The thromboxane receptor antagonist NTP42 promotes beneficial adaptation and preserves cardiac function in experimental models of right heart overload

Figures

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  FIGURE 1. Effect of NTP42:KVA4 treatment on RV hypertrophy and pathology in the MCT-PAH model. Normal control rats (“No MCT—Placebo”) and rats injected with MCT (60 mg/kg) were treated twice daily orally (PO BID) with either drug placebo (“MCT—Placebo”) or NTP42:KVA4 (1 mg/kg PO BID), starting from Day 7 following administration of MCT. (A–F) show: (A) RVSP in the “No MCT—Placebo,” “MCT—Placebo,” and NTP42:KVA4 groups [n = 5, 11, 7], (B) Fulton’s Index [n = 5, 9, and 8, respectively]; (C) RV cardiomyocyte size [n = 5, 11, 8]; (D) RV vascularization [n = 5, 11, 8]; (E) RV adaptation index [n = 6, 11, 8], and (F) RV fibrosis [n = 5, 11, 7]. Data presented are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. “MCT—Placebo,” according to one-way analysis of variance (ANOVA) with Holm-Šídák correction applied for multiple comparisons. (G,H) Show representative photomicrographs, selected from a random animal from each treatment group, of: (G) Anti-CD31-stained RV tissue captured at 200 × magnification (scale bars represent 40 μm), and (H) Masson’s trichrome-stained RV tissue captured at 200 × magnification (scale bars, 40 μm). Note that while Supplementary Table 1 provides…

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  FIGURE 2. Effect of NTP42:KVA4 treatment on right heart dimensions and geometry in the PAB model. Normal control rats (“Sham—Placebo”) and rats subjected to pulmonary arterial banding (PAB) were treated with either drug placebo (“PAB—Placebo”) or NTP42:KVA4 (1 mg/kg PO BID), starting from Day 2 following PAB. (A–D) Show ECHO-derived measurements of: (A) RVFWT in the “Sham—Placebo,” “PAB—Placebo,” and NTP42:KVA4 groups [n = 6, 8, and 7, respectively]; (B) RVEDD [n = 6, 8, 8]; (C) RVEDA [n = 7, 8, 7], and (D) RA area (RAA) [n = 6, 8, 7]. Data presented are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. “PAB—Placebo,” according to one-way ANOVA with Holm-Šídák correction. (E) Shows representative ECHO images from M-mode recordings and from 2D parasternal short-axis (PSAX) and apical four-chamber (A4C) views selected from a random animal from each treatment group and where blue and orange shading and lines delineate the RV and RA, respectively.

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  FIGURE 3. Effect of NTP42:KVA4 treatment on RV pressure, volume and function in the PAB model. Hemodynamic measurements of: (A) HR in the “Sham—Placebo,” “PAB—Placebo,” and NTP42:KVA4 groups [n = 5, 8, and 7, respectively]; (B) mAP [n = 6, 8, 7]; (C) RV ESP [n = 5, 8, 8]; (D) RV EDP [n = 6, 8, 8]; (E) RV ESV [n = 6, 8, 8]; (F) RV EDV [n = 6, 8, 8]; (G) CO [n = 6, 8, 8]; (H) RV EF [n = 6, 7, 8]; (I) RV Ees [n = 6, 8, 7], and (J) RV Eed [n = 6, 8, 7]. Data presented are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. “PAB—Placebo,” according to one-way ANOVA with Holm-Šídák correction. (K) shows representative RV PV loops from PAB study animals. The linear end-systolic and exponential end-diastolic PV relationships within each group are displayed as thick dashed or solid lines, respectively. Maximum/minimum PV points on the displayed representative loops, and the PV relationships plotted thereon, were adjusted to correspond approximately with the average values determined within treatment group.

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  FIGURE 4. Effect of NTP42:KVA4 treatment on RV hypertrophy and pathology in the PAB model. (A–H) show: (A) Fulton’s Index in the “Sham—Placebo,” “PAB—Placebo,” and NTP42:KVA4 groups [n = 6, 8, and 7, respectively]; (B) RV cardiomyocyte size [n = 7, 8, 7]; (C) RV vascularization [n = 6, 7, 8]; (D) RV adaptation index [n = 7, 8, 8], (E) RV ANP mRNA expression [n = 7, 8, 7], (F) RV BNP mRNA expression [n = 7, 8, 8], (G) RV fibrosis [n = 7, 8, 8], and (H) RV collagen type I/III ratio, calculated from the RV collagen type I and III mRNA expression for each animal, and expressing the ratio in arbitrary units (a.u.) [n = 7, 8, 6]. Data presented are the mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. “PAB—Placebo,” according to one-way ANOVA with Holm-Šídák correction. (I,J) show representative photomicrographs, selected from a random animal from each treatment group, of: (I) (Upper panel): Anti-CD31-stained RV tissue captured at 400 × magnification (scale bars represent 40 μm), (Lower panel): Image analysis overlay showing annotated cardiomyocyte cross-sectional area (gray outline) and CD31+ vessels (green dots), and (J) Masson’s trichrome-stained RV tissue captured at 400 ×…

