Right Ventricular Myocardial Stiffness in Experimental Pulmonary Arterial Hypertension: Relative Contribution of Fibrosis and Myofibril Stiffness

Abstract

Background: The purpose of this study was to determine the relative contribution of fibrosis-mediated and myofibril-mediated stiffness in rats with mild and severe right ventricular (RV) dysfunction.

Methods and results: By performing pulmonary artery banding of different diameters for 7 weeks, mild RV dysfunction (Ø=0.6 mm) and severe RV dysfunction (Ø=0.5 mm) were induced in rats. The relative contribution of fibrosis- and myofibril-mediated RV stiffness was determined in RV trabecular strips. Total myocardial stiffness was increased in trabeculae from both mild and severe RV dysfunction in comparison to controls. In severe RV dysfunction, increased RV myocardial stiffness was explained by both increased fibrosis-mediated stiffness and increased myofibril-mediated stiffness, whereas in mild RV dysfunction, only myofibril-mediated stiffness was increased in comparison to control. Histological analyses revealed that RV fibrosis gradually increased with severity of RV dysfunction, whereas the ratio of collagen I/III expression was only elevated in severe RV dysfunction. Stiffness measurements in single membrane-permeabilized RV cardiomyocytes demonstrated a gradual increase in RV myofibril stiffness, which was partially restored by protein kinase A in both mild and severe RV dysfunction. Increased expression of compliant titin isoforms was observed only in mild RV dysfunction, whereas titin phosphorylation was reduced in both mild and severe RV dysfunction.

Conclusions: RV myocardial stiffness is increased in rats with mild and severe RV dysfunction. In mild RV dysfunction, stiffness is mainly determined by increased myofibril stiffness. In severe RV dysfunction, both myofibril- and fibrosis-mediated stiffness contribute to increased RV myocardial stiffness.

Keywords: collagen; fibrosis; heart failure; hypertension; right ventricular dysfunction.

Figure 1.

Right ventricular (RV) myocardial stiffness in skinned trabecular strips. A, Schematic representation of skinned trabecular strip measurements. B, Representative examples of RV myocardial stiffness measurements in skinned trabecular strips of control, mild RV dysfunction, and severe RV dysfunction before (solid line) and after incubation with KCl/KI. C, Total RV myocardial stiffness was significantly increased in both mild RV dysfunction (blue bars) and severe RV dysfunction (red bars) in comparison to controls (white bars). In severe RV dysfunction, increased RV myocardial stiffness could be explained by both increased fibrosis- and myofibril-mediated stiffness, whereas in mild RV dysfunction only myofibril-mediated stiffness was increased in comparison to control. Data are presented as mean±SEM. Controls: n=5, mild RV dysfunction: n=5, and severe RV dysfunction: n=5. **P<0.01 vs control.

Figure 2.

Right ventricular (RV) fibrosis and collagen I/III ratio. A, RV histology sections were stained for collagen using a Picrosirius red staining and analyzed under double-polarized light, results from Andersen et al. A gradual increase in RV fibrosis was found in mild and severe RV dysfunction in comparison to control. Data presented as percentage of controls. B, Collagen type I and III mRNA expression was determined by qPCR, and the collagen I/III ratio was calculated. Collagen I/III expression was significantly increased in severe RV dysfunction. Data presented as mean±SEM. Controls: n=13, mild RV dysfunction: n=5, and severe RV dysfunction: n=5. *P<0.05, ^#P<0.05.

Figure 3.

Skinned cardiomyocytes. A and B, Right ventricular (RV) cardiomyocyte stiffness was gradually increased in mild and severe RV dysfunction in comparison to control. C and D, Protein kinase A (PKA) incubation significantly decreased cardiomyocyte stiffness in mild and severe RV dysfunction but remained significantly elevated in comparison to controls. Data are presented as mean±SEM. Controls: n=5, mild RV dysfunction: n=5, and severe RV dysfunction: n=5. **P<0.01, ***P<0.001 vs control. The P_interaction value represents the response of passive tension on increase in sarcomere length or PKA incubation.

Figure 4.

Right ventricular (RV) titin isoform composition and titin phosphorylation. A, Titin isoform ratio determined on 1% agarose gels stained with Coomassie Blue by dividing the N2BA isoform content to the more abundant N2B isoform and expressed in percentages. N2BA/total titin ratio was similar in control and severe RV dysfunction, whereas elevated in mild RV dysfunction. B, Total titin phosphorylation was determined by ProQ staining for phosphorylation divided by Sypro staining for total protein content. Titin phosphorylation was lower both in mild and severe RV dysfunction. C, Example of ProQ and Sypro staining of titin. Data are presented as mean±SEM. Controls: n=5, mild RV dysfunction: n=5, and severe RV dysfunction: n=5. *P<0.05. A.u. indicates arbitrary units.

Figure 5.

Components of trabecular stiffness. Schematic representation of the relative contribution of fibrosis and cardiomyocytes to trabecular stiffness. RV indicates right ventricular.