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Disruption of Rho signaling results in progressive atrioventricular conduction defects while ventricular function remains preserved ¹

LEI WEI^*,,,,||,2, GEORGE E. TAFFET^*,,||, DIRAR S. KHOURY^¶, JACQUELINE BO^*,,||, YI LI^||, ATSUKO YATANI^#, M. CRAIG DELAUGHTER^*, RAISA KLEVITSKY^**, TIMOTHY E. HEWETT^**, JEFFREY ROBBINS^**, LLOYD H. MICHAEL^*,,||, MICHAEL D. SCHNEIDER^*,,, MARK L. ENTMAN^*,,|| and ROBERT J. SCHWARTZ^*,,,,||

^* Departments of Medicine,
Molecular and Cellular Biology,
Section of Cardiovascular Sciences,
Center for Cardiovascular Development,
^|| The DeBakey Heart Center, and
^¶ Section of Cardiology, Baylor College of Medicine and The Methodist Hospital, Houston, Texas, USA;
^# UMDNJ-New Jersey Medical School, Cardiovascular Research Institute, Newark, New Jersey, USA; and
^** Molecular Cardiovascular Biology, Children’s Hospital Medical Center, Cincinnati, Ohio, USA

²Correspondence: Cardiovascular Sciences Section, Department of Medicine, Room 506D, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: lwei{at}bcm.tmc.edu

SPECIFIC AIMS

The aim of the present study was to explore the necessity for Rho GTPase activity in normal cardiac development and function through inhibition of Rho GTPase activity in the heart with cardiac-specific overexpression of Rho GDI, an endogenous specific GDP dissociation inhibitor for Rho family proteins, using -myosin heavy chain promoter.

PRINCIPAL FINDINGS

1. Increased cardiac expression of Rho GDI inhibited activity of Rho family GTPases and revealed a negative feedback mechanism for gene regulation of Rho family proteins
The study was focused on two transgenic lines (M2 and L2), in which Rho GDI expression level is 7- and 3-fold higher than the endogenous level in adult ventricles. In adult hearts of the M2 mouse line, content of all three Rho family proteins (RhoA, Rac1 and Cdc42) in the membrane fraction (active form) was significantly decreased vs. nontransgenic littermates (60 to 80%). In contrast, their total protein level was markedly increased in transgenic hearts compared with nontransgenic littermates (10-fold).

2. Increased cardiac expression of Rho GDI led to atrial enlargement and mild ventricular hypertrophy
The M2 mouse line (100%) showed abnormal atrial morphogenesis with varying degrees of severity from the embryonic stage. The left atrium showed finger-like extensions and the right atrium had sac-like structures. L2 transgenic mice (50%) exhibited mild atrial morphological defect and some (50%) had apparently normal atrial morphology. At 4 months of age, both M2 and L2 transgenic mice displayed significant increase in atrial weight (64% and 33%, respectively, vs. nontransgenic mice). A small, but statistically significant increase in ventricular weight (less than 15%) was detected in M2, but not in L2 transgenic mice at 4 months of age.

3. Increased cardiac expression of Rho GDI did not significantly affect cardiac output
Doppler echocardiography performed at 4 wk and at 4 and 7 months of age in the M2 line showed that left ventricular systolic and diastolic functions were largely preserved before and after development of cardiac hypertrophy indicating that Rho GTPases are not required to maintain ventricular contractile function under basal physiological condition.

4. Increased cardiac expression of Rho GDI resulted in progressive AV conduction defects
At 1 wk of age, 75% of transgenic mice showed first-degree AV block. At 4 wk of age, 100% transgenic mice demonstrated first-degree AV block, and some mice also showed second-degree AV block (Fig. 1 ). At 7 months of age, severe atrial arrhythmias were observed at baseline in transgenic mice, including transient sinus arrest, sinus bradycardia, atrial tachycardia and atrial fibrillation, and varying degrees of AV block. Heart rates of transgenic mice were not significantly different compared with controls at 4 wk of age, but were significantly decreased at 4 and 7 months of age. We also observed progressive impairment in AV nodal activation and recovery at an early stage and progressive impairment of atrial recovery in the late stage in transgenic mice. These observations indicate progressive impairment of AV conduction defects in the first 4 wk after birth and progressive impairment of SA node function and atrial conduction in the late stage. Ventricular conduction was normal at all ages. All homozygotes (but no heterozygotes) of L2 line mice at two months of age showed first-degree AV block, indicating that severity of AV conduction defects was proportional to Rho GDI expression level.

View larger version (20K): [in this window] [in a new window]   Figure 1. Rho GDI transgenic hearts presented abnormal AV conduction at young age.A) Body surface ECG (Lead I) in 4-wk-old nontransgenic (NTG) and M2 line transgenic (TG) mice. Top panel: normal conduction in NTG mice. Second panel: first-degree AV block in TG mice revealed by prolonged PR interval. Third panel: second-degree AV block in TG mice with Wenckebach AV conduction revealed by progressive prolongation of the PR interval until AV block was reached. Bottom panel: second-degree AV block with 2:1 AV conduction. B)Incidence of first- and second-degree AV block at baseline in transgenic and nontransgenic mice at 1 and 4 wk of age. First-degree AV block was defined as PR interval > 35 ms. Second-degree AV block consisted of Wenckebach AV conduction and 2:1 AV conduction.

5. Down-regulation of gap junction protein connexin 40 (Cx40) in Rho GDI transgenic heart
Western blot analysis showed marked reduction of Cx40 expression (more than 80%) in the atrium of transgenic mice at 4 wk and 4 months of age, but not at 1 wk of age (Fig. 2 A), indicating that Cx40 expression was gradually decreased in the first 4 wk after birth. Cx40 mRNA levels in atria were similar in transgenic and nontransgenic mice at 1 wk , but were also markedly down-regulated at 4 wk of age (50%) (Fig. 2B ). Immunofluorescent staining showed that expression of Cx40 expression in the atrium and ventricular conduction system was markedly decreased in transgenic mice at 4 wk (Fig. 2C ). Expression of Cx40 was reduced in homozygotes of L2 line mice (50%), but not in heterozygotes of L2 line mice, at 2 months (Fig. 2D ), indicating that reduced Cx40 expression was associated with impairment of AV conduction in transgenic mice.

View larger version (45K): [in this window] [in a new window]   Figure 2. Reduced expression of Cx40 in Rho GDI transgenic hearts. A) Western blot of Cx40 and Cx43 in the atrium of M2 line and nontransgenic (NTG) mice at 1 wk , 4 wk and 4 months of age. B) Real time quantitative RT-PCR analysis of Cx40 and Cx43 in the atrium of M2 line and nontransgenic mice at 1 and 4 wk of age. The level for Cx40 or Cx43 was normalized to that of GAPDH. Normalized level for Cx40 or Cx43 transcript in the transgenic heart sample is shown relative that in the nontransgenic heart sample. *P < 0.05 vs. 1 wk . C)Immunofluorescent staining of Cx40 (Alexa-568) and sarcomeric -actinin (Alexa-488) in the atrium of M2 line and nontransgenic mice at 4 wk of age. Cx40 in the transgenic atrium was dramatically reduced at 4 wk of age compared with nontransgenic hearts. Cx40 expression in Purkinje fibers was also reduced at 4 wk of age. Scale bars are as indicated. D) Western blot of Cx40 in the atrium of heterozygotes or homozygotes of L2 line and nontransgenic (NTG) mice at 2 months of age.

CONCLUSIONS AND SIGNIFICANCE

Using a transgenic approach for cardiac-specific inhibition of Rho family proteins by expressing their endogenous inhibitor, Rho GDI, the present study provides important new insights into physiological function of Rho family proteins in regulating cardiac conduction (Fig. 3 ). We observed that transgenic hearts exhibited AV conduction abnormalities at a young age, and developed bradycardia and severe atrial arrhythmias at old age, while ventricular structure and function were largely preserved in both young and old age. This information will be useful in designing clinical trials directed at inhibiting Rho GTPases in order to treat cardiovascular diseases, including cardiac hypertrophy and failure.

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic diagram of biological roles of Rho GTPases in cardiac development and function revealed by cardiac-specific inhibition of Rho GTPases. In a previous study, we observed that first generation (F1) transgenic mice expressing highest levels of the transgene died around E10.5, and that heart tube looping and ventricular maturation were disrupted in these transgenic embryos. In the present study, we showed that middle-copy number line and low-copy number line mice displayed abnormal atrial morphogenesis. Increased expression of Rho GDI led to electrophysiological alterations resulting in atrial arrhythmias and AV conduction abnormalities, while ventricular contractile function was largely preserved. We have also observed that Cx40 expression was markedly down-regulated in transgenic hearts.

Comparison of the present mouse model to that of cardiac-specific overexpression of RhoA (which resulted in marked sinus and AV dysfunction) suggests that both inhibition and activation of RhoA signaling may alter cardiac rhythm and conduction. Alterations in heart rate or rhythm have been observed in several other murine transgenic models such as angiotensin AT1 receptor and G_q transgenic mice. As both angiotensin II and 1-adrenergic agonists have been shown to activate RhoA signaling pathway in cardiomyocytes, electrophysiological alterations observed in these transgenic models may be related with activation of RhoA signaling pathway.

These observations raise important questions about the molecular basis contribution to AV conduction abnormalities and atrial electrical remodeling in Rho GDI transgenic mice. We observed that Cx40 expression was marked down-regulated in the first 4 wk of age in transgenic mice. Cx40 is expressed specifically in the atrium and the conduction system, and AV conduction and ventricular conduction abnormalities were reported in Cx40 homozygous deficient mice. Marked reduction of Cx40 (50% at RNA level, more than 80% at protein level in the atrium) may contribute in part to progressive AV conduction defects in the first 4 wk of age in Rho GDI transgenic mice, and additional effects of the transgene are also involved. These observations suggest that Rho GDI transgene induces AV conduction defects through regulating expression and/or activity of cardiac proteins (including Cx40) involved in regulating propagation of electrical activity.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/10.1096/fj.03-0664fje;

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