Pathological effects of chronic myocardial infarction on peripheral neurons mediating cardiac neurotransmission

Abstract

Objective: To determine whether chronic myocardial infarction (MI) induces structural and neurochemical changes in neurons within afferent and efferent ganglia mediating cardiac neurotransmission.

Methods: Neuronal somata in i) right atrial (RAGP) and ii) ventral interventricular ganglionated plexi (VIVGP), iii) stellate ganglia (SG) and iv) T1-2 dorsal root ganglia (DRG) bilaterally derived from normal (n=8) vs. chronic MI (n=8) porcine subjects were studied. We examined whether the morphology and neuronal nitric oxide synthase (nNOS) expression in soma of RAGP, VIVGP, DRG and SG neurons were altered as a consequence of chronic MI. In DRG, we also examined immunoreactivity of calcitonin gene related peptide (CGRP), a marker of afferent neurons. Chronic MI increased neuronal size and nNOS immunoreactivity in VIVGP (but not RAGP), as well as in the SG bilaterally. Across these ganglia, the increase in neuronal size was more pronounced in nNOS immunoreactive neurons. In the DRG, chronic MI also caused neuronal enlargement, and increased CGRP immunoreactivity. Further, DRG neurons expressing both nNOS and CGRP were increased in MI animals compared to controls, and represented a shift from double negative neurons.

Conclusions: Chronic MI impacts diverse elements within the peripheral cardiac neuraxis. That chronic MI imposes such widespread, diverse remodeling of the peripheral cardiac neuraxis must be taken into consideration when contemplating neuronal regulation of the ischemic heart.

Keywords: Autonomic nervous system; CGRP; Chronic myocardial infarction; Dorsal root ganglia; Intrinsic cardiac nervous system; Stellate ganglion; nNOS.

Figure 1. Myocardial Infarct Model

The induction and location of anteroapical myocardial infarction is shown in the upper and lower panels. The course of the left anterior descending (LAD) oronary artery is indicated by the black arrows in panel A. Injection of microspheres is shown in panel B. Folllowing microsphere injection, absence of flow in the LAD is indicated by the black arrows in panel C. Panel D shows a 3-dimensional reconstruction of an infarcted heart, with the region of anterior infaction indicated by the black arrow heads. Panels E and F show axial and coronal contrast-enhanced magnetic resonance slices of an infarcted heart respectively, with the infarct indicated by the white arrow heads. Panels G and H show the methodology of infarct segmentation, and the quantifications of left ventricular (LV) volume and infarct volume. Data in panel H are mean±standard deviation.

Figure 1. Myocardial Infarct Model

The induction and location of anteroapical myocardial infarction is shown in the upper and lower panels. The course of the left anterior descending (LAD) oronary artery is indicated by the black arrows in panel A. Injection of microspheres is shown in panel B. Folllowing microsphere injection, absence of flow in the LAD is indicated by the black arrows in panel C. Panel D shows a 3-dimensional reconstruction of an infarcted heart, with the region of anterior infaction indicated by the black arrow heads. Panels E and F show axial and coronal contrast-enhanced magnetic resonance slices of an infarcted heart respectively, with the infarct indicated by the white arrow heads. Panels G and H show the methodology of infarct segmentation, and the quantifications of left ventricular (LV) volume and infarct volume. Data in panel H are mean±standard deviation.

FIgure 2. Differential Neuronal Enlargement and Increases nNOS Expression in Cardiac Ganglionated Plexi

Representative immunohistochemical stains of neurons in the right atrial (RAGP) and ventral interventricular ganglionated plexi (VIVGP) are shown in panel A (scale bar: 50 µm). The larger neurons in the VIVGP can be appreciated, as well as the intensity of neuronal nitric oxide synthanse (nNOS). The percentage of nNOS positive neurons in the RAGP and VIVGP are shown in panel B. The relationship between neuronal morphology and nNOS immunoreactivity for RAGP and VIVGP neurons is shown in panel C.

Figure 3. Impact of chronic ischemia on morphologic and neurochemical profiles of sympathetic afferent neurons

In Panel A, representative immunohistochemical stains of neuronal nitric oxide synthase (nNOS) and calcitonin gene related peptide (CGRP) in dorsal root ganglion (DRG) neurons is shown. The graphical quantifications of nNOS and CGRP immunoreactivity is shown in panel B. The distribution of neuronal sizes by CGRP and nNOS immunoreactivity is shown in panel C. CGRP and nNOS co-labeling in DRG neurons is shown in panel D in control and MI states. The decrease in double negative neurons, and increase in double positive neurons can be readily appreciated. Panel E shows the the intensity of CGRP and nNOS staining in controls and MI animals (scale bar: 50 µm).

Figure 3. Impact of chronic ischemia on morphologic and neurochemical profiles of sympathetic afferent neurons

In Panel A, representative immunohistochemical stains of neuronal nitric oxide synthase (nNOS) and calcitonin gene related peptide (CGRP) in dorsal root ganglion (DRG) neurons is shown. The graphical quantifications of nNOS and CGRP immunoreactivity is shown in panel B. The distribution of neuronal sizes by CGRP and nNOS immunoreactivity is shown in panel C. CGRP and nNOS co-labeling in DRG neurons is shown in panel D in control and MI states. The decrease in double negative neurons, and increase in double positive neurons can be readily appreciated. Panel E shows the the intensity of CGRP and nNOS staining in controls and MI animals (scale bar: 50 µm).

Figure 4. Myocardial Infarction Induces Increased nNOS Expression with Neuronal Enlargement in Stellate Ganglia

The morphologic profile and neuronal nitirc oxide synthase (nNOS) immunoreactivity of left stellate ganglion (LSG) neurons are shown in panel A. The Neurons can be appreciated to be larger, and nNOS immunoreacitivity greater. Panels B and C show the fraction of nNOS immunoreactivity, and the distribution of neuronal sizes based on nNOS staining in stellate ganglion neurons, respectively (scale bar: 50 µm).