Accuracy of echocardiographic estimates of left ventricular mass in mice.

Genetically modified mice have created the need for accurate noninvasive left ventricular mass (LVM) measurements. Recent technical advances provide two-dimensional images adequate for LVM calculation using the area-length method, which in humans is more accurate than M-mode methods. We compared the standard M-mode and area-length methods in mice over a wide range of LV sizes and weights (62-210 mg). Ninety-one CD-1 mice (38 normal, 44 aortic banded, and 9 inherited dilated cardiomyopathy) were imaged transthoracically (15 MHz linear transducer, 120 Hz). Compared with necropsy weights, area-length measurements showed higher correlation than the M-mode method (r = 0.92 vs. 0.81), increased accuracy (bias +/- SD: 1.4 +/- 27.1% vs. 36.7 +/- 51.6%), and improved reproducibility. There was no significant difference between end-systolic and end-diastolic estimates. The truncated ellipsoid estimation produced results similar in accuracy to the area-length method. Whereas current echocardiographic technology can accurately and reproducibly estimate LVM with the two-dimensional, area-length formula in a variety of mouse models, additional technological improvements, rather than refinement of geometric models, will likely improve the accuracy of this methodology.

The left ventricular stress-velocity relation in transgenic mice expressing a dominant negative CREB transgene in the heart.

OBJECTIVE: CREB(A133) transgenic mice that express a dominant negative CREB transcription factor in cardiomyocytes develop a dilated cardiomyopathy that is anatomically, physiologically, and clinically similar to human idiopathic dilated cardiomyopathy. The goals of this study were to quantitate left ventricular (LV) contractility and measure cardiac reserve in CREB(A133) mice by using the relation of end-systolic wall stress to the velocity of fiber shortening. METHODS: A total of 37 adult CD-1 mice (including both nontransgenic and CREB(A133) transgenic mice) were studied with simultaneously acquired high-fidelity instantaneous aortic pressures and 2-dimensionally targeted M-mode echocardiograms. RESULTS: CREB(A133) mice displayed significantly lower values of LV fiber shortening velocities over a wide range of afterloads, and they displayed smaller dobutamine-induced shifts from baseline contractility relations. Counterbalancing effects of differences in LV geometry and aortic pressures resulted in comparable levels of LV wall stress during ejection in both groups. CONCLUSION: These results demonstrate directly that CREB(A133) mice display reduced LV contractility at baseline and decreased cardiac reserve.

Impaired cardiomyocyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart.

1. To assess the specific functions of the cardiac isoform of troponin I (cTnI), we produced transgenic mice that expressed slow skeletal troponin I (ssTnI) specifically in cardiomyocytes. Cardiomyocytes from these mice displayed quantitative replacement of cTnI with transgene-encoded ssTnI. 2. The ssTnI transgenic mice were viable and fertile and did not display increased mortality or detectable cardiovascular histopathology. They exhibited normal ventricular weights and heart rates. 3. Permeabilized transgenic cardiomyocytes demonstrated an increased Ca2+ sensitivity of tension and a lack of contractile responsiveness to cAMP-dependent protein kinase (PKA). Isolated cardiomyocytes from transgenic mice had normal velocities of unloaded shortening but unlike wild-type controls exhibited no enhancement of the velocity of shortening in response to treatment with isoprenaline. Transgenic cardiomyocytes exhibited greater extents of shortening than non-transgenic cardiomyocytes at baseline and after treatment with isoprenaline. 4. The rates of rise of intracellular [Ca2+] and the peak amplitudes of the intracellular [Ca2+] transients were similar in transgenic and wild-type myocytes. However, the half-time of intracellular [Ca2+] decay was significantly greater in the transgenic myocytes. This change in decay of intracellular [Ca2+] was correlated with an increase in the re-lengthening time of the transgenic cells. 5. These changes in cardiomyocyte function in vitro were manifested in vivo as impaired diastolic function both at baseline and after stimulation with isoprenaline. 6. Thus, cTnI has important roles in regulating the Ca2+ sensitivity of cardiac myofibrils and controlling cardiomyocyte relaxation and cardiac diastolic function. cTnI is also required for the normal responsiveness of cardiomyocytes to beta-adrenergic receptor stimulation.

Quantitative evaluation of left ventricular function in a TransgenicMouse model of dilated cardiomyopathy with 2-dimensional contrast echocardiography.

