Introduction

Lymphatic system regulates many processes involved in cardiac physiology and pathology, such as inflammatory reactions¹, tissue fluid balance² and atherosclerosis^3,4 which can change the function of myocardium. Vascular endothelial growth factor receptor 3 (VEGFR3) is one of the key molecules in the development and maintenance of lymphatic system. T₂ relaxation time increase has related to edema⁵ and increased water content which is associated with necrosis in the acutely ischemic myocardium. Increased longitudinal rotating frame relaxation time (T_1ρ) has been related with later tissue remodeling i.e. granulation tissue formation and fibrosis^6,7. Novel rotating frame relaxation time method to decrease specific absorption rate is relaxation along a fictitious field in n^th rotating frame (RAFFn) which has shown to increase at myocardial infarct (MI) area compared to remote myocardium⁸. Here we applied T₂, T_1ρ and RAFFn relaxation time measurements T_RAFF2 and T_RAFF4 to study alterations of cardiac lymphatic system, its effects on MI and the role of VEGFR3 in cardiac lymphatics. We measured left ventricular (LV) functional parameters and support our findings with histology.

Methods

Approximately 13-17 week old female and male VEGFR3 mice (n=8) expressing lymphatic insufficiency were used with 12 normal mice serving as a control group. MI was induced to all mice (n=20) by ligating the left anterior descending (LAD) artery. All experiments were done using 9.4 T magnet interfaced with Bruker BioSpec console. Gradient echo cine (TR=8.0ms, TE=1.9ms, thickness=1.0mm, matrix=192x192, number of cine frames=10-11) were acquired and double adiabatic Hahn echo (TE=0.05-14ms) was applied to measure T₂ relaxation at 0, 8 and 42 days after MI. Adiabatic continuous wave T_1ρ (pulse power 1250Hz, durations 0-57ms), T_RAFF2 and T_RAFF4 (RF powers: 1250Hz and 648Hz, respectively duration 2.26ms, pulse train lengths=0-36.2ms) relaxation times were measured 8 days after MI (n=5). Fast imaging with steady state precession readout (TR=5ms, TE=2ms) after weighting pulse was used.

Results

A significant difference in T_RAFF4 between VEGFR3 and control mice were found at day 8 (Figure 1a). T_1ρ, T_RAFF2 and T₂ relaxation times increased significantly after infarct in both VEGFR3 and control mice groups but no difference was found between the groups at day 8 (Figure 1b-d). Dilation of the LV was detected in both groups (Figure 2a). Also, increased end systolic volume and end diastolic volume caused decreased stroke volume and ejection fraction at 8 days after infarct (Figure 2b-d).

Discussion

Elevated T₂ at days 8 and 42 are most likely due to long-lasting edema in myocardium. Presence of edema has been shown to increase T₂ and modify the development of fibrosis. Based on similar T₂ at day 8, decreased lymph flow in VEGFR3 has not have an impact on long-lasting edema in infarct area. Furthermore, significant increase of T_RAFF4 in VEGFR3 group compared to control group cannot be due to difference in edema, but more likely due to increased fibrosis. Histology showed that VEGFR3 mice had different composition of fibrotic scar compared to control group since VEGFR3 had less histological staining of collagen I and III compared to control group. Additionally, extracellular volumes (ECV) were higher at VEGFR3 group (16%) compared to control group (12%). These results together with increased levels of extracellular matrix proteins (e.g. α-SMA) in VEGFR3 group, supports the increased fibrosis in VEGFR3 mice compared to control mice. Increased levels of extracellular matrix proteins lead to stiffness of myocardium after MI which is reflected as a decreased ejection fraction. All of these findings supports elevated fibrosis in VEGFR3 group and may explain why rotating frame relaxation times were elevated in VEGFR3 mice at 8 days after MI.

Conclusion

RAFF4 relaxation time T_RAFF4 can be used to determine and to gain information of altered fibrosis in lymphatic insufficient myocardial infarction mouse model. Furthermore, alterations in lymphatic morphology after myocardial infarct are reflected in cardiac function parameters.

Acknowledgements

This study was supported by Finnish Academy Center of Excellence, Finnish Foundation for Cardiovascular Research, ERC Advanced Grant, Finnish Cultural Foundation (Kymenlaakso Regional Fund), The Paolo Foundation and Urho Känkänen Foundation.