RGS4 causes increased mortality and reduced cardiac hypertrophy in response to pressure overload (original) (raw)
Generation of RGS4-myc transgenic mice. We have shown previously that the RGS3 and RGS4 genes are expressed in the heart (30). We demonstrated recently that overexpression of RGS4 can inhibit the action of phenylephrine and endothelin-1, but not basic fibroblast growth factor, in cultured cardiomyocytes (32). To determine whether RGS4 could inhibit cardiac hypertrophy in an animal model system, we generated transgenic mice with a construct that contained the α-MHC promoter that has been demonstrated previously to direct modest embryonic and increased postnatal ventricular gene transcription (33). The α-MHC promoter was linked to the coding region of rat RGS4 that contained a 3′-triple-myc-1 epitope tag (RGS4-myc). Two founder mice were obtained and used to generate independent lines. In 1 line, there was integration of 5 copies of the transgene (5x-RGS4-myc), and in the second line there was integration of 8 copies of the transgene (8x-RGS4-myc).
Expression of the RGS4-myc fusion protein was analyzed in mice, using both an anti-RGS4 mAb and an anti–myc-1 epitope mAb (data not shown), revealing that ventricular tissue isolated from 5x-RGS4-myc mice contained 4- to 5-fold excess RGS4 protein and, despite a higher copy number, 8x-RGS4-myc mice contained only 2- to 3-fold excess RGS4 protein. The levels of a control protein, 14-3-3β, were identical in 5x-RGS4-myc mice, 8x-RGS4-myc transgenic mice, and their nontransgenic littermates (Figure 1). All 5x-RGS4-myc and 8x-RGS4-myc heterozygote transgenic mice appeared grossly normal at birth and lived for at least 6 months in the absence of experimental intervention. Baseline echocardiographic parameters were identical in 5x-RGS4-myc mice and nontransgenic littermates (Table 1). The 8x-RGS4-myc mice also had normal left ventricular (LV) systolic function, chamber size, and wall thickness. The hearts of 5x-RGS4-myc and 8x-RGS4-myc mice appeared grossly normal. Histologic analysis of 5x-RGS4-myc and 8x-RGS4-myc mouse ventricular tissue revealed normal cardiomyocyte appearance compared with nontransgenic littermates (data not shown).
Characteristics of RGS4-myc cardiac tissue. Increased ventricular RGS4 protein levels in 5x-RGS4-myc (5x) and 8x-RGS4-myc (8x) mice compared with nontransgenic littermate mice (NTG). The 5x-RGS4-myc mice express 4- to 5-fold excess protein, whereas the 8x-RGS4-myc mice express only 2- to 3-fold excess protein. A hamster monoclonal anti-RGS4 antibody and a rabbit polyclonal anti–14-3-3β antibody (to confirm equal loading) were used. Similar results were obtained in 6 hearts in each group.
In vivo echocardiographic assessment, tight TAC
Response of RGS4-myc mice to tight TAC. We next examined the ability of mice to develop cardiac hypertrophy in response to provocative stimulation by using tight TAC. In this procedure, a 60–70% stenosis in the transverse aorta is created by surgical ligation (35–37). In nontransgenic littermates, TAC was tolerated, and nearly 80% of animals survived for at least 1 week (Figure 2). After 1 week, most mice developed significant cardiac hypertrophy that was easily detected by echocardiographic study.
Decreased survival of RGS4-myc transgenic mice after tight TAC. Survival rates after tight TAC in 8x-RGS4-myc mice, 5x-RGS4-myc mice, nontransgenic littermates of 5x-RGS4-myc mice (NTG littermates), and nontransgenic congenic mice (NTG C57BL × SJL TAC).
In contrast, 5x-RGS4-myc transgenic mice did not tolerate tight TAC, and most died within hours to 2 days after the procedure (Figure 2). Echocardiography of premorbid 5x-RGS4-myc animals within 1 to 2 days after tight TAC demonstrated ventricular dilatation, wall thinning, depressed cardiac function, and no appreciable increase in LV mass (Table 1 and Figure 3).
