Diffuse myocardial fibrosis (DMF) is a common histological feature of the failing heart. It is presented in many conditions, ranging from advanced aging to hypertension or hypertrophic cardiomyopathy, causing myocardial stiffness and diastolic dysfunction.¹ Recent advances in cardiovascular MRI (CMR) technologies such as T1-mapping or extracellular volume (ECV) fraction (derived from T1-mapping)² offer robust techniques to assess diffuse fibrosis in patients with myocardial infarction and heart failure. In the present study, CMR assessment of myocardial fibrosis and hypertrophy was evaluated in an isoproterenol infusion model in Balb/c mice.

Study Design: Cardiac remodeling induced by isoproterenol injection has been an established model.³ Male Balb/c mice were utilized in the study. The study contained 2 groups: 1) iso-group implanted with isoproterenol infusion mini-pump, sc, 30 mg/kg per day, n=8; and 2) sham group implanted with mini-pump of vehicle (saline+0.02% ascortic acid), sc, n=10. Mice were dosed for 21 days prior to imaging study. The Infusion pumps were removed under anesthesia on the imaging day. Mice were anesthetized with 0.5~2.0% inhaled isoflurane while maintained at a core body temperature of approximately 36.9°C during the entire imaging session. The electrocardiogram (ECG), rectal temperature, and respiration were constantly monitored. Catheter was inserted i.p. for administration of Gd-DTPA (Magnevist) at a dose of 0.5 mmol/kg for the post contrast image acquisitions. After imaging, the mouse hearts were dissected, weighted, and prepared for measurement of hydroxyproline (collagen content). Blood samples from a satellite group (n=3) were collected for hematocrit (HCT) measurement to estimate ECV.

MRI Method: CMR T1-mapping technique was implemented on the on a Bruker Biospec 7T 20-cm horizontal bore system (Bruker, Billerica, MA) equipped with a 72mm ID whole body RF volume coil as the transmit and a mouse surface coil as the receive coil. Short axis CINE images were acquired using the ECG-gated fast gradient echo FLASH cine sequence, with TR = 10ms per cardiac phase, TE = 2.5ms, and number of averages 8. The T1-mapping sequence is implemented based on the modified look-locker inversion recovery sequence (MOLLI) ⁴, with TR/TE = ~3000ms/3.5ms; flip angle 10°; 20~24 inversion pulses; and an inversion pulse interval determined by the R-R interval. The imaging slice-thickness is 1mm, with a field of view 25.6×25.6 mm2, 128×128 matrix, and 2 averages. The small animal MOLLI sequence allows the multi-slice acquisitions, and imaging were performed before and after Gd-DTPA injection. The waiting time for the post-Gd small animal MOLLI imaging is 15-20 minutes. The total scan time was approximately 60 minutes.

Data Analysis: Cine images were used for ejection fratoin assessment. Pixel-wise and regional T1 measurements were calculated using the three-parameter curve fitting to: M = A×(1-B×exp(-t/T1*)),⁵ where M is the signal intensity, A the scaling factor for equilibrium magnetization M0, B the correction factor for imperfect inversion, and t the effective inversion time. T1 was calculated from the resulting T1*, A, and B by applying the equation T1 = T1*×((B/A)-1).⁵ ECV was estimated using: ECV = λ×(100-HCT), where λ = (1/T1myo-post–1/T1myo-pre)/(1/T1blood-post–1/T1blood-pre), and HCT=48%. The image quantifications were preformed using the customized MATLAB (Mathworks, Natick, MA) based image analysis toolkit.

Constant isoproterenol infusion increased LV mass indicating the induction of cardiac hypertrophy in the mosue model, which was confirmed by the heart weight of 133±2.7g in the sham group and 192±12.4g in the iso-group (P<0.01). Fig. 1 demonstrates the calculated ECV maps in a sham and an isoproterenol induced disease mouse, ECV is increased in the myocardium of the disease mouse (Fig 1c, red arrow) compared to that of the sham mouse (Fig. 1b). The mean ECV values in the myocardium of adjacent slices are close, which are %18.3±1.95 and %26.6±1.3 in the middle slice (Fig. 1a), and %19.2±1.6 and %25.8±0.97 in the adjacent slice toward the base, for the sham group and iso-group, respectively. Left ventricle ejection fraction (EF) was significantly reduced (Fig. 2a, p<0.01) in the iso-group. Native T1 and ECV in myocardium of the iso-group are significantly increased (Fig. 2b and 2c, p<0.01), which is consistent with the significant increase of hydroxyproline conternt detected in the iso-group (Fig 2d, 25.5±4.4 ug/mg) compared to that in the sham group (11.2±1.1 ug/mg) to confirm the isoproterenol induced cardiac fibrosis. Our CMR results demonstrated elevated myocardial ECV and reduced left ventricular ejection fraction in the isoproterenol infusion mouse group. CMR techniques including T1-mapping and the ECV quantification provide a translational non-invasive imaging marker to assess diffuse myocardial fibrosis, and the potential to evaluate efficacy of anti-fibrosis treatment which has been hypothesized as a viable treatment strategy for heart failure (HF).