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  FIGURE 5. Effect of NTP42:KVA4 treatment on isolated cardiomyocyte force transduction and calcium-handling protein expression in the PAB model. (A–D) show: (A) Steady-state PT measured at increasing sarcomere lengths (1.8–2.3 μm) from cardiomyocytes isolated from animals in the “Sham—Placebo,” “PAB—Placebo,” and NTP42:KVA4 groups [n = 6, 6, and 6, respectively]; (B) Maximum AT development at [Ca2+] 31.6 μmol/L [n = 6, 6, 6]; (C) Relative AT development in response to increasing submaximal free Ca2+ concentration ([Ca2+], 1–31.6 μmol/L) [n = 6, 6, 6], where calcium response curves were fitted using non-linear regression, and (D) Calcium sensitivity (pCa50) determined from individual regression analyses [n = 6, 6, 6]. (E,F) Show western blot protein expression of: (E) NCX1 and (F) SERCA2a, where representative RV lysates are displayed from 3 random animals from each treatment group and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels were used as loading control. The relative positions of the molecular size markers (kDa) are indicated to the right of the panels and the positions of the observed degraded fragments of NCX1 and SERCA2a are marked with red arrowheads to the…

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  FIGURE 6. RV expression of the TP in preclinical models of PAH and RV dysfunction. (A,B) Show two representative photomicrographs, selected from a random animal from each treatment group, of anti-TP-stained RV tissue from the: (A) MCT-PAH model, and (B) PAB model, where images were captured at 400 × magnification (scale bars represent 40 μm). (C) Shows relative immunohistochemical (IHC) expression of the TP in the MCT-PAH model in RV tissue from animals of the “No MCT—Placebo,” “MCT—Placebo,” and NTP42:KVA4 groups [n = 5, 11, and 8, respectively]. (D–E) Show: (D) relative TP IHC expression in the PAB model in RV tissue from animals of the “Sham—Placebo,” “PAB—Placebo,” and NTP42:KVA4 groups [n = 7, 8, and 7, respectively], and (E) RV TP mRNA expression [n = 7, 8, 7]. (F–H) Show representative photomicrographs, selected from a random animal from each treatment group, of anti-TP-stained RV tissue from: (F) Control groups of a previously described SuHx-PAH model (28), namely “No SuHx” and “SuHx,” where normal control rats (“No SuHx”) and rats treated with SuHx (“SuHx”) were treated PO BID with vehicle upon removal from hypoxia and continuing in normoxia until Day 49 (i.e., 4 weeks duration);…

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  FIGURE 7. RV expression of the TP in human PAH and other RV pathologies. (A–C) show three representative photomicrographs of anti-TP-stained human RV tissue obtained from: (A) Healthy donors; (B) PAH patients, and (C) DCM patients, where all images were captured at 150 × magnification (scale bars represent 50 μm). (D) Shows relative RV TP mRNA expression levels in Healthy (n = 5) or RV Failure patients (n = 5, data for PAH and DCM combined). Data presented are the mean ± SEM. **P < 0.01 according to unpaired Student’s t-test.

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  FIGURE 8. Specific antagonism of TP signaling may alleviate key mechanisms involved in cardiac dysfunction in PAH and other cardiac conditions. Schematic representation focusing on the potential mechanisms through which the TXA2/TP signaling axis may lead to cardiac dysfunction in PAH and other cardiac conditions. Signaling through the Gq-coupled TP leads to PLCβ activation, liberating IP3 and DAG from PIP2. IP3 release mobilizes Ca2+ from the sarco/endoplasmic reticulum via IP3R activation. IP3-mobilized Ca2+, along with extracellular Ca2+ influx via LTCCs, triggers a larger Ca2+ release via RyRs. LTCC-mediated Ca2+ influx may also occur via a TP-mediated PKC mechanism. Chronically elevated Ca2+ leads to hypertrophic signaling via NFAT. Ca2+ overload also activates the protease calpain, which degrades myofibrillar proteins including myosin and titin, as well as Ca2+-handling proteins including NCX1 and SERCA2a. This study has demonstrated decreased degradation of both NCX1 and SERCA2a following NTP42:KVA4 treatment. Furthermore, PKC-mediated phosphorylation of titin leads to increased stiffness, and it is hypothesized that the decreased cardiomyocyte stiffness observed herein following TP…