Quantification of cardiomyocyte hypertrophy by cardiac magnetic resonance: implications for early cardiac remodeling

Abstract

Background: Cardiomyocyte hypertrophy is a critical precursor to the development of heart failure. Methods to phenotype cellular hypertrophy noninvasively are limited. The goal was to validate a cardiac magnetic resonance-based approach for the combined assessment of extracellular matrix expansion and cardiomyocyte hypertrophy.

Methods and results: Two murine models of hypertension (n=18, with n=15 controls) induced by l-N(G)-nitroarginine methyl ester (L-NAME) and pressure overload (n=11) from transaortic constriction (TAC) were imaged by cardiac magnetic resonance at baseline and 7 weeks after L-NAME treatment or up to 7 weeks after TAC. T1 relaxation times were measured before and after gadolinium contrast. The intracellular lifetime of water (τic), a cell size-dependent parameter, and extracellular volume fraction, a marker of interstitial fibrosis, were determined with a model for transcytolemmal water exchange. Cardiomyocyte diameter and length were measured on FITC-wheat germ agglutinin-stained sections. The τic correlated strongly with histological cardiomyocyte volume-to-surface ratio (r=0.78, P<0.001) and cell volume (r=0.75, P<0.001). Histological cardiomyocyte diameters and cell volumes were higher in mice treated with L-NAME compared with controls (P<0.001). In the TAC model, cardiac magnetic resonance and histology showed cell hypertrophy at 2 weeks after TAC without significant fibrosis at this early time point. Mice exposed to TAC demonstrated a significant, longitudinal, and parallel increase in histological cell volume, volume-to-surface ratio, and τic between 2 and 7 weeks after TAC.

Conclusion: The τic measured by contrast-enhanced cardiac magnetic resonance provides a noninvasive measure of cardiomyocyte hypertrophy. Extracellular volume fraction and τic can track myocardial tissue remodeling from pressure overload.

Keywords: hypertrophy; magnetic resonance imaging.

Figure 1

Illustration of cardiomyocyte hypertrophy determination. (A) Cardiomyocytes have an elongated shape with the ratio of the major-to-minor cell-diameter on the order of 4:1. (B) The intracellular lifetime (τ_ic) of a water molecule undergoing diffusional motion within the cell is proportional to the volume-to-surface ratio (V/S), a measure of cell size. (C) CMR measurements of the longitudinal relaxation rate constant (R₁) before and after administration of gadolinium contrast were used to determine τ_ic. The relation between R₁ in myocardial tissue and R₁ of blood starts out linear, with a slope proportional to the extracellular volume fraction. With increasing R₁ in blood, τ_ic ceases to be sufficiently short relative to the extra-to-intracellular R₁ difference. The degree of deviation from linear dependence is sensitive to τ_ic. Red data circles are from measurements in a mouse at 7 weeks after transverse aortic constriction. The solid line is the fit to the data with a two-space water-exchange model, using orthogonal distance regression. The τ_ic for the best fit was 0.4 s. The line with long dashes illustrates how the model fit changes with an increase of τ_ic to 0.8 s, causing a larger deviation from the straight line. (D) Cardiomyocyte length (D_maj) and diameter (D_min) were measured on digitized images of tissue slices stained with fluorescein isothiocyanate-conjugated (FITC-) wheat germ agglutinin. For D_min a cell cross-section with approximately circular shape was chosen (highlighted by red arrow). D_maj corresponded to the distance between intercalated discs, highlighted by the red arrows.

Figure 2

Cellular hypertrophy and interstitial fibrosis in L-NAME and TAC mice. Short-axis, mid-level LV sections of cardiac tissue from mice treated with placebo (panel A), L-NAME (panel B), and TAC (panel C). L-NAME and TAC are representative tissues after 7 weeks of exposure. Panels were stained with fluorescein isothiocyanate-conjugated (FITC-) wheat germ agglutinin to delineate cell membranes and scanned at a resolution equivalent to 1.0 μm/pixel to measure minor and major cell diameters in 15 fields within 4 myocardial segments. (A)-(B) are representative illustrations of larger short-axis diameters in the L-NAME and TAC groups relative to controls. Adjacent short-axis sections from the same mice, stained with Masson’s trichrome stains in (D)-(F) illustrate the absence of interstitial fibrosis in the control group and higher levels of interstitial fibrosis (blue) in L-NAME compared to TAC mice.

Figure 3

Intracellular lifetime of water and ECV in L-NAME mice. (A) The intracellular lifetime of water (τ_ic) increased significantly in mice treated with L-NAME, and was significantly higher than in placebo-treated controls. (B) In mice exposed for 7 weeks to L-NAME the extracellular volume fraction increased also significantly, compared to baseline, and was also significantly higher than in placebo-treated controls.

Figure 4

Longitudinal assessment of intracellular lifetime of water and cell size in mice exposed to TAC. A) Mice were imaged at approximately 2 weeks after transverse aortic constriction, and followed-up for up to 46 days before harvesting of the heart. Over this time the intracellular lifetime increased significantly (0.0581 s/week; P=0.0002) as illustrated by the solid line, calculated with a linear mixed effects (LME) model. Dashed lines connect repeated measurements within the same mouse. B) The volume-to-surface ratio calculated from the histologic major and minor cell diameters showed a significant association with the time between TAC and histological examination (0.79±0.18 μm/week; P=0.001). The solid line represents the linear regression line estimate for all mice, based on the fixed effects in the LME model.

Figure 5

Association between intracellular lifetime of water and cell volume-to-surface ratio. For a diffusion-based exchange the intracellular lifetime of water is expected to be proportional to the cellular volume-to-surface ratio. The volume-to-surface ratio (V/S) was highly correlated (r=0.78; P<0.001) with the intracellular lifetime of water obtained from the T1 measurements before and after administration of an extracellular gadolinium contrast agent.