The study of transgenic mouse models of human cardiovascular disease has been limited by the small size and high heart rate of the mouse heart. Advances in digital echocardiographic imaging equipment have provided the high spatial and temporal resolution necessary for 2-dimensional (2D) in vivo imaging of the mouse heart. The goal of this study was to test the use of contrast-enhanced 2D echocardiography to quantitatively assess left ventricular (LV) size and function in normal and transgenic mice with dilated cardiomyopathy. Images were obtained with a 12-MHz broadband transducer in the parasternal short-axis view in 8 control mice and 8 transgenic mice with dilated cardiomyopathy resulting from expression of a dominant-negative CREB transcription factor in the heart. LV opacification was achieved with injections of human albumin microspheres, injectable suspension (Optison) (15 to 30 microliter bolus). LV area was measured throughout the cardiac cycle with manual frame-by-frame tracing of the endocardial boundary. End-systolic and end-diastolic areas (ESA and EDA) were measured and fractional area change (FAC) calculated in both groups at baseline and during administration of dobutamine (40 microgram/kg/min intravenously). High-quality 2D images, which yielded LV area over time waveforms, were obtained in all mice. Under baseline conditions, ESA was significantly higher and FAC lower in the transgenic mice compared with their controls. During administration of dobutamine, normal mice had significantly smaller ESA and significantly larger FAC compared with baseline conditions, whereas this trend did not reach significance in the transgenic mice. In summary, quantitative assessment of LV size and function may be achieved with contrast-enhanced 2D echocardiographic imaging. This technique promises to facilitate studies of pathophysiology in murine models of human cardiovascular disease.

Dilated cardiomyopathy in transgenic mice expressing a dominant-negative CREB transcription factor in the heart.

Idiopathic-dilated cardiomyopathy (IDC) is a common primary myocardial disease of unknown etiology characterized by progressive biventricular failure, cardiac dilatation, and premature mortality. Here we show that transgenic mice expressing a dominant-negative form of the CREB transcription factor (CREBA133) under the control of the cardiac myocyte-specific alpha-MHC promoter develop dilated cardiomyopathy that closely resembles many of the anatomical, physiological, and clinical features of human IDC. Between 2 and 20 wk of age, these mice develop four chamber cardiac dilatation, decreased systolic and diastolic left ventricular function, and attenuated contractile responses to the beta-adrenergic agonist, isoproterenol. Histologically, the CREBA133 hearts demonstrated both atrophic and hypertrophied fibers as well as significant interstitial fibrosis. These anatomical and hemodynamic changes were associated with hepatic congestion and peripheral edema, intracardiac thrombi, and premature mortality. Taken together, these results implicate CREB as an important regulator of cardiac myocyte function and provide a genetic model of dilated cardiomyopathy which should facilitate studies of both the pathogenesis and therapy of this clinically important disorder.

Evaluation of ventricular and arterial hemodynamics in anesthetized closed-chest mice.

Transgenic and knock-out mice with cardiovascular phenotypes have created the need for methods to measure murine arterial and ventricular mechanics. The aims of this study were (1) to develop a method for the assessment of wall stress (sigma es)-rate corrected velocity of fiber shortening (Vcfc) relation and (2) to assess the feasibility of quantifying global arterial function in normal mice. This method can thus serve as a reference for future studies in genetically altered mice by establishing normal values for comparison. Ten anesthetized closed-chest mice were studied with targeted M-mode echocardiography of the left ventricle recorded simultaneously with high-fidelity aortic pressures. Data were acquired at baseline and during infusions of methoxamine and isoproterenol. Tracings were digitized to obtain end-systolic wall stress (sigma es) and rate-corrected velocity of fiber shortening (Vcfc) relationships and plots of systolic meridional wall stress. Instantaneous aortic pressures and continuous wave aortic Doppler velocities were digitized to study arterial hemodynamics. The Vcfc-sigma es relationship was inverse and linear in all mice studied with a median value of r2 = 0.94. Isoproterenol resulted in an upward shift from the baseline contractility line obtained with methoxamine (mean shift = 2.0 +/- 0.3 circ/sec). Relative to baseline the integral of wall stress decreased with isoproterenol and increased with methoxamine. Methoxamine increased mean arterial pressure and total vascular resistance and decreased heart rate, cardiac output, and arterial compliance. Isoproterenol decreased total vascular resistance and increased cardiac output. Stress-shortening relationships, systolic wall stress, and evaluation of vascular function can be obtained in a closed-chest mouse model.

A novel collection of accessory factors associated with yeast RNA polymerase II.

A relatively simple subset of general transcription factors is sufficient for transcript initiation by RNA polymerase II. However, a recently identified "holoenzyme" contains additional accessory proteins required for mediating signals from some activators (Y-J. Kim et al., 1994, Cell 77, 599-608; A. Koleske and R. Young, 1994, Nature 368, 466-469). By immobilizing RNA polymerase II and associated proteins (RAPs) from a transcriptionally active yeast extract, we have identified a novel collection of proteins distinct from those found in the holoenzyme. The eluted RAP fraction did not contain the holoenzyme components Srb2,4,5 + 6p, Gal11p, or Sug1p, but did include the known transcription factors TFIIB and TFIIS and the three subunits of yeast TFIIF (Ssu71p/Tfg1p, Tfg2p, and Anc1p/Tfg3p). Also isolated as RAPs are two proteins (Cdc73p and Paf1p) with interesting connections to gene expression. Mutations in CDC73 and PAF1 affect cell growth and the abundance of transcripts from a subset of yeast genes (X. Shi et al., Mol. Cell. Biol., 1996 16, 669-676). The RAP fraction may therefore define one or more functional forms of RNA polymerase II distinct from the activator-mediating holoenzyme.