Furosemide Promotes Inflammatory Activation and Myocardial Fibrosis in Swine with Tachycardia-Induced Heart Failure

Figures

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  Figure 1. MAPK signaling molecules in the progression of heart failure. Expression in the furosemide-treated animals for proteins in the MAPK family shows an elevation in all the proteins. (A) Western blot analyses and densitometric quantitation of p38 MAPK and MEK-1 images from the infrared scanner. (B) Western blot analyses and densitometric quantitation of ERK1/2. (C) Western blot analyses and densitometric quantitation of JNK1, JNK2, and Ras in the LV homogenate of Normal, HF furosemide, and HF saline swine. The graphs of individual proteins demonstrate the relative intensity mean values, shown as mean ± SEM. The HF furosemide group for all proteins was found to be significantly different from both the control and HF saline groups, p ≤ 0.05 (n = 7). The saline HF means were also significantly different from the control animals (* = statistically different from control group, # = statistically different between furosemide HF and saline HF group).

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  Figure 2. Proteins associated with the TGF-β1 pathway and inflammatory response. Western blots of a variety of proteins relating to heart failure from the tachycardia-induced pacing are depicted in (A). Western blot expression in furosemide-treated animals found elevated TGF-β1 and TGF- β receptor1 endogenously (B,C) and plasma level of TGF-β1 proteins to be elevated shown in (D), and the data shown as mean ± SEM, p ≤ 0.05. Similarly, a Western blot data for Smad-2 and Smad-3 are shown in (E–G), which are significantly increased in furosemide-treated animals compared with saline-treated and control animals, and the data shown as mean ± SEM, p ≤ 0.05, (* = statistically different from control group, # = statistically different between furosemide HF and saline HF group).

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  Figure 3. Biomarkers related to growth factor regulation in ECM remodeling. Western blot results for several growth factors involved in the regulation of growth on the extracellular matrix. Endogenous expression of galectin-3 (A,B), PAI-1 (A,C), CTGF (E,F) and GDF-15 (E,G), are significantly elevated in furosemide-treated animals compared to control and saline-treated HF animals (data shown as mean ± SEM, p ≤ 0.05). ELISAs were performed on plasma from a limited subset of the animals (control = 4, HF furosemide = 6, and HF saline = 8). Endothelin-1 has been associated with possibly inducing pro-inflammatory mechanisms in the heart. Endothelin-1 in plasma was found to be significantly elevated in the HF animals as compared to controls ((D), data shown as mean ± SEM, p ≤ 0.05), but not significantly different between HF groups. The level of CTGF found to be significantly elevated in the left ventricular tissue is also elevated in the plasma (H) for the heart failure animals compared to control animals, plus furosemide HF-treated animals were significantly higher than the saline-treated HF animals (data shown as mean ± SEM, p ≤ 0.05). (* = statistically different from control group, # =…

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  Figure 4. Level of matrix metalloproteins in tachycardia-induced heart failure. Western blot results for several proteins involved in the regulation of growth on the extracellular matrix. MMP-14 and TIMP-1 are competitors in the remodeling of the heart. MMP-14, which is activated in response to inflammation and injury, is significantly elevated in the furosemide animals as compared to both controls and saline HF animals (A–C, data shown as mean ± SEM, p ≤ 0.05). The saline HF animals are also significantly elevated as compared to the control animals, p ≤ 0.05. TIMP-1 is an inhibitor of the MMPs; it is also involved in the remodeling process and is elevated when there is tissue damage. The plasma levels of MMP-13 and TIMP-2 results show significantly elevated levels of MMP-13 and TIMP-2 levels in the furosemide-treated animals compared with control. There is no significant elevation of MMP-13 is seen in furosemide treatment when compared with saline treatment, but the TIMP-2 level is significantly increased in furosemide-treated when compared with saline administration (D,E, data shown as mean ± SEM, p ≤ 0.05). (* = statistically different from control group, # = statistically different…

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  Figure 5. Immunohistochemistry of collagens I, II, and VI and tissue histology. Collagens I, II, and VI were assessed with immunohistochemistry on 5 µm slices obtained from the frozen blocks of left ventricular tissue (A–C). The bar graphs (D–F) show the relative fluorescent intensities of the collagens, presented as mean ± SEM, p ≤ 0.05 (n = 5). All of the collagens show significantly elevated in the furosemide animals as compared to both controls and saline HF animals. Also, the saline HF animals are significantly elevated, as compared to the control animals, p ≤ 0.05. (* = statistically different from control group, # = statistically different between furosemide HF and saline HF group).

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  Figure 6. Trichrome staining (Masson’s): Quantification of the proportion of myocardial fibrosis area using Masson’s trichrome staining was performed on 5 µm thin slices from paraffin-embedded histological blocks of left ventricular tissue. Representative images from each of the groups are shown in (A–C). The yellow arrows are pointing to the blue fibrotic tissue in the ECM. The heart has three main layers of tissue running in different orientations. The amount of fibrosis varies in the different layers and locations. Independent samples Kruskal–Wallis test results, shown in the table and graph, found a significant difference between the furosemide group and the controls, but not between the control and saline groups, nor the furosemide and saline groups. Group means and standard error are reported on the graph.