Pulmonary Vein Ganglia Are Remodeled in the Diabetic Heart

Abstract

Background Cardiac autonomic neuropathy is thought to cause adverse cardiovascular effects in diabetes mellitus. Pulmonary vein ganglia ( PVG ), which have been implicated in normal and abnormal heart rhythm regulation, have not been fully investigated in type 1 diabetes mellitus (T1D). We examined the functional and anatomical effects of T1D on PVG and studied the details of T1D-induced remodeling on the PVG structure and function. Methods and Results We used a mouse model of T1D (Akita mouse), immunofluorescence, isolated Langendorff-perfused hearts, and mathematical simulations to explore the effects of T1D on PVG . Whole-mount atrial immunofluorescence of choline acetyltransferase and tyrosine hydroxylase labeling showed that sympathetic and parasympathetic somas of the PVG neurons were significantly hypotrophied in T1D hearts versus wild type. Stimulation of PVG in isolated Langendorff-perfused hearts caused more pronounced P-P interval prolongation in wild type compared with Akita hearts. Propranolol resulted in a comparable P-P prolongation in both phenotypes, and atropine led to more pronounced P-P interval shortening in wild type compared with Akita hearts. Numerical modeling using network simulations revealed that a decrease in the sympathetic and parasympathetic activities of PVG in T1D could explain the experimental results. Conclusions T1D leads to PVG remodeling with hypotrophy of sympathetic and parasympathetic cell bodies and a concomitant decrease in the PVG sympathetic and parasympathetic activities.

Keywords: autonomic nervous system; diabetes mellitus; pulmonary vein.

Figure 1

Immunofluorescence of murine atrial intrinsic ganglia. Whole‐mount atrial preparation from a wild‐type heart stained with tyrosine hydroxylase (green) and choline acetyltransferase (red). The pulmonary vein ganglion boxed in white is shown at higher magnification in Figure 2. Ao indicates aorta; IVC, inferior vena cava; LA, left atrium; PV, pulmonary veins; RA, right atrium; SAN, sinoatrial node; SVC, superior vena cava.

Figure 2

Quantification of pulmonary vein ganglia (PVG) soma size. A, Immunofluorescence of mouse PVG. Tyrosine hydroxylase (TH, green) and choline acetyltransferase (ChAT, red). The PVG boxed in white in Figure 1 is shown at higher magnification. B, The white arrows point to nerve containing ChAT‐ and TH‐positive fibers. Cumulative frequency distribution of ganglionic neuronal cell body areas of ChAT‐positive and of biphenotypic (ChAT‐ and TH‐positive) cells. Cells that are only TH positive were not common. The left panel shows cumulative frequency distribution of ChAT‐positive cell body area, where the mean soma size was 130.2±0.22 μm², R ²=0.99 (N=3 hearts, n=160 cells) for wild‐type (WT) and 118.4±0.25 μm², R ²=0.99 (N=3, n=161 cells) for Akita hearts (P<0.001). The right panel shows cumulative frequency distribution of biphenotypic cell body areas, where the mean soma size was 141.7±0.161 μm², R ²=0.99 (N=3, n=152 cells) for WT and 127±0.36 μm², R ²=0.99 (N=3, n=150 cells) for Akita hearts (P<0.001).

Figure 3

Acetylcholinesterase staining of wild‐type (WT) and Akita (Akt) atria. A and B, Stained WT and Akita hearts respectively. Pulmonary vein ganglia (PVG) are indicated by white double arrows. Triple arrowheads indicate nerves projecting from PVG to the sinoatrial node (SAN). C and E, Enlarged views of the SAN area boxed in white in WT and Akita hearts. D and F, ×20 magnification of the PVG (white double arrows) in panels A and B. Ao indicates aorta; LA, left atrium; PLA, posterior left atrium; RA, right atrium; SVC, superior vena cava.

Figure 4

Modulation of the P‐P interval in the isolated Langendorff‐perfused wild‐type (WT) and Akita (Akt) mouse hearts by pulmonary vein ganglia (PVG) stimulation. A, ECG traces from WT and Akita hearts. The P‐P intervals of the 3 beats before and after stimulation were measured. Stim=700‐ms stimulation. B, P‐P intervals of the 20 beats following 700‐ms stimulation of the PVG in control (WT, n=7; Akt, n=7), propranolol (WT, n=8; Akt, n=8), or atropine (WT, n=5; Akt, n=5). *P<0.01, WT vs Akita hearts, Wilcoxon rank sum test. C, Average of 3 P‐P intervals preceding PVG stimulation (pre) and the average of the 3 P‐P intervals immediately following PVG stimulation of the experiments shown in panel B during control and treatment with propranolol or atropine. ^# P<0.05, *P<0.01, pre vs post, Wilcoxon signed rank test.

Figure 5

Average change in P‐P interval of the first 3 beats after stimulation. Change in P‐P interval after stimulation with 300‐, 500‐, 700‐, and 1200‐ms stimulus trains in (A) control (wild type [WT], n=7; Akita [Akt], n=7), (B) propranolol (WT, n=8; Akt, n=8), (C) atropine (WT, n=5; Akt, n=5), and (D) propranolol plus atropine (WT, n=4; Akt, n=4). *P<0.05, **P<0.01, WT vs Akita hearts, t test. CL indicates cycle length.

Figure 6

Numerical model. A, A ganglion was modeled as having 15 choline acetyltranferase positive cells, and 8 biphenotypic (tyrosine hydroxylase and choline acetyltranferase positive cells). The nerve projecting from the ganglion to the sinoatrial node (SAN) cell has sympathetic and parasympathetic fibers. B, Action potentials from the different components of the circuit. Stim shows the time when stimulation is on. ChAT and ChAT plus TH populations increased their firing rates in response to stimulation, and SAN cell action potential firing slowed down. C, Simulated SAN action potential cycle length changes (Δ P‐P) in wild‐type (WT) and Akita (Akt) cases without and with propranolol or atropine. D, Experimental data from Figure 5 (700‐ms stimulus duration) showing good agreement between numerical and experimental data. ChAT indicates choline acetyltransferase; TH, tyrosine hydroxylase.