Pressure overload induces early morphological changes in the heart

Abstract

Cardiac hypertrophy, whether pathological or physiological, induces a variety of additional morphological and physiological changes in the heart, including altered contractility and hemodynamics. Events exacerbating these changes are documented during later stages of hypertrophy (usually termed pathological hypertrophy). Few studies document the morphological and physiological changes during early physiological hypertrophy. We define acute cardiac remodeling events in response to transverse aortic constriction (TAC), including temporal changes in hypertrophy, collagen deposition, capillary density, and the cell populations responsible for these changes. Cardiac hypertrophy induced by TAC in mice was detected 2 days after surgery (as measured by heart weight, myocyte width, and wall thickness) and peaked by day 7. Picrosirius staining revealed increased collagen deposition 7 days after TAC; immunostaining and flow cytometry indicated a concurrent increase in fibroblasts. The findings correlated with angiogenesis in TAC hearts; a decrease in capillary density was observed at day 2, with recovery to sham-surgery levels by day 7. Increased pericyte levels, which were observed 2 days after TAC, may mediate this angiogenic transition. Gene expression suggests a coordinated response in growth, extracellular matrix, and angiogenic factors to mediate the observed morphological changes. Our data demonstrate that morphological changes in response to cardiovascular injury occur rapidly, and the present findings allow correlation of specific events that facilitate these changes.

Figure 1

Induction of pressure overload cardiac hypertrophy. Left ventricular wall thickness was increased 2 days after transverse aortic constriction (TAC) and was fully realized by day 7 (A), as measured by echocardiography and heart weight (B), normalized to body weight. Measurement of myocyte width indicated significant myocyte hypertrophy present 7 days after TAC (C). In addition, a trend in lower ejection fraction at day 2 and 28 after TAC was observed (D), implying impaired cardiac function at the acute and chronic stages of cardiac hypertrophy. *P ≤ 0.05 TAC versus sham surgery at the same time point; **P ≤ 0.05 TAC day 2 versus TAC at other time points. n ≥ 6.

Figure 2

Picrosirius-Fast Green staining revealed a significant increase in collagen deposition 7 days after TAC. Left ventricular and septal regions contained basal levels of collagen in day 2 sham-surgery (A) and TAC (B) hearts and in day 7 sham-surgery (C) hearts, whereas a significant increase in fibrosis was observed in day 7 TAC hearts (D). E: Collagen measurements were quantified as the ratio of collagen to protein. *P ≤ 0.05. n ≥ 4. Scale bar = 25 μm.

Figure 3

Increased ECM deposition and myocyte hypertrophy in TAC hearts. Transmission electron microscopic images of left ventricular regions in day 2 sham-surgery (A and C) and TAC (B and D) hearts and in day 7 sham-surgery (E and G) hearts revealed similar levels of dense ECM, whereas a significant increase in ECM deposition was observed in day 7 TAC hearts (F and H). Scale bar = 2 μm.

Figure 4

Immunofluorescent staining suggests alterations in cell populations and vasculature in the hypertrophic heart. Immunofluorescent imaging of day 2 sham-surgery (A–D) and TAC (E-H) and day 7 sham-surgery (I-L) and TAC (M-P) heart sections. CD31 staining (A, E, I, and M) and subsequent quantification (Q) identified a significant decrease in capillary density in day 2 TAC hearts, which was accompanied by an increase in 3G5-labeled pericytes (B, F, J, N, and R). The fibroblast-specific marker 1611 demonstrated an increase in the fibroblast cell population in day 7 TAC hearts (C, G, K, O, and S). The merged images (D, H, L, and P) suggest that localization of cells was altered during the early remodeling process. Interestingly, pericytes localized near great vessels (arrows) in day 2 sham-surgery and TAC hearts and in day 7 sham-surgery hearts were not present in day 7 TAC samples. In addition, the increased fibroblast population 7 days after TAC localized near capillary beds, suggesting a shift from pericyte-regulated, early vascular regression to fibroblast-mediated capillary growth between days 2 and 7 after TAC. In merged images, F-actin was stained by phalloidin (green); nuclei were stained by DAPI (blue). Scale bar = 100 μm; n ≥ 4; *P ≤ 0.05.

Figure 5

Cell proliferation and growth are induced by pressure overload. Cell growth and proliferation were identified by p-histone H3 staining in left ventricular and septal walls of day 2 sham-surgery (A) and TAC (B) hearts, day 7 sham-surgery (C) and TAC (D) hearts, and day 28 sham-surgery (E) and TAC (F) hearts. Quantification of positive nuclei demonstrated a significant increase in proliferation after 2 days in TAC hearts, which was further augmented by day 7 and then returned back to sham-surgery levels by day 28 (G). Although a significant increase in nonmyocyte proliferating cells is present in both day 2 and day 7 TAC mice, no significant number of growing myocytes was present until day 7 in TAC mice, after which numbers returned to sham-surgery levels by day 28 (E). In addition, day 7 TAC hearts had significantly more nonmyocyte proliferating cells, compared with day 2 or 28 TAC mice, and consequently had significantly more proliferating nonmyocytes than growing myocytes (E), indicating that the increased proliferative rate in day 7 TAC mice is due mainly to expanding of resident nonmyocyte populations. F-actin was stained by phalloidin (green); nuclei were stained by DAPI (blue) and p-histone-positive nuclei were stained pink. *P ≤ 0.05 in TAC versus sham surgery at the same time point; **P ≤ 0.05 in TAC day 7 versus TAC at other time points; ***P ≤ 0.05 in myocyte versus nonmyocyte populations. n ≥ 4. Scale bar = 50 μm.

Figure 6

Cell populations in the heart are altered early in response to pressure overload. Fluorescence-activated flow cytometry was used to label and quantify cell populations of the heart at 3, 7, and 14 days after TAC. Equal numbers of cells were analyzed in each group; for percentage calculation, the absolute quantity of cell type was divided by total cells within each sample. *P ≤ 0.05 versus sham surgery and day 2 TAC; **P ≤ 0.05 versus all other groups. n = 4.

Figure 7

Diagram of cell changes in the heart over a time course after pressure overload in a TAC model. N/C, no change; SMC, smooth muscle cells; TAC, transverse aortic constriction.