Vagally-mediated heart block after myocardial infarction associated with plasticity of epicardial neurons controlling the atrioventricular node

FIGURE 1. MI hearts are more susceptible to AV conduction block during stimulation of the left vagus nerve. (A) Cardiac responsiveness to cervical vagal stimulation was assessed in anesthetized, open chest pigs, after bilateral transection of the vagus nerves. RVNS, right vagus nerve stimulation; LVNS, left vagus nerve stimulation. (B) Example tracings of ventricular rate from a control (top) and MI (bottom) animal during stimulation of the left cervical vagus nerve (LVNS) at 5, 10, and 20 Hz. Note the augmented bradycardic response in MI animal due to AV block (inset) during 20 Hz stimulation. This was typical for all MI animals tested. (C) Summary changes in atrial and ventricular rate during stimulation of the right (RVNS) and left (LVNS) cervical vagus. In MI animals, 20 Hz LVNS stimulation produced a significantly greater decrease in ventricular rate due to propensity for AV node block. * Indicates P < 0.05, Šidák’s multiple comparisons test. (D) Baseline heart rate, animal weights, and current intensity for LVNS or RVNS were not different between groups.

FIGURE 2. Neuroanatomy of the IVC-ILA ganglionated plexus. (A) Area of the IVC-ILA is circumscribed by the hatched line on a photograph of the dorsal surface of the porcine heart. LA, left atrium; RA, right atrium; CS, coronary sinus; LV, left ventricle; RV, right ventricle. (B) Stitched confocal image (10×) showing the network of interganglionic nerves and intrinsic cardiac ganglia within the boxed area in (A). scale bar: 1 mm (C) Representative examples of intrinsic cardiac ganglia isolated from control (left four panels) and MI (right four panels) pig hearts. Scale bars: 100 μm.

FIGURE 3. Depressed excitability of ICNs from MI animals shown by intracellular assessment of ICN membrane physiology. (A) Schematic overlay of the stimulation and recording configuration over a stitched confocal image (40×) of an individual intrinsic cardiac ganglion. Bipolar stimulating electrodes (labeled S1 and S2) were placed on interganglionic nerves. The magenta cell was backfilled with neurobiotin from the recording electrode. Scale bar is 500 μm. (B) Split images of boxed region in (A), showing all cells (left) and single labeled cell (right). Scale bar 50 μm for both panels. (C) The cross-sectional areas of ICNs from pigs with MI were larger than controls. * Indicates P < 0.05, Fisher’s exact test. (D) The steady state membrane voltage in response to intracellularly injected current is plotted for MI and control ICNs. Input resistances were similar between groups but MI cells tended to have greater depolarizations in response to 400 and 500 pA currents. * Indicates P < 0.05, Šidák’s multiple comparisons test. (E) Membrane potential responses to long duration (500 ms), intracellular depolarizing current pulses from a control and MI neuron. Note that ICNs from control animals…

FIGURE 4. PACAP increased excitability of control and MI ICNs. (A) PACAP-immunoreactivity was localized to pericellular varicosities surrounding some, but not all ICNs. (B) Extracellular application of PACAP (20 nM) increased membrane excitability as evident by a greater number of action potentials in response to depolarizing current steps. Dash indicates 0 mV. Resting membrane potential = 58 mV. (C) The change in excitability for the response shown in B is plotted. Cells tended to fire only a single action potential before PACAP. After PACAP application this cell fired a maximum of five action potentials. (D) The percentage of ICNs from MI animals which fired a greater number of action potentials after PACAP application was similar to controls. (E,F) The summary excitability curves show mean changes in excitability before and after PACAP application for control ICNs (E) and MI ICNs (F). The increase in excitability after PACAP was greater at control ICNs. * Indicates P < 0.05, Šidák’s multiple comparisons test. † Indicates P < 0.05 MI vs control, Šidák’s multiple comparisons test.

FIGURE 5. Muscarinic slow potentials are more prevalent and of greater amplitude after MI. (A) Representative tracings of a slow-EPSPs evoked from one cell before and after administration of hexamethonium (Hex). Hex blocked fast-synaptic potentials elicited by acetylcholine activation of ganglion nicotinic receptors. In the presence of Hex, the slow-EPSP could be elicited reproducibly, as long as the interval between trains was greater than 200 ms. (B) In a separate cell, in the presence of Hex, 20 Hz stimulation of an interganglionic nerve bundle caused a slow depolarization (∼13 mV) of the postsynaptic membrane. The slow depolarization was inhibited by application of the muscarinic receptor inhibitor atropine. (C) A significantly greater fraction of MI ICNs responded with muscarinic s-EPSPs. (D) In addition to being observed more frequently, the amplitudes of the s-EPSPs were greater in MI ICNs.

FIGURE 6. Inhibition of M-current by bath application of BaCl2 identifies role of M-current in regulation of pig ICN excitability. (A,B) Membrane potential tracing showing a typical response from a control ICN in control PSS and after extracellular addition of BaCl2 (1 mM). Barium application increased membrane excitability, evident by a greater number of action potentials in response to membrane depolarization. (C) In total (n = 7), barium caused a modest but significant change in the excitability of the cardiac neuron membrane.