Reversibility of PRKAG2 glycogen-storage cardiomyopathy and electrophysiological manifestations

Abstract

Background: PRKAG2 mutations cause glycogen-storage cardiomyopathy, ventricular preexcitation, and conduction system degeneration. A genetic approach that utilizes a binary inducible transgenic system was used to investigate the disease mechanism and to assess preventability and reversibility of disease features in a mouse model of glycogen-storage cardiomyopathy.

Methods and results: Transgenic (Tg) mice expressing a human N488I PRKAG2 cDNA under control of the tetracycline-repressible alpha-myosin heavy chain promoter underwent echocardiography, ECG, and in vivo electrophysiology studies. Transgene suppression by tetracycline administration caused a reduction in cardiac glycogen content and was initiated either prenatally (Tg(OFF(E-8 weeks))) or at different time points during life (Tg(OFF(4-16 weeks)), Tg(OFF(8-20 weeks)), and Tg(OFF(>20 weeks))). One group never received tetracycline, expressing transgene throughout life (Tg(ON)). Tg(ON) mice developed cardiac hypertrophy followed by dilatation, ventricular preexcitation involving multiple accessory pathways, and conduction system disease, including sinus and atrioventricular node dysfunction.

Conclusions: Using an externally modifiable transgenic system, cardiomyopathy, cardiac dysfunction, and electrophysiological disorders were demonstrated to be reversible processes in PRKAG2 disease. Transgene suppression during early postnatal development prevented the development of accessory electrical pathways but not cardiomyopathy or conduction system degeneration. Taken together, these data provide insight into mechanisms of cardiac PRKAG2 disease and suggest that glycogen-storage cardiomyopathy can be modulated by lowering glycogen content in the heart.

Figure 1

Modulation of transgene expression. A, Tetracycline-controlled transcriptional activator (tTA) system under the control of a cardiac-specific myosin heavy chain promoter (α-MHC) was used to regulate expression of the PRKAG2 transgene, which carried the disease-causing Asn488Ile mutation (PRKAG2^N488I). Disease-causing transgene expression is suppressed in the presence of tetracycline (or doxycycline), which binds to the tTA protein. Pro indicates promoter; P_CMV, human cytomegalovirus promoter IE; VP16, viral protein 16; tetR, tetracycline repressor; and tetO, tetracycline operator. B through E, Histological heart sections from a WT mouse (B) and transgenic (C through E) mice, stained with periodic acid Schiff for glycogen detection in purple, show decreased glycogen content in Tg^OFF (D: Tg^{OFF(E-8 weeks)}; E: Tg^{OFF(4–16 weeks)}) compared with Tg^ON (C) hearts. Bar=50 μm.

