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. 2011 Apr;61(2):109-18.

Capacity for resolution of Ras-MAPK-initiated early pathogenic myocardial hypertrophy modeled in mice

Bih-Rong Wei  1 , Philip L Martin, Shelley B Hoover, Elizabeth Spehalski, Mia Kumar, Mark J Hoenerhoff, Julian Rozenberg, Charles Vinson, R Mark Simpson
Affiliations

Capacity for resolution of Ras-MAPK-initiated early pathogenic myocardial hypertrophy modeled in mice

Bih-Rong Wei et al. Comp Med. 2011 Apr.

Abstract

Activation of Ras signaling in cardiomyocytes has been linked to pathogenic myocardial hypertrophy progression and subsequent heart failure. Whether cardiomyopathy can regress once initiated needs to be established more fully. A 'tet-off' system was used to regulate expression of H-Ras-G12V in myocardium to examine whether Ras-induced pathogenic myocardial hypertrophy could resolve after removal of Ras signaling in vivo. Ras activation at weaning for 2 wk caused hypertrophy, whereas activation for 4 to 8 wk led to cardiomyopathy and heart failure. Discontinuing H-Ras-G12V transgene expression after cardiomyopathy onset led to improved survival and cardiomyopathy lesion scores, with reduced heart:body weight ratios, demonstrating the reversibility of early pathogenic hypertrophy. Activation of Ras and downstream ERK 1/2 was associated with elevated expression of proliferating cell nuclear antigen and cyclins B1 and D1, indicating cell-cycle activation and reentry. Coordinate elevation of broad-spectrum cyclin-dependent kinase inhibitors (p21, p27, and p57) and Tyr15 phosphorylation of cdc2 signified the activation of cell-cycle checkpoints; absence of cell-cycle completion and cardiomyocyte replication were documented by using immunohistochemistry for mitosis and cytokinesis markers. After resolution of cardiomyopathy, cell-cycle activators and inhibitors examined returned to basal levels, a change that we interpreted as exit from the cell cycle. Cardiac cell-cycle regulation plays a role in recovery from pathogenic hypertrophy. The model we present provides a means to further explore the underlying mechanisms governing cell-cycle capacity in cardiomyocytes, as well as progression and regression of pathogenic cardiomyocyte hypertrophy.

