Cinaciguat prevents the development of pathologic hypertrophy in a rat model of left ventricular pressure overload

Figures

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  Figure 1. Representative echocardiographic images from the 6th week in diastole. White bars represent walls, and red bars show cavities. Note the difference among groups in the length of the bars.

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  Figure 2. Cinaciguat normalises increased contractility in pressure overload. Baseline characteristics of pressure-volume relations did not differ in the AABCo and AABCin groups (a). P-V loops recorded during occlusion of the inferior caval vein (b) and the load independent indices of contractility derived from these measurements (c) show, however, that Cinaciguat significantly decreased the increase in contractility following abdominal aortic banding. PRSW: preload recruitable stroke work; Ees: end systolic elastance; dP/dtmax-EDV: maximum rate of pressure change – end diastolic volume relationship; pint: interaction p value *p < 0.05 vs. ShamCo; #p < 0.05 vs. AABCo.

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  Figure 3. Histological alterations in pressure overload are blunted by Cinaciguat. Differences between the groups are illustrated in representative photomicrographs of left ventricular (LV) myocardial sections with haematoxylin-eosin (a) and Picrosirius red staining (b), TUNEL (c) and cGMP immunohistochemistry (d). Average cardiomyocyte diameter (e) and collagen area of subendocardial LV myocardium (f) was significantly elevated in the AABCo group compared to ShamCo, both of which alterations were significantly decreased following Cinaciguat treatment. TUNEL staining revealed a significant increase in the number of apoptotic cell nuclei in the AABCo group compared to ShamCo and AABCin (g). cGMP score (h) was significantly higher in the AABCin group than in the AABCo animals. pint: interaction p value *p < 0.05 vs. ShamCo; #p < 0.05 vs. AABCo.

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  Figure 4. Effects of Cinaciguat on cGMP signalling in pressure overload. The strong sGC activating effect of Cinaciguat during pathologic conditions was confirmed by measuring plasma level of cGMP, which was significantly elevated in AABCin rats. cGMP activates PKG, which then phosphorylates VASP and Pln, phosphorylation ratio of which are widely used markers of PKG activity. Both of these were markedly increased despite the unchanged amount of PKG in the AABCin group, indicating increased PKG activity in these animals. Representative Western blot bands are shown for each group and investigated protein on the right. PKG: protein kinase G; Pln: phospholamban; VASP: vasodilator stimulated phosphoprotein; pint: interaction p value *p < 0.05 vs. ShamCo; #p < 0.05 vs. AABCo.

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  Figure 5. Gene expression changes are prevented in response to Cinaciguat treatment. (a) Aortic banding resulted in the reactivation of the foetal gene program, as evidenced by the elevated expression of ANP, MHCβ, and NOS3, the decreased expression of MHCα. Expression of NOS3 and MHCα along with the MHC isoform expression ratio was normalised by the Cinaciguat treatment following aortic banding. The SERCA2a/Pln expression ratio was significantly increased in the AABCin rats compared to the AABCo animals. (b) Both HSP70 and Bcl-2 expression was markedly elevated in the AABCin group, indicating reinforced anti-apoptotic signalling in these animals. ANP: atrial natriuretic peptide; Bcl-2: B-cell lymphoma 2; HSP70: 70 kDa heat shock protein; MHCα/β: α and β isoform of myosin heavy chain; NOS3: endothelial nitric oxide synthase; Pln: phospholamban; SERCA2a: sarcoplasmic and endoplasmic reticulum Ca2+ ATPase isoform 2a; pint: interaction p value *p < 0.05 vs. ShamCo; #p < 0.05 vs. AABCo.