Figure 2

Echocardiographic and ECG parameters. A, Cardiac hypertrophy seen in Tg^ON mice starting at 8 weeks of age (*,** WT vs Tg^ON: P<0.001) is delayed by prenatal (*Tg^ON vs Tg^{OFF(E-8 weeks)}, P<0.001) and early (*Tg^ON vs Tg^{OFF(4–16 weeks)}, P<0.001) transgene suppression and reversed by transgene suppression later in life (Tg^ON at 8 weeks vs Tg^{OFF(8–20 weeks)} at 16 weeks, P=0.022, paired t test). B, Left ventricular dilation starting at 8 weeks of age in Tg^ON mice with enlarged left ventricular end-diastolic diameters (*WT vs Tg^ON, P<0.001; **WT vs Tg^ON, P=0.025; ***WT vs Tg^ON, P=0.006) is delayed by early transgene suppression (*Tg^ON vs Tg^{OFF(E-8 weeks)}, P=0.032; Tg^ON vs Tg^{OFF(4–16 weeks)}, P=0.021; Tg^ON vs Tg^{OFF(8–20 weeks)}, P<0.001) and reversed by transgene suppression at 20 weeks of age (***Tg^ON vs Tg^{OFF(>20 weeks)}, P=0.014). C, Cardiac dysfunction as shown in percentage of fractional shortening begins at 8 weeks of age in Tg^ON mice (*WT vs Tg^ON, P<0.001; **WT vs Tg^ON, P<0.001; ***WT vs Tg^ON, P=0.004). Transgene suppression early in life preserves (*Tg^ON vs Tg^{OFF(E-8 weeks)}, P<0.001; Tg^ON vs Tg^{OFF(4–16 weeks)}, P=0.002) and later in life restores (*Tg^ON vs Tg^{OFF(8–20 weeks)}, P=0.002) cardiac function. *,**,***Independent t tests; data shown as mean and SD. D, Half of Tg^ON but no WT mice develop ventricular preexcitation as seen on surface ECG starting at 4 weeks of age (*WT vs Tg^ON, P<0.001). No Tg^{OFF(E-8 weeks)} mice ever develop ventricular preexcitation. Transgene suppression starting at 4 weeks of life causes a loss of ventricular preexcitation (***Tg^ON at 4 weeks vs Tg^{OFF(4–16 weeks)} at 8 weeks, P<0.001, paired t test; **Tg^ON vs Tg^{OFF(4–16 weeks)}, P<0.001), whereas transgene suppression after 8 weeks of age does not reverse signs of ventricular preexcitation on surface ECG.

Figure 3

CCS disease. A, Tg^ON mice display ventricular preexcitation on ECG. The right ventricular intracardiac recording (RV IECG) demonstrates short and varying distances between the atrial (A) and ventricular (V) activation signal, which suggests multiple electrical accessory pathways. B and C, Optical mapping of the left ventricle in WT mice shows normal electrical activation initiating at the apex (B), whereas there are multiple areas of preexcitation evident at the base of the heart (arrows) in Tg^ON mice (C). D, Later in life (>20 weeks old), Tg^ON mice develop severe conduction system disease with sinus bradycardia, sinus block, and varying degrees of AV block (arrowheads). Fifty percent of these mice exhibit recurrent long runs of supraventricular tachycardia (SVT).

Figure 4

Histopathology. A, WT mice demonstrate an intact annulus fibrosis separating the interatrial (IAS) from the interventricular (IVS) septum on histopathology with Masson trichrome–stained sections in a 4-chamber view. B, The same anatomic region in Tg^ON mice appears interrupted (arrows) and is enriched with glycogen-containing vacuolated cells (arrowheads). C, Tg^{OFF(E-8 weeks)} mice develop a normal annulus fibrosis without disorganization or interruption, even after transgene reexpression. D, Transgene suppression starting at 4 weeks of life in Tg^{OFF(4–16 weeks)} diminishes the amount of vacuole-containing myocytes within the annulus fibrosis. Bar=100 μm.

Figure 5

Prevention of ventricular preexcitation but not conduction system disease by transgene suppression during prenatal and early postnatal development. A, Right ventricular (RV) intracardiac recordings of Tg^{OFF(E-8 weeks)} mice show clear His signals with normal conduction times. B, Optical mapping of the left ventricle shows normal elliptical “anisotropic” pattern (arrowhead). C, Adenosine administration with atrial pacing provokes AV block without evidence for accessory AV nodal connections. D, At 28 weeks of age, Tg^{OFF(E-8 weeks)} mice show conduction system disease that includes higher-degree AV block (arrows).

Figure 6

Reversibility of ventricular preexcitation with transgene suppression. A and B, Surface ECG lead I recordings of 2 different Tg^{OFF(4–16 weeks)} mice at 3 time points. There was a loss of preexcitation on ECG after transgene suppression. C, Intracardiac electrophysiological testing revealed conduction via accessory pathways on atrial pacing in some of these Tg^{OFF(4–16 weeks)} mice. Transgene reexpression caused reoccurrence of preexcitation in some (A) and no WPW recurrence in others (B). RV indicates right ventricular.