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Figures

Figure 1.
Figure 1.
Study design, experimental animal groups, and hypertrophic phenotype induced by Ras activation. (A) Cardiac specific expression of the Ras-12V transgene was activated for different periods of time through use of a tet-off system in M/R mice (Ras-on, solid arrow). Mice experienced 2 wk or 4 wk of Ras activation, after which Ras activation was discontinued for 0, 1, or 4 wk (Ras-off, dashed arrow). A group of mice receiving 8 consecutive weeks of Ras-12V expression served as late-stage disease controls for mice afforded 4 wk of recovery after 4 wk of Ras activation. Additional control mice within each treatment included wild type (WT), M, R, and M/R mice with no Ras-12V transgene expression. Numbers (n) of animals in each group (M/R, control [C]) are indicated. (B) Gross photograph of representative hearts from an M/R mouse with heart enlargement (left) and a WT mouse. (C, D) Representative photomicrographs of biventricular short-axis (transverse) tissue sections from (C) an M/R heart (4 wk Ras-on) with pathogenic myocardial hypertrophy and (D) a WT heart. Hematoxylin and eosin stain; bar, 1 mm. (E) Hypertrophic cardiomyocytes with multiple enlarged nuclei (karyomegaly, black arrows) interspersed among less affected cardiomyocytes (red arrows) in a tissue section from a representative M/R mouse heart (4 wk Ras-on). Interstitial cellularity was increased. Hematoxylin and eosin stain; bar, 15 μm. (F) Essentially normal cardiomyocytes from a representative control mouse, shown for comparison. Hematoxylin and eosin stain; bar, 15 μm. (G) Immunohistochemical labeling of active caspase 3 in hypertrophic cardiomyocytes, indicating ongoing apoptosis during advanced-stage cardiomyopathy in M/R mice with 8 wk of Ras activation. Positively labeled myocytes (brown chromogen) are featured by vacuolization and loss of striations. Mononuclear cells infiltrate amid degenerate cardiomyocytes in areas often associated with fibrosis and positive active caspase 3 labeling. Chromogen, 3,3′-diaminobenzidine tetrahydrochloride. (Insert photomicrograph): Serial tissue section of heart shown in G, with antibody preabsorbed with active caspase 3 derived immunogenic peptide, used as control. Chromogen, 3,3′-diaminobenzidine tetrahydrochloride. Bar, 25 μm.
Figure 2.
Figure 2.
Criteria developed for assigning semiquantitative histopathologic myocardial lesion scores for M/R and control mice include 4 parameters: (1) affected myocardial area: assessment of affected area represented estimates of affected surface area made from examining 5 microscopic fields by using the 10× objective lens, thereby permitting evaluation of the entire cross-sectional area of tissue sections taken from the midregions through both ventricles; (2) pathologic hypertrophic myocardial phenotype: phenotypes included myofiber hypertrophy, anisocytosis, anisokaryosis, and karyomegaly distinguishable from that of control mouse hearts, which may have had occasional enlarged nuclei within normal cardiac myofiber cytomorphology; (3) cardiac myocyte degeneration: signs of cardiac myocyte degeneration included myofiber vacuolization, fragmentation, and loss of cross striations. Other features included occasional necrosis or apoptosis and mineralization of cells; and (4) inflammation and fibrosis: evaluations for inflammation and fibrosis included assessments for mononuclear leukocyte infiltration and interstitial connective tissue. *, Combinations of mild infrequent hypertrophy accompanied by limited inflammation and fibrosis were assigned a lesion score 1 and interpreted to represent some degree of resolution of pathogenic myocardial hypertrophy. These changes were included with more mild hypertrophic changes in the absence of any significant inflammation and fibrosis within lesion score 1, observed during induction.
Figure 3.
Figure 3.
Survival of M/R mice with continuous Ras activation (Ras-on, solid line, 8 wk, n = 21) or with 4 wk Ras activation (solid line) followed by an additional 4 wk subsequent to discontinuation of Ras (dashed line, n = 27). Ras activation was begun at weaning (week 0). Mice were removed from study once they developed advanced heart failure or at study end (week 8).
Figure 4.
Figure 4.
Extent of pathogenic myocardial hypertrophy development in M/R mice during and after Ras activation. Percentage of mice exhibiting cardiac lesion scores of 1 through 3 with (A) 2 wk or (B) 4 wk of Ras activation (Ras-on). Each bar represents mice given different periods during which Ras activation was discontinued for 0, 1, or 4 wk (Ras-off). Lesion scores (described in Table 1) of 1 (light gray), 2 (hatched), and 3 (black) are indicated for mice in each group. Significant difference was shown between the groups with Ras discontinuation for 1 wk (peak disease severity) and 4 wk. C) Heart weight (HW) to body weight (BW) ratios for M/R mice with 4 wk of Ras activation, followed by 0, 1, or 4 wk of Ras-off (hatched bars), as compared with matched control mice (solid bars). All HW:BW data are expressed relative to those of the 4-wk control mice (4 wk Ras-on, 0 wk Ras-off group [*]) set at a value of 1.0 (that is, the norm-referenced HW:BW ratio). Each group of control mice was matched individually for age and time on study for each respective M/R treatment group. Numbers of mice in groups are shown in Figure 1. Statistical analysis was performed between M/R mice and matched controls as well as between M/R groups; P values are indicated.
Figure 5.
Figure 5.
Ras and pERK1/2 levels in M/R hearts. Representative immunohistochemistry labeling of (A) Ras and (B) pERK1/2 in serial sections of myocardium from an M/R mouse with 2 wk of Ras activation. In M/R hearts with 2-wk Ras activation, immunohistochemistry revealed both nuclear and cytoplasmic localization of pERK1/2 (arrows). Mostly cytoplasmic accumulation of pERK1/2 was observed after 4 wk of Ras activation (inset photomicrograph). (C) Ras immunohistochemistry on an M/R control heart lacking Ras-12V transgene induction revealed minimal labeling. Double immunofluorescent labeling of (D) Ras and (E) pERK1/2 was used to further demonstrate representative colocalization of Ras and pERK1/2 within a hypertrophic M/R heart with 4 wk of Ras activation. F) Optically merged photomicrograph to document coexpression of Ras and pERK1/2 in the same hypertrophic cardiomyocytes (yellow). (G through I) Representative (G) Ras and (H) pERK1/2 expression in serial sections from an M/R heart with 4 wk Ras-on and subsequent 4 wk Ras-off. Minimal Ras production and residual pERK occur in myocardium lacking hypertrophic myocytes. (I) A negative control lacking primary antibody. Overall, Ras and pERK1/2 immunohistochemical assays were performed on 49 mice, including 35 M/R mice, and were distributed among the various treatment groups. Bar, 50 μm.
Figure 6.
Figure 6.
Western blot analysis of Ras, pERK1/2, ANF, and cell-cycle regulators in individual hearts of M/R and control (C) mice. Samples during and after induction of Ras-12V expression are included. Representative results are shown for mice with 2 and 4 wk of Ras activation (Ras-on), as well as for mice that had Ras discontinued for either 0, 1, or 4 wk after initial induction (Ras-off). Lesion score of each heart (as defined in Figure 2) is indicated. Analyses are shown for Ras, pERK1/2, total ERK1/2, ANF, and cell-cycle associated proteins, PCNA, cyclin B1, cyclin D1, pY15-cdc2, and the cyclin-dependent kinase inhibitors p21, p27, and p57.
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