Relaxin suppresses atrial fibrillation in aged rats by reversing fibrosis and upregulating Na+ channels

Abstract

Background: Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly patients and has been correlated with enhanced age-dependent atrial fibrosis. Reversal of atrial fibrosis has been proposed as therapeutic strategy to suppress AF.

Objective: To test the ability of relaxin to reverse age-dependent atrial fibrosis and suppress AF.

Methods: Aged F-344 rats (24 months old) were treated with subcutaneous infusion of vehicle or relaxin (0.4 mg/kg/day) for 2 weeks. Rat hearts were excised, perfused on a Langendorff apparatus, and stained with voltage and Ca(2+) indicator dyes. Optical mapping and programmed electrical stimulation was used to test arrhythmia vulnerability and changes in electrophysiological characteristics. Changes in protein expression and Na(+) current density (INa) were measured by tissue immunofluorescence and whole-cell patch clamp technique.

Results: In aged rats, sustained AF was readily induced with a premature pulse (n = 7/8) and relaxin treatment suppressed sustained AF by a premature impulse or burst pacing (n = 1/6) (P < .01). Relaxin significantly increased atrial action potential conduction velocity and decreased atrial fibrosis. Relaxin treatment increased Nav1.5 expression (n = 6; 36% ± 10%) and decreased total collagen and collagen I (n = 5-6; 55%-66% ± 15%) in aged atria (P < .05) and decreased collagen I and III and TGF-β1 mRNA (P < .05). Voltage-clamp experiments demonstrated that relaxin treatment (100 nM for 2 days) increased atrial INa by 46% ± 4% (n = 12-13/group, P < .02).

Conclusion: Relaxin suppresses AF through an increase in atrial conduction velocity by decreasing atrial fibrosis and increasing INa. These data provide compelling evidence that relaxin may serve as an effective therapy to manage AF in geriatric patients by reversing fibrosis and modulating cardiac ionic currents.

Keywords: Aging; Atrial fibrillation; Fibrosis; Relaxin; Sodium channel.

Figure 1. Inducibility of atrial fibrillation (AF) in aged rats treated with relaxin (RLX) or vehicle

Aged rats were readily inducible into sustained AF while RLX treated aged rats were considerably more refractory to inducible AF.

Figure 2. Relaxin treatment (RLX) significantly decreases AF inducibility in aged rats

A: Representative action potential (AP) tracing in an aged rat. A premature pulse (S1–S2 interval 45 ms) elicits a transient episode of AF. B/C: When a shorter S1–S2 interval of 40 ms is delivered, sustained AF is induced. D: Representative AP tracing in an RLX treated aged rat. A premature pulse (S1–S2 interval 45 ms) is longer than the refractory period and captures. E: When an S1–S2 impulse which is shorter than the refractory period is delivered, no capture occurs. F: Burst pacing was unable to elicit AF in RLX treated aged rats.

Figure 3. Restitution kinetics (RK) of conduction velocity (CV) during S1–S2 (A) and S1 (B) pacing

RK measured for CV from the left and right atrium (LA and RA), respectively. All values are reported as mean ± SEM.

Figure 4. Effect of Relaxin on Atrial Fibrosis

The top panels are atrial immunohistological sections (20×) of aged rat hearts treated with vehicle (A) or relaxin (B). Phalloidin is represented in red and collagen I in green. The middle panels are aged rat atrial sections (40×) stained with picrosirius red. Dark red staining denotes fibrosis. Images C&D are from vehicle treated aged rats imaged under non-polarized (C) and polarized light (D). Images E&F are from relaxin treated aged rats under non-polarized (E) and polarized light (F). G and H quantify the collagen I or collagen to tissue ratio.

Figure 5. Relaxin treatment decreases expression of fibrosis related transcripts

Relative expression of profibrotic transcripts in relaxin treated aged rats relative to untreated controls isolated from atria tissue. All values are reported as mean ± SEM; *P<0.05.

Figure 6. Relaxin (RLX) treatment of F-344 rat atrial myocytes increases expression of voltage-gated Na⁺ channels

A: F-344 rat atrial myocytes were co-incubated with 100 nM RLX for 48 hours. As measured by immunofluorescence, the relative expression of Nav1.5 is increased by RLX. B: The left and right panels (40×) show representative rat atrial myocytes stained with Nav1.5 antibody without and with 100 nM relaxin, respectively. C: Current-to-voltage (I–V) plots for control and RLX-treated rat atrial myocytes demonstrate an upregulation of Na⁺ current density. All values are reported as mean ± SEM.

Figure 7. Relaxin increases expression of voltage-gated Na⁺ channels in the atria of aged rats

A representative image of left atrial tissue from an aged rat treated with vehicle (A) or relaxin (B). As measured by immunofluorescence, the relative expression of Nav1.5 is increased by relaxin (C). All values are reported as mean ± SEM.