Analysis of cardiac function in 5x-RGS4-myc transgenic mice by M-mode echocardiography. Representative transthoracic M-mode echocardiographic tracings in a 5x-RGS4-myc mouse and a nontransgenic littermate (NTG) at baseline. TAC images shown for nontransgenic (1 week after tight TAC) and 5x-RGS4-myc (premorbid, 1 day after TAC) mice.
χ2 analysis revealed that the decrease in survival observed in 5x-RGS4-myc transgenic mice (11%) 7 days after tight TAC was statistically significant when compared with either nontransgenic littermates (78%) (P = 0.0001) or to nontransgenic congenic C57BL × SJL animals (63%) (P = 0.0001). To exclude the possibility that 5x-RGS4-myc transgenic mice were uniquely sensitive to anesthesia or thoracotomy, sham operations were performed that were identical to the TAC procedure, except that the aorta was not ligated after it was identified by dissection. All 6 sham-operated 5x-RGS4-myc mice tolerated the procedure and survived more than 7 days.
Tight TAC was performed on 8x-RGS4-myc mice, and they also did not tolerate TAC. All 8x-RGS4-myc mice (14:14) died within 3 days of tight TAC (Figure 2), and echocardiography on premorbid mice demonstrated findings identical to those found in premorbid 5x-RGS4-myc mice after tight TAC.
Response of RGS4-myc mice to loose TAC. Given the high mortality rate observed in 5x-RGS4-myc and 8x-RGS4-myc mice following tight TAC, to increase postoperative survival we adopted a modified form of the TAC procedure where a less restrictive band was placed on the transverse aorta, resulting in a stenosis of approximately 40–50%. Either this modified form of TAC (loose TAC) or a sham operation was performed on 5x-RGS4-myc mice and also on nontransgenic mice. All mice were approximately 12 weeks of age and were matched by body weight (Table 2). Cardiac catheterization was performed on all surviving mice at 1 week after surgery to confirm that loose TAC resulted in a physiologically significant stenosis. Catheterization revealed that the mean ascending aortic systolic blood pressure (SBP) was 131 ± 6 mmHg in sham-operated nontransgenic animals and 195 ± 19 mmHg 7 days after loose TAC. In 5x-RGS4-myc animals, the SBP was 127 ± 7 mmHg in the sham-operated group and 176 ± 5 mmHg 7 days after loose TAC (Table 2). Mice were excluded from analysis after loose TAC if the ascending aortic SBP was not at least 2 SDs greater than the mean ascending aortic SBP in sham-operated animals.
In vivo echocardiographic assessment, loose TAC
Seven days after loose TAC, nontransgenic mice had a survival rate of 75%, and 5x-RGS4-myc mice had an improved survival rate of 33%, as compared with a 11% survival rate after tight TAC. The decrease in survival observed in 5x-RGS4-myc transgenic mice following loose TAC compared with nontransgenic mice at 7 days was statistically significant by χ2 analysis (P = 0.0001) (Figure 4).
Decreased survival of RGS4-myc transgenic mice after loose TAC. Survival rates after loose TAC in 5x-RGS4-myc mice (5x-RGS4-myc TAC) and nontransgenic congenic C57BL × SJL mice (C57BL × SJL TAC) and after a sham operation in 5x-RGS4-myc mice (5x-RGS4-myc sham).
