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.

Conflict of interest statement

Disclosures: None.

Figures

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 (τic) 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 τic. 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, τic ceases to be sufficiently short relative to the extra-to-intracellular R1 difference. The degree of deviation from linear dependence is sensitive to τic. 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 τic for the best fit was 0.4 s. The line with long dashes illustrates how the model fit changes with an increase of τic to 0.8 s, causing a larger deviation from the straight line. (D) Cardiomyocyte length (Dmaj) and diameter (Dmin) were measured on digitized images of tissue slices stained with fluorescein isothiocyanate-conjugated (FITC-) wheat germ agglutinin. For Dmin a cell cross-section with approximately circular shape was chosen (highlighted by red arrow). Dmaj 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 (τic) 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.