Introduction:

Current diagnosis of hypertrophic cardiomyopathy (HCM) is based on unexplained thickening of ventricular walls detected by non-invasive imaging and further supported by invasive histopathology. This morphological change does not allow assessing the underlying subclinical ultra-structural myocardial changes including the relations of hypertrophy, fibrosis and microvasculature deficits that usually dictate disease prognosis. Notwithstanding the substantial progress of cardiac MRI (CMR) in assessing changes in the myocardial microstructure, in vivo evaluation of the kinetics of such microstructural changes and their mechanisms underlying disease progression is still missing. This significantly limits the prognosis of HCM and disease management. Based on our recent findings in patients with hypertrophic cardiomyopathy (HCM) [1], we hypothesize that factors related to impairment in myocardial perfusion may contribute to the microstructural changes in HCM. To test this hypothesis, we used state-of-the-art arterial spin labeling (ASL) CMR free of contrast agents to quantify the myocardial blood flow (MBF) throughout the cardiac cycle. We tracked patho/physiological MBF variations in a HCM mouse model before showing the overt left ventricular functional impairments. We then validated our imaging results with gold standard histological collagen staining to identify myocardial fibrosis.

Methods:

We used DBA2J/D2 mouse strain that naturally carries genetic variants and bear key features of HCM [2]. Three months-old male D2 (n=3) and the age matched reference strain C57BL/6 (B6; n=3) were scanned using a 9.4T animal MR scanner (Biospec 94/20, Bruker, Germany). During in vivo CMR, mice were received a dose of 1.2% isofluorane mixed with pure oxygen to prevent hypoxia. Short axis stacks (SAX) of CINE images were acquired for cardiac function assessment using self-gated FLASH (IntraGate, TE/TR= 1.58/8.5ms, FA = 20°, BW = 98kHz, FOV=11×22mm², matrix=192×384, thickness=0.8mm, cardiac frames= 16) [3]. Myocardial blood flow (MBF) was quantified using a CINE-ASL technique [4] (TE/TR= 1.12/7.6ms, FA=6°, FOV=25× 25mm², matrix=128×64, slice thickness = 1mm. ECG signal was monitored with a small-animal dedicated module (SA Instruments). CINE images were analyzed to measure ventricular thickness and ejection fraction (EF) using CMR42 (Circle CVI, Canada). Perfusion maps were calculated using Interactive Data Language (IDL). We averaged 2 cardiac phases to obtain the mean MBF in mid ventricular slice in both end-diastolic and end-systolic phases. After in vivo CMR, mouse hearts were fixed with 4% PFA. For detecting fibrosis, Sirius Red (stain for collagen) was stained on heart paraffin sections. We tested the significance of our results using t-test. Differences were considered significant where p < 0.05.

Results:

In 3 month old mice, left ventricular wall was significantly thicker in D2 than in B6 mice (LVwall thickness=1.27±0.06mm vs 0.99±0.04mm, P<0.01) (Figure 1). Left ventricular EF in D2 mice is 70.2±5.7% and 73.8±0.7% in control B6 mice (no difference). Mean perfusion in the mid-ventricular slice is significantly lower in D2 than in B6 mice (end-diastole: 6.8±1.0 vs 10.1±1.7 mL g^-1 min^-1, P<0.05; end-systole: 4.6±0.5 vs 9.1±1.8 mL g^-1 min^-1, P<0.01) (Figure 2). Sirius Red staining revealed a normal expression of collagen deposition in B6 myocardium, while mild fibrosis was shown in D2 mice (Figure 3).

Conclusion and Discussion:

Our study is the first one to measure myocardial perfusion in a mouse model with HCM features. We showed that measuring the dynamic MBF over cardiac cycle can quantify the changes in myocardial perfusion in the condition of cardiomyopathy. This perfusion change associates with the signs of myocardial remodeling such as hypertrophy and fibrosis. In human studies, myocardial fibrosis is progressive in some HCM patients and the perfusion abnormality is one of the mechanisms of the fibrotic process [5]. Furthermore, the progression of myocardial remodeling is usually associated with an increased risk of subsequent clinical events in HCM. Our work provides adequate ground for measuring perfusion deficit in HCM. The MBF differences between cardiac phases suggest that cardiac phase needs to be considered when interpreting CMR-derived MBF values in the HCM. Our results will be validated with more sophisticated microvasculature analysis in the HCM mouse model. To conclude, our results help to fill in the missing link between in vivo detection of subclinical perfusion deficits and histological hallmarks of myocardial remodeling. Ultimately, we wish to establish effective CMR marker to better assess disease progression and translate our knowledge into improving early cardiomyopathy treatment.

Acknowledgements

No acknowledgement found.

References

[1] Huelnhagen T et al., Myocardial Effective Transverse Relaxation Time T₂* is Elevated in Hypertrophic Cardiomyopathy: A 7.0 T Magnetic Resonance Imaging Study. Sci Rep. 2018 Mar 5;8(1):3974. [2] Zhao W et al., A Murine Hypertrophic Cardiomyopathy Model: The DBA/2J Strain. PLoS One. 2015;10:e0133132. [3] Ku MC et al., Cardiac MRI in Small Animals. Methods Mol Biol. 2018;1718:269. [4] Troalen T et al., Cine-ASL: a steady-pulsed arterial spin labeling method for myocardial perfusion mapping in mice. Theoretical model and sensitivity optimization. Magn Reson Med. 2013; 70(5):1389. [5] Raman B et al., Progression of myocardial fibrosis in hypertrophic cardiomyopathy: mechanisms and clinical implications. Eur Heart J Cardiovasc Imaging. 2018 Oct 24[Epub ahead of print]