Echocardiographic assessment of 5x-RGS4-myc survivors 7 days after loose TAC revealed that they exhibited preserved LV function but had significantly less LV hypertrophy than nontransgenic survivors. Specifically, fractional shortening and LV end-diastolic and end-systolic dimensions were not statistically different in nontransgenic and 5x-RGS4-myc mice 1 week after loose TAC (Table 2). However, indices of LV hypertrophy such as end-diastolic posterior wall and intraventricular septal thickness, relative wall thickness, and M-mode derived LV mass were significantly less in 5x-RGS4-myc animals when compared with nontransgenic mice (Table 2). LV mass/body weight ratios (LVMI) were determined gravimetrically. The LVMI was 3.06 ± 0.10 mg/g in sham-operated nontransgenic mice (n = 5) and 4.24 ± 0.33 mg/g in nontransgenic banded mice 7 days after loose TAC (n = 4; P = 0.0002). In the loose TAC group, the LVMI in nontransgenic animals increased by 39% compared with that in sham-operated mice (Table 2 and Figure 5). In contrast, the LVMI in 5x-RGS4-myc animals increased by only 18% after loose TAC compared with sham-operated 5x-RGS4-myc mice. The LVMI for sham-operated 5x-RGS4-myc mice was 3.00 ± 0.08 mg/g (n = 8) and was 3.53 ± 0.27 mg/g in 5x-RGS4-myc mice (n = 4) 7 days after loose TAC (P = 0.04) (Table 2 and Figure 5). The increase in LVMI observed in 5x-RGS4-myc mice (18%) was significantly smaller than that observed in nontransgenic mice (39%) after loose TAC (P = 0.02) despite similar mean ascending aortic SPBs.
Reduced hypertrophic response to TAC in 5x-RGS4-myc mice. The LV weight/body weight ratio (LVW/BW), an index of LV mass, was determined 7 days after loose TAC or after a sham operation in nontransgenic congenic C57BL × SJL mice (NTG) or in 5x-RGS4-myc mice. Mice were excluded from LVW/BW analysis after TAC if the ascending aortic SBP was less than 2 SDs greater than the mean ascending aortic SBP obtained in sham-operated animals. The error bars represent the SEM.
Histologic analysis of LV tissue of 5x-RGS4-myc and nontransgenic littermates was performed on survivors 7 days after TAC. Nontransgenic animals exhibited typical myofibrillar disarray and fibrosis after TAC, whereas transgenic animals had little or no fibrosis and relatively preserved myofibrillar architecture (Figure 6).
Histologic analysis of cardiac morphology after TAC. Ventricular tissue sections from 5x-RGS4-myc (RGS4-myc) and nontransgenic littermates (NTG) of 5x-RGS4-myc mice were stained with Masson’s trichrome 1 week after loose TAC (original magnification ×200). Note the increased extracellular matrix content (blue color), cardiomyocyte enlargement, and disarray in the nontransgenic cardiac tissue.
Examination of the cardiac hypertrophy gene regulatory program in RGS4-myc mice after loose TAC. In wild-type mice, previous work has established that provocative stimulation by TAC induces the expression of “embryonic” genes such as ANF and MLC-2 (35) and reduces the expression of genes involved in the mitochondrial fatty acid oxidation pathway, such as MCAD (41). Loose TAC was performed on 5x-RGS4-myc mice and nontransgenic C57BL/6 × SJL mice, and ventricular tissue was collected 7 days after the procedure. Northern blot analysis of RNA obtained from ventricular tissue revealed that loose TAC induced a marked increase in ANF expression in nontransgenic, but not 5x-RGS4-myc, animals (Figure 7a). Normalized ANF levels were significantly reduced in 5x-RGS4-myc mice when compared with nontransgenic animals after TAC (Figure 7b) (P = 0.03). Loose TAC caused a marked decrease in MCAD expression in nontransgenic, but not 5x-RGS4-myc, mice. Loose TAC did not affect RGS4-myc mRNA levels in 5x-RGS4-myc animals (Figure 7a).
Northern blot analysis of cardiac gene expression after TAC. (a) Northern blot analysis of ANF, MCAD, RGS4, and GAPDH gene expression after loose TAC. Loose TAC (+) or a sham operation (–) was performed on 5x-RGS4-myc or nontransgenic (NTG) congenic C57BL × SJL mice. (b) Quantitative analysis of cardiac ANF gene expression 7 days after loose TAC. The relative intensities of the resultant bands were quantified in their linear range by automated 2-dimensional computer densitometry. The graph depicts normalized ANF mRNA levels in ventricular tissue obtained from 5x-RGS4-myc or nontransgenic congenic C57BL × SJL mice (NTG). RGS4 mRNA levels were normalized by GAPDH mRNA. Data are presented in arbitrary units and error bars reflect the SE of 4 determinations.
Response of RGS4-myc mice to α-adrenergic and β-adrenergic stimulation. To determine whether the ability of RGS4-myc ventricular tissue to respond to ligands that activate Gi or Gq proteins was impaired, hearts were infused with the α-adrenergic ligand phenylephrine, and intracellular MAP kinase activation was evaluated. Previous work has demonstrated that intraventricular injection of phenylephrine results in a rapid (90 seconds) increase in cardiac MAP kinase activity (22). Nontransgenic littermates, but not 5x-RGS4-myc transgenic mice, exhibited increased MAP kinase activity in response to intracardiac phenylephrine infusion as measured by anti–active MAP kinase (ERK-1) immunoblotting (Figure 8).
Reduced RGS4-myc cardiac response to the Gi/Gq-coupled ligand phenylephrine. The p44 MAP kinase activity was assessed in the ventricular tissue of RGS4-myc mice or their nontransgenic littermates 90 seconds after intracardiac infusion of phenylephrine (or control buffer). Immunoblots of cytosolic extracts were analyzed using an anti–active ERK-1 MAP kinase mAb. Equal amounts of total protein were loaded in each lane. Densitometric analysis of 3 separate experiments was performed using NIH Image software, and data are expressed as the mean signal intensity ± SEM.
To determine whether 5x-RGS4-myc transgenic mice could respond normally to ligands that activate Gs proteins in contrast to phenylephrine, which activates Gi and Gq proteins, we performed in vivo echocardiography and hemodynamic evaluations after graded infusions of the β-adrenergic agonist dobutamine (40, 42). Augmentation of LV contractility, assessed by peak LV +dP/dt and heart rate in 5x-RGS4-myc mice, was comparable to nontransgenic mice, and there was no statistically significant difference in either peak LV +dP/dt or heart rate at any level of dobutamine infusion (Figure 9). This is consistent with the inability of RGS4 to inactivate Gs proteins.
Preserved inotropic and chronotropic response of 5x-RGS4-myc mice to dobutamine. Peak LV +dP/dt (a) and heart rate (b) are shown at baseline and after progressive infusion of dobutamine in 5x-RGS4-myc mice (squares; n = 3) or nontransgenic C57BL × SJL mice (diamonds; n = 3). Peak +dP/dt, maximal first derivative of LV pressure. P = NS between 5x-RGS4-myc mice and nontransgenic mice at any level of dobutamine infusion.
Examination of apoptosis in 5x-RGS4-myc nonsurvivors and survivors after TAC. To investigate whether apoptotic mechanisms play a role in the acute lethality or reduced hypertrophy phenotypes after TAC, ventricular tissue was isolated from 5x-RGS4-myc mice that expired less than 2 days after TAC or that survived 7 days after TAC. Ventricular tissue was also collected from nontransgenic mice that survived 7 days after TAC. Staining of nuclei with a TdT assay revealed a paucity of apoptotic nuclei, and there was no statistically significant difference in apoptotic indices of 5x-RGS4-myc mice that died in less than 2 days or that survived to 1 week after TAC, compared with nontransgenic survivors at 1 week after TAC. Specifically, apoptotic indices were as follows: nontransgenic mice at 1 week after loose TAC, 0.87 ± 0.43%; 5x-RGS4-myc mice at 1 week after loose TAC, 1.1 ± 0.59%; 5x-RGS4-myc mice that expired less than 24 hours after TAC, 1.43 ± 0.87% (Figure 10).
Apoptotic indices are not increased in 5x-RGS4-myc mice after TAC. All figures are of LV myocardium (original magnification ×400), and are representative of TdT assays performed. (a) Positive control: tissue incubated in DNase demonstrating pan staining of nucleic DNA by TdT-labeling assay. (b) 5x-RGS4-myc 1 week after loose TAC. Note paucity of apoptotic nuclei shown by the arrow. (c) 5x-RGS4-myc, death less than 24 hours after TAC. Note absence of apoptotic nuclei. Nonapoptotic nuclei are counterstained with methyl green.