Homeobox protein Hop functions in the adult cardiac conduction system

Abstract

Hop is an unusual homeobox gene expressed in the embryonic and adult heart. Hop acts downstream of Nkx2-5 during development, and Nkx2-5 mutations are associated with cardiac conduction system (CCS) defects. Inactivation of Hop in the mouse is lethal in half of the expected null embryos. Here, we show that Hop is expressed strongly in the adult CCS. Hop-/- adult mice display conduction defects below the atrioventricular node (AVN) as determined by invasive electrophysiological testing. These defects are associated with decreased expression of connexin40. Our results suggest that Hop functions in the adult CCS and demonstrate conservation of molecular hierarchies between embryonic myocardium and the specialized conduction tissue of the mature heart.

Figure 1

Hop expression in the murine CCS. A through C, β-Galactosidase staining of Hop^−/− hearts, demonstrating staining of the ventricular myocardium with strong staining in the CCS (arrowheads). Left and right ventricles (lv, rv) are marked. B, Enlargement of box in A, demonstrating the atrioventricular node and His-bundle (avn/hb) and left bundle branch (lbb, arrowheads). C, Right ventricular septal view of another Hop^−/− heart, showing strong staining of the right bundle branch (rbb, arrowheads). D through F, Microscopic evaluation of the CCS in Hop^+/− (D and F) and Hop^−/− (E) adult mice. F, β-Galactosidase staining of the His-bundle (hb) and right and left bundle branches (rbb, lbb) of a Hop^+/− mouse heart. Homozygous mice appeared similar (data not shown). G and H, Frozen sections of the hearts of newborn Hop^+/− (G) and Hop^−/− (H) littermates, stained for β-galactosidase activity after sectioning. These sections demonstrate a more restrictive pattern of expression as compared with the global cardiac expression seen at E12.5. There is more prominent staining of the endocardium and proximal CCS (arrowhead) as compared with the rest of the myocardium. This myocardial staining is more pronounced than that seen in adults (A through F). lv indicates left ventricular cavity. I and J, β-Galactosidase activity in Hop mutant atria. Grossly stained hearts from adult Hop^+/− (I) and Hop^−/− (J) mice, demonstrating strong β-galactosidase activity in the atria. There was no observed area of increased activity within the atria, such as the region of the sinoatrial node. K through M, Hop expression compared with AchE activity. K, Proximal His-bundle (arrowhead) of the heart from an adult Hop^−/− mouse stained for AChE activity (brown). L, β-Galactosidiase expression (blue) in an adjacent section of the heart from (K). There is subtle staining of the muscle and very strong staining of the His-bundle (arrowhead). M, Overlay of images from K and L, demonstrating colocalization of the AChE and β-galactosidase activities in the His bundle (arrowhead).

Figure 2

Conduction defects in adult Hop^−/− mice. A and B, Surface ECG leads I, II, and III. aVR, aVL, and aVF from a Hop^−/− (A) and Hop^+/+ (B) mouse show QRS-complex widening, QT-interval prolongation, and p-wave widening in the absence of Hop. Right axis deviation is present in the Hop^−/− mouse vs the Hop^+/+ mouse. C and D, Intracardiac electrophysiological recording, with surface ECG leads I, II, aVF, the right atrial electrogram (RAE), and His-bundle electrogram (HBE) from a Hop^−/− (C) and Hop^+/+ mouse (D), demonstrating prolongation of the Hisioventricular (HV) interval in the absence of Hop. Hop^−/− mouse has a HV-interval of 15 ms vs 10 ms for the Hop^+/+ mouse. Vertical scale bar=1 mV; Horizontal scale bar=100 ms.

Figure 3

Connexin40 expression in Hop mutant mice. A and B, Western blot analysis of connexin40 expression in the superior septum. A, Sample blot with Hop genotypes as indicated. B, Expression of connexin40 in three separate blots normalized to α-tubulin expression, ± SEM. There is a reduction in the relative expression of connexin40 in Hop^−/− mice as compared with Hop^+/+ and Hop^+/− littermates. C through H, Immunohistochemistry of the CCS in Hop mutant mice. Serial frozen sections of newborn Hop^+/− (C through E) and Hop^−/− (F through H) hearts were stained for β-galactosidase activity (C and F) and connexin40 (D and E, G and H). Areas of the CCS are outlined. Although there was strong expression of connexin40 seen in the Hop^+/− CCS (D), the Hop^−/− CCS showed expression of connexin40 in a much smaller area that did not extend beyond the proximal AVN and His-bundle (G). This pattern was noted on several serial sections. E and H, Connexin40 staining in Hop mutant atria. Frozen sections of newborn Hop^+/− (E) and Hop^−/− (H) mice stained together show reduced intensity of staining in the Hop^−/− atrium as compared with Hop^+/− mice. This finding was observed throughout the atria. All sections were photographed at the same magnification (20× CCS, 10× atria).