Hypertrophic gene expression induced by chronic stretch of excised mouse heart muscle

Abstract

Altered mechanical stress and strain in cardiac myocytes induce modifications in gene expression that affects cardiac remodeling and myocyte contractile function. To study the mechanisms of mechanotransduction in cardiomyocytes, probing alterations in mechanics and gene expression has been an effective strategy. However, previous studies are self-limited due to the general use of isolated neonatal rodent myocytes or intact animals. The main goal of this study was to develop a novel tissue culture chamber system for mouse myocardium that facilitates loading of cardiac tissue, while measuring tissue stress and deformation within a physiological environment. Intact mouse right ventricular papillary muscles were cultured in controlled conditions with superfusate at 95% O2/ 5% CO2, and 34 degrees C, such that cell to extracellular matrix adhesions as well as cell to cell adhesions were undisturbed and both passive and active mechanical properties were maintained without significant changes. The system was able to measure the induction of hypertrophic markers (BNP, ANP) in tissue after 2 hrs and 5 hrs of stretch. ANP induction was highly correlated with the diastolic load of the muscle but not with developed systolic load. Load induced ANP expression was blunted in muscles from muscle-LIM protein knockout mice, in which defective mechanotransduction pathways have been predicted.

Figure 1

The RV papillary muscle culture chamber and mechanics apparatus. Muscle is secured between a basket and titanium hook. Muscle length is adjusted using a Newport CM-12CC actuator. Muscle forces are measured using an Isometric Harvard Apparatus force transducer (724490). Culture media is continuously circulated into and out of the muscle bath through inlet and outlet chamber ports. An ECM2 magnetic stirrer provides for a well stirred environment within the chamber for optimal diffusion of nutrients, oxygen, and wastes. The culture media is maintained at 34°C with a heated stage insert with temperature controller (World Precision Instrument: WPI-14471).

Figure 2

Stable systolic tissue mechanics indicate tissue viability was preserved for duration of experiment. (A) Average time course of systolic tension developed over first 5 hours of culture. Data was normalized by initial developed systolic tension. (B) Time course of time from activation to peak tension developed (ATP), time from activation to 50% relaxation (AT50), and 50% relaxation time (RT50) of right ventricular papillary muscles stretched to 90% of Lmax. (n=4) Data are mean +/− SEM.

Figure 3

Mechanical properties of mouse RV papillary muscles. (A) Average twitch kinetics of mouse RV papillary muscles. (n=9) Data was normalized by unloaded cross sectional area. (B) Video image of mouse RV papillary muscle that has been marked with titanium dioxide in the loaded stretched state. (C) Average peak systolic stress strain relationship and diastolic stress strain relationship of mouse RV papillary muscles. (n=4) Data are mean +/− SEM.

Figure 4

Increase in diastolic strain and/ or diastolic stress regulates natriuretic peptide gene induction. (A) Relative average gene expression data calculated from real time PCR results using the ΔΔCt. Muscles were stretched to 90% or 10% of Lmax and were induced to contract by electrical stimulation. Muscles were cultured for 2 or 5 hour duration. (B) Normalized ANP gene expression as a function of developed systolic tension. (n=6) (C) Normalized ANP gene expression as a function of diastolic tension. (n=6) (D) Normalized ANP gene expression as a function of diastolic strain. (n=6) Multiple linear regression model (R²=0.91, P=0.01) was computed for ANP gene expression as a function of developed systolic (β₁ =0.002, P=0.07) and diastolic tension (β₂=0.012, P=0.01). Simple linear regression models were computer for ANP as a function of diastolic strain (R²=0.84, P=0.01) and ANP as a function of diastolic stress (R²=0.82, P=0.01). (E) Relative average ANP gene expression of electrically unstimulated mouse RV papillary muscles. Muscles were stretched to 90% or 10% of Lmax. (n=3) Muscles were cultured for 5 hour duration. (F) Effect of sustained stretch (90% Lmax) on the developed systolic and diastolic tension of electrically unstimulated muscles. (n=3) * Indicates statistical significance (P<0.05). ** Indicates high statistical significance (P<0.005). Data are mean +/− SEM.

Figure 5

Chronic culture of MLPKO RV papillary muscles. (A) Relative average ANP gene expression data calculated from real time PCR results using the ΔΔCt. Muscles from two week old mice were stretched to 90% of Lmax and induced to contract by electrical stimulation. (n=3) (B) Average time course of systolic tension developed by MLPKO muscles. (n=3) Data was normalized by initial developed systolic tension. * Indicates statistical significance (P<0.05). Data are mean +/− SEM.