Pulmonary Hypertension-Associated Right Ventricular Cardiomyocyte Remodelling Reduces Treprostinil Function

Abstract

(1) Pulmonary hypertension (PH)-associated right ventricular (RV) failure is linked to a reduction in pulmonary vasodilators. Treprostinil has shown effectiveness in PAH patients with cardiac decompensation, hinting at potential cardiac benefits. We investigated treprostinil's synergy with isoprenaline in RV and LV cardiomyocytes. We hypothesised that disease-related RV structural changes in cardiomyocytes would reduce contractile responses and cAMP/PKA signalling activity. (2) We induced PH in male Sprague Dawley rats using monocrotaline and isolated their ventricular cardiomyocytes. The effect of in vitro treprostinil and isoprenaline stimulation on contraction was assessed. FRET microscopy was used to study PKA activity associated with treprostinil stimulation in AKAR3-NES FRET-based biosensor-expressing cells. (3) RV cells exhibited maladaptive remodelling with hypertrophy, impaired contractility, and calcium transients compared to control and LV cardiomyocytes. Combining treprostinil and isoprenaline failed to enhance inotropy in PH RV cardiomyocytes. PH RV cardiomyocytes displayed an aberrant contractile behaviour, which the combination treatment could not rectify. Finally, we observed decreased PKA activity in treprostinil-treated PH RV cardiomyocytes. (4) PH-associated RV cardiomyocyte remodelling reduced treprostinil sensitivity, inotropic support, and impaired relaxation. Overall, this study highlights the complexity of RV dysfunction in advanced PH and suggests the need for alternative therapeutic strategies.

Keywords: cardiomyocytes; cell length deflection; pulmonary hypertension; right ventricle; sarcomere shortening; treprostinil.

Figure 1

Characterisation of pulmonary hypertension (PH) and Sham rats. (a) Representative traces of the body weight changes in PH and Sham rats. The dotted line indicates the time point of MCT or vehicle administration. (b) Normalised body weight (BW) of PH and Sham rats. (c) Normalised heart weight of PH and Sham rats. (d) Representative images of explanted lungs from PH (red box) and Sham (black box) rats post-treatment. The lungs from PH animals exhibited lung damage (indicated with white arrows). (e) Heart weight (HW)/tibia length (TL) ratios of PH and Sham rats. (f) Lung weight (LW)/TL ratios of PH and Sham rats. PH animals (black square); Sham animals (white square). Data presented as mean ± SEM, n = 5; * p < 0.05, **** p < 0.0001, by Mann–Whitney test.

Figure 2

Characterisation of RV and LV cardiomyocytes from pulmonary hypertension (PH) and Sham animals. (a) Representative images of the PH and Sham RV and LV cardiomyocyte; scale bar = 20 μm. Mean cardiomyocyte cell width and length (b), aspect ratio (c), and power of sarcomere regularity (d). PH RV cardiomyocytes (black dots), PH LV cardiomyocytes (black triangles), Sham RV cardiomyocytes (white dots), Sham LV cardiomyocytes (white tringles). Data presented as mean ± SEM, n = 5; ** p < 0.01, **** p < 0.0001 by nested t-test.

Figure 3

Remodelling of ventricular cardiomyocyte contraction and calcium transients (CaTs) in pulmonary hypertension (PH). Representative calcium transients (a), Sarcomere Shortening (b), and Cell Length Deflections (c) in PH (red) and Sham (black) right (RV) and left (LV) ventricular cardiomyocytes.

Figure 4

Characterisation of the effects of treprostinil, isoprenaline, and the combination of treprostinil and isoprenaline on Ca²⁺ transients (CaTs) in PH ventricular cardiomyocyte populations. (a) Representative traces of CaTs in RV and LV cardiomyocytes treated with treprostinil (black solid line), isoprenaline (red dashed line), and the combination of treprostinil and isoprenaline (blue dotted line). Treatment-associated normalised mean CaT parameters of PH (red) and Sham (black) RV the (b) and LV (c) cardiomyocytes. PH RV cardiomyocytes (black dots), PH LV cardiomyocytes (black triangles), Sham RV cardiomyocytes (white dots), Sham LV cardiomyocytes (white tringles). Data expressed as mean ± SEM; n = 5 rats; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 based on one-way ANOVA with Tukey’s multiple comparisons.

Figure 5

Characterisation of effects of treprostinil, isoprenaline, and the combination of treprostinil and isoprenaline on PH (red) and Sham (black) RV cardiomyocytes. Representative traces (a) and normalised mean percentage Sarcomere Shortening (b) and Cell Length Deflection (c). PH RV cardiomyocytes (black dots), Sham RV cardiomyocytes (white dots). Data expressed as mean ± SEM, n = 5 rats; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by nested one-way ANOVA with Tukey’s multiple comparisons.

Figure 5

Characterisation of effects of treprostinil, isoprenaline, and the combination of treprostinil and isoprenaline on PH (red) and Sham (black) RV cardiomyocytes. Representative traces (a) and normalised mean percentage Sarcomere Shortening (b) and Cell Length Deflection (c). PH RV cardiomyocytes (black dots), Sham RV cardiomyocytes (white dots). Data expressed as mean ± SEM, n = 5 rats; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by nested one-way ANOVA with Tukey’s multiple comparisons.

Figure 6

Characterisation of effects of treprostinil, isoprenaline and the combination of treprostinil and isoprenaline on PH (red) and Sham (black) LV cardiomyocytes. Representative traces (a) and normalised mean percentage Sarcomere Shortening (b) and Cell Length Deflection (c). PH animals (black square); Sham animals (white square). Data expressed as mean ± SEM, n = 5 rats; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by nested one-way ANOVA with Tukey’s multiple comparisons.

Figure 6

Characterisation of effects of treprostinil, isoprenaline and the combination of treprostinil and isoprenaline on PH (red) and Sham (black) LV cardiomyocytes. Representative traces (a) and normalised mean percentage Sarcomere Shortening (b) and Cell Length Deflection (c). PH animals (black square); Sham animals (white square). Data expressed as mean ± SEM, n = 5 rats; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by nested one-way ANOVA with Tukey’s multiple comparisons.

Figure 7

Nuclear FRET responses in PH and Sham ventricular cardiomyocytes expressing AKAR3-NES FRET sensor. (a) Pseudo-colour images of RV cardiomyocytes with nuclear localisation of AKAR3-NES FRET-based sensor. Representative normalised FRET curves (Sham—solid black line; PH—red dashed line) and Log(agonist) vs. response Line chart (Sham white symbol/black line; PH—black symbol/red line) to treprostinil (0.01, 0.1, 1 and 10 μM) stimulation followed by Saturator (10 μM Forskolin, 100 μM IBMX) of RV (circle symbol) (b) and LV (triangle symbol) (c). Data expressed as mean ± SEM; n = 30 from 5 animals; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 based on two-way ANOVA with Šídák’s multiple comparisons.