Novel Roles of GATA4/6 in the Postnatal Heart Identified through Temporally Controlled, Cardiomyocyte-Specific Gene Inactivation by Adeno-Associated Virus Delivery of Cre Recombinase

Abstract

GATA4 and GATA6 are central cardiac transcriptional regulators. The postnatal, stage-specific function of the cardiac transcription factors GATA4 and GATA6 have not been evaluated. In part, this is because current Cre-loxP approaches to cardiac gene inactivation require time consuming and costly breeding of Cre-expressing and “floxed” mouse lines, often with limited control of the extent or timing of gene inactivation. We investigated the stage-specific functions of GATA4 and GATA6 in the postnatal heart by using adeno-associated virus serotype 9 to control the timing and extent of gene inactivation by Cre. Systemic delivery of recombinant, adeno-associated virus 9 (AAV9) expressing Cre from the cardiac specific Tnnt2 promoter was well tolerated and selectively and efficiently recombined floxed target genes in cardiomyocytes. AAV9:Tnnt2-Cre efficiently inactivated Gata4 and Gata6. Neonatal Gata4/6 inactivation caused severe, rapidly lethal systolic heart failure. In contrast, Gata4/6 inactivation in adult heart caused only mild systolic dysfunction but severe diastolic dysfunction. Reducing the dose of AAV9:Tnnt2-Cre generated mosaics in which scattered cardiomyocytes lacked Gata4/6. This mosaic knockout revealed that Gata4/6 are required cell autonomously for physiological cardiomyocyte growth. Our results define novel roles of GATA4 and GATA6 in the neonatal and adult heart. Furthermore, our data demonstrate that evaluation of gene function hinges on controlling the timing and extent of gene inactivation. AAV9:Tnnt2-Cre is a powerful tool for controlling these parameters.

Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo

“…I) was constructed by transferring a cTNT-Cre cassette from AAV-TNT-Cre plasmid 11 to PX552 plasmid 12 . A second U6gRNA was further added to generate AAV-U6gRNA 1 -U6gRNA 2 -cTNT-Cre (Addgene #87682).…”

Myocardial transcription factors in diastolic dysfunction: clues for model systems and disease

“…Mice with cardiac-specific overexpression of FOG-2 display normalsized ventricles with enlarged atria; a significant downregulation of MYH6 and SERCA2a was found in ventricular myocardium of FOG-2 transgenic mice [57]. Although unproven as yet, it is tempting to speculate that a severe decrease in GATA-4 and GATA-6 transcriptional activity, which could lead to diastolic dysfunction [51], might be associated, at least in part, with overexpression of FOG-2 in the stressed heart.…”

“…There is a good reverse correlation between these results and the data from conditional GATA-4-/GATA-6-knockout models: Mutant mice with mid-to-late fetal cardio-specific deletion of GATA-4 [50] or combined deletion of GATA-4 and GATA-6 [49] develop dilated cardiomyopathy with severe systolic dysfunction in adulthood. Similarly, the simultaneous loss of both GATA-4 and GATA-6 in perinatal cardiomyocytes causes progressive systolic dysfunction and ventricular dilatation [51].…”

“…Recently, the functions of GATA-4 and GATA-6 in adult heart have been highlighted using mouse models of temporally controlled, cardiomyocyte-specific gene inactivation [51]. In the adult heart, simultaneous cardiomyocytespecific deletion of GATA-4 and GATA-6 leads to dramatically attenuated diastolic function, whereas systolic performance is only slightly impaired.…”

Cardiac Regeneration

“…Our recent study of the postnatal roles of transcription factors GATA4 and GATA6 offers clues as to how regulation of CM maturation can be dissected in vivo. 210 Mosaic gene knockout, achieved by administering a low dose of Cre-expressing adeno-associated virus serotype 9 that only transduced a fraction of CMs, enabled the study of mutant CMs in functionally healthy hearts. In this context, GATA4/6 double knockout CMs were dramatically smaller and less mature than control CMs, indicating that GATA4 and GATA6 are crucial cell autonomous regulators of postnatal cardiomyocyte growth and maturation.…”