plasticity occurs on a much greater scale than previously appreciated in the mammalian auditory midbrain.

Operational verification of a 40-MHz annular array transducer

An experimental system to take advantage of the imaging capabilities of a 5-ring polyvinylidene fluoride (PVDF)-based annular array is presented. The array has a 6-mm total aperture and a 12-mm geometric focus. The experimental system is designed to pulse a single element of the array and then digitize the received data of all array channels simultaneously. All transmit/receive pairs are digitized and then the data are post-processed with a synthetic-focusing technique to achieve an enhanced depth of field (DOF). The performance of the array is experimentally tested with a wire phantom consisting of 25-mum diameter wires diagonally spaced at 1-mm by 1-mm intervals. The phantom permitted the efficacy of the synthetic-focusing algorithm to be tested and was also used for two-way beam characterization. Experimental results are compared to a spatial impulse response method beam simulation. After synthetic focusing, the two-way echo amplitude was enhanced over the range of 8 to 19 mm and the 6-dB DOF spanned from 9 to 15 mm. For a wire at a fixed axial depth, the relative time delays between transmit/receive ring pairs agreed with theoretical predictions to within plusmn2 ns. To further test the system, B-mode images of an excised bovine eye were rendered