Relaxin Inhibits Ventricular Arrhythmia and Asystole in Rats With Pulmonary Arterial Hypertension

Figures

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  Figure 1. Effects of RLX on RV hypertrophy and pulmonary pressure in PAH. Sugen/Hypoxia significantly increased RV hypertrophy as measured by (A) Fulton Index and (B) RV/Tibia ratio. Low (+RLX-LD) and high (+RLX-HD) had no effect on RV hypertrophy. (C) Sugen/Hypoxia significantly increased systolic pressure and Relaxin failed to alter it. (D) Heart rate did not significantly change in all four groups. (E) SuHx hearts had a small but significant increase in myocyte cross-sectional area (CSA) (WGA-label) which was prevented by RLX-HD but not RLX-LD; n > 100 cells/group. *Indicates p < 0.05 vs. control. #Indicates p < 0.05 vs. SuHx.

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  Figure 2. Relaxin significantly altered Doppler flow patterns. (A) Doppler flow velocities from the RV outflow-track exhibited marked mid-systolic notching (indicative of decreased pulmonary compliance) and an increase in PFV in SuHx compared to control (Ctrl) rats [(A) top panels]. SuHx+ low-dose of RLX (RLX-LD) prevented the increase in PFV but did not alter the notching (bottom right panel). High-dose of RLX (RLX-HD) prevented the notching but not PFV (bottom left panel). PAH (SuHx) rats had no significant changes in (B) heart rate, and a tendency to increases in (C) mean Velocity, (D) Stroke Distance, (E) Cardiac Output and (F) Stroke Volume compared to controls. RLX-LD had a tendency to prevent the increase in these parameters caused by SuHx (D–F) but these reductions did not reach statistical significance. RLX-HD was less effective at preventing the higher mean velocity, stroke distance, cardiac output and stroke volume in SuHx compared to RLX-LD.

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  Figure 3. Effects of RLX on RV fibrosis and cell-cell coupling. (A) PAH (SuHx) hearts developed an increase in fibrosis that was prevented by treating rats with SuHx + RLX-HD [(A): RLX-HD panel], but not in rats treated with SuHx + RLX-LD [(A): RLX-LD panel]. (B) RV cell-cell junctions were disrupted by SuHx treatment compared to controls, as measured by β-catenin expression and β-catenin localization to intercalated disk (Ctrl). PAH rats (SuHx) had a marked reduction of β-catenin expression [(B): SuHx panel] compared to control rats [(B): Ctrl panel]. PAH rats treated with RLX-LD [(B): RLX-LD panel] did not show appreciable differences in β-catenin compared to rats treated with SuHx, alone. In PAH rats, RLX-HD treatment significantly prevented the reduction of β-catenin expression [(B): RLX-HD panel] (n ≥ four animals/per group. *Indicates p < 0.05 vs. control. #Indicates p < 0.05 vs. SuHx.

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  Figure 4. Effects of RLX on CV and Restitution Kinetics (RK). (A): Plots of CV vs. cycle length show a 50% decrease in CV in the RV (left) and LV (right) of SuHx hearts compared to controls. RLX treatments partially prevented CV slowing in a dose-dependent manner. (B): RK plots the CV of the premature AP as a function of S1-S2 inter-pulse interval. CV RK curves of SuHx hearts have a steeper slope and are reduced compared to controls in the RV and LV, and RLX reduces the slope of CV RK curves and increases CVs in a dose-dependent manner. *Indicates p < 0.05 vs. SuHx.

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  Figure 5. Arrhythmia Phenotypes of PAH hearts. Optical mapping revealed that SuHx hearts exhibited two equally prevalent phenotypes (A,B). (A): SuHx hearts were in bradycardia (i) and could be briefly paced at higher rates (ii) which was followed by cardiac arrest (iii) (n = 4/11). Once in cardiac arrest, electrical stimuli (10X threshold, 1–10 ms) failed to capture. (B): Other SuHx hearts exhibited spontaneous ventricular fibrillations (VF) (i) which increased in durations, were self-terminating (ii) and transitioned to a sustained VF, in this illustration in ~ 3 min) (iii) (n = 5/11). (C): In the SuHX+RLX LD (RLX LD) group, Relaxin at low dose suppressed both sustained VF and cardiac arrest (n = 0/8): (i) AP recordings in sinus rhythm, followed by 2 s of rapid pacing and a premature impulse at S1-S2 of 85 ms which failed to elicit a VF. (ii) In another SuHx+RLX-LD heart, AP record a bradycardia, followed by rapid pacing and a premature impulse (S1-S2 = 70 ms). Here, the premature impulse elicits a brief transient arrhythmia (4 beats) and a return to sinus rhythm. (iii) An example of burst pacing (4 s) which elicits a transient VF that self terminates in <3 s. (D): In the SuHX+RLX HD (RLX…

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  Figure 6. Summary of Experimental Arrhythmia Phenotypes. (A) Histogram of the onset of VT/VF. (B) Histogram of the Percentage of Hearts that exhibited Transient VF lasting > 10 s, sustained VF or sustained Asystole. (C) Histogram of the onset of Bradycardia/Asystole.

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  Figure 7. Dithiothreitol (DTT) rescues PAH hearts. (A): Dual optical mapping of voltage (blue) and Ca2+ (red) illustrates an asystolic SuHx heart under sinus rhythm that fails to capture during pacing (S1–S1 = 300 ms) with an occasional transient salvo of APs but is otherwise not excitable. (B): The same heart was rescued by perfusing with 1 mM DTT (2–3 min), as electrical impulses now successfully capture at each stimulus (n = 2/2 hearts). (C): Voltage and Calcium mapping of a SuHx heart in sinus rhythm illustrates spontaneous bursts of tachycardia which typically progress to sustained VF. (D): Perfusion of the same heart with DTT (1 mM) stabilized the intrinsic sinus rhythm for the remainder of the experiment (n = 2/2).

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  Figure 8. Relaxin prevents oxidative stress in PAH hearts. Relaxin quantitatively prevents the overexpression of Nrf2 and Glutathione Transferase but not Glutathione Reductase. Each lane was loaded with 20 μg of protein and the variability of loaded proteins was due to high protein levels in some tissue samples resulting in the addition of small volumes (2 μl) in some lanes. Data were analyzed by ANOVA followed by Tukey's multiple comparisons test. Only statistically significant differences are noted in the graph (n = 4 animals per condition; each sample was run at least in duplicate in separate gels; significance: *p < 0.05; **p < 0.01; ***p < 0.001).