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TGF[Beta] family members are implicated in cardiac organogenesis, growth control, and positional information, including the direction of cardiac looping. However, genetic analysis of TGF[Beta] signaling in mice has been confounded, in some cases, by noncardiac and generalized defects. Hence, deciphering TGF[Beta] function in myocardium would benefit from cardiac-restricted mutations. We developed a constitutively activated type I receptor, [ALK5.sup.L193A,P194A,T204D], and directed it to embryonic myocardium in transgenic mice. Expression of the activated ALK5 gene arrests looping morphogenesis and causes a linear, dilated, hypoplastic heart tube, despite normal expression of Nkx2.5 and dHAND, cardiogenic transcription factors whose absence provokes a similar phenotype. Ventricular hypoplasia was associated with precocious induction of the cyclin-dependent kinase inhibitor, p21. Thus, an ALK5-sensitive pathway mediates looping, perhaps through control of cardiac myocyte proliferation. Key Words: ALK5; cardiac development; looping; p21; TGF[Beta].

Ultrasound (US), with or without microbubbles, enhances gene transfer in cultured cells, but the effect is modest. We tested if attaching DNA to albumin microbubbles during bubble synthesis could enhance gene expression. Plasmid DNA was loaded on the albumin shell over a range of concentrations (500 to 10,000 μg/mL). Optimal gene expression occurred with loading doses of 4000 μg DNA/mL (4k-loaded bubbles). These microbubbles had diameters of 2.4 ± 0.7 μm and carried 40 pg DNA/microbubble. DNA-loaded microbubbles had optimal transfection at higher delivered doses of DNA than unloaded bubbles mixed with plasmid. The 4k-loaded bubbles demonstrated a fivefold (p = 0.0003) increase in luciferase reporter expression over that with unloaded bubbles. Similarly, transfection efficiency was better for 4k-loaded microbubbles than unloaded microbubbles (41 ± 3% vs. 9 ± 3%, p < 0.0001). DNA loading of microbubbles enhances gene expression and transfection efficiency in US-targeted transfection in vitro and may represent an improved avenue for therapeutic gene delivery in vivo. (E-mail: peter.frenkel@utsouthwestern.edu) [Copyright &y& Elsevier]

: ObjectivesThis study was undertaken to optimize echocardiographic parameters for successful gene delivery to the heart and to extend the method from adenoviral to plasmid deoxyribonucleic acid (DNA).: BackgroundWe have previously shown that ultrasound-targeted microbubble destruction can direct tissue expression of adenoviral transgenes to the heart. The optimal echocardiographic parameters for this technique have not been reported.: MethodsAdenoviral or plasmid DNA encoding the luciferase reporter gene was incorporated into liposome microbubbles and infused intravenously into anesthetized rats. We systematically evaluated the effects of ultrasound parameters known to influence microbubble destruction, including electrocardiogram (ECG) triggering, ultrasound frequency, mode of ultrasound, and mechanical index, on gene expression in rat myocardium four days after treatment. In addition, gene expression in heart, liver, and skeletal muscle were compared between adenoviral and plasmid DNA.: ResultsOptimal ultrasound parameters for this technique include low-transmission frequency (1.3 MHz), maximal mechanical index, and ECG triggering to allow complete filling of the myocardial capillary bed by microbubbles. No difference was seen between ultraharmonics and power Doppler mode. Using adenoviral DNA, optimal ultrasound parameters yielded myocardial luciferase activity on the order of 104 relative light units/mg protein/min but with even higher liver activity. Plasmid DNA was expressed in rat myocardium at similar levels but without detectable liver expression.: ConclusionsUltrasound-targeted microbubble destruction can be used to deliver adenoviral or plasmid DNA to the myocardium. This technique holds great promise in applying the rapidly expanding repertoire of gene therapies being developed for cardiac disease. [Copyright &y& Elsevier]