43 4.3 The Use of rHC in Myocardial Infarction

Experimental Design

The study evaluated the rHC therapeutic potential for treating MI in mice. They used C57BL/6 mouse models which is the strain used for immunological studies. MI was induced by ligating the left anterior descending coronary artery just below its emergence from the left atrium. After a week post MI, 3-15 mice were randomly assigned to one of the treatment groups and received intramyocardial injections (Figure6). There were three treatment groups in total. In these three groups, mice were injected with either PBS, rHCI or rHCIII, with PBS being saline solution and this was the control group.

  Figure 6. The components and properties of the injected material used in the study (McLaughlin et al, 2019)

The following video demonstrated the process of rHC matrix intramyocardial injection in mice model. Please watch it for a better understanding of the delivery method.

Post-MI Cardiac Function and Morphology 

The primary outcome was cardiac function determined by echocardiography 28 days after treatment since this is when scar tissue has already formed, and ventricular remodeling occurs. To assess improvement in cardiac function, they measured factors that are clinical predictors of heart failure and survival after MI, including left ventricular ejection fraction (LVEF), stroke volume (SV), cardiac output (CO), tensile elasticity and contractility.

The results of measured parameters were consistent throughout. As shown in Figure 7a, at 28 days after treatment, LVEF was greater in hearts with rHC treatment compared with PBS treated hearts, the same trend was observed in SV and CO (Figure 7b,c), with both treatment groups having improved fold change. The tensile elasticity of the infarcted myocardium was shown to be largely restored by rHCI and rHCIII treatments at 2 days post injection, and this was maintained up to 28 days for rHCI (Figure 7d). Moreover, the rHCI injection also improves contractility in the LV wall where it was injected as compared with PBS-treated mice (Figure 7e). Overall, measures of cardiac function were higher in the treatment groups compared to the control.

Figure 7. rHC matrix and post-MI cardiac function. a. left ventricular ejection fraction (LVEF) b. stroke volume (SV) c. cardiac output (CO) d. tensile elasticity and e. contractility Image adapted from McLaughlin et al, 2019.

In advanced stages post-MI, cardiac remodeling can lead to enlargement of the heart. Thus, the treated hearts of mice were then harvested to visualize rHCI and rHCIII distribution within the hearts. While mice that received PBS treatment had enlarged hearts, the effect was prevented in mice whose hearts were treated with rHCI matrix (Figure 8a). Mass over tibia length ratio was used to assess the heart size since the measurement is commonly done to qualify cardiac hypertrophy. The same result can be observed in Figure 8b, which are the image of hearts harvested from mice.

Figure 8. rHC matrix and post-MI cardiac morphology. a. Mass/tibia length ratio measured 28 days after injection. b. image of hearts harvested at 28 days post treatment. Image adapted from McLaughlin et al, 2019

Post-MI Vascularity and cardiomyocyte preservation 

Immunohistochemistry was performed to assess vascularity and cardiomyocyte preservation after treatment.

The number of capillaries was increased in the border zone of the hearts for both treatment groups, and rHCI in particular increased the cardiomyocytes preservation. They measured two parameters, one being troponin I, which is a cardiomyocyte marker. It is a regulatory protein that controls the calcium mediated interaction between actin and myosin. There was clearly greater expression of troponin I in the rHCI treatment group compared to other groups (Figure 9a). To test the connectivity between cardiomyocytes, the major protein of cardiac ventricular gap junctions, connexin 43, was stained and observed to be highly expressed in the rHCI treated group (Figure 9b). It demonstrated that there is greater connections for the rHCI matrix group compared with control.

Figure 9. Immunohistochemisty analysis and quantification. a. troponin 1 (red) staining in the border zone. b. connexin 43 (green) staining in the remote area. Image adapted from McLaughlin et al, 2019

Mononuclear cell recruitment 

Then immunohistochemistry was used to detect the amount of M2 macrophages in blood and in the heart to assess the severity of inflammation. Results showed that rHC matrix injection upregulated the mononuclear cell recruitment post-MI, with an increase in the amount of M2 macrophages in the scar region in the rHCI treatment group compared with the PBS group. By evaluating the mononuclear cells in wild type mice, an increase in the overall number of circulating monocytes in the rHCIII-treated mice was observed.

In vitro assessment of rHC matrices

In vitro assessments testing the effect of rHC matrices on macrophage differentiation was also conducted.

The results indicate that both matrices promote the polarization of the M2 wound healing phenotype, with rHCIII promoting a greater effect than rHCI. In addition, a negative effect during post-MI is oxidative stress. Macrophages cultured on rHCI or III matrices were exposed to hydrogen peroxide to mimic the presence of oxidative stress. The results indicate that cells cultured on both rHC matrices were protected from H₂O₂-induced death.