Effect of trehalose on heart functions in rats model after myocardial infarction: assessment of novel intraventricular pressure and heart rate variability

Figures

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  Figure 1. (A) All groups had the mitral inflow evaluated using pulsed-wave Doppler echocardiography from the left apical four-chamber view. (B) Apical four-chamber view CMME was employed to trace the path of the inflow from the left atrium (LA) to the apex of the left ventricle (LV) with the cursor parallel to the mitral inflow. (C) MATLAB software was used for further processing of the photos for IVPD and IVPG calculation. (D) CMME analysis with MATLAB software generated 3D temporal and spatial profiles of the IVPG based on Euler's equation over a certain portion of the systolic duration starting from the total closure of the aortic valve to the end of diastole. The z-axis represents the distance (mm) from the apex to the mitral valve (base).

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  Figure 2. A two-dimensional graph of the temporal profile of the IVPG over a certain percentage of systole from the time of closure of the aortic valve to the end of diastole is displayed, highlighting the peak total IVPG and segmental IVPGs respectively (1). Additionally, a two-dimensional spatial profile of the IVPG from the apex to the base (MV) of the heart is presented, with the red line symbolizing the spatial profile at peak total IVPG which generally occurs at the level of the MV. The top and bottom blue lines signify the inertial and convective IVPGs, respectively. Determining the basal and mid-apical segmental IVPGs was based on the LV segmentation rule (one-third for basal and two-thirds for mid-apical) (2).

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  Figure 3. Bar charts showed the IVPG indices for the different groups, with the data represented as mean ± SD (n = 8, for each group). Mid to Apical IVPG and Apical IVPG exhibited no significant difference between the various groups. The Total IVPG, Basal IVPG, and Mid IVPG were significantly lower in the MI group than the Sham group. Trehalose seemed to improve these changes, presenting a non-significant distinction for the same parameters when compared to the Sham group apart from Mid IVPG (indicated by an asterisk for statistical significance, Ns P > 0.05, *P < 0.05).

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  Figure 4. Bar graphs showed the heart rate variability parameters in the different groups represented by mean ± SD (n = 8, for each group). Abbreviations included standard deviation of RR intervals (SDRR), coefficient of variation of RR intervals (CVRR), square root of the mean of the squared differences between adjacent RR intervals (RMSSD), percentage of differences higher than 50 ms in RR intervals (pRR50), very low-frequency power (VLF), low-frequency power (LF), and high-frequency power (HF). The values for VLF, LF, and pRR50 showed no significant variations among the various study groups, while the HF was significantly lower in the MI group compared to the Sham one. The Trehalose-treated group demonstrated significantly higher SDRR and CVRR when compared to the Sham group, and a significantly higher RMSSD than the MI group (indicated by asterisk for statistical significance, Ns P > 0.05, *P < 0.05, **P < 0.01).

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  Figure 5. Gross examination of the myocardium in the three experimental groups revealed a significant difference in infarct size and density compared to each other: (A) the sham group had no myocardial lesion; (B) the MI group showed a characteristic, thickened epicardial lesion; and (C) the MI + TRE group had an obvious decrease in infarct size with resolution in the pathognomonic lesion as compared to the model group. (D) Analyzing data with Image J software, including Mean Gray Value, Integrated Density, and Median, among the three groups showed that the prescribed parameters were significantly higher compared to the Sham group. There was no significant difference in the measured values between the Sham and Treated (MI + TRE) groups (indicated by asterisk for statistical significance, ****P < 0.0001) (n = 8, for each group).

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  Figure 6. Histological examination of the heart tissue in the various groups observed at two levels of magnification (10× and 40×) indicated that the sham group had normal physical characteristics and maintained the structure of its tissue. The MI group, however, exhibited a high infiltration of inflammatory cells and intense ischemic necrotic tissue. Interstitial edema distorted cardiac muscle fibers, as well as disassembled cardiomyocytes with a wavy appearance, were also evident. The Trehalose-treated group showed that the overall inflammatory state was improved following the treatment with Trehalose, represented by decreased mononuclear cell extravasation and lack of edema. (J) The graph revealed the histopathological score between the three groups; the histopathological score was significantly lower in the Trehalose-treated group compared to the MI group (indicated by asterisk for statistical significance, ****P < 0.0001) (n = 8, for each group).

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  Figure 7. Cardiac tissue fibrosis evaluation. The severity of interstitial and perivascular fibrosis in the left ventricular myocardium was evaluated using Masson's trichrome staining on tissue sections, followed by examination under optical microscopy. The intensity of light blue staining indicates the extent of cardiac fibrosis. (A) shows peripheral myocardial interstitial fibrosis in the sham, MI, and MI + TRE groups, while (B) illustrates perivascular fibrosis in these three groups. After four weeks of acute MI, the MI group exhibited severe fibrosis at both interstitial and perivascular levels, in contrast to the treated group where moderate light blue staining was observed at both levels.

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  Figure 8. High-magnification photomicrographs illustrating immunohistochemistry of myocardial tissue following an infarction in rats. The images depict brown staining, indicating CD3-positive endothelial cells. The myocardium of the MI group exhibited a higher number of CD3+ cells, distinguished as brown particles under the microscope, compared to both the sham and MI + TRE groups. Quantitatively, the percentage of CD3+ cells in the MI group was significantly elevated four weeks post-surgery in comparison to the sham-operated and trehalose-treated rats. Statistical significance is denoted by an asterisk (****P < 0.0001).

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  Figure 9. Bar graphs depicted the results of real-time PCR in the heart tissues from the study groups. (A) Levels of pro-inflammatory cytokines IL-1B and IL-18 increased significantly in the MI group compared to Sham rats. (B) In MI rats, The Bcl-2 family also exhibited a decrease in Bcl-2, an increase in Bax expression, and a decrease in the Bcl-2/Bax ratio relative to the sham group. (C) Superoxide dismutase isoenzymes SOD1, SOD2, and SOD3 had decreased expression levels in the MI group vs. those of the sham group, The asterisk is used to indicate the significance, Ns P > 0.05, *P < 0.05 (n = 